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Introduction: Research on snakebite has mostly been conducted on settled populations and current risk factors and potential interventions are therefore most suited for these populations. There is limited epidemiological data on mobile and nomadic populations, who may have a higher risk of snakebite. Methods and results: We conducted a scoping review to gather evidence on survey methods used in nomadic populations and compared them with contemporary survey methods used for snakebite research. Only 16 (10.5%) of 154 articles reportedly conducted on pastoralist nomadic populations actually involved mobile pastoralists. All articles describing snakebite surveys (n = 18) used multistage cluster designs on population census sampling frames, which would not be appropriate for nomadic populations. We used geospatial techniques and open-source high-resolution satellite images to create a digital sampling frame of 50,707 households and used a multistage sampling strategy to survey nomadic and semi-nomadic populations in Samburu County, Kenya. From a sample of 900 geo-located households, we correctly identified and collected data from 573 (65.4%) households, of which 409 were in their original locations and 164 had moved within 5km of their original locations. We randomly sampled 302 (34.6%) households to replace completely abandoned and untraceable households. Conclusion: Highly mobile populations require specific considerations in selecting or creating sampling frames and sampling units for epidemiological research. Snakebite risk has a strong spatial component and using census-based sampling frames would be inappropriate in nomadic populations. We propose using open-source satellite imaging and geographic information systems to improve the conduct of epidemiological research in these populations. Author summary: Nomadic populations are at increased risk of snakebites, but interventions will not be developed or improved without reliable data on this risk. Highly mobile populations require specific considerations in selecting or creating sampling frames and sampling units for epidemiological research, and using census-based sampling frames would be inappropriate. We conducted scoping reviews to gather evidence on survey methods used in nomadic populations and compared them with contemporary survey methods used for snakebite research, which we used to inform our methodology. We describe the creation of a sampling frame of 50,707 households, the selection and geo-tagging of a sample of 900 households, and the identification and enrolment of participants from 875 households between August 2019 and March 2020. We propose using open-source satellite imaging and geographic information systems to improve the conduct of epidemiological research in these populations. [ABSTRACT FROM AUTHOR]

Rabies is a lethal zoonotic encephalomyelitis that causes an estimated 59,000 human deaths yearly worldwide. Although developing countries of Asia and Africa bear the heaviest burden, surveillance and disease detection in these countries is often hampered by the absence of local laboratories able to diagnose rabies and/or the difficulties of sample shipment from low-access areas to national reference laboratories. Filter papers offer a convenient cost-effective alternative for the sampling, shipment, and storage of biological materials for the diagnosis of many pathogens including rabies virus, yet the properties of diagnostic tests using this support have not been evaluated thoroughly. Sensitivity and specificity of molecular diagnosis of rabies infection using a reverse transcription followed by a hemi-nested polymerase chain reaction (RT-hn-PCR) either directly on brain tissue or using brain tissue dried on filter paper were assessed on 113 suspected field animal samples in comparison to the direct fluorescent antibody test (FAT) recommended by the World Health Organization as one of the reference tests for rabies diagnosis. Impact of the duration of the storage was also evaluated. The sensitivity and the specificity of RT-hn-PCR i) on brain tissue were 96.6% (95% CI: [88.1–99.6]) and 92.7% (95% CI: [82.4–98.0]) respectively and ii) on brain tissue dried on filter paper 100% (95% CI: [93.8–100.0]) and 90.9% (95% CI: [80.0–97.0]) respectively. No loss of sensitivity of RT-hn-PCR on samples of brain tissue dried on filter paper left 7 days at ambient temperature was detected indicating that this method would enable analyzing impregnated filter papers sent to the national reference laboratory at ambient temperature within a 1-week shipment time. It could therefore be an effective alternative to facilitate storage and shipment of samples from low-access areas to enhance and expand rabies surveillance. Author summary: Dogs are responsible for 99% of human rabies deaths. Facilitating diagnostic of rabid dogs can help further identify i) people who were exposed to rabies in order to encourage them to seek post exposure prophylaxis and ii) other exposed animals to break the transmission chain (i.e. prevent them from further transmitting the virus to other people and animals). Yet, the reference diagnostic method for animal rabies requires brain samples to be collected post-mortem and shipped under temperature-controlled conditions. The shipping of such samples is complex in low-access areas, especially in low income countries, which are often those that bear the heaviest rabies burden. Filter papers offer a convenient alternative for biological sample shipment and storage. Here the efficiency of a molecular diagnostic method applied to brain tissue dried on filter paper is compared to one of the reference methods, the direct fluorescent antibody test. Our results show it has an excellent sensitivity (it does not miss any positive samples), even when filter papers are left 7 days at ambient temperature. These results let us foresee a cost-effective alternative facilitating shipment, storage and testing of samples from rabies suspected animals from low-access areas. This could considerably enhance and expand rabies surveillance in low-income countries, allowing a more comprehensive evaluation of rabies burden, and thus reinforcing arguments for allocating funds to rabies control policies. [ABSTRACT FROM AUTHOR]

Background: Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings: In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions: B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations. Author summary: Brucellosis is one of the most important zoonosis in the world and has caused tremendous economic losses in agriculture and animal husbandry in many countries. Developing rapid, sensitive and specific diagnostic methods is very important for early detection and treatment of brucellosis patients. In this study, a novel diagnostic technique, nano-ZnO modified paper ELISA, was established. The antigen used in this technique was a fusion protein containing multiple B cell epitopes, which were predicted from Brucella major outer membrane proteins such as Bp26, Omp31, Omp16, Omp2b and Omp25. Comparing to traditional LPS antigen, this multiepitope based antigen displayed considerably higher sensitivity and higher specificity in laboratory. With the strategy described in this paper, more efficient epitopes and protein antigen can be identified in the future. Currently, LPS antigen is only prepared from live Brucella, while protein antigen can be produced in large quantities in prokaryotic expression system. In addition to nano-p-ELISA, this protein antigen can also be used for development other methods such as fluorescent polarization assay (FPA) and immunochromatographic assay (ICA) to meet the varied demand for brucellosis testing. [ABSTRACT FROM AUTHOR]

Background: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. Methodology: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. Principal findings: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. Conclusions: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations. Author summary: Human cutaneous leishmaniasis affects low-income populations living in places far from health services. The early sampling and detection of the parasite are necessary for timely treatment, however there are no uniform sampling criteria, thus the sensitivity of molecular tests may vary due to various factors such as the type of sample, the time of the disease and the parasite load in the lesion. In this study, we compared the performance of three sampling methods for molecular identification of the genus and Leishmania (Viannia) species in Peru. Several analytical methods, including composite reference standards and latent class models, suggested that lancet scraping might be the best approach for parasite genus detection by kDNA-PCR and for parasite species determination by FRET probes-based Nested Real-Time PCR. [ABSTRACT FROM AUTHOR]

Background: Global estimates for cholera annually approximate 4 million cases worldwide with 95,000 deaths. Recent outbreaks, including Haiti and Yemen, are reminders that cholera is still a global health concern. Cholera outbreaks can rapidly induce high death tolls by overwhelming the capacity of health facilities, especially in remote areas or areas of civil unrest. Recent studies demonstrated that stool specimens preserved on filter paper facilitate molecular analysis of Vibrio cholerae in resource limited settings. Specimens preserved in a rapid, low-cost, safe and sustainable manner for sequencing provides previously unavailable data about circulating cholera strains. This may ultimately provide new information to shape public policy response on cholera control and elimination. Methodology/Principal findings: Whole genome sequencing (WGS) recovered close to a complete sequence of the V. cholerae O1 genome with satisfactory genome coverage from stool specimens enriched in alkaline peptone water (APW) and V. cholerae culture isolates, both spotted on filter paper. The minimum concentration of V. cholerae DNA sufficient to produce quality genomic information was 0.02 ng/μL. The genomic data confirmed the presence or absence of genes of epidemiological interest, including cholera toxin and pilus loci. WGS identified a variety of diarrheal pathogens from APW-enriched specimen spotted filter paper, highlighting the potential for this technique to explore the gut microbiome, potentially identifying co-infections, which may impact the severity of disease. WGS demonstrated that these specimens fit within the current global cholera phylogenetic tree, identifying the strains as the 7th pandemic El Tor. Conclusions: WGS results allowed for mapping of short reads from APW-enriched specimen and culture isolate spotted filter papers this provided valuable molecular epidemiological sequence information on V. cholerae strains from remote, low-resource settings. These results identified the presence of co-infecting pathogens while providing rare insight into the specific V. cholerae strains causing outbreaks in cholera-endemic areas. [ABSTRACT FROM AUTHOR]

Background: Clinical and laboratory diagnosis of cutaneous leishmaniasis (CL) is hampered by under-ascertainment of direct microscopy. Methods: This study compared the diagnostic accuracy of qPCR on DNA extracted from filter paper to the accuracy of direct smear slide microscopy in participants presenting with a cutaneous lesion suspected of leishmaniasis to 16 rural healthcare centers in the Ecuadorian Amazon and Pacific regions, from January 2019 to June 2021. We used Bayesian latent class analysis to estimate test sensitivity, specificity, likelihood ratios (LR), and predictive values (PV) with their 95% credible intervals (95%CrI). The impact of sociodemographic and clinical characteristics on predictive values was assessed as a secondary objective. Results: Of 320 initially included participants, paired valid test results were available and included in the diagnostic accuracy analysis for 129 from the Amazon and 185 from the Pacific region. We estimated sensitivity of 68% (95%CrI 49% to 82%) and 73% (95%CrI 73% to 83%) for qPCR, and 51% (95%CrI 36% to 66%) and 76% (95%CrI 65% to 86%) for microscopy in the Amazon and Pacific region, respectively. In the Amazon, with an estimated disease prevalence among participants of 73%, negative PV for qPCR was 54% (95%CrI 5% to 77%) and 44% (95%CrI 4% to 65%) for microscopy. In the Pacific, (prevalence 88%) the negative PV was 34% (95%CrI 3% to 58%) and 37% (95%CrI 3% to 63%). The addition of qPCR parallel to microscopy in the Amazon increases the observed prevalence from 38% to 64% (+26 (95%CrI 19 to 34) percentage points). Conclusion: The accuracy of either qPCR on DNA extracted from filter paper or microscopy for CL diagnosis as a stand-alone test seems to be unsatisfactory and region-dependent. We recommend further studies to confirm the clinically relevant increment found in the diagnostic yield due to the addition of qPCR. Author summary: Cutaneous leishmaniasis is caused by the parasite Leishmania and is treated when a microscopy test confirms the presence of the parasite in a sample of the lesion. This test, however, is known to miss patients with cutaneous leishmaniasis. DNA diagnostic tests (like PCR) that detect the parasite's genetic material in the lesion, have been proposed to improve diagnosis. Filter paper preserves DNA at room temperature and allows samples to be transported from remote health centers to the PCR laboratory. The ability of microscopic and DNA testing to recognize leishmaniasis patients in real-life is complex to evaluate. We compared the performance of both tests using a statistical method that can evaluate both tests simultaneously without assuming that either test works perfectly. We found that PCR will be positive 68% of the time in a participant with leishmaniasis in the Amazon and 73% of the time in the Pacific region. In the Amazon, microscopy detects one out of every two cases, while it does in three out of every four cases in the Pacific. The addition of the PCR test can improve the number of participants with a diagnosis of leishmaniasis, mostly in the Amazon region. [ABSTRACT FROM AUTHOR]

Background: The use of filter paper as a simple, inexpensive tool for storage and transportation of blood, ‘Dried Blood Spots’ or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA). Methodology and Principal Findings: Sample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009–15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25–30°C showed 81.6% (65.7–92.3 95%CI) positive agreement, 96.8% (91.0–99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70–0.92 95%CI). Conclusions/Significance: The novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids. [ABSTRACT FROM AUTHOR]

Background: Human African trypanosomiasis (HAT) is a neglected tropical disease that usually occurs in rural areas in sub-Saharan Africa. It caused devastating epidemics during the 20th century. Sustained, coordinated efforts by different stakeholders working with national sleeping sickness control programmes (NSSCPs) succeeded in controlling the disease and reducing the number of cases to historically low levels. In 2012, WHO targeted the elimination of the disease as a public health problem by 2020. This goal has been reached and a new ambitious target was stated in the WHO road map for NTDs 2021–2030 and endorsed by the 73rd World Health Assembly: the elimination of gambiense HAT transmission (i.e. reducing the number of reported cases to zero). The interruption of transmission was not considered as an achievable goal for rhodesiense HAT, as it would require vast veterinary interventions rather than actions at the public health level. Methodology/principal findings: Data reported to WHO by NSSCPs were harmonized, verified, georeferenced and included in the atlas of HAT. A total of 802 cases were reported in 2021 and 837 in 2022. This is below the target for elimination as a public health problem at the global level (< 2000 HAT cases/year); 94% of the cases were caused by infection with T. b. gambiense. The areas reporting ≥ 1 HAT case/10 000 inhabitants/year in 2018–2022 cover a surface of 73 134 km2, with only 3013 km2 at very high or high risk. This represents a reduction of 90% from the baseline figure for 2000–2004, the target set for the elimination of HAT as a public health problem. For the surveillance of the disease, 4.5 million people were screened for gambiense HAT with serological tests in 2021–2022, 3.6 million through active screening and 0.9 million by passive screening. In 2021 and 2022 the elimination of HAT as a public health problem was validated in Benin, Uganda, Equatorial Guinea and Ghana for gambiense HAT and in Rwanda for rhodesiense HAT. To reach the next goal of elimination of transmission of gambiense HAT, countries have to report zero cases of human infection with T. b. gambiense for a period of at least 5 consecutive years. The criteria and procedures to verify elimination of transmission have been recently published by WHO. Conclusions/significance: HAT elimination as a public health problem has been reached at global level, with seven countries already validated as having reached this goal. This achievement was made possible by the work of NSSCPs, supported by different public and private partners, and coordinated by WHO. The new challenging goal now is to reach zero cases by 2030. To reach this goal is crucial to maintain the engagement and support of donors and stakeholders and to keep the involvement and coordination of all partners. Along with the focus on elimination of transmission of gambiense HAT, it is important not to neglect rhodesiense HAT, which is targeted for elimination as a public health problem in the WHO road map for NTDs 2021–2030. Author summary: Human African trypanosomiasis (HAT), also known as "sleeping sickness, is a neglected tropical disease (NTD) transmitted by the bite of infected tsetse flies mainly affecting rural areas in sub-Saharan Africa. During the 20th century it caused enormous suffering in the endemic areas with a last reported peak in the late 1990s. This situation triggered a successful coordinated control effort that allowed, in 2012, the targeting of elimination of the disease as a public health problem by 2020. This paper follows previous papers to present the achievements towards HAT elimination. Annually reported cases have been kept below 1000 cases per year. HAT surveillance is maintained through active screening in endemic areas but also within a network of health facilities able to detect the disease. The World Health Organization (WHO), as defined in its 2021–2030 road map for NTDs, is now targeting the elimination of transmission of gambiense HAT and the elimination of rhodesiense HAT as a public health problem. The commitment of national health authorities and the international community will be essential to achieve these ambitious targets. [ABSTRACT FROM AUTHOR]

Background: Dengue virus (DENV) is endemic to many parts of the world and has serious health and socioeconomic effects even in high-income countries, especially with rapid changes in the climate globally. We explored the literature on dengue vector control methods used in high-income, city settings and associations with dengue incidence, dengue prevalence, or mosquito vector densities. Methods: Studies of any design or year were included if they reported effects on human DENV infection or Aedes vector indices of dengue-specific vector control interventions in high-income, city settings. Results: Of 24 eligible sources, most reported research in the United States (n = 8) or Australia (n = 5). Biocontrol (n = 12) and chemical control (n = 13) were the most frequently discussed vector control methods. Only 6 sources reported data on the effectiveness of a given method in reducing human DENV incidence or prevalence, 2 described effects of larval and adult control on Aedes DENV positivity, 20 reported effectiveness in reducing vector density, using insecticide, larvicide, source reduction, auto-dissemination of pyriproxyfen and Wolbachia, and only 1 described effects on human-vector contact. Conclusions: As most studies reported reductions in vector densities, rather than any effects on human DENV incidence or prevalence, we can draw no clear conclusions on which interventions might be most effective in reducing dengue in high-income, city areas. More research is needed linking evidence on the effects of different DENV vector control methods with dengue incidence/prevalence or mosquito vector densities in high-income, city settings as this is likely to differ from low-income settings. This is a significant evidence gap as climate changes increase the global reach of DENV. The importance of community involvement was clear in several studies, although it is impossible to tease out the relative contributions of this from other control methods used. Author summary: Dengue virus is present in many parts of the world and has serious health and socioeconomic effects even in high-income countries, especially with rapid changes in the climate globally. In this study, we explored the literature on dengue vector control methods used in high-income, city settings and associations with number of human dengue infections, and the density of mosquitoes. We found 24 papers with relevant results. Most of these described studies in the United States or Australia. Most were about various forms of biological or chemical control of the mosquitoes. Few papers discussed effects on human dengue infection. We were unable to draw any clear conclusions on how effective mosquito control methods were as we could identify little research on this subject in this specific setting. More research is needed on this topic, particularly as climate change will make more areas of the world vulnerable to dengue infection. [ABSTRACT FROM AUTHOR]

Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M. mycetomatis is able to cause this disease. However, the genetic toolbox for M. mycetomatis is limited. To date, no method is available to genetically modify M. mycetomatis. In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M. mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M. mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies. Author summary: Madurella mycetomatis is the main cause of mycetoma, a Neglected Tropical Disease characterized by large tumorous swellings. Currently little is known about the role of the pathogen in mycetoma formation. Furthermore, there are only a few drugs which are able to inhibit this fungus. In order to gain insight in the disease mycetoma and identify novel drug targets we need a method to genetically modify M. mycetomatis. Up till now this was not available for M. mycetomatis. In this paper, we developed a method in which we for the first time were able to genetically modify Madurella mycetomatis. We did this by removing the M. mycetomatis cell wall and introduced a plasmid into the fungal cell which made the fungus express GFP. The reported methodology can be adapted to introduce other reporters in M. mycetomatis and will be invaluable to explore the pathogenicity of M. mycetomatis. Key Points: A genetic transformation method for Madurella mycetomatis has been developed. Hygromycin is a suitable selection marker for Madurella mycetomatis. GFP can be used as a reporter for Madurella mycetomatis. [ABSTRACT FROM AUTHOR]

Background: Dengue, Zika, and chikungunya, whose viruses are transmitted mainly by Aedes aegypti, significantly impact human health worldwide. Despite the recent development of promising vaccines against the dengue virus, controlling these arbovirus diseases still depends on mosquito surveillance and control. Nonetheless, several studies have shown that these measures are not sufficiently effective or ineffective. Identifying higher-risk areas in a municipality and directing control efforts towards them could improve it. One tool for this is the premise condition index (PCI); however, its measure requires visiting all buildings. We propose a novel approach capable of predicting the PCI based on facade street-level images, which we call PCINet. Methodology: Our study was conducted in Campinas, a one million-inhabitant city in São Paulo, Brazil. We surveyed 200 blocks, visited their buildings, and measured the three traditional PCI components (building and backyard conditions and shading), the facade conditions (taking pictures of them), and other characteristics. We trained a deep neural network with the pictures taken, creating a computational model that can predict buildings' conditions based on the view of their facades. We evaluated PCINet in a scenario emulating a real large-scale situation, where the model could be deployed to automatically monitor four regions of Campinas to identify risk areas. Principal findings: PCINet produced reasonable results in differentiating the facade condition into three levels, and it is a scalable strategy to triage large areas. The entire process can be automated through data collection from facade data sources and inferences through PCINet. The facade conditions correlated highly with the building and backyard conditions and reasonably well with shading and backyard conditions. The use of street-level images and PCINet could help to optimize Ae. aegypti surveillance and control, reducing the number of in-person visits necessary to identify buildings, blocks, and neighborhoods at higher risk from mosquito and arbovirus diseases. Author summary: The strategies to control Ae. aegypti require intensive work and considerable financial resources, are time-consuming, and are commonly affected by operational problems requiring urgent improvement. The PCI is a good tool for identifying higher-risk areas; however, its measure requires a high amount of human and material resources, and the aforementioned issues remain. In this paper, we propose a novel approach capable of predicting the PCI of buildings based on street-level images. This first work combines deep learning-based methods with street-level data to predict facade conditions. Considering the good results obtained with PCINet and the good correlations of facade conditions with PCI components, we could use this methodology to classify building conditions without visiting them physically. With this, we intend to overcome the high cost of identifying high-risk areas. Although we have a long road ahead, our results show that PCINet could help to optimize Ae. aegypti and arbovirus surveillance and control, reducing the number of in-person visits necessary to identify buildings or areas at risk. [ABSTRACT FROM AUTHOR]

Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that displays considerable genetic diversity. Infections result in a range of pathological outcomes, and different strains can exhibit a wide spectrum of anti-parasitic drug tolerance. The genetic determinants of infectivity, virulence and therapeutic susceptibility remain largely unknown. As experimental tools to address these issues, we have generated a panel of bioluminescent:fluorescent parasite strains that cover the diversity of the T. cruzi species. These reporters allow spatio-temporal infection dynamics in murine models to be monitored in a non-invasive manner by in vivo imaging, provide a capability to detect rare infection foci at single-cell resolution, and represent a valuable resource for investigating virulence and host:parasite interactions at a mechanistic level. Importantly, these parasite reporter strains can also contribute to the Chagas disease drug screening cascade by ensuring that candidate compounds have pan-species in vivo activity prior to being advanced into clinical testing. The parasite strains described in this paper are available on request. Author summary: Chagas disease results from infection with the protozoan parasite Trypanosoma cruzi and is a major public health problem throughout Latin America. T. cruzi is a genetically diverse species and infection can result in a wide range of pathological outcomes, mainly associated with the heart and/or digestive tract. Research on Chagas disease, ranging from fundamental biology to drug development, has been greatly aided by the availability of genetically modified parasite reporter strains that express bioluminescent:fluorescent fusion proteins. In combination with mouse models and imaging technology, these strains allow infections to be monitored in real-time, with high sensitivity, and infection foci to be visualised at single-cell resolution. Here, we describe an extensive panel of bioluminescent and fluorescent strains that cover the diversity of the T. cruzi species. These reporter strains, that are available on request, should have wide utility in many areas of Chagas disease research. In particular, as part of the drug development screening programme, they can be used to ensure that candidate compounds have in vivo activity across the species prior to being advanced into clinical testing. [ABSTRACT FROM AUTHOR]

Critical scientific questions remain regarding infection with Mycobacterium ulcerans, the organism responsible for the neglected tropical disease, Buruli ulcer (BU). A controlled human infection model has the potential to accelerate our knowledge of the immunological correlates of disease, to test prophylactic interventions and novel therapeutics. Here we present microbiological evidence supporting M. ulcerans JKD8049 as a suitable human challenge strain. This non-genetically modified Australian isolate is susceptible to clinically relevant antibiotics, can be cultured in animal-free and surfactant-free media, can be enumerated for precise dosing, and has stable viability following cryopreservation. Infectious challenge of humans with JKD8049 is anticipated to imitate natural infection, as M. ulcerans JKD8049 is genetically stable following in vitro passage and produces the key virulence factor, mycolactone. Also reported are considerations for the manufacture, storage, and administration of M. ulcerans JKD8049 for controlled human infection. Non-technical author summary: In this paper, we progress our research towards establishing a controlled human infection model (CHIM) of Buruli ulcer, caused by Mycobacterium ulcerans. It is crucial that the challenge agent used for such experiments is thoroughly characterised; not only is this important to ensure standardisation between participants, but also to reinforce their safety. Using established criteria, we systematically demonstrate why the isolate M. ulcerans JKD8049 may be a suitable challenge strain. This strain is non-genetically modified, susceptible to antibiotics, can be cultured in relatively simple media, and can be filtered and preserved as individual bacteria to ensure accurate dosing of participants. Even after repeatedly culturing the organism for months, it remains genetically stable, so significant mutations are not expected to develop during manufacture. After preserving the isolate at -80°C, it can be revived and diluted for human inoculation at very low doses. Importantly, it produces the toxin mycolactone, so it is expected to recreate typical Buruli ulcer disease in participants, which will be important to properly test vaccines and other therapeutics. We also demonstrate a set of principles to ensure the produced batch is free from contamination. [ABSTRACT FROM AUTHOR]

The article offers information on catalyzing Neglected Tropical Diseases (NTD) gender and equity research. Topics discussed include significance of the NTD program predicated opportunity to treat disease, prevent morbidity and disability, and interrupt infection transmission among large populations; addresses the issue of gender equity, organizational, policy, and practice activities; and sex and gender roles can profound effect on healthcare access and health outcomes.

There is a need for novel chemical matter for phenotypic and target-based screens to find starting points for drug discovery programmes in neglected infectious diseases and non-hormonal contraceptives that disproportionately affect Low- and Middle-Income Countries (LMICs). In some disease areas multiple screens of corporate and other libraries have been carried out, giving rise to some valuable starting points and leading to preclinical candidates. Whilst in other disease areas, little screening has been carried out. Much screening against pathogens has been conducted phenotypically as there are few robustly validated protein targets. However, many of the active compound series identified share the same molecular targets. To address the need for new chemical material, in this article we describe the design of a new library, designed for screening in drug discovery programmes for neglected infectious diseases. The compounds have been selected from the Enamine REAL (REadily AccessibLe) library, a virtual library which contains approximately 4.5 billion molecules. The molecules theoretically can be synthesized quickly using commercially available intermediates and building blocks. The vast majority of these have not been prepared before, so this is a source of novel compounds. In this paper we describe the design of a diverse library of 30,000 compounds from this collection (graphical abstract). The new library will be made available to laboratories working in neglected infectious diseases, subject to a review process. The project has been supported by the Bill & Melinda Gates Foundation and the Wellcome Trust (Wellcome). Author summary: Screening of diverse compound libraries is a powerful way to find chemical start points for drug discovery programmes. There is a lack of such chemical libraries available for neglected infectious diseases and other neglected areas such as non-hormonal contraception. In this paper we describe the design of such a library of 30,000 compounds (graphical abstract). The aim was to develop a library of diverse chemical matter unlikely to have been screened in these disease areas. The compounds were selected from the REAL (REadily AccessibLe) Enamine library, a virtual library of 4.5 billion compounds. The methodology for selection of the compounds is described, along with a detailed analysis of the physicochemical properties of the compounds and comparison to other libraries that we have assembled. This shows that the library contains hit/lead-like compounds suitable as chemical start points, and whilst it covers a similar space to some other libraries, the material within the library is mainly different to other libraries. Copies of the library will be made available for screening; the process for gaining access to this library is described. [ABSTRACT FROM AUTHOR]

Background: The impact of climate change has led to variations in various biological processes, leading to altered transmission dynamics of infectious diseases, including snail-borne diseases (SBDs). Fascioliasis is one of the neglected zoonotic tropical snail-borne diseases caused by the trematode of the genus Fasciola. This review focused on laboratory experimental and model studies that evaluate the potential effect of temperature change on the ecology and biology of the intermediate host snails (IHS) of Fasciola. Methods: A literature search was conducted on Google Scholar, EBSCOhost, and PubMed databases using predefined medical subject heading terms, Boolean operators, and truncation symbols in combination with direct keywords: Fasciolosis AND Temperature, Lymnaea OR Austropeplea OR Radix OR Galba OR Fossaria OR Pseudosuccinea AND growth, fecundity, AND survival at the global scale. Other search terms used were (Fascioliasis AND Temperature), (Lymnaea AND Temperature), (Austropeplea AND Temperature), (Fossaria AND Temperature), (Galba AND Temperature), (Pseudosuccinea AND Temperature), and (Radix AND Temperature). Results: The final synthesis included thirty-five published articles. The studies reviewed indicated that temperature rise may alter the distribution, and optimal conditions for breeding, growth, and survival of IHS, ultimately resulting in changing the transmission dynamics of fascioliasis. The literature also confirmed that the life history traits of IHS and their interaction with the liver fluke parasites are driven by temperature, and hence climate change may have profound outcomes on the population size of snails, parasite density, and disease epidemiology. Conclusion: We concluded that understanding the impact of temperature on the growth, fecundity, and survival of IHS may broaden our knowledge of the possible effects of climate change and hence inform fascioliasis control programs. Author summary: Fascioliasis is a neglected tropical disease caused by flatworms or liver flukes of the genus Fasciola. The review paper focuses on the effect of temperature on the life history traits of intermediate hosts of fascioliasis. The authors aim to explore the impact of temperature on the growth, survival, and reproduction of the host snails and the development of parasites inside intermediate hosts. Fascioliasis is considered a major public health problem in many countries, affecting both humans and livestock. The disease is transmitted through the consumption of contaminated water or vegetation containing the infective stage of metacercaria. The review paper highlights that temperature plays a crucial role in the life history traits of these intermediate hosts. The authors gathered and analyzed various studies conducted on different intermediate host snails of Fasciola species to investigate the relationship between temperature and the development, survival, reproduction, and infectivity of the parasites within them. Their findings reveal that the temperature can significantly influence the life history traits of these intermediate hosts. Higher temperatures generally promote faster development, increased reproduction, and higher infectivity rates. However, there is a limit to the beneficial effects of temperature, beyond which the survival and fitness of the intermediate hosts start to decline. The authors also emphasize the relevance of understanding the impact of temperature on fascioliasis in the context of climate change. As global temperatures continue to rise, the distribution and prevalence of fascioliasis may change, affecting the transmission dynamics of the disease. This knowledge is crucial for the design and implementation of effective control strategies to mitigate the impact of fascioliasis on human and animal health. [ABSTRACT FROM AUTHOR]

Background: This paper identifies opportunities and challenges for leishmaniasis control and elimination in Colombia, emphasizing the role of pooled procurement of essential medicines and supplies. Colombia is among the countries most affected by leishmaniasis globally, and also faces the dual challenge of procuring critically needed medicines in the context of limited national resources. It recently renewed its commitment to the control and elimination of leishmaniasis under its 2022–2031 Public Health Plan (PDSP) through a comprehensive public health approach. Methodology/Principal findings: The methodology comprises a comprehensive literature review and key informant interviews with leishmaniasis experts from the Colombian national control program and PAHO/WHO, focusing on cutaneous, mucocutaneous, and visceral leishmaniasis. Leishmaniasis is endemic throughout Colombia, with over 11 million people at risk, many of whom live in poverty-stricken, remote and isolated rural areas with limited access to health services. Leishmaniasis care, including medicines, is provided free of charge, but many barriers were nonetheless identified at environmental, population, and health system levels, including the supply of quality-assured medicines. Opportunities to alleviate these barriers were identified, including the support of the PAHO Strategic Fund. Within the context of the sustainable development goals and international leishmaniasis control and elimination targets, Colombian officials have established their own priorities, the highest of which is the reduction of deaths from visceral leishmaniasis. Conclusions/Significance: The elimination of leishmaniasis as a public health problem presents significant challenges, given its biological complexity and diversity, physical and clinical manifestations, social and economic impacts, frequently burdensome treatment regimens, and insufficient supply of necessary medicines. However, rigorous prevention and control efforts through strong political commitment and a highly motivated workforce can dramatically reduce its burden. Colombia's new PDSP, which highlights leishmaniasis control, is an opportunity for a revitalized health system response through committed leadership, intersectoral actions, and partnerships with international organizations that share a common vision. Author summary: Leishmaniasis is a vector-borne disease transmitted by female sand flies to humans and animals, causing infections ranging from asymptomatic and self-healing cutaneous leishmaniasis to visceral leishmaniasis which is highly lethal if untreated. Although all forms of leishmaniasis are treatable and curable with early diagnosis and treatment, the disease presents many challenges for affected populations, many of whom live in remote rural areas of the developing world, with limited access to public health facilities. Health services also face major constraints in supplying critically needed, yet costly medicines, in the context of limited financial resources. This paper presents the case of Colombia which is among the countries most affected by leishmaniasis globally, and discusses its many challenges in responding to the disease, including the guaranteed supply of necessary medicines. Our results uncover new opportunities for a revitalized health system response through committed leadership, intersectoral actions, such as a One Health approach, and partnerships with like-minded international organizations. These include Colombia's new 10-Year Public Health Plan that contains specific time-bound goals for leishmaniasis control and elimination as a public health problem, prioritizing a reduction in deaths from visceral leishmaniasis, and potential cost-savings from the acquisition of essential medicines and supplies through the Strategic Fund of the Pan American Health Organization. [ABSTRACT FROM AUTHOR]

According to the World Health Organization (WHO), dengue is the most common acute arthropod-borne viral infection in the world. The spread of dengue and other infectious diseases is closely related to human activity and mobility. In this paper we analyze the effect of introducing mobility restrictions as a public health policy on the total number of dengue cases within a population. To perform the analysis, we use a complex metapopulation in which we implement a compartmental propagation model coupled with the mobility of individuals between the patches. This model is used to investigate the spread of dengue in the municipalities of Caldas (CO). Two scenarios corresponding to different types of mobility restrictions are applied. In the first scenario, the effect of restricting mobility is analyzed in three different ways: a) limiting the access to the endemic node but allowing the movement of its inhabitants, b) restricting the diaspora of the inhabitants of the endemic node but allowing the access of outsiders, and c) a total isolation of the inhabitants of the endemic node. In this scenario, the best simulation results are obtained when specific endemic nodes are isolated during a dengue outbreak, obtaining a reduction of up to 2.5% of dengue cases. Finally, the second scenario simulates a total isolation of the network, i.e., mobility between nodes is completely limited. We have found that this control measure increases the number of total dengue cases in the network by 2.36%. Author summary: For the World Health Organization, dengue is a disease of public health concern. In recent years there is an increasing trend in the number of dengue cases despite existing prevention and control campaigns. The mobility of the population is considered an important factor in dengue dispersion. In this paper, we are interested in addressing how restrictions to human mobility might affect the incidence of dengue in a region. Our research is relevant because the model can be adapted to other regions or scales, and the mobility control measures can be taken into account for the development of public health policies in endemic regions. [ABSTRACT FROM AUTHOR]

Background: Mozambique has one of the highest burdens of neglected tropical diseases in Africa. Lymphatic filariasis, schistosomiasis and soil-transmitted helminths are being targeted for elimination as part of integrated mass drug administration campaigns. The progress made towards interruption of transmission has been affected by recent conflict in Cabo Delgado province. The aim of this paper was to determine the potential impact of this crisis on the neglected tropical diseases programme and the challenges in reaching the elimination goals of 2030. Methodology: A desk-based secondary data analysis was conducted on publicly available sources of neglected tropical diseases, conflict incidents, internally displaced persons and geographical access between 2020 and 2022. Data were summarised and mapped using GIS software. A combined risk stratified assessment at district level was developed with five classifications i) Very high-risk; ii) High-risk; iii) Medium to high-risk; iv) Medium risk; and v) Not at risk due to conflict absence but co-endemic. Results: Lymphatic filariasis, schistosomiasis and soil-transmitted helminths were co-endemic in 115 out of 156 (74%) districts. Between 2020 and 2022 a total of 1,653 conflict-related incidents were reported, most of them in Cabo Delgado province (n = 1,397, 85%). A five-fold increase of internally displaced persons was recorded from April 2020 (n = 172,186) to November 2022 (n = 935,130). Geographical accessibility also deteriorated across the province with an increase from five (29%) in 2021 to seven (41%) districts in 2022 classified as hard-to-reach. The combined risk stratification identified that most districts (n = 7; 41%) in Cabo Delgado province had medium to high-risk; very high-risk (n = 5, 29%); medium risk (n = 3, 18%); high-risk (n = 2, 12%). Conclusion: Most of the districts of Cabo Delgado were considered to be at risk of not meeting the neglected tropical diseases road map 2021–2030 targets due to the humanitarian crisis ongoing. There is the need for practical strategies and funding to overcome these hostile challenges. Author summary: Neglected tropical diseases (NTDs) are a group of diseases that usually affect the most vulnerable population in tropical countries. Mozambique has been tackling lymphatic filariasis, schistosomiasis and soil-transmitted helminths for two decades with the aim of eliminating them through large-scale distribution of drugs to interrupt transmission. In recent years, internal conflict has emerged and led to an escalating humanitarian crisis in the endemic province of Cabo Delgado which has hampered elimination efforts. This study aimed to shed light on the situation and to determine the potential impact on the Mozambique NTDs' programme. We used a range of open-source data on NTDs, conflict, internally displaced persons and geographical access to summarise, describe, and map the situation at the district level in the most affected province. We used this data to also examine their geographical overlap and to develop a risk assessment tool to highlight the high risk and most vulnerable areas. We found that between 2021–2022 more than 1,300 conflict incidents occurred, and almost 1 million people were displaced in Cabo Delgado province with many districts having limited geographical access which impacted the NTDs programme's ability to work. The study highlights the important challenges of conflict and displacement of vulnerable populations, and the need for support and set of guidelines for NTDs endemic programmes to use to help to overcome key barriers and meet the targets of elimination set by the World Health Organization for 2030. [ABSTRACT FROM AUTHOR]

Background: Pythiosis is a rare disease with high mortality, with over 94% of cases reported from Thailand and India. Prompt diagnosis and surgery improves patient outcomes. Therefore, continuing professional development (CPD) is essential for early recognition. However, a needs assessment related to a pythiosis CPD program has not been performed. Objectives: We conducted a needs assessment to develop a pythiosis CPD program. Patients/Methods: We conducted a survey study with 267 King Chulalongkorn Memorial Hospital residents (141 internal medicine (IM) residents and 126 surgery residents). A 30-item survey consisting of a knowledge assessment, demographic section, and an attitudes portion was distributed both electronically and via paper. The data was analyzed with descriptive and inferential statistics. Results: Sixty-seven percent completed the survey (110/141 IM residents, 70/126 surgery residents). The mean score [95% confidence interval] on the knowledge assessment was 41.67% [39.64%-43.69%] across all objectives. The three domains with the highest scores were pythiosis risk factors (67.22% correct), microbiologic characteristics (50.83%), and radiographic interpretation (50.56%). The three domains with the lowest scores were laboratory investigation (15.00%), epidemiology (29.17%), and symptomatology (30.83%). Most participants noted that the program should be online with both synchronous and asynchronous sessions, with a preferred length of 60–90 minutes per session. Conclusion: The pythiosis CPD program should emphasize education regarding symptomatology, laboratory investigation, and epidemiology, all of which are critical for the early detection of pythiosis to decrease mortality from this devastating disease. Most respondents felt this program was necessary and should be implemented in a virtual blended format. Author summary: Researchers conducted a needs assessment to develop a Continuing Professional Development (CPD) program on pythiosis, a rare and often fatal disease prevalent in Thailand and India. The study surveyed 267 residents at King Chulalongkorn Memorial Hospital, revealing a 67% completion rate. The residents exhibited a mean knowledge score of 41.67%, with the highest proficiency in pythiosis risk factors (67.22%), microbiologic characteristics (50.83%), and radiographic interpretation (50.56%). Conversely, lower scores were observed in laboratory investigation (15.00%), epidemiology (29.17%), and symptomatology (30.83%). Participants expressed a preference for an online CPD program with both synchronous and asynchronous sessions, each lasting 60–90 minutes. The study emphasizes the need for education on symptomatology, laboratory investigation, and epidemiology to enhance early pythiosis detection and reduce mortality. Respondents widely supported the implementation of the proposed virtual blended CPD program. [ABSTRACT FROM AUTHOR]

Background: Lymphatic filariasis (LF) is a parasitic disease transmitted by mosquitoes, causing severe pain, disfiguring, and disabling clinical conditions such as lymphoedema and hydrocoele. LF is a global public health problem affecting 72 countries, primarily in Africa and Asia. Since 2000, the World Health Organization (WHO) has led the Global Programme to Eliminate Lymphatic Filariasis (GPELF) to support all endemic regions. This paper focuses on the achievements of the Malawi LF Elimination Programme between 2000 and 2020 to eliminate LF as a public health problem, making it the second sub-Saharan country to receive validation from the WHO. Methodology/principal findings: The Malawi LF Programme addressed the widespread prevalence of LF infection and disease across the country, using the recommended WHO GPELF strategies and operational research initiatives in collaboration with key national and international partners. First, to stop the spread of infection (i.e., interrupt transmission) and reduce the circulating filarial antigen prevalence from as high as 74.4% to below the critical threshold of 1–2% prevalence, mass drug administration (MDA) using a two-drug regime was implemented at high coverage rates (>65%) of the total population, with supplementary interventions from other programmes (e.g., malaria vector control). The decline in prevalence was monitored and confirmed over time using several impact assessment and post-treatment surveillance tools including the standard sentinel site, spot check, and transmission assessment surveys and alternative integrated, hotspot, and easy-access group surveys. Second, to alleviate suffering of the affected populations (i.e., control morbidity) the morbidity management and disability prevention (MMDP) package of care was implemented. Specifically, clinical case estimates were obtained via house-to-house patient searching activities; health personnel and patients were trained in self-care protocols for lymphoedema and/or referrals to hospitals for hydrocoele surgery; and the readiness and quality of treatment and services were assessed with new survey tools. Conclusions: Malawi's elimination of LF will ensure that future generations are not infected and suffer from the disfiguring and disabling disease. However, it will be critical that the Malawi LF Elimination programme remains vigilant, focussing on post-elimination surveillance and MMDP implementation and integration into routine health systems to support long-term sustainability and ongoing success. Summary: Lymphatic filariasis, also known as elephantiasis, is a disabling, disfiguring, and painful disease caused by a parasite that infected mosquitoes transmit to millions of people worldwide. Since 2000, the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has supported endemic countries such as Malawi in south-eastern Africa, to eliminate the disease as a public health problem. The Malawi National LF Elimination Programme has worked tirelessly over the past two decades to implement the GPELF recommended strategies to interrupt the transmission with a two-drug regime, and to alleviate suffering in patients with lymphoedema and/or hydrocoele through morbidity management and disability prevention. Additionally, the LF Programme has collaborated with national and international stakeholders to implement a range of supplementary operational research projects to address outstanding knowledge gaps and programmatic barriers. In 2020, the World Health Organisation validated that Malawi had successfully eliminated LF as a public health problem, making it the second country in sub-Saharan Africa to achieve this, which is remarkable given that Malawi previously had very high infection rates. The LF Programme now remains vigilant, putting its efforts towards post-elimination surveillance and the continued implementation of care for patients with chronic conditions. Malawi's elimination of LF will ensure that future generations are not affected by this devastating disease. [ABSTRACT FROM AUTHOR]

Objectives: Deworming programmes of soil-transmitted helminths are generally monitored and evaluated by aggregating drug coverage and infection levels at a district level. However, heterogeneity in drug coverage at finer spatial scales means indicators may remain above thresholds for elimination as a public health problem or of transmission in some areas. This paper aims to highlight the misleading information that aggregating data at larger spatial scales can have for programme decision making. Methods: Drug coverage data from the Geshiyaro project were compared at two spatial scales with reference to the World Health Organisation's targets. District (woreda) and village (kebele) level were compared. The association between infection levels and drug coverage was analysed by fitting a weighted least-squares function to the mean intensity of infection (eggs per gram of faeces) against drug coverage. Results: The data show clearly that when the evaluation of coverage is aggregated to the district level, information on heterogeneity at a finer spatial scale is lost. Infection intensity decreases significantly (p = 0.0023) with increasing drug coverage. Conclusion: Aggregating data at large spatial scales can result in prematurely ceasing deworming, prompting rapid infection bounce-back. There is a strong need to define context-specific spatial scales for monitoring and evaluating intervention programmes. Author summary: For soil-transmitted helminths (STH), control is centred around mass drug administration (MDA) of eligible subgroups without prior diagnosis, aiming to achieve 75% drug coverage. MDA programmes are monitored by assessing whether drug coverage and infection prevalence and intensity, aggregated to a spatial scale, typically an administrative unit such as a district, have met programmatic targets. Spatial heterogeneity in drug coverage, prevalence, and intensity is commonplace and well documented. However, little attention has been paid to the spatial scales of this heterogeneity and the implications it has on providing the basis to inform the cessation or continuation of prophylactic mass treatment. We present a clear analysis showing that fine scale spatial heterogeneity in drug coverage is commonplace even in well implemented MDA programmes. We show that aggregating data at the district level, which is routine for deworming programmes, can give misleading information on which to base future treatment decisions, specifically the premature reduction in frequency, or cessation of MDA, because fine scale heterogeneity is not captured by larger aggregations. This work highlights the critical need to define context specific spatial scales for monitoring and evaluating interventions implemented in different transmission settings. [ABSTRACT FROM AUTHOR]

Background: Onchocerciasis is a neglected tropical disease with 217.5 million people globally at risk of having the infection. In both settled and semi-nomadic communities of Massangam Health District in Cameroon, Sightsavers has been carrying out test-and-treat with doxycycline and twice-yearly ivermectin distribution. This paper focuses on the cost of test-and-treat with doxycycline in the two community contexts of settled and semi-nomadic. Methods: For the valuation, a combination of gross or micro-costing was used to identify cost components, as well as bottom-up and top-down approaches. The opportunity costs of vehicle and equipment use were estimated and included. Not included, however, were the opportunity costs of building use and Ministry of Public Health staff salaries. We only captured the incremental costs of implementing test-and-treat activities as part of a functional annual community-directed treatment with the ivermectin programme. Results: We estimate the economic cost per person tested and cost per person treated in Massangam to be US$135 and US$667 respectively. Total implementation cost in the settled community was US$79,409, and in the semi-nomadic community US$69,957. Overall, the total economic cost of implementing the doxycycline test-and-treat strategy for onchocerciasis elimination in Massangam came to US$168,345. Financial costs represented 91% of total costs. Conclusions: Unit costs of test-and-treat in both settled and semi-nomadic communities are higher than unit costs of community-directed treatment with ivermectin. However, it is critical to note that a two-year implementation shows a significantly larger reduction in infection prevalence than the preceding 20 years of annual community-directed treatment with ivermectin. Test-and-treat with doxycycline may be a cost-effective intervention in places where the prevalence of microfilaria is still high, or in hard-to-reach areas where community-directed treatment with ivermectin and MDA coverage are not high enough to stop transmission or where marginalised populations consistently miss treatment. Author summary: Onchocerciasis, also known as river blindness, is a neglected tropical disease (NTD) caused by a parasitic nematode, Onchocerca volvulus. It is transmitted to humans through the bites of infected blackflies of the genus Simulium, which breed in fast-flowing rivers and streams. To eliminate transmission, a yearly distribution of ivermectin is conducted in infected areas. This medication kills the microfilaria but not the adult nematode, which is killed by doxycycline. Despite over 20 years of ivermectin mass distribution, the disease is still prevalent in the Massangam Health District in Cameroon. Therefore, alternative strategies have been discussed to stop transmission, including treatment of positive tested persons with doxycycline in both settled and nomadic communities. This paper estimated the total cost of implementation at US$168,345 in Massangam district, in order to compare it with the current elimination strategy and to demonstrate the potential effectiveness of the intervention–especially in high infection, hard-to-reach and loa loa co-infection settings. [ABSTRACT FROM AUTHOR]

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the 'Mary kDNA PCR' and a newly designed 'LC kDNA PCR' for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available. Author summary: Cutaneous leishmaniasis is a neglected tropical disease and causing a public health problem in Ethiopia. Microscopy is still the standard method for detection of the parasite in Ethiopia, and also in many other low resource settings. A more sensitive method is needed for timely diagnosis and treatment. In this study, we compared five molecular methods on samples collected from patients with a skin lesion suspected of cutaneous leishmaniasis to advice on optimal diagnosis of L. aethiopica. We collected two sample types from the same lesion (skin scrapings and lesion fluid on filter paper) and isolated both DNA and RNA of them. Majority (90.1%) of the samples from skin scrapings were positive in two or more methods and the molecular methods had a higher sensitivity than the conventional methods. Interestingly, we evaluated for the first time a new molecular method designed to improve L. aethiopica detection. Also, we showed that RNA detection performed well for samples that were collected under difficult field conditions. Samples collected on filter paper showed less positive results than skin scraped samples, but could still be the method of choice for easy sampling and transport in resource-limited settings as it performed better than microscopy. [ABSTRACT FROM AUTHOR]

Background: Snake bite envenoming (SBE) is one neglected tropical disease that has not received the needed attention. The sequelae of burdensome disability and mortality impact the socioeconomic life of communities adversely with little documentation of SBE in health facility records in Ghana. This study details SBE and snake distribution, habits/habitats, type of venom expressed and clinical manifestations. Methodology: We conducted a structured thematic desk review of peer reviewed papers, books and reports from repositories including PubMed, WHO and WCH using bibliographic software EndNote and search engine Google Scholar with the following key words; snakes, medical importance, snake bites, venom and venom type, envenomation, symptoms and signs, vaccines, venom expenditure, strike behaviour and venom-metering + Ghana, West Africa, Africa, World. We also reviewed data from the District Health Information Management System (DHIMS) of the Ghana Health Service (GHS). Outcome variables were organized as follows: common name (s), species, habitat/habit, species-specific toxin, clinical manifestation, antivenom availability, WHO category. Findings: Snake bites and SBE were grouped by the activity of the expressed venom into neurotoxic, cardiotoxic, haemorrhagic, cytotoxic, myotoxic, nephrotoxic and procoagulants. Neurotoxic snake bites were largely due to elapids. Expressed venoms with cardiotoxic, haemorrhagic, nephrotoxic and procoagulant activities principally belonged to the family Viperidae. Snakes with venoms showing myotoxic activity were largely alien to Ghana and the West African sub-region. Venoms showing cytotoxic activity are expressed by a wide range of snakes though more prevalent among the Viperidae family. Snakes with neurotoxic and haemorrhagic venom activities are prevalent across all the agro-ecological zones in Ghana. Conclusion/Significance: Understanding the characteristics of snakes and their venoms is useful in the management of SBE. The distribution of snakes by their expressed venoms across the agro-ecological zones is also instructive to species identification and diagnosis of SBE. Author summary: We observed rudimentary data on snake bites and a paucity of venom characterization in the health records of patients in health facilities in Ghana. Knowledge of the distribution of snake species across the agro-ecological zones of the country was also limited. We set out to complement this data by doing a structured thematic desk review of peer reviewed papers, books and reports from repositories including PubMed, WHO and the District Health Information Management System (DHIMS) of the Ghana Health Service (GHS). Our findings revealed that, snake bites were largely grouped according to the activity of the expressed venom into neurotoxic, cardiotoxic, haemorrhagic, cytotoxic, myotoxic and in some documentations nephrotoxic and procoagulants. Neurotoxic snake bites were largely due to cobras and mambas. Snake expressing venoms with cardiotoxic, haemorrhagic, nephrotoxic and procoagulant activities were principally vipers. Snakes with venoms showing myotoxic activity were largely alien to Ghana and the West African sub-region. Venoms showing cytotoxic activity are expressed by a wide range of snakes though more prevalent among vipers. In Ghana, snakes expressing venoms with neurotoxic and haemorrhagic activities are prevalent across all the agro-ecological zones of the country. We identified 3 antivenoms registered by the Ghana Food and Drug Authority. [ABSTRACT FROM AUTHOR]

Introduction: Traditionally, health ministries implement mass drug administration programmes for each neglected tropical disease (NTD) as separate and distinct campaigns. Many NTDs have overlapping endemicity suggesting co-administration might improve programme reach and efficiency, helping accelerate progress towards 2030 targets. Safety data are required to support a recommendation to undertake co-administration. Methodology: We aimed to compile and summarize existing data on co-administration of ivermectin, albendazole and azithromycin, including both data on pharmacokinetic interactions and data from previous experimental and observational studies conducted in NTD-endemic populations. We searched PubMed, Google Scholar, research and conference abstracts, gray literature, and national policy documents. We limited the publication language to English and used a search period from January 1st, 1995 through October 1st, 2022. Search terms were: azithromycin and ivermectin and albendazole, mass drug administration co-administration trials, integrated mass drug administration, mass drug administration safety, pharmacokinetic dynamics, and azithromycin and ivermectin and albendazole. We excluded papers if they did not include data on co-administration of azithromycin and both albendazole and ivermectin, or azithromycin with either albendazole or ivermectin alone. Results: We identified a total of 58 potentially relevant studies. Of these we identified 7 studies relevant to the research question and which met our inclusion criteria. Three papers analyzed pharmacokinetic and pharmacodynamic interactions. No study found evidence of clinically significant drug-drug interactions likely to impact safety or efficacy. Two papers and a conference presentation reported data on the safety of combinations of at least two of the drugs. A field study in Mali suggested the rates of adverse events were similar with combined or separate administration, but was underpowered. A further field study in Papua New Guinea used all three drugs as part of a four-drug regimen also including diethylcarbamazine; in this setting, co-administration appeared safe but there were issues with the consistency in how adverse events were recorded. Conclusion: There are relatively limited data on the safety profile of co-administering ivermectin, albendazole and azithromycin as an integrated regimen for NTDs. Despite the limited amount of data, available evidence suggests that such a strategy is safe with an absence of clinically important drug-drug interactions, no serious adverse events reported and little evidence for an increase in mild adverse events. Integrated MDA may be a viable strategy for national NTD programmes. Author summary: Treatment of the whole community (mass drug administration, MDA) has been a major intervention strategy against many neglected tropical diseases (NTDs) over the last decade. Normally health ministries deliver individual MDA rounds targeting specific NTDs. This multiplies the training, transport and time burden for local health service personnel in districts in which several NTDs are present, imposing considerable financial and human resource costs to health ministries and their partners, and causing requiring repeated disruption to the daily life of communities receiving MDA. Delivering MDA for several NTDs at one time could improve the efficiency of NTD programmes. We reviewed existing data on the safety and feasibility of combining MDA of albendazole, ivermectin and azithromycin into a single co-delivered MDA. Several studies had evaluated if taking these drugs at the same time changed drug levels in recipients' blood; these studies concluded that there was not an important difference in blood drug levels comparing instances when the medicines were taken separately to instances when they were taken at the same time. Two non-randomised studies assessed side effects experienced by people taking combinations of the three drugs and suggested doing so was safe. One small study in Mali had assessed combining all three drugs and also suggested this was safe but was too small to give a definitive answer. Two studies in Papua New Guinea assessed all three drugs being taken together in combination with a fourth drug, diethylcarbamazine. These studies also suggest co-administration was safe overall. Most of the identified studies had some methodological shortcomings, such as small sample sizes or issues with the way adverse events were recorded. Overall, the data suggest co-administration of azithromycin, ivermectin and albendazole is viable, but larger safety studies are needed. [ABSTRACT FROM AUTHOR]

Introduction: Dengue is a major public health challenge and a growing problem due to climate change. The release of Aedes aegypti mosquitoes infected with the intracellular bacterium Wolbachia is a novel form of vector control against dengue. However, there remains a need to evaluate the benefits of such an intervention at a large scale. In this paper, we evaluate the potential economic impact and cost-effectiveness of scaled Wolbachia deployments as a form of dengue control in Vietnam–targeted at the highest burden urban areas. Methods: Ten settings within Vietnam were identified as priority locations for potential future Wolbachia deployments (using a population replacement strategy). The effectiveness of Wolbachia deployments in reducing the incidence of symptomatic dengue cases was assumed to be 75%. We assumed that the intervention would maintain this effectiveness for at least 20 years (but tested this assumption in the sensitivity analysis). A cost-utility analysis and cost-benefit analysis were conducted. Results: From the health sector perspective, the Wolbachia intervention was projected to cost US$420 per disability-adjusted life year (DALY) averted. From the societal perspective, the overall cost-effectiveness ratio was negative, i.e. the economic benefits outweighed the costs. These results are contingent on the long-term effectiveness of Wolbachia releases being sustained for 20 years. However, the intervention was still classed as cost-effective across the majority of the settings when assuming only 10 years of benefits Conclusion: Overall, we found that targeting high burden cities with Wolbachia deployments would be a cost-effective intervention in Vietnam and generate notable broader benefits besides health gains. Author summary: Dengue is a major public health challenge and a growing problem due to climate change. The release of Aedes aegypti mosquitoes infected with the intracellular bacterium Wolbachia is a novel form of vector control against dengue. However, there remains a need to evaluate the health and economic benefits of such an intervention at a large scale, as well as its value for money. In this paper, we evaluate the potential economic impact and cost-effectiveness of scaled Wolbachia deployments as a form of dengue control in Vietnam–targeted at the highest burden urban areas. Ten settings within Vietnam were identified as priority locations for potential future Wolbachia deployments (using a population replacement strategy). We assumed that the effectiveness of Wolbachia deployments in reducing the incidence of symptomatic dengue cases would be 75%. We found that targeting high burden cities with Wolbachia deployments would be a cost-effective intervention in Vietnam and generate notable broader benefits besides health gains. Overall, this work highlights the value of investment in the scaled implementation of Wolbachia deployments as an effective and cost-effective tool for dengue control in Vietnam, and more generally for addressing the global challenge of dengue control. [ABSTRACT FROM AUTHOR]

Introduction: Schistosomiasis is a parasitic disease in Tanzania affecting over 50% of the population. Current control strategies involve mass drug administration (MDA) campaigns at the district level, which have led to problems of over- and under-treatment in different areas. WHO guidelines have called for more targeted MDA to circumvent these problems, however a scarcity of prevalence data inhibits decision makers from prioritizing sub-district areas for MDA. This study demonstrated how geostatistics can be used to inform planning for targeted MDA. Methods: Geostatistical sub-district (ward-level) prevalence estimates were generated through combining a zero-inflated poisson model and kriging approach (regression kriging). To make predictions, the model used prevalence survey data collected in 2021 of 17,400 school children in six regions of Tanzania, along with several open source ecological and socio-demographic variables with known associations with schistosomiasis. Results: The model results show that regression kriging can be used to effectively predict the ward level parasite prevalence of the two species of Schistosoma endemic to the study area. Kriging was found to further improve the regression model fit, with an adjusted R-squared value of 0.51 and 0.32 for intestinal and urogenital schistosomiasis, respectively. Targeted treatment based on model predictions would represent a shift in treatment away from 193 wards estimated to be over-treated to 149 wards that would have been omitted from the district level MDA. Conclusions: Geostatistical models can help to support NTD program efficiency and reduce disease transmission by facilitating WHO recommended targeted MDA treatment through provision of prevalence estimates where data is scarce. Author summary: In Tanzania, schistosomiasis is a vast public health problem treated through mass drug administration (MDA) campaigns that target large groups of the population. Such mass drug administration (MDA) campaigns require significant amounts of resources challenging the capacity of chronically underfunded schistosomiasis control programs to sustain annually. The way in which MDA campaigns have been conducted, by targeting whole districts believed to be endemic, have not had optimal results for reducing disease transmission, have facilitated problems of under-treating in many population groups while over-treating other non-endemic areas, and have placed a significant strain on the limited resources available. To circumvent such problems, the World Health Organization (WHO) has recommended that treatment efforts need to be more targeted to endemic communities and administrative areas within districts, and that MDA campaigns should be conducted at a sub-district level. The WHO also provides explicit guidelines for treatments of communities based on parasitological prevalence of schistosomiasis. While there is sound rationale in making this switch, most schistosomiasis endemic countries including Tanzania do not have adequate surveillance data to make such informed decisions at this level. Limited data on schistosomiasis prevalence inhibits disease control programs from making data informed decision on where to treat, as well as adhering to WHO recommendations for conducting targeted treatment at the sub-district level. Geostatistical models are spatial analysis tools that have been used in the past to help predict the likelihood of disease prevalence in nearby areas where there is limited data. They are particularly helpful in predicting diseases like schistosomiasis that have strong associations with environmental and socio-demographic elements that we do have data on widespread across much of the world. Publications on the use geostatistical models to predict schistosomiasis prevalence in different settings date back more than 20 years, but have not yet been integrated to aid the decision process of making of national programs, nor have they been specifically advocated for by the WHO. The study seeks to examine the implications of using model predictions to guide targeted, sub-district level treatment, in terms of populations requiring treatments when compared with conventional district level approaches. This publication demonstrates that use of geostatistical models for predicting schistosomiasis is possible, can be a valuable tool to inform where to treat. It furthermore estimates that the populations eligible for targeted treatment are considerably different than the populations who would be receiving treatment under conventional district level treatment approached, placing emphasis on the importance of switching to targeted treatment. The paper argues that uptake of geostatistical models can enable targeted planning below the district level, a major step to facilitate a systematic change that the WHO believes will drive down transmission of the disease, the importance of which cannot be understated in an area that is so historically burdened by the disease and its associated health complications. [ABSTRACT FROM AUTHOR]

Vector-borne pathogens (VBPs) causing vector-borne diseases (VBDs) can circulate among humans, domestic animals, and wildlife, with cattle in particular serving as an important source of exposure risk to humans. The close associations between humans and cattle can facilitate the transmission of numerous VBPs, impacting public health and economic security. Published studies demonstrate that cattle can influence human exposure risk positively, negatively, or have no effect. There is a critical need to synthesize the information in the scientific literature on this subject, in order to illuminate the various ecological mechanisms that can affect VBP exposure risk in humans. Therefore, the aim of this systematic review was to review the scientific literature, provide a synthesis of the possible effects of cattle on VBP risk to humans, and propose future directions for research. This study was performed according to the PRISMA 2020 extension guidelines for systematic review. After screening 470 peer-reviewed articles published between 1999–2019 using the databases Web of Science Core Collection, PubMed Central, CABI Global Health, and Google Scholar, and utilizing forward and backward search techniques, we identified 127 papers that met inclusion criteria. Results of the systematic review indicate that cattle can be beneficial or harmful to human health with respect to VBDs depending on vector and pathogen ecology and livestock management practices. Cattle can increase risk of exposure to infections spread by tsetse flies and ticks, followed by sandflies and mosquitoes, through a variety of mechanisms. However, cattle can have a protective effect when the vector prefers to feed on cattle instead of humans and when chemical control measures (e.g., acaricides/insecticides), semio-chemicals, and other integrated vector control measures are utilized in the community. We highlight that further research is needed to determine ways in which these mechanisms may be exploited to reduce VBD risk in humans. Author summary: Vector-borne diseases (VBDs) are caused by pathogens spread by blood-feeding arthropods from an infected to an uninfected organism. These infections may be caused by pathogenic bacteria, viruses, or protozoans and arthropod vectors may spread these pathogens to humans, domestic animals, and wildlife. Humans and cattle spend a significant amount of time in close proximity with each other through various activities such as agriculture, animal husbandry, trading, and animal farming, which can potentially increase risk to human health. Previously published studies indicated cattle can impact vector-borne pathogen (VBP) transmission both positively and negatively, however, there has not been a recent synthesis of the scientific literature on this subject. Through this global systematic review of the scientific literature, we found that cattle could have either harmful or beneficial impacts on human health when it comes to VBDs, but most often increase exposure risk to VBPs in humans. We identified various mechanisms from the scientific literature by which cattle can impact VBP risk in humans. Further research is needed to better understand specific ecological mechanisms by which cattle impact human health and develop measures that will prevent and reduce VBP exposure risk in humans. [ABSTRACT FROM AUTHOR]

Introduction: Act to End NTDs | West, a USAID-funded program that supports national governments to eliminate or control five neglected tropical diseases (NTDs) in West Africa including trachoma, lymphatic filariasis (LF), onchocerciasis, schistosomiasis and soil-transmitted helminthiasis, conducted a gender and social inclusion analysis to determine how NTDs differentially impact various populations and how gender and social norms impact NTD programs to inform future programming. Methods: The study used a mixed methods approach including a literature review; primary qualitative data collection; and monitoring data in Côte d'Ivoire, Sierra Leone, and Ghana. Results: Women and girls face additional health risks from many NTDs compared to men and boys. In addition to differential health burden, the social and economic impacts of NTD-related disability or infertility can be particularly dire for women and girls. Men were somewhat less likely to participate in mass drug administration (MDAs) due to: lack of information about campaigns, lack of access due to work, and higher levels of mistrust of the government and concerns about side effects of the medicines. Pregnant and breastfeeding women were sometimes excluded by community drug distributors (CDDs) from certain types of MDAs for which they are eligible. Training participation rates for CDDs and supervisors were universally higher for men than women, even though feedback on the effectiveness of female CDDs was overwhelmingly positive, and female CDDs often have more access to other women in conservative households. The role of a CDD can lead to career and social opportunities for women. However, challenges faced by CDDs were seen as a greater barrier for women, including transportation, safety, household responsibilities, lower education levels, and low or lack of wages. Discussion: Programs to address NTDs can promote equity and improve programming by increasing women's participation as CDDs and providing financial compensation. Additionally, programs should prioritize inclusive training for CDDs, and inclusive messaging about MDA for communities. Author summary: The Act to End NTDs | West program supports USAID's efforts to control or eliminate trachoma, lymphatic filariasis (LF), onchocerciasis, schistosomiasis and soil-transmitted helminthiasis in 11 West African countries using mass drug administration.Act | West conducted a gender and social inclusion analysis to inform a strategy for the program to improve outcomes and more meaningfully engage and empower women and marginalized individuals in program implementation. Our objective was to identify how gender and social norms and stigma differentially impact men's and women's adoption of healthy practices around NTD prevention, use of NTD services, participation in programing, and opportunities to engage in identifying solutions, using an intersectional lens inclusive of disability, ethnic identity, and geographic location. Several studies have looked at gender dimensions of staffing for NTD programming, analyzed differential risk factors and prevalence of NTDs between males and females, or presented data on gender and national mass drug administration (MDA) coverage to combat NTDs. This paper aims to consolidate these findings through a literature review as well as a qualitative study conducted in Côte d'Ivoire, Ghana, and Sierra Leone, to provide a comprehensive look at gender and social inclusion within NTD programming across various contexts and diseases. This study found that women and girls face additional health risks from five NTDs compared to men and boys. In addition to the health burden, the social and economic impacts of NTD-related disability or infertility can be particularly severe for women and girls. Additionally, men were somewhat less likely to participate in mass drug administration (MDAs) and pregnant and breastfeeding women were sometimes excluded by community drug distributors (CDDs) from certain types of MDAs for which they are eligible due to misunderstanding on the part of CDDs and community members on eligibility criteria. Training participation rates for CDDs and supervisors were universally higher for men than women, even though feedback on the effectiveness of female CDDs was overwhelmingly positive, and female CDDs often have more access to other women in conservative households. Additionally, challenges faced by CDDs were seen as a greater barrier for women to serve in that capacity. [ABSTRACT FROM AUTHOR]

Background: Neglected tropical diseases (NTDs) affect most impoverished communities in developing countries, like Myanmar in Southeast Asia. NTDs have been understudied and underreported in Myanmar. Methods: A systematic review of published and grey literature (1900–2023) on neglected tropical diseases (NTDs) in Myanmar was conducted. The literature search included five international databases: PubMed, EMBASE, Ovid Global Health, and Web of Science Core Collection and one national database: the Myanmar Central Biomedical Library (locally published papers and grey literature). The selection criteria included articles with all types of study designs of current or previous infections conducted in humans, that reported NTDs, recognised by WHO, US CDC, and listed in PLoS NTDs. We included melioidosis and rickettsioses which we consider also meet the definition of an NTD. Results: A total of 5941 records were retrieved and screened, of which, 672 (11%) met the selection criteria and were included in this review. Of the included articles, 449 (65%) were published after 2000 and 369 (55%) were from two regions (Yangon and Mandalay) of Myanmar. Of the included articles, 238 (35%) reported bacterial NTDs, 212 (32%) viral NTDs, 153 (23%) helminth NTDs, 25 (4%) protozoal NTDs and 39 (6%) reported more than one aetiology. Based on reported frequency in descending order, the bacterial NTDs were leprosy, Escherichia coli enteritis, salmonellosis, cholera, shigellosis, melioidosis, leptospirosis and rickettsioses; the viral NTDs were dengue, chikungunya and Japanese encephalitis virus (JEV) infection; the protozoal NTDs were amoebiasis, giardiasis and leishmaniasis, and the helminth NTDs were ascariasis, trichuriasis, hookworm disease, filariasis and strongyloidiasis. Conclusion: This review summarises NTDs reported in Myanmar over the past 100 years. The findings suggest that most NTDs are likely to be under reported, especially from the majority of the country which is far from academic centres. Research capacity building together with strengthening of laboratory systems would lead to better understanding of the true burden of NTDs in Myanmar. Trial registration: PROSPERO registration ID: CRD42018092627. Author summary: Neglected tropical diseases (NTDs) are a group of preventable and treatable diseases common in tropical countries primarily affecting poor rural communities. Myanmar is a tropical country in Southeast Asia in which 70% of the population live in rural areas. It is one of the top ten countries with a high burden of NTDs in term of incidence and prevalence. We conducted a systematic review of all available literature on infectious NTDs in Myanmar to collate all reported NTDs published in international as well as local journals including theses and dissertations. Our review found bacterial and viral NTDs being the most commonly reported NTDs followed by helminthiasis and protozoal NTDs. More than half of the studies were located in two out of fifteen states and regions of Myanmar. This review summarises reported NTDs in Myanmar over 100 years and indicates gaps in knowledge about certain diseases to inform future research directions related to NTDs in Myanmar. [ABSTRACT FROM AUTHOR]

In recent years, the number of reported Human African Trypanosomiasis (HAT) cases caused by Trypanosoma brucei (T.b.) gambiense has been markedly declining, and the goal of 'elimination as a public health problem' is within reach. For the next stage, i.e. interruption of HAT transmission by 2030, intensive screening and surveillance will need to be maintained, but with tools and strategies more efficiently tailored to the very low prevalence. We assessed the sequential use of ELISA and Immune Trypanolysis (ITL) on dried blood spot (DBS) samples as an alternative to the traditional HAT field testing and confirmation approach. A cross-sectional study was conducted in HAT endemic and previously endemic zones in Kongo Central province, and a non-endemic zone in Haut Katanga province in the Democratic Republic of the Congo (DRC). Door-to-door visits were performed to collect dried blood spot (DBS) samples on filter paper. ELISA/T.b. gambiense was conducted followed by ITL for those testing positive by ELISA and in a subset of ELISA negatives. In total, 11,642 participants were enrolled. Of these, 11,535 DBS were collected and stored in appropriate condition for ELISA testing. Ninety-seven DBS samples tested positive on ELISA. In the endemic zone, ELISA positivity was 1.34% (95%CI: 1.04–1.64). In the previously endemic zone and non-endemic zone, ELISA positivity was 0.34% (95% CI: 0.13–0.55) and 0.37% (95% CI: 0.15–0.60) respectively. Among the ELISA positives, only two samples had a positive ITL result, both from the endemic zone. One of those was from a former HAT patient treated in 2008 and the other from an individual who unfortunately had deceased prior to the follow-up visit. Our study showed that a surveillance strategy, based on DBS samples and centralized testing with retracing of patients if needed, is feasible in DRC. ELISA seems well suited as initial test with a similar positivity rate as traditional screening tests, but ITL remains complex. Alternatives for the latter, also analyzable on DBS, should be further explored. Author summary: Human African Trypanosomiasis (HAT), also known as sleeping sickness is a parasitic disease, transmitted by tsetse flies, that is usually fatal if untreated. The number of cases have been rapidly declining over the past years indicating that elimination of the disease as a public health problem is within reach. To achieve the next stage, i.e. interruption of HAT transmission by 2030, intensive screening and surveillance will need to be maintained, but with tools and strategies more efficiently tailored to the very low prevalence. In contrast to the traditional approach of sending laboratory expertise to the field, we assessed an alternative approach based on the collection of dried blood samples on filter paper that were tested in a regional laboratory. Samples were taken in endemic, previously endemic and non-endemic villages and tested by ELISA and Immune Trypanolysis. The ELISA positivity rates were similar to those of other screening techniques currently used and Immune Trypanolysis was highly specific. Hence for surveillance in HAT endemic areas, collecting dried blood samples followed by centralized testing could become an alternative to the current strategy of active screening by mobile teams with on the spot confirmation. It has also potential for post-elimination surveillance to monitor resurgence and for exploratory surveillance in historic foci. Though highly specific, Immune Trypanolysis remains too complex for use in intermediate level laboratories, to further expand this strategy an alternative second step test is required. [ABSTRACT FROM AUTHOR]

Reference 1 Yin D, Bai Q, Wu X, Li H, Shao J, Sun M, et al. (2021) Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein. The last author's name is spelled incorrectly. [Extracted from the article]

Human infection with the intestinal nematode Strongyloides stercoralis is persistent unless effectively treated, and potentially fatal in immunosuppressed individuals. Epidemiological data are lacking, partially due to inadequate diagnosis. A rapid antigen detection test is a priority for population surveillance, validating cure after treatment, and for screening prior to immunosuppression. We used a targeted analysis of open access 'omics' data sets and used online predictors to identify S. stercoralis proteins that are predicted to be present in infected stool, Strongyloides-specific, and antigenic. Transcriptomic data from gut and non-gut dwelling life cycle stages of S. stercoralis revealed 328 proteins that are differentially expressed. Strongyloides ratti proteomic data for excreted and secreted (E/S) proteins were matched to S. stercoralis, giving 1,057 orthologues. Five parasitism-associated protein families (SCP/TAPS, prolyl oligopeptidase, transthyretin-like, aspartic peptidase, acetylcholinesterase) were compared phylogenetically between S. stercoralis and outgroups, and proteins with least homology to the outgroups were selected. Proteins that overlapped between the transcriptomic and proteomic datasets were analysed by multiple sequence alignment, epitope prediction and 3D structure modelling to reveal S. stercoralis candidate peptide/protein coproantigens. We describe 22 candidates from seven genes, across all five protein families for further investigation as potential S. stercoralis diagnostic coproantigens, identified using open access data and freely-available protein analysis tools. This powerful approach can be applied to many parasitic infections with 'omic' data to accelerate development of specific diagnostic assays for laboratory or point-of-care field application. Author summary: The worm Strongyloides stercoralis causes infectious disease in people throughout tropical and sub-tropical regions, leading to an extensive reduction in quality of life and even death. Millions of people are at risk of infection with this parasite and improved diagnostic and control methods and technologies are urgently required. Currently, most diagnosis is carried out through methods involving visual inspection of patient's faeces, which has a number of drawbacks, particularly its poor sensitivity. This paper presents a new method to develop improved diagnostic tests for S. stercoralis, by computational analysis of publicly available gene and protein sequences to predict proteins that may be detectable in faeces. This would enable the development of rapid diagnostic tests in the form of lateral flows or dipsticks, with better predictive ability and fewer drawbacks than current diagnostic methods. A number of potential proteins, predicted to have all the desired characteristics for use in such tests were found through the new method and have been presented in this paper. With validation, new diagnostic tests for S. stercoralis could be developed from these results and the computational approach could be used to target other parasitic diseases. [ABSTRACT FROM AUTHOR]

Background: Chagas disease or American trypanosomiasis, a neglected tropical disease, is a persistent Public Health problem in Latin America and other, non-endemic, countries. Point-of-care (POC) sensitive methods are still needed to improve and extend early diagnosis in acute infections such as congenital Chagas disease. The objective of this study was to analytically evaluate in the lab the performance of a qualitative POC molecular test (Loop-mediated isothermal amplification (LAMP), Eiken, Japan) for rapid diagnosis of congenital Chagas disease employing FTA cards or Whatman 903 filter paper as solid supports for small-scale volumes of human blood. Methodology/principal findings: We used human blood samples artificially infected with cultured T. cruzi strains to assess the analytical performance of the test in comparison with liquid blood anticoagulated with heparin. The DNA extraction process was evaluated using the ultrarapid purification system PURE manufactured by Eiken Chemical Company (Tokio, Japan) over artificially infected liquid blood or different amounts of dried blood spot (DBS) 3- and 6-mm pieces of FTA and Whatman 903 paper. LAMP was performed on a AccuBlock (LabNet, USA) heater or in the Loopamp LF-160 incubator (Eiken, Japan), and visualization of results was either done at naked eye, using the LF-160 device or P51 Molecular Fluorescence Viewer (minipcr bio, USA). Best conditions tested showed a limit of detection (LoD) with 95% accuracy (19/20 replicates) of 5 and 20 parasites/mL, respectively for heparinized fluid blood or DBS samples. FTA cards showed better specificity than Whatman 903 filter paper. Conclusions/significance: Procedures to operate LAMP reactions from small volumes of fluid blood or DBS in FTA were standardized for LAMP detection of T. cruzi DNA. Our results encourage prospective studies in neonates born to seropositive women or oral Chagas disease outbreaks to operationally evaluate the method in the field. Author summary: Chagas disease is a global health problem in endemic and non-endemic regions due to migration. Early diagnosis of newborns to seropositive mothers is of utmost importance to Public Health, enabling prompt access to highly effective anti-parasitic treatment. For this, it is essential to develop rapid and sensitive methods at point-of-care (POC). The aim of this work was to estimate the analytical performance of the molecular amplification technique named Loop-mediated isothermal amplification (LAMP) as POC strategy to detect Trypanosoma cruzi in small volumes of blood collected in dried blood spots. The procedure was carried out in human blood samples spiked with T. cruzi strains on Whatman 903 or FTA commercial filter papers in comparison with fluid blood in heparin as anticoagulant. We used an ultra-rapid DNA extraction method, tested the LAMP reaction in two different heaters and visualized the results either by the naked eye or using fluorescence viewers. FTA cards showed excellent sensitivity and specificity; LAMP could be performed in a simple heater and results easily visualized with fluorescence viewers. The entire process entails handling basic laboratory devices that will enable POC diagnosis of congenital infection and other acute cases of Chagas disease such as those derived from oral outbreaks. [ABSTRACT FROM AUTHOR]

Cholera continues to inflict high rates of morbidity and mortality. Prompt identification of cholera cases facilitates rapid outbreak responses in the short term while providing reliable surveillance data to guide long-term policies and interventions. Microbiological stool culture, the current recognized gold standard for diagnosing cholera, has significant limitations. Rapid diagnostic tests (RDTs) represent promising alternatives for diagnosing cholera in areas with limited laboratory infrastructure. However, studies conducted with the current cholera RDTs demonstrated wide variations in sensitivity and specificity. To address this gap in the diagnosis of cholera, we developed a simple, rapid, and sensitive diagnostic assay, "Rapid LAMP based Diagnostic Test (RLDT)." With a novel, simple sample preparation method directly from the fecal samples along with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, cholera toxin gene (ctxA) and O1 (O1rfb) gene could be detected in less than an hour. Cholera RLDT assay is cold chain and electricity-free. To avoid any end-user bias, a battery-operated, handheld reader was used to read the RLDT results. The performance specifications of the cholera RLDT assay, including analytical sensitivity and specificity, were evaluated using direct fecal samples, dried fecal samples on filter paper, and environmental water samples spiked with cholera strain. The limit of detection (LOD) was ~104 CFU/gm of stool for both ctxA and O1 genes, corresponding to about 1 CFU of Vibrio cholerae per reaction within 40 minutes. The LOD was 10 bacteria per ml of environmental water when tested with RLDT directly, without enrichment. Being simple, RLDT has the potential to be applied in resource-poor endemic settings for rapid, sensitive, and reliable diagnosis of cholera. Author summary: Cholera is an acute diarrheal infection caused by Vibrio cholerae. Cholera continues to severely threaten the health of the impoverished and vulnerable populations in the endemic regions. The present laboratory-based cholera detection methods take time and require well-equipped facilities and well-trained staff that are often not available in the resource-poor settings. The available rapid diagnostic tests have a wide range of sensitivity and specificity. A rapid, simple, sensitive, and reliable diagnostic for cholera is required, which would identify cholera cases early on in outbreaks to facilitate a rapid response and provide reliable surveillance data. We developed a simple, rapid, and sensitive diagnostic assay, "Rapid LAMP based Diagnostic Test (RLDT)," to detect cholera in low-resource settings. Using RLDT, cholera could be detected directly from stool, dried stool on filter paper, and environmental water in less than one hour with minimum hands-on time. The RLDT assay does not require maintaining a cold chain and is electricity-free, facilitating point-of-care testing. The RLDT assay is sensitive, simple to use, and can be scaled up for rapid detection of cholera and surveillance in low-resource endemic settings. [ABSTRACT FROM AUTHOR]

Background: The Geshiyaro project aims to assess the feasibility of interrupting transmission of soil-transmitted helminths (STH) and schistosome (SCH) infection in the Wolaita zone of southern Ethiopia through high coverage community-wide mass drug administration (MDA), in combination with improved water, sanitation, and hygiene services and behaviour change communication delivered through the existing health care infrastructure. To accurately measure treatment coverage a population census was conducted enrolling individuals with biometric fingerprinting and barcoded ID cards. This paper details the baseline census and parasitology surveys conducted before the start of any interventions. Methods: The census was conducted in five of the 15 Wolaita districts between October 2018 and December 2019, enrolling all consenting participants from every household. Simultaneously, a cross-sectional parasitology survey was conducted in 130 out of 361 randomly selected communities from all 15 districts, with 100 individuals across all age groups (infant to adult) per community providing stool and urine for analysis by duplicate Kato-Katz and a point-of-care circulating cathodic antigen (POC-CCA) to test for Schistosoma mansoni and STH, and microhaematuria and urine filtration for Schistosoma haematobium. Of the 130 communities, 30 were randomly selected for annual, longitudinal parasitological monitoring, with 150 randomly selected individuals from infant to adult providing two days of stool and urine samples for analysis by the same diagnostic tests per community. Results: In total 97,919 households participated in the baseline census enrolling 466,071 individuals, with parasitological data obtained from 10,785 people. At baseline, 15.5% were infected with at least one STH species, with Ascaris lumbricoides (9.5%), followed by hookworm (7.2%) and Trichuris trichiura (1.8%). Substantial heterogeneity in STH prevalence was observed between communities ranging from 0% to 61% where most infections were low intensity. Schistosoma mansoni infection was the dominant schistosome infection (0.85% by Kato-Katz and 13.3% by POC-CCA trace negative and 21.5% trace positive), with few Schistosoma haematobium infections identified (2.77% haematuria positive and 0.13% positive by urine filtration). Conclusions: While the national control program in Ethiopia has made good progress in reducing prevalence of STH and SCH in Wolaita since it was launched in 2015, there remain areas of persistent infection suggesting the existence of environmental or behavioural risk factors that contribute to ongoing transmission. This project aims to identify the most efficient intervention strategies to reduce community burden and reach interruption of transmission. Author summary: The standard method of control for intestinal worms, a common public health problem in sub-Saharan Africa, is distribution of deworming treatment on a mass scale. These infections are difficult to eliminate as re-infection is common due a combination of people not being treated and high exposure to the parasites through poor access to sanitation and clean water. The Geshiyaro project in Ethiopia has the ambitious goal of breaking the transmission cycle of such infections through treatment in combination with provision of water and sanitation services. The impact of these interventions is being measured using a unique approach, that is, using finger printing to evaluate prevalence of infection with compliance to treatment and access water or sanitation. [ABSTRACT FROM AUTHOR]

Long non-coding (lnc)RNAs are a class of eukaryotic RNA that do not code for protein and are linked with transcriptional regulation, amongst a myriad of other functions. Using a custom in silico pipeline we have identified 6,436 putative lncRNA transcripts in the liver fluke parasite, Fasciola hepatica, none of which are conserved with those previously described from Schistosoma mansoni. F. hepatica lncRNAs were distinct from F. hepatica mRNAs in transcript length, coding probability, exon/intron composition, expression patterns, and genome distribution. RNA-Seq and digital droplet PCR measurements demonstrated developmentally regulated expression of lncRNAs between intra-mammalian life stages; a similar proportion of lncRNAs (14.2%) and mRNAs (12.8%) were differentially expressed (p<0.001), supporting a functional role for lncRNAs in F. hepatica life stages. While most lncRNAs (81%) were intergenic, we identified some that overlapped protein coding loci in antisense (13%) or intronic (6%) configurations. We found no unequivocal evidence for correlated developmental expression within positionally correlated lncRNA:mRNA pairs, but global co-expression analysis identified five lncRNA that were inversely co-regulated with 89 mRNAs, including a large number of functionally essential proteases. The presence of micro (mi)RNA binding sites in 3135 lncRNAs indicates the potential for miRNA-based post-transcriptional regulation of lncRNA, and/or their function as competing endogenous (ce)RNAs. The same annotation pipeline identified 24,141 putative lncRNAs in F. gigantica. This first description of lncRNAs in F. hepatica provides an avenue to future functional and comparative genomics studies that will provide a new perspective on a poorly understood aspect of parasite biology. Author summary: With increased understanding of genomes and transcriptomes it has become clear that not all RNAs code for protein, in contrast to the Central Dogma of molecular biology. Nevertheless, many such "non-coding" RNAs have functions in their own right. For example, long non-coding (lnc)RNAs comprise a large proportion of the human genome and control expression of some human protein coding genes. In parasites, lncRNAs remain understudied and poorly understood. This paper provides the first description of the lncRNA complement of the liver fluke, Fasciola hepatica. We show that lncRNA expression changes as parasites develop, in a similar manner to protein-coding mRNA, suggesting important roles in parasite growth and development. Some lncRNAs show correlated developmental expression with mRNAs coding for important parasite proteins, suggesting that they may interact, suggesting regulatory functions for fluke lncRNAs. A larger subset of lncRNA may interact with micro (mi)RNAs, another type of non-coding RNA that are important regulators of gene expression in eukaryotes. These interactions are, again, important additions to our understanding of fluke lncRNA biology. Our ultimate goal is to discover ways to inhibit these interactions that could be used for new medicines for fluke infections which are a global scourge on the farming industry. [ABSTRACT FROM AUTHOR]

Diagnosis of arbovirus infection or exposure by antibody testing is becoming increasingly difficult due to global expansion of arboviruses, which induce antibodies that may (cross-)react in serological assays. We provide a systematic review of the current knowledge and knowledge gaps in differential arbovirus serology. The search included Medline, Embase and Web of Science databases and identified 911 publications which were reduced to 102 after exclusion of studies not providing data on possible cross-reactivity or studies that did not meet the inclusion criteria regarding confirmation of virus exposure of reference population sets. Using a scoring system to further assess quality of studies, we show that the majority of the selected papers (N = 102) provides insufficient detail to support conclusions on specificity of serological outcomes with regards to elucidating antibody cross-reactivity. Along with the lack of standardization of assays, metadata such as time of illness onset, vaccination, infection and travel history, age and specificity of serological methods were most frequently missing. Given the critical role of serology for diagnosis and surveillance of arbovirus infections, better standards for reporting, as well as the development of more (standardized) specific serological assays that allow discrimination between exposures to multiple different arboviruses, are a large global unmet need. Author summary: Arboviruses are notorious for triggering antibodies that may cross-react with (closely) related arboviruses, complicating interpretation of serology-based arbovirus diagnostics and of epidemiological surveys. This is further complicated by the rapid global spread of arboviruses, that may lead to increasing co-circulation of antigenically related arboviruses, and by the lack of detailed background information on the population tested, methods used and background arbovirus exposures such as prior infections or vaccinations. Here, we aim to provide a better understanding of the complex cross-reactivity patterns as well as the current knowledge and knowledge gaps in differential arbovirus serology. We have done a systematic analysis of reported cross-reactivity patterns from published peer-reviewed articles, using a system that allows assessment of the quality of reported cross-reactivity results. We show that using this approach, the majority of studies lack details needed to reliably assess whether reactivity to antigens from different, related arboviruses is caused by antibody cross-reactivity, or if it reflects antibodies from multiple exposures to these different viruses. Furthermore, there is a strong need for standardization of assays and study designs in future serological studies to compare cross-reactivity results from different studies. Tackling this may lead to improved diagnosis and surveillance of (newly) emerging and/or rapidly spreading arboviruses. [ABSTRACT FROM AUTHOR]

Background: Moxidectin is a macrocyclic lactone registered for the treatment of human onchocerciasis. The drug has a good safety profile, large volume of distribution and a long elimination half-life. This paper reports tolerability data from the first use of moxidectin in persons with Wuchereria bancrofti infection. Methods: In this randomized, open-label, masked-observer superiority trial, adults with Wuchereria bancrofti microfilaremia in Côte d'Ivoire were randomized to 1 of 4 treatment arms: ivermectin + albendazole (IA), moxidectin + albendazole (MoxA), ivermectin + diethylcarbamazine (DEC) + albendazole (IDA), or moxidectin + DEC + albendazole (MoxDA). As part of a larger efficacy trial, all participants were closely monitored for 7 days after treatment. Results: One hundred sixty-four individuals were treated, and monitored for treatment emergent adverse events (TEAE). Eighty-seven participants (53%) experienced one or more mild (grade 1) or moderate (grade 2) TEAE. Four participants had transient Grade 3 hematuria after treatment (3 after IDA and 1 after IA). There were no serious adverse events. There were no significant differences in frequency or types of TEAE between treatment groups (IA = 22/41 (53%), MoxA = 24/40 (60%), IDA = 18/41 (44%), MoxDA = 15/42 (36%), p = 0.530). Fifty-nine participants (36%) had multiple TEAE, and 8.5% had a one or more grade 2 (moderate) TEAE. Grade 2 TEAE were more frequent after triple drug treatments (IDA, 14.6%; MoxDA, 9.5%) than after two-drug treatments (IA, 7.3%; MoxA, 2.5%). There was no difference in TEAEs based on baseline Mf counts (OR 0.69 (0.33, 1.43), p-value 0.319). Conclusion: All treatment regimens were well tolerated. We observed no difference in safety parameters between regimens that contained ivermectin or moxidectin. Trial registration: Clinicaltrials.gov, NCT04410406. Author summary: Lymphatic Filariasis (LF) is a mosquito-borne parasitic infection caused predominantly by Wuchereria bancrofti. Infection can lead to significant lymphatic dysfunction, including hydroceles and lymphedema, which can progress to elephantiasis. The World Health Organization's Global Programme to Eliminate LF (GPELF) recommends mass drug administration (MDA) in endemic populations to eliminate the disease. GPELF recommendations for MDA include ivermectin (IVM) plus albendazole (IA) in sub-Saharan Africa where onchocerciasis is present. In 2018 the US Food and Drug Administration approved use of moxidectin for treatment of onchocerciasis. Moxidectin is a macrocytic lactone, similar to IVM, but more lipophilic, with a larger volume of distribution and longer half-life. Onchocerciasis studies found moxidectin to be superior to IVM for clearance of microfiladermia in people with onchocerciasis, with a treatment emergent adverse event (TEAE) profile similar to that of ivermectin. Moxidectin has not yet been studied, alone or in combination with other antihelminthic drugs. This is a safety evaluation of the first trial of moxidectin combination therapy for LF, which shows that moxidectin combination regimens are well tolerated in Wuchereria bancrofti-infected patients, with a TEAE profile comparable to standard ivermectin containing regimens. [ABSTRACT FROM AUTHOR]

Long-term immune evasion by the African trypanosome is achieved through repetitive cycles of surface protein replacement with antigenically distinct versions of the dense Variant Surface Glycoprotein (VSG) coat. Thousands of VSG genes and pseudo-genes exist in the parasite genome that, together with genetic recombination mechanisms, allow for essentially unlimited immune escape from the adaptive immune system of the host. The diversity space of the "VSGnome" at the protein level was thought to be limited to a few related folds whose structures were determined more than 30 years ago. However, recent progress has shown that the VSGs possess significantly more architectural variation than had been appreciated. Here we combine experimental X-ray crystallography (presenting structures of N-terminal domains of coat proteins VSG11, VSG21, VSG545, VSG558, and VSG615) with deep-learning prediction using Alphafold to produce models of hundreds of VSG proteins. We classify the VSGnome into groups based on protein architecture and oligomerization state, contextualize recent bioinformatics clustering schemes, and extensively map VSG-diversity space. We demonstrate that in addition to the structural variability and post-translational modifications observed thus far, VSGs are also characterized by variations in oligomerization state and possess inherent flexibility and alternative conformations, lending additional variability to what is exposed to the immune system. Finally, these additional experimental structures and the hundreds of Alphafold predictions confirm that the molecular surfaces of the VSGs remain distinct from variant to variant, supporting the hypothesis that protein surface diversity is central to the process of antigenic variation used by this organism during infection. Author summary: The African trypanosome is a single-celled parasite that causes African Sleeping Sickness in humans and related diseases in many animals. It reproduces in the blood of an infected organism, completely exposed to the immune system, but survives due to its ability to repeatedly disguise itself with new surface coat proteins that fool the antibody response. The African trypanosome has thousands of these Variant Surface Glycoproteins (VSGs) in its genome to switch onto the parasite surface. Therefore, trypanosomiasis is centered on the antibody-VSG interaction and how it varies over time. At the most basic level, understanding the possibilities and constraints of VSG protein architecture is a critical foundation to understand the interaction of the pathogen with the host immune system. The work in this paper thoroughly characterizes this genomic collection of VSGs at the molecular level, classifying the coat proteins into related families based on several important criteria, thereby providing the groundwork for developing an understanding of how this organism is able to evade immunity and thrive during infection. [ABSTRACT FROM AUTHOR]

Mycetoma is one of the six Neglected Tropical Diseases that are prevalent in Turkana County (northwest Kenya). The aim of the study was to estimate the prevalence of mycetoma in the county, as well as to describe the main causative agents involved in the disease using methods affordable locally. Based on the data collected by the team of cooperative medicine Cirugia en Turkana (Surgery in Turkana), a specific study for mycetoma was started during the 16th humanitarian medicine campaign in February 2019. Patients with suspected mycetoma were studied at the Lodwar County Referral Hospital (LCRH). After informing the patient and getting their consent, the lesions were examined and sampled (mainly by biopsy) and clinical data were recorded. Samples were washed in sterile saline solution and cut in fragments. Some of these were inoculated on Sabouraud Dextrose Agar, Malt Extract Agar, and diluted Nutrient Agar plates. One fragment of each sample was used for DNA extraction. The DNA and the rest of the fragments of samples were kept at -20°C. All cultures were incubated at room temperature at the LCRH laboratory. The DNA obtained from clinical samples was submitted to PCR amplification of the ITS-5.8S and the V4-V5 16S rRNA gene region, for the detection and identification of fungi and bacteria respectively. From February 2019 till February 2022, 60 patients were studied. Most of them were men (43, 74,1%) between 13 and 78 y.o. (mean age 37). Half of the patients were herdsmen but, among women 40% (6) were housewives and 26.7% (4) charcoal burners. Lesions were mainly located at the feet (87.9%) and most of the patients (54; 93.1%) reported discharge of grains in the exudate, being 27 (46.6%) yellow or pale colored and 19 (32.8%) of them dark grains. Culture of clinical samples yielded 35 fungal and bacterial putative causative agents. Culture and molecular methods allowed the identification of a total of 21 causative agents of mycetoma (39.6% of cases studied). Most of them (17) corresponded to fungi causing eumycetoma (80.9%) being the most prevalent the genus Madurella (7; 41.2%), with two species involved (M. mycetomatis and M. fahalii), followed by Aspergillus (2; 11.8%). Other minority genera detected were Cladosporium, Fusarium, Acremonium, Penicillium, and Trichophyton (5.9% each of them). Actinobacteria were detected in 19.1% of samples, but only Streptomyces somaliensis was identified as a known agent of mycetoma, the rest being actinobacteria not previously described as causative agents of the disease, such as Cellulosimicrobium cellulans detected in two of the patients. Although Kenya is geographically located in the mycetoma belt, to our knowledge this is the first report on mycetoma in this country from 1973, and the first one for Turkana County. Author summary: In this paper we describe the medical situation of people affected by mycetoma in Turkana County, (north-west Kenya). This is a neglected disease, suffered by the poorest people in the world due to lack of education and access to health services. In Turkana there is a high incidence of this disease, especially among young men working in the fields, where they get infected by fungi or bacteria in the soil or on plants and thorns. The lack of nearby health care means that these patients' injuries develop into severe disability and even death. The study of the tissues of patients detecting and studying the microorganisms involved, led to a more specific knowledge about the disease in this area of the world. Our work aims to contribute to the global knowledge of mycetoma to favor the implementation of means for the diagnosis and treatment of this problem, which will allow a better management and the implementation of satisfactory medical solutions. [ABSTRACT FROM AUTHOR]

Background: The Democratic Republic of the Congo (DRC) accounts for the majority of the reported gambiense human African trypanosomiasis (HAT) cases. Kongo Central province in the DRC reports a relatively low, yet steady number of cases, and forms a transboundary focus with Angola and the Republic of Congo. This paper describes an intervention aimed at reducing the case burden in Kongo Central by improving passive case detection, complemented with reactive screening. Methodology/Principal findings: At the initiation of this programme in August 2015, 620 health facilities were identified and equipped with Rapid Diagnostic Tests (RDTs) for HAT screening. Of these, 603 (97%) reported use of RDTs, and 584 (94%) that continued to use RDTs to the last quarter of 2016 were used in the analysis going forward. Among all health facilities involved, 23 were equipped to confirm HAT by microscopy, and 4 of the latter were equipped to perform molecular testing with loop-mediated isothermal amplification (LAMP). Patients clinically suspected of HAT were tested with an RDT and those with a positive RDT result were referred to the nearest microscopy facility for confirmatory testing. If RDT positive patients were negative by microscopy, they were tested by LAMP, either on fresh blood or blood that was dried on filter paper and transported to a facility performing LAMP. This network of diagnostic facilities reduced the median distance for a patient to travel to a screening facility from 13.7km when the classical card agglutination test for trypanosomiasis (CATT) was used as a screening test in the past, to 3.4km. As a consequence, passive case detection was improved by between 30% and 130% compared to the period before. Furthermore, the proportion of HAT cases detected in early stage disease by passive screening increased from 27% to 64%. Reactive screening took place in 20 villages where cases were reported by passive screening, and in 45 villages in the neighbourhood of these villages. Reactive screening was responsible for detection of 40% of cases, of which, 90% were in first stage of the disease. Conclusions: This programme has demonstrated that it is possible to deploy passive screening for HAT at sub-country or country levels in the DRC, and this is made more effective when supplemented with reactive screening. Results and achievements showed an increase in the number of HAT cases detected, the majority of them in early disease, demonstrating that this strategy enables better population coverage and early detection of cases, which is critical in removing the HAT reservoir and interrupting transmission, and could contribute to HAT elimination in regions where it is implemented. Author summary: A number of diagnostic tests for HAT have recently been developed, to improve case detection. We report on the use of these technologies in a strategy to increase coverage and early detection of HAT cases in Kongo Central province of DRC. All 620 health facilities in the focus were equipped with RDTs to test patients presenting with symptoms suggestive of HAT. Among these health facilities, 23 were upgraded to perform confirmatory testing, for a final diagnosis. This strategy has reduced the distance a patient travels to a facility screening for HAT, from 13.7km to 3.4km. From August 2015 to December 2016, the proportion of HAT cases detected, adjusted annually, increased by between 30% and 130% compared to the previous two years, and 64% of them were in early stage disease, compared to 27% previously. This strategy has enabled better population coverage, and when supplemented with reactive screening, the identification of local outbreaks and early detection of most cases, which is critical in removing the HAT reservoir and interrupting transmission, thus contributing to elimination of the disease. [ABSTRACT FROM AUTHOR]

Background: Improved education on water-related diseases in schools could help to reduce disease burden. This paper presents specific results on knowledge, attitudes and practices (KAP) of a cluster-randomized controlled trial to reduce diarrheal disease and dengue entomological risk factors in rural primary schools in Colombia. The aim was to investigate whether enhanced educational interventions on dengue and diarrheal disease in schools could improve KAP scores related to these diseases in students and teachers in rural primary schools, as well as the students' parents. Methodology/Principal findings: A factorial cluster-randomized controlled trial was carried out in 35 rural primary schools in two municipalities in Cundinamarca, central Colombia. Schools were randomized into four arms: interventions related to diarrheal disease (DIA), dengue (DEN), both (DIADEN), or no interventions (control, CON). Both educational and physical interventions to reduce risk factors of dengue and diarrhea were implemented. Comprehensive teachers' manuals were developed and deployed to guide the learning activities. The intervention was carried out over two school years. The knowledge scores of students receiving dengue interventions (DEN, DIADEN) increased by 1.16 point score (0.75–1.56, p<0.001) and those receiving diarrhea interventions (DIA, DIADEN) increased by 1.15 point score (0.67–1.63, p<0.001). The attitude and practice scores of students receiving the diarrhea interventions increased (Attitudes: 0.41 [0.11–0.71, p = 0.01]; Practices: 0.33 [0.01–0.65, p = 0.042]), but not for those receiving the dengue interventions (p = 0.31 and p = 0.08, respectively). Conclusions/Significance: There were increases in knowledge scores among students, their teachers and their parents for both diseases. However, the attitudes and practices components were not affected to the same extent. The hypothesis that the students would disseminate knowledge acquired from the educational interventions to their parents was confirmed for dengue, but not for diarrhea. Trial registration: ISRCTN40195031 The trial is registered in the Current Controlled Trials under Infections and Infestations category. Author summary: The transmission of both dengue and diarrheal diseases depends on water. For dengue, the mosquitoes that transmit the infection lay their eggs in containers that hold water, and this same water may contain microorganisms that cause diarrheal diseases. Improved education on these diseases in schools could help reduce their burden. In this paper we present the results of an integrated intervention on dengue and diarrhea in terms of knowledge, attitudes and practices of students, teachers and parents in 35 rural primary schools in central Colombia. The schools were randomized into four groups: interventions related to diarrheal disease alone, dengue alone, both, or neither. For two years, both educational and physical interventions to reduce risk factors were implemented. The interventions increased knowledge scores among students, their teachers and their parents for both diseases. However, the attitudes and practices components were not affected to the same extent. There was evidence that students disseminated their new knowledge about dengue to their parents, although this was not the case for diarrhea. [ABSTRACT FROM AUTHOR]

Background: Phlebotomus (Paraphlebotomus) sergenti is a widespread proven vector of Leishmania pathogens causing anthroponotic cutaneous leishmaniasis (ACL), due to L. tropica, in the old world. The activity of P. (Par.) sergenti is seasonal and sensitive to general variations in climate. Phenological data sets can thus provide a baseline for continuing investigations on P. (Par.) sergenti population dynamics that may impact future leishmaniasis transmission and control scenarios. Methods/Principal finding: A systematic review of the seasonality of P. (Par.) sergenti was undertaken globally. Sixty-eight scientific papers were identified, and data were extracted from 35 ones, with informative data on sand fly seasonal dynamics on trapping performed from 1992 to December 2021 on 63 sites from 12 countries. Morocco, Saudi Arabia, Iraq, Iran, Pakistan, Palestine, Turkey, Spain, Portugal, Italy, Cyprus, and Georgia. The data extracted from the literature survey were further normalized. Our analysis recorded that the highest P.(Par.) sergenti activity occurs during the hot and dry seasons, primarily in July and August, whatever the location studied. We noticed a relationship between the latitude of sites and sand fly presence (from early April to June) and the type of density trend, varying from a single peak to multiple peaks. On a geographical scale, P. (Par.) sergenti concentrates between 32–37° in latitude in a large interval following the longitude and the highest number of sites with high P. (Par.) sergenti activity is located at the latitude 32°. We also quoted a similar seasonal dynamic and geographic distribution with Phlebotomus (Phlebotomus) papatasi, a proven vector of L. major that causes cutaneous infection. No apparent risk for ACL occurred from December to March, at least in the years and geographic areas considered in this survey. Altogether, knowing that high P. (Par.) sergenti activity would be linked with an increased risk of leishmaniasis transmission, and our study provides information that can be used for control programs on ACL transmission. Conclusions: Despite variations, we found a relatively homogeneous pattern of P. (Par.) sergenti potential behavior in sites whose data are published. A higher risk for L. tropica transmission was identified in the June-October period. Still, such risk was not equally distributed throughout the area since density waves of adults occurred earlier and were more frequent in some territories, like Saudi Arabia. Author summary: Phlebotomus (Paraphlebotomus) sergenti is the most widespread vector of Leishmania pathogens causing anthroponotic cutaneous leishmaniasis (ACL) in the old world. This paper undertook a systematic review of seasonality on a global geographical scale. This systematic analysis allowed us to identify 608 scientific papers, 35 of which 63 sites were explored in 12 countries from 1992 to December 2021. In the current review, Iran (Southern Asia) is the most influential country regarding the number of studies published on P. (Par.) sergenti seasonality with available data. Furthermore, the highest P. (Par.) sergenti activity occurs in all studied locations during the hot and dry periods (with a density that peaked during the hot season, primarily in July and August). Strikingly, we recorded a similar seasonal dynamic and geographic distribution with Phlebotomus papatasi, a proven vector of L. major causing cutaneous leishmaniasis. On a geographical scale, sites with P. (Par.) sergenti activity concentrate between 32–37° in latitude in a large interval following the longitude, with the highest number of sites with high P. (Par.) sergenti activity is located at the latitude 32°. Altogether, knowing that high P. (Par.) sergenti activity would be linked with an increased risk of leishmaniasis transmission. Our study provides information that can be used for control programs on ACL transmission. [ABSTRACT FROM AUTHOR]

Mass drug administration (MDA), targeted at school-aged children (SAC) is recommended by the World Health Organization for the control of morbidity induced by soil-transmitted helminth (STH) infection in endemic countries. However, MDA does not prevent reinfection between treatment rounds, and research suggests that only treating SAC will not be sufficient to interrupt transmission of STH. In countries with endemic infection, such as Ethiopia, the coverage, community-groups targeted, and rates of reinfection will determine how effective MDA is in suppressing transmission in the long-term. In this paper, individually-linked longitudinal data from three epidemiological STH surveys conducted between November 2018 and November 2020 in the Wolaita region of Ethiopia are analysed to determine how STH prevalence and intensity changes according to individual level treatment data collected over two rounds of MDA. This study demonstrates that while community-wide MDA successfully reduces overall infection intensity across the villages treated, the observed levels of non-compliance to treatment by individuals acts to maintain levels of parasite abundance whereby transmission interruption is not possible at to, despite reasonable levels of MDA coverage in the communities studied (ranging from 65% to 84% of the village populations). This quantifies with substantial data the often-postulated difference between coverage (accepting treatment) and compliance (swallowing of treatment), the latter impacting the former to a previously unquantified level. The paper highlights the need to focus treatment to partially treated, or never treated groups of individuals within existing community wide MDA control activities to interrupt the transmission of STH, and to reduce the basic reproductive number, R0, of the parasites to less than unity in value. Author summary: Soil-transmitted helminths (STH) are a group of parasites that remain a public health challenge across developing countries. The high number of global infections is ongoing despite significant financial investment in reducing the disease burden in endemic countries. In recent years WHO recommendations have shifted morbidity reduction to the possibility of transmission elimination through MDA and WaSH measures. One concern with attempting this route towards transmission interruption remains the impact that compliance and coverage has on effectiveness of community-wide treatment. The problem of STH reinfection means that a sustainable break in transmission remains difficult to achieve across an entire community. Despite repeated rounds of treatment, non-compliant individuals can shed infective eggs into the environment, essentially exposing treated individuals to become re-infected. This study highlights this problem, despite the overall effectiveness in reducing infection intensity across communities in the study area, prevalence remains at levels that would not indicate transmission interruption. This in part is due to the non-compliant individuals remaining viable reservoirs for infection that will lead to reinfection of that community before repeated round of MDA. This study indicates the importance of understanding treatment compliance and tools to combat the problem in settings where transmission interruption is being sought. [ABSTRACT FROM AUTHOR]

Background: This study describes the spatial and temporal distribution between 2005 and 2020 of human and animal leishmaniasis by Leishmania infantum in European countries reporting autochthonous cases, and highlights potential activities to improve disease control. Methodology/Principal findings: It was based on a review of the scientific literature and data reported by the World Health Organization (WHO), the World Organization for Animal Health (WOAH) and the Ministries of Health, including hospital discharges in some countries. Autochthonous infections were reported in the scientific literature from 22 countries, including 13 and 21 countries reporting human and animal infections, respectively. In contrast, only 17 countries reported autochthonous human leishmaniasis cases to the WHO and 8 countries animal infections to the WOAH. The number of WOAH reported cases were 4,203, comprising 4,183 canine cases and 20 cases in wildlife. Of 8,367 WHO reported human cases, 69% were visceral leishmaniasis cases—of which 94% were autochthonous—and 31% cutaneous leishmaniasis cases—of which 53% were imported and mostly in France. The resulting cumulative incidence per 100,000 population of visceral leishmaniasis between 2005–2020, was highest in Albania (2.15 cases), followed by Montenegro, Malta, Greece, Spain and North Macedonia (0.53–0.42), Italy (0.16), Portugal (0.09) and lower in other endemic countries (0.07–0.002). However, according to hospital discharges, the estimated human leishmaniasis incidence was 0.70 in Italy and visceral leishmaniasis incidences were 0.67 in Spain and 0.41 in Portugal. Conclusions/Significance: Overall, there was no evidence of widespread increased incidence of autochthonous human leishmaniasis by L. infantum in European countries. Visceral leishmaniasis incidence followed a decreasing trend in Albania, Italy and Portugal, and peaked in Greece in 2013, 2014 and 2017, and in Spain in 2006–2007 and 2011–2013. Animal and human cutaneous leishmaniasis remain highly underreported. In humans, hospital discharge databases provide the most accurate information on visceral leishmaniasis and may be a valuable indirect source of information to identify hotspots of animal leishmaniasis. Integrated leishmaniasis surveillance and reporting following the One Health approach, needs to be enhanced in order to improve disease control. Author summary: Leishmaniasis caused by the protozoan parasite Leishmania infantum, is a zoonotic sand fly-transmitted disease endemic in southern Europe, of great medical and veterinary importance. Environmental and human-related changes affecting the risk of exposure to vectors, such as travelling of infected animals, urbanization of endemic rural areas and climate change are some examples of drivers of the epidemiology of leishmaniasis. In this paper, we review the distribution and assess the evidence of emergence of human and animal leishmaniasis between 2005 and 2020 in endemic European countries. We found autochthonous infections in every Mediterranean and Balkan countries with a highly variable incidence. However, we detected no evidence of a widespread increased incidence across Europe. Notwithstanding this, underreporting is very high for animal and cutaneous human leishmaniasis. Moreover, human hospital discharges are the most accurate source of data for estimating the incidence of severe human visceral leishmaniasis, and this could be used to identify hotspots of leishmaniasis in animal reservoirs of infection. Consequently, we encourage integrated surveillance and accurate notification of cases following the One Health approach to adopt effective strategies resulting in a better control of leishmaniasis. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic disrupted essential health services, including those provided by national neglected tropical disease (NTD) programs. Most mass drug administration (MDA) programs were postponed for 6–12 months following World Health Organization guidance released in April 2020 to temporarily halt NTD programs and launch necessary COVID-19 precautions. While NTD-endemic countries have since resumed MDA activities, it is critical to understand implementers' perspectives on the key challenges and opportunities for program relaunch, as these insights are critical for maximizing gains towards disease control and elimination during public health emergencies. Using data from using online surveys and focus group discussions, this mixed-methods study sought perspectives from Ministry of Health NTD Program Managers and implementing partners from non-governmental organizations working in sub-Saharan Africa. Data analysis revealed that findings converged around several main themes: disruptions for MDA programs included resource shortages due to prioritization of pandemic response, challenges adhering to COVID-19 safety protocols, and community hesitancy due to coronavirus transmission fears. Identified solutions for restarting MDA programs focused on adapting intervention delivery and packaging to minimize disease transmission, embracing technology to optimize intervention planning and delivery, and identifying opportunities to promote program integration between pandemic response strategies and NTD campaign delivery. Findings identifies key challenges due to disruptions to NTD program delivery and provide strategic recommendations for endemic countries to build resilient programs that can continue to perform during and beyond global pandemics. Author summary: In April 2020, global guidelines suspended mass drug administration (MDA) programs used in the control and elimination of neglected tropical diseases (NTDs) due to the ongoing COVID-19 pandemic. While the guidance aimed to reduce the risk of coronavirus exposure and transmission among stakeholders involved in MDA delivery and recipient community members, it risked impeding the gains made by MDA programs towards NTD control and elimination, leaving billions at risk of these infectious diseases. This study summarizes the perspectives of Ministry of Health NTD Program Managers and representatives from non-governmental organizations across sub-Saharan African working in NTDs regarding challenges imposed on MDA programs by the COVID-19 pandemic and identify opportunities to improve planning and implementation during the relaunch of MDA programs. Respondents noted key disruptions for MDA programs, including resource shortages, challenges adhering to COVID-19 safety protocols, and community hesitancy due to coronavirus transmission fears. Specific solutions for restarting MDA programs included adaptations to MDA strategies to incorporate safer, low-contact drug delivery techniques, incorporating technology to optimize MDA planning and delivery, and identifying opportunities to promote integration between NTD campaign delivery and pandemic response strategies. This paper includes suggestions for building resilient programs that can continue to perform in the face of public health emergencies. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine the diagnostic efficacy of Xpert MTB/RIF assay for rapid diagnosis of Tuberculosis (TB) and detection of rifampicin (RIF) resistance in patients suspected of having EPTB, assessing it against traditional culture and drug susceptibility test (DST) by proportional method, and the ability to predict multidrug resistance TB by Xpert MTB/RIF assay. In this study, the Xpert MTB/RIF assay was applied to 1,614 extrapulmonary specimens. Compared with TB culture and Composite Reference Standard (CRS), the Xpert MTB/RIF assay had a high sensitivity and specificity for detection of EPTB. Depending on the culture method or CRS as the standard, sensitivity of the Xpert MTB/RIF assay for detection of MTB in pleural effusion, cerebrospinal fluid, thoracic drainage fluid and throat swabs specimens were lower than that of other specimens. According to the experimental results, we have reason to believe that Xpert MTB/RIF assay is a rapid and simple technique with high sensitivity and specificity for diagnosing EPTB and detecting drug resistance in variety of specimens. Xpert MTB/RIF assay combined with DST maybe identify more cases of multi-drug resistant tuberculosis (MDR-TB). Author summary: Tuberculosis (TB) is the second most deadly infectious disease after COVID-19. Improving the accessibility of WHO recommended rapid molecular diagnostic tests to achieve earlier and more accurate diagnosis of TB is an important part of strengthening TB laboratory work in the strategy of ending the epidemic of tuberculosis. Extrapulmonary tuberculosis (EPTB) accounts for 15% of all tuberculosis cases. Due to the unique biological samples of different classified EPTB and the occult nature of their onset, their diagnosis is more challenging. Smear microscopy is often insensitive and culture methods are prolonged. With the introduction of Xpert MTB/RIF assay (Cepheid Inc.), this has changed TB diagnostics by providing a rapid accessible platform to diagnose TB and identify rifampicin(RIF) resistance within 2h. Xpert MTB/RIF assay is a suite of semi-nested real-time fluorescent quantitative PCR in vitro diagnostic technologies with good detection efficiency. In recent years, WHO has limited recommended Xpert MTB/RIF assay to detect various types of samples other than sputum for preliminary diagnosis of EPTB. The purpose of this paper is to systematically evaluate the practical application effect of Xpert MTB/RIF assay in the clinical diagnosis of EPTB, and provide scientific guidance for clinicians.ű [ABSTRACT FROM AUTHOR]

This paper summarises the lessons learnt in dengue epidemiology, risk factors, and prevention in Singapore over the last half a century, during which Singapore evolved from a city of 1.9 million people to a highly urban globalised city-state with a population of 5.6 million. Set in a tropical climate, urbanisation among green foliage has created ideal conditions for the proliferation of Aedes aegypti and Aedes albopictus, the mosquito vectors that transmit dengue. A vector control programme, largely for malaria, was initiated as early as 1921, but it was only in 1966 that the Vector Control Unit (VCU) was established to additionally tackle dengue haemorrhagic fever (DHF) that was first documented in the 1960s. Centred on source reduction and public education, and based on research into the bionomics and ecology of the vectors, the programme successfully reduced the Aedes House Index (HI) from 48% in 1966 to <5% in the 1970s. Further enhancement of the programme, including through legislation, suppressed the Aedes HI to around 1% from the 1990s. The current programme is characterised by 4 key features: (i) proactive inter-epidemic surveillance and control that is stepped up during outbreaks; (ii) risk-based prevention and intervention strategies based on advanced data analytics; (iii) coordinated inter-sectoral cooperation between the public, private, and people sectors; and (iv) evidence-based adoption of new tools and strategies. Dengue seroprevalence and force of infection (FOI) among residents have substantially and continuously declined over the 5 decades. This is consistent with the observation that dengue incidence has been delayed to adulthood, with severity highest among the elderly. Paradoxically, the number of reported dengue cases and outbreaks has increased since the 1990s with record-breaking epidemics. We propose that Singapore's increased vulnerability to outbreaks is due to low levels of immunity in the population, constant introduction of new viral variants, expanding urban centres, and increasing human density. The growing magnitude of reported outbreaks could also be attributed to improved diagnostics and surveillance, which at least partially explains the discord between rising trend in cases and the continuous reduction in dengue seroprevalence. Changing global and local landscapes, including climate change, increasing urbanisation and global physical connectivity are expected to make dengue control even more challenging. The adoption of new vector surveillance and control tools, such as the Gravitrap and Wolbachia technology, is important to impede the growing threat of dengue and other Aedes-borne diseases. Author summary: A densely populated, highly urban tropical city-state with long-established populations of Aedes aegypti and Ae. albopictus mosquitoes, plus travel and trade links to all corners of the world, Singapore is ideally suited for dengue transmission. Singapore's experience and rich surveillance data provide important insights for an increasingly large number of territories at risk of dengue epidemics. Decades of vector control efforts, focused on source reduction and surveillance, have successfully lowered the vector population, with accompanying reductions in the resident population's seroprevalence to dengue. We propose that Singapore's vulnerability to outbreaks is due to low levels of immunity in the population, constant introduction of viral variants, expanding urban centres, and increasing human density. The discord between the rising trend in reported cases and falling seroprevalence could be at least partly attributed to improved diagnostics and surveillance. Singapore's evidence-based vector-control programme, which involves strong partnership between the public and private sectors, will continue to adapt to future challenges in this space. [ABSTRACT FROM AUTHOR]