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Community prevalence of infection is a widely used, standardized metric for evaluating malaria endemicity. Conventional methods for measuring prevalence include light microscopy and rapid diagnostic tests (RDTs), but their detection thresholds are inadequate for diagnosing low-density infections. The significance of submicroscopic malaria infections is poorly understood in Madagascar, a country of heterogeneous malaria epidemiology. A cross-sectional community survey in the western foothills of Madagascar during the March 2014 transmission season found malaria infection to be predominantly submicroscopic and asymptomatic. Prevalence of Plasmodium infection diagnosed by microscopy, RDT, and molecular diagnosis was 2.4%, 4.1%, and 13.8%, respectively. This diagnostic discordance was greatest for Plasmodium vivax infection, which was 98.5% submicroscopic. Village location, insecticide-treated bednet ownership, and fever were significantly associated with infection outcomes, as was presence of another infected individual in the household. Duffy-negative individuals were diagnosed with P. vivax , but with reduced odds relative to Duffy-positive hosts. The observation of high proportions of submicroscopic infections calls for a wider assessment of the parasite reservoir in other regions of the island, particularly given the country's current focus on malaria elimination and the poorly documented distribution of the non- P. falciparum parasite species.

Background: Microscopic examination of Giemsa-stained blood films remains a major form of diagnosis in malaria case management, and is a reference standard for research. However, as with other visualization-based diagnoses, accuracy depends on individual technician performance, making standardization difficult and reliability poor. Automated image recognition based on machine-learning, utilizing convolutional neural networks, offers potential to overcome these drawbacks. A prototype digital microscope device employing an algorithm based on machine-learning, the Autoscope, was assessed for its potential in malaria microscopy. Autoscope was tested in the Iquitos region of Peru in 2016 at two peripheral health facilities, with routine microscopy and PCR as reference standards. The main outcome measures include sensitivity and specificity of diagnosis of malaria from Giemsa-stained blood films, using PCR as reference., Methods: A cross-sectional, observational trial was conducted at two peripheral primary health facilities in Peru. 700 participants were enrolled with the criteria: (1) age between 5 and 75 years, (2) history of fever in the last 3 days or elevated temperature on admission, (3) informed consent. The main outcome measures included sensitivity and specificity of diagnosis of malaria from Giemsa-stained blood films, using PCR as reference., Results: At the San Juan clinic, sensitivity of Autoscope for diagnosing malaria was 72% (95% CI 64-80%), and specificity was 85% (95% CI 79-90%). Microscopy performance was similar to Autoscope, with sensitivity 68% (95% CI 59-76%) and specificity 100% (95% CI 98-100%). At San Juan, 85% of prepared slides had a minimum of 600 WBCs imaged, thus meeting Autoscope's design assumptions. At the second clinic, Santa Clara, the sensitivity of Autoscope was 52% (95% CI 44-60%) and specificity was 70% (95% CI 64-76%). Microscopy performance at Santa Clara was 42% (95% CI 34-51) and specificity was 97% (95% CI 94-99). Only 39% of slides from Santa Clara met Autoscope's design assumptions regarding WBCs imaged., Conclusions: Autoscope's diagnostic performance was on par with routine microscopy when slides had adequate blood volume to meet its design assumptions, as represented by results from the San Juan clinic. Autoscope's diagnostic performance was poorer than routine microscopy on slides from the Santa Clara clinic, which generated slides with lower blood volumes. Results of the study reflect both the potential for artificial intelligence to perform tasks currently conducted by highly-trained experts, and the challenges of replicating the adaptiveness of human thought processes.

Background: Zoonotic infections with epidemic potential, as non-human primate malaria and yellow fever (YF), can overlap geographically. Optimizing a small blood sample for diagnosis and surveillance is of great importance. Blood are routinely collected for YF diagnosis and blood clots usually discarded after serum obtention. Aiming to take sample advantage, the sensitivity of a PCR using extracted DNA from long-term frozen clots from human and non-human primates for detection of Plasmodium spp. in low parasitaemia conditions was assayed., Results: Malaria diagnosis with DNA extracted from blood clots generated results in agreement with samples obtained with whole blood, including mixed Plasmodium vivax/simium and Plasmodium malariae/brasilianum infections., Conclusion: Blood clots from human and non-human primates may be an important and low cost source of DNA for malaria surveillance in the Atlantic Forest.

Background: Malaria in Equatorial Guinea remains a major public health problem. The country is a holo-endemic area with a year-round transmission pattern. In 2016, the prevalence of malaria was 12.09% and malaria caused 15% of deaths among children under 5 years. In the Continental Region, 95.2% of malaria infections were Plasmodium falciparum, 9.5% Plasmodium vivax, and eight cases mixed infection in 2011. The main strategy for malaria control is quick and accurate diagnosis followed by effective treatment. Early and accurate diagnosis of malaria is essential for both effective disease management and malaria surveillance. The quality of malaria diagnosis is important in all settings, as misdiagnosis can result in significant morbidity and mortality. Microscopy and RDTs are the primary choices for diagnosing malaria in the field. However, false-negative results may delay treatment and increase the number of persons capable of infecting mosquitoes in the community. The present study analysed the performance of microscopy and RDTs, the two main techniques used in Equatorial Guinea for the diagnosis of malaria, compared to semi-nested multiplex PCR (SnM-PCR)., Results: A total of 1724 samples tested by microscopy, RDT, and SnM-PCR were analysed. Among the negative samples detected by microscopy, 335 (19.4%) were false negatives. On the other hand, the negative samples detected by RDT, 128 (13.3%) were false negatives based on PCR. This finding is important, especially since it is a group of patients who did not receive antimalarial treatment., Conclusions: Owing to the high number of false negatives in microscopy, it is necessary to reinforce training in microscopy, the "Gold Standard" in endemic areas. A network of reference centres could potentially support ongoing diagnostic and control efforts made by malaria control programmes in the long term, as the National Centre of Tropical Medicine currently supports the National Programme against Malaria of Equatorial Guinea to perform all of the molecular studies necessary for disease control. Taking into account the results obtained with the RDTs, an exhaustive study of the deletion of the hrp2 gene must be done in EG to help choose the correct RDT for this area.

The effectiveness of malaria screening and treatment highly depends on the low-cost access to the highly sensitive and specific malaria test. We report a real-time fluorescence nucleic acid testing device for malaria field detection with automated and scalable sample preparation capability. The device consists a compact analyzer and a disposable microfluidic reagent compact disc. The parasite DNA sample preparation and subsequent real-time LAMP detection were seamlessly integrated on a single microfluidic compact disc, driven by energy efficient non-centrifuge based magnetic field interactions. Each disc contains four parallel testing units which could be configured either as four identical tests or as four species-specific tests. When configured as species-specific tests, it could identify two of the most life-threatening malaria species (P. falciparum and P. vivax). The NAT device is capable of processing four samples simultaneously within 50 min turnaround time. It achieves a detection limit of ~0.5 parasites/µl for whole blood, sufficient for detecting asymptomatic parasite carriers. The combination of the sensitivity, specificity, cost, and scalable sample preparation suggests the real-time fluorescence LAMP device could be particularly useful for malaria screening in the field settings., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Competing Interests: Competing interests: None declared.

Background: Malaria remains an endemic disease in Pakistan with an estimated healthcare burden of 1.6 million cases annually, with Plasmodium vivax accounting for 67% of reported cases. P. vivax is the most common species causing malaria outside of Africa, with approximately 13.8 million reported cases worldwide., Method: We report a series of P. vivax cases with cerebral involvement that presented at Aga Khan University Hospital, Karachi, Pakistan., Results: The majority of the patients presented with high-grade fever accompanied by projectile vomiting and abnormal behaviour, seizures, shock and unconsciousness. Seven of 801 patients with P. vivax monoinfection presented or developed cerebral complications. P. vivax infections were diagnosed based on peripheral smears and rapid diagnostic testing., Conclusion: P. vivax infection can lead to severe complications, although not with the frequency of Plasmodium falciparum infection. Current cases highlight an increasing trend of cerebral complications caused by P. vivax., Competing Interests: No financial or competing interests declared

Congenital malaria (CM) is uncommon in both malaria-endemic and non-endemic countries. It may be caused by any Plasmodium spp., although Plasmodium falciparum and Plasmodium vivax are the more frequent etiologic agents. We report a case of delayed diagnosis of CM by P. vivax in a newborn of an Eritrean primigravida . The mother developed pregnancy-related immunodepression and varicella-zoster viral infection 9 days before natural delivery; therefore, the child was admitted in the neonatal intensive care unit (NICU) to administer specific varicella-zoster immunoglobulin prophylaxis and for clinical monitoring. During the NICU stay, the newborn presented a febrile syndrome with vomiting, anemia, and thrombocytopenia. A P. vivax severe malaria diagnosis was made by detecting trophozoites in the thick and thin blood smears. The infant was successfully treated with intravenous artesunate and clindamycin. Our experience suggests that malaria diagnostic tests need to be included in routine blood analyses in newborns with febrile syndrome from mothers with an epidemiologic link to malaria-endemic areas.

Conventional molecular methods, such as nested polymerase chain reaction (PCR), are very sensitive for detection of malaria parasites, but require advanced laboratory equipment and trained personnel. Real-time loop-mediated isothermal amplification (RealAmp), a loop-mediated isothermal amplification-based molecular tool (LAMP), facilitates rapid target amplification at a single temperature setting, reducing the need for sophisticated equipment. We evaluated the performance of a field-adapted RealAmp assay for malaria diagnosis in Cruzeiro do Sul, Acre State, Brazil, a remote area in Brazil with limited laboratory capabilities. We enrolled 1,000 patients with fever (axillary temperature ≥ 37.5 C) or history of fever in last 24 h presenting for malaria diagnosis from February through June 2015. DNA was extracted from dried blood spots using a boil and spin method (heat treatment) at the sample processing site, and also using commercial kits at a Brazilian national reference laboratory. RealAmp was performed for Plasmodium genus, P. falciparum, and P. vivax identification. In addition, Giemsa-stained blood smears were prepared and examined by two independent well-trained study microscopists. A combination of Real-time PCR and nested PCR was used as reference test. The sensitivity and specificity of RealAmp in the field site laboratory were 94.1% (95% confidence interval [CI]: 90.1-96.8) and 83.9% (95% CI: 81.1-86.4), respectively. The sensitivity and specificity of local microscopy were 87.7% (95% CI: 82.6-91.7) and 98.9% (95% CI: 97.8-99.4), respectively, while study microscopy showed sensitivity of 96.4% (95% CI: 93.0-98.4) and specificity of 98.2% (95% CI: 97.0-99.0). None of the three tests detected 20 P. falciparum and P. vivax mixed infections identified by the reference test. Our findings highlight that it is possible to implement simple molecular tests in facilities with limited resources such as Cruzeiro do Sul in Brazil. RealAmp sensitivity was similar to that of microscopy performed by skilled professionals; both RealAmp and study microscopy performed poorly in detection of mixed infection. Attempts to develop and evaluate simpler molecular tools should continue, especially for the detection of malaria infection in remote areas., Competing Interests: The authors have declared that no competing interests exist.

In the Peruvian North Coast (PNC), the number of Plasmodium vivax malaria cases increased steadily from 2007 to 2010 despite a significant decline in the overall number of cases in Peru during the same period. To better understand the transmission dynamics of P. vivax populations in the PNC and the neighboring Ecuadorian Amazon Basin (EAB), we studied the genetic variability and population structure of P. vivax in these areas. One hundred and twenty P. vivax isolates (58 from Piura and 37 from Tumbes in the PNC collected from 2008 to 2010 and 25 from the EAB collected in Pastaza from 2001 to 2004) were assessed by five polymorphic microsatellite markers. Genetic variability was determined by expected heterozygosity ( He ) and population structure by Bayesian inference cluster analysis. We found very low genetic diversity in the PNC ( He = 0-0.32) but high genetic diversity in the EAB ( He = 0.43-0.70). Population structure analysis revealed three distinct populations in the three locations. Six of 37 (16%) isolates from Tumbes had an identical haplotype to that found in Piura, suggesting unidirectional flow from Piura to Tumbes. In addition, one haplotype from Tumbes showed similarity to a haplotype found in Pastaza, suggesting that this could be an imported case from EAB. These findings strongly suggest a minimal population flow and different levels of genetic variability between these two areas divided by the Andes Mountains. This work presents molecular markers that could be used to increase our understanding of regional malaria transmission dynamics, which has implications for the development of strategies for P. vivax control.

Background: This retrospective analysis assessed all recorded malaria cases in Bulgaria after 1965, when the country was certified as malaria-free by the World Health Organization (WHO), and evaluated the readiness of the public health system to interrupt an outbreak of local transmission in case of infection importation., Methods: The cases were analyzed according to causative species; geographic origin of the imported case; and the citizenship, age, and gender of the infected individuals., Results: In the 50-year study period (1966-2015), there were a total of 3011 cases of malaria imported to Bulgaria from different regions of the world. The majority of the cases originating in Africa were caused by Plasmodium falciparum (65.5%), while most of these originating in Asia were caused by P. vivax (80.9%). The potential season for malaria transmission in Bulgaria is from April to October, and 58.5% of the malaria cases were imported during that time of the year., Conclusions: The increasing movement of people to and from areas endemic for malaria requires the health authorities of countries with appropriate conditions for reintroduction to conduct enhanced measures for surveillance and control of this potentially deadly disease., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We measured the prevalence of malaria in pregnancy and estimated its impact on birth weight and length and maternal hemoglobin in 1,180 women from Juruá Valley, the main malaria hotspot in Brazil. Antenatal malaria episodes, 74.6% of them due to Plasmodium vivax , were microscopically diagnosed in 8.0% of the women and were associated with an average reduction in birth weight z -scores of 0.35 (95% confidence interval [CI] = 0.14-0.57) and in birth length z -scores of 0.31 (95% CI = 0.08-0.54), compared with malaria-free pregnancies. Affected mothers had a mean decrease in hemoglobin concentration at delivery of 0.33 g/100 mL (95% CI = 0.05-0.62 g/100 mL); 51.6% were anemic. The timing and frequency of antenatal infections influenced pregnancy outcomes and first- or second-trimester infections were not associated with decreased birth weight and length and maternal hemoglobin at delivery. Although repeated antenatal vivax infections were associated with poorer birth outcomes, even a single vivax malaria episode was associated with a significant reduction in birth weight and length and maternal hemoglobin. Overall, 7.5% women had the parasite's DNA found in peripheral blood at delivery. Most (83.1%) of these 89 perinatal infections were due to P. vivax and only 7.9% of them progressed to symptomatic disease after delivery. Plasmodium vivax and Plasmodium falciparum DNA was found in 0.6% and 0.3% of 637 cord blood samples examined, respectively, but only one newborn developed clinical neonatal malaria. Our results further challenge the notion that vivax malaria is relatively benign during pregnancy and call for better strategies for its prevention.

Malaria is an infectious disease caused by an intracellular parasite, Plasmodium, which is transmitted to humans after the bite of an Anopheles mosquito. This disease has been prevalent for decades. It has caused great epidemics in history and has also delayed social and economic development. It is endemic in the Eastern Mediterranean and Southeastern Anatolia regions of our country. The most common plasmodium in our country is P. vivax. In P. vivax infections, patients should be treated with primaquine to eradicate hypnozoites. Here, we present a case of relapse with P. vivax, and we emphasize the importance of primaquine in the treatment.

More than 80% of the global burden of the Plasmodium vivax is contributed by mainly three countries (India, Indonesia, and Pakistan). Reports from last decades have highlighted the occurrence of severe P. vivax malaria which was earlier considered to be benign. The recent trends of increasing P. vivax -associated morbidity and mortality emphasizes the need for early and accurate diagnosis of P. vivax malaria for the timely management of patients. Microscopy is considered a gold standard but needs experienced laboratory technologists. Over the last few years, Polymerase chain reaction (PCR) is being used as a highly sensitive and specific test but it requires expensive equipment which limits its use in the field. Therefore, in the present study, utility of visually improved loop-mediated isothermal amplification (LAMP) for the detection of P. vivax was evaluated targeting 18SrRNA gene in 145 microscopically confirmed P. vivax and 20 P. vivax negative patients. Sensitivity and specificity of LAMP was assessed with respect to microscopy and multiplex nested PCR (nPCR). Results of the LAMP assay was also correlated with rapid diagnostic test, multiplex nPCR and real-time PCR results. Overall, sensitivity and specificity of P. vivax -specific LAMP compared with microscopy were found to be 100% and 85%, respectively. Furthermore, detection limit for LAMP was found to be 0.8 copies/μL and it was also able to detect three complicated cases of P. vivax which were missed by microscopy. This study showed a LAMP assay to be a rapid and very sensitive method for the early diagnosis of both complicated and uncomplicated P. vivax malaria.

Malaria is a vector-borne infectious disease, caused by five different species of the genus Plasmodium, and is endemic to many tropical and sub-tropical countries of the globe. At present, malaria diagnosis at the primary health care level in India is conducted by either microscopy or rapid diagnostic test (RDT). In recent years, molecular diagnosis (by PCR assay), has emerged as the most sensitive method for malaria diagnosis. India is highly endemic to malaria and shoulders the burden of two major malaria parasites, Plasmodium falciparum and P. vivax. Previous studies using PCR diagnostic assay had unraveled several interesting facts on distribution of malaria parasites in India. However, these studies had several limitations from small sample size to limited geographical areas of sampling. In order to mitigate these limitations, we have collected finger-prick blood samples from 2,333 malaria symptomatic individuals in nine states from 11 geographic locations, covering almost the entire malaria endemic regions of India and performed all the three diagnostic tests (microscopy, RDT and PCR assay) and also have conducted comparative assessment on the performance of the three diagnostic tests. Since PCR assay turned out to be highly sensitive (827 malaria positive cases) among the three types of tests, we have utilized data from PCR diagnostic assay for analyses and inferences. The results indicate varied distributional prevalence of P. vivax and P. falciparum according to locations in India, and also the mixed species infection due to these two species. The proportion of P. falciparum to P. vivax was found to be 49:51, and percentage of mixed species infections due to these two parasites was found to be 13% of total infections. Considering India is set for malaria elimination by 2030, the present malaria epidemiological information is of high importance.

To explore epidemiological characteristics of imported acute infectious diseases between 2011 and 2016 in Zhejiang province, China. Data of imported infectious diseases from 2011 to 2016 was collected from the China Information System for Disease Control and Prevention in Zhejiang province, and subsequently analyzed for epidemiological characteristics. A survey was conducted to investigate clinicians' abilities to diagnose these diseases in Zhejiang province. From 2011 to 2016, 1,241 cases of imported acute infectious disease were reported in Zhejiang province, including 1,078 malaria cases, 156 dengue cases, three chikungunya fever cases and four Zika cases. Between 2011 and 2016, incidences of these diseases increased ( P < 0.001). For malaria, male adults for labor export were the most affected group. Seasonal fluctuation was not obvious. Plasmodium falciparum was the main malaria type (822 cases) and most cases were acquired from African Region (791/822, 96.1%). Plasmodium vivax cases (194 cases) were mainly from African Region (78/194, 40.2%) and South-East Asia Region (51/194, 26.3%). Meanwhile, for dengue, adults and tourists were the most affected groups. The incidence of dengue was particularly high in August and October. The percent of correct clinician responses in the survey of diagnosis knowledge was 54.6% (standard deviation = 21.0%); this percentage was particularly low in general practitioners and clinicians from township hospitals. The capabilities of clinicians to diagnose these diseases were low and should be improved. Efforts should be made in improving and disseminating proper preventive measures of high-risk populations, surveillance of imported cases, and prevention and control of local epidemics.

Malaria-related hemophagocytic lymphohistiocytosis is a rare, potentially fatal, hyperinflammatory disease entity which can be challenging to diagnose and treat. It is usually associated with Plasmodium falciparum infection. It is less frequently associated with Plasmodium vivax. Here we report an unusual case of a 23-year-old healthy Nigerian man who presented with fever, microangiopathic hemolytic anemia, acute renal failure, and confusion, and was diagnosed as having cerebral malaria-related hemophagocytic lymphohistiocytosis caused by P. vivax infection. He was successfully treated with intravenous artesunate and doxycycline with dramatic clinical improvement., (Copyright © 2016 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.)

Severe congenital malaria associated with Plasmodium vivax is uncommon. In Indonesia, most congenital malaria cases are due to Plasmodium falciparum infections. Most cases of congenital or neonatal malaria in endemic areas are diagnosed from peripheral smear as part of routine sepsis workup. Differentiating congenital and acquired neonatal malaria is very difficult. The case presented in this study describes severe P. vivax malaria with cholestatic jaundice and sepsis-like signs and symptoms in neonates. The mother was asymptomatic and the neonate was successfully treated with intravenous artesunate. Severe P. vivax malaria with cholestatic jaundice in neonates is an uncommon condition that should be included in the differential diagnosis of infants displaying hemolytic anemia, thrombocytopenia, cholestatic jaundice, and hepatosplenomegaly in malaria-endemic zones. Early diagnosis can prevent the use of unnecessary antibiotics and mortality of neonates.

There is a critical need for better malaria rapid diagnostic tests to discriminate Plasmodium falciparum and Plasmodium vivax infection given the recent observation of HRP2 deletions in P. falciparum parasites. We previously identified a DNA aptamer, 2008s, that targets P. falciparum lactate dehydrogenase (PfLDH) and developed a sensitive aptamer-tethered enzyme capture (APTEC) assay. Here, we characterise two different LDH-binding DNA aptamers in their species-specific activities, then integrate within biochemical diagnostic assays and test in clinical samples. An enzyme-linked oligonucleotide assay demonstrated that aptamer pL1 bound with high affinity to both PfLDH and P. vivax lactate dehydrogenase (PvLDH), whereas aptamer 2008s was specific to PfLDH. An aptamer-tethered enzyme capture (APTEC) assay confirmed the specificity of 2008s in binding and capturing the enzyme activity of PfLDH which could be observed colorimetrically. In malaria patient samples, the 2008s APTEC assay was specific for P. falciparum blood samples and could discriminate against P. vivax blood samples. An aptamer for specific detection of falciparum malaria holds promise as a new strategy for species-specific malaria diagnosis rather than the conventional HRP2 immuno-assay., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

A distinctive feature of Plasmodium vivax infections is the overall low parasite density in peripheral blood. Thus, identifying asymptomatic infected individuals in endemic communities requires diagnostic tests with high sensitivity. The detection limits of molecular diagnostic tests are primarily defined by the volume of blood analysed and by the copy number of the amplified molecular marker serving as the template for amplification. By using mitochondrial DNA as the multi-copy template, the detection limit can be improved more than tenfold, compared to standard 18S rRNA targets, thereby allowing detection of lower parasite densities. In a very low transmission area in Brazil, application of a mitochondrial DNA-based assay increased prevalence from 4.9 to 6.5%. The usefulness of molecular tests in malaria epidemiological studies is widely recognized, especially when precise prevalence rates are desired. Of concern, however, is the challenge of demonstrating test accuracy and quality control for samples with very low parasite densities. In this case, chance effects in template distribution around the detection limit constrain reproducibility. Rigorous assessment of false positive and false negative test results is, therefore, required to prevent over- or under-estimation of parasite prevalence in epidemiological studies or when monitoring interventions.

Background: Trenggalek district is a hypoendemic malaria area with mainly imported cases brought by migrant workers from islands outside Java. During malaria surveillance in 2015, no malaria cases were found microscopically, but some cases were positive by PCR. Therefore, a study was conducted to prove that local malaria transmission still occur., Methods: The adult villagers were invited to the house of the head of this village to be screened for malaria using aseptic venipuncture of 1 mL blood upon informed consent. Thin and thick blood films as well as blood spots on filter paper were made for each subject. The blood films were stained with Giemsa and the blood spots were used to extract DNA for polymerase chain reaction (PCR) amplification to determine the malaria infection. In addition, the history of malaria infection and travel to malaria endemic areas were recorded. Entomologic survey to detect the existence of anopheline vector was also conducted., Results: Of the total 64 subjects that participated in the survey, no malaria parasites were found through microscopic examination of the blood films. The PCR analysis found six positive cases (two Plasmodium falciparum, one Plasmodium vivax and two mixed infection of both species), and two of them had no history of malaria and have never travelled to malaria endemic area. Entomologic survey using human bait trap detected the existence of Anopheles indefinitus that was found to be positive for P. vivax by PCR., Conclusions: The results indicated that although we did not find any microscopically slide positive cases, six PCR positive subjects were found. The fact that 2 of the 6 malaria positive subjects have never travelled to malaria endemic area together with the existence of the vector confirm the occurence of local transmission of malaria in the area.

Plasmodium vivax is the most common specie causing malaria outside Africa with approximately 13.8 million reported cases worldwide. We report case of P. vivax infection with cerebral involvement. A nine year old boy presented with high grade fever accompanied by projectile vomiting and abnormal behavior later he developed seizures, shock, and unconsciousness. P. vivax monoinfection was diagnosed based on peripheral smears and PCR. After antimalarial therapy, patient made full recovery. Current case highlights increasing trend of cerebral complications caused by P. vivax.

Objective: To evaluate the importance of external quality assessment program on malaria microscopic diagnosis., Results: A total of 3148 slides were collected in 4 consecutive external quality assessment rounds and blindly rechecked at Amhara Public Health Institute. The average agreement between health facility and APHI slide readers was 96.6%. The percent agreement for parasite detection and species identification for P. falciparum became improved in four consecutive EQA rounds from 93.88 to 99.24% and 92.67 to 97.35% respectively. The rates of false positive and false negative were also dramatically decreased in each round from 10.5 to 0.79% and 2.14 to 0.74% respectively. Therefore, we recommend that malaria EQA program should maintain and expand in all malaria diagnostic health facilities in the region to provide accurate and reliable malaria microscopic service.

Background: Although the malaria burden in the Lao PDR has gradually decreased, the elimination of malaria by 2030 presents many challenges. Microscopy and malaria rapid diagnostic tests (RDTs) are used to diagnose malaria in the Lao PDR; however, some studies have reported the prevalence of sub-microscopic Plasmodium infections or asymptomatic Plasmodium carriers in endemic areas. Thus, highly sensitive detection methods are needed to understand the precise malaria situation in these areas., Methodology/principal Findings: A cross-sectional malaria field survey was conducted in 3 highly endemic malaria districts (Xaysetha, Sanamxay, Phouvong) in Attapeu province, Lao PDR in 2015, to investigate the precise malaria endemicity in the area; 719 volunteers from these villages participated in the survey. Microscopy, RDTs and a real-time nested PCR were used to detect Plasmodium infections and their results were compared. A questionnaire survey of all participants was also conducted to estimate risk factors of Plasmodium infection. Numbers of infections detected by the three methods were microscopy: P. falciparum (n = 1), P. vivax (n = 2); RDTs: P. falciparum (n = 2), P. vivax (n = 3); PCR: Plasmodium (n = 47; P. falciparum [n = 4], P. vivax [n = 41], mixed infection [n = 2]; 6.5%, 47/719). Using PCR as a reference, the sensitivity and specificity of microscopy were 33.3% and 100.0%, respectively, for detecting P. falciparum infection, and 7.0% and 100.0%, for detecting P. vivax infection. Among the 47 participants with parasitemia, only one had a fever (≥37.5°C) and 31 (66.0%) were adult males. Risk factors of Plasmodium infection were males and soldiers, whereas a risk factor of asymptomatic Plasmodium infection was a history of ≥3 malaria episodes., Conclusions/significance: There were many asymptomatic Plasmodium carriers in the study areas of Attapeu province in 2015. Adult males, probably soldiers, were at high risk for malaria infection. P. vivax, the dominant species, accounted for 87.2% of the Plasmodium infections among the participants. To achieve malaria elimination in the Lao PDR, highly sensitive diagnostic tests, including PCR-based diagnostic methods should be used, and plans targeting high-risk populations and elimination of P. vivax should be designed and implemented.

Background: The scale-up of effective malaria control in the last decade has resulted in a substantial decline in the incidence of clinical malaria in many countries. The effects on the proportions of asymptomatic and submicroscopic infections and on transmission potential are yet poorly understood., Methods: In Papua New Guinea, vector control has been intensified since 2008, and improved diagnosis and treatment was introduced in 2012. Cross-sectional surveys were conducted in Madang Province in 2006 (with 1280 survey participants), 2010 (with 2117 participants), and 2014 (with 2516 participants). Infections were quantified by highly sensitive quantitative polymerase chain reaction (PCR) analysis, and gametocytes were quantified by reverse-transcription qPCR analysis., Results: Plasmodium falciparum prevalence determined by qPCR decreased from 42% in 2006 to 9% in 2014. The P. vivax prevalence decreased from 42% in 2006 to 13% in 2010 but then increased to 20% in 2014. Parasite densities decreased 5-fold from 2006 to 2010; 72% of P. falciparum and 87% of P. vivax infections were submicroscopic in 2014. Gametocyte density and positivity correlated closely with parasitemia, and population gametocyte prevalence decreased 3-fold for P. falciparum and 29% for P. vivax from 2010 to 2014., Conclusions: Sustained control has resulted in reduced malaria transmission potential, but an increasing proportion of gametocyte carriers are asymptomatic and submicroscopic and represent a challenge to malaria control., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Recurrence rates of Plasmodium vivax infections differ across various geographic regions. Interestingly, South-East Asia and the Asia-Pacific region are documented to exhibit the most frequent recurrence incidences. Identifying patients at a higher risk for recurrences gives valuable information in strengthening the efforts to control P. vivax infections. The aim of the study was to develop a tool to identify P. vivax- infected patients that are at a higher risk of recurrence in Malaysia., Methods: Patient data was obtained retrospectively through the Ministry of Health, Malaysia, from 2011 to 2016. Patients with incomplete data were excluded. A total of 2044 clinical P. vivax malaria cases treated with primaquine were included. Data collected were patient, disease, and treatment characteristics. Two-thirds of the cases (n = 1362) were used to develop a clinical risk score, while the remaining third (n = 682) was used for validation., Results: Using multivariate analysis, age (p = 0.03), gametocyte sexual count (p = 0.04), indigenous transmission (p = 0.04), type of treatment (p = 0.12), and incomplete primaquine treatment (p = 0.14) were found to be predictors of recurrence after controlling for other confounding factors; these predictors were then used in developing the final model. The beta-coefficient values were used to develop a clinical scoring tool to predict possible recurrence. The total scores ranged between 0 and 8. A higher score indicated a higher risk for recurrence (odds ratio [OR]: 1.971; 95% confidence interval [CI]: 1.562-2.487; p ≤ 0.001). The area under the receiver operating characteristic (ROC) curve of the developed (n = 1362) and validated model (n = 682) was of good accuracy (ROC: 0.728, 95% CI: 0.670-0.785, p value < 0.001, and ROC: 0.766, 95% CI: 0.700-0.833, p-value < 0.001, respectively). In both the developed and validated models, area under the ROC curves showed no significant difference in predicting recurrence based on the constructed scoring mechanism (p = 0.399; Z-value: -0.8441; standard error: 0.045)., Conclusions: The developed model to predict recurrence was found to be of good accuracy and could be a useful tool in targeting patients at a higher risk for recurrence for closer monitoring during follow-up, after treatment with primaquine.

Malaria is a major public health problem in India and in the Chhattisgarh state. The diagnosis of malaria presents a major challenge in remote areas The prevalence of malaria in Darbha and Kilepal Community Health Centers (CHCs) of the Jagdalpur district, Chhattisgarh affected by conflict was determined using microscopy and polymerase chain reaction (PCR). In the year 2015, 29.4% and 21.5% cases were found to be positive for malaria at the Darbha and Kilepal CHCs, respectively, by microscopy, and 7.4% and 1.6% of cases had mixed infections, respectively. Among the suspected cases of mixed infections and doubtful diagnoses, 21% had mixed infections with two or more species at the Darbha CHC, and 17% from the Kilepal CHC, as determined by PCR. Both the P. vivax subspecies Pv210 (56%) and Pv247 (44%) and the P. ovale curtisi subspecies were found in this area. The high proportion of mixed malaria parasitic infections detected in this study indicate the need to adequately train health staff involved in diagnosing malaria. This study showed that there is a need for site-specific data to understand the epidemiological picture and to develop appropriate intervention strategies and management guidelines for controlling and eliminating malaria in India.

Plasmodium lactate dehydrogenase (pLDH) is a strong target antigen for the determination of infection with Plasmodium species specifically. However, a more effective antibody is needed because of the low sensitivity of the current antibody in many immunological diagnostic assays. In this study, recombinant Plasmodium vivax LDH (PvLDH) was experimentally constructed and expressed as a native antigen to develop an effective P. vivax-specific monoclonal antibody (mAb). Two mAbs (2CF5 and 1G10) were tested using ELISA and immunofluorescence assays (IFA), as both demonstrated reactivity against pLDH antigen. Of the 2 antibodies, 2CF5 was not able to detect P. falciparum, suggesting that it might possess P. vivax-specificity. The detection limit for a pair of 2 mAbs-linked sandwich ELISA was 31.3 ng/ml of the recombinant antigen. The P. vivax-specific performance of mAbs-linked ELISA was confirmed by in vitro-cultured P. falciparum and P. vivax-infected patient blood samples. In conclusion, the 2 new antibodies possessed the potential to detect P. vivax and will be useful in immunoassay.

The surveillance of malaria is generally undertaken on the assumption that samples passively collected at health facilities are comparable to or representative of the broader Plasmodium reservoir circulating in the community. Further characterization and comparability of the hidden asymptomatic parasite reservoir are needed to inform on the potential impact of sampling bias. This study explores the impact of sampling strategy on molecular surveillance by comparing the genetic make-up of Plasmodium falciparum and Plasmodium vivax isolates collected by passive versus active case detection. Sympatric isolates of P. falciparum and P. vivax were collected from a large community survey and ongoing clinical surveillance studies undertaken in the hypomesoendemic setting of Mimika District (Papua, Indonesia). Plasmodium falciparum isolates were genotyped at nine microsatellite loci and P. vivax at eight loci. Measures of diversity and differentiation were used to compare different patient and parasitological sample groups. The results demonstrated that passively detected cases (symptomatic) had comparable population diversity to those circulating in the community (asymptomatic) in both species. In addition, asymptomatic patent infections were as diverse as subpatent infections. However, a significant difference in multiplicity of infection (MOI) and percentage of polyclonal infections was observed between actively and passively detected P. vivax cases (mean MOI: 1.7 ± 0.7 versus 1.4 ± 1.4, respectively; P = 0.001). The study findings infer that, in hypomesoendemic settings, passive sampling is appropriate for molecular parasite surveillance strategies using the predominant clone in any given infection; however, the findings suggest caution when analyzing complexity of infection. Further evaluation is required in other endemic settings.

Malaria infections may be symptomatic, leading to treatment, or "asymptomatic," typically detected through active surveillance, and not leading to treatment. Malaria elimination may require purging both types of infection. Using detection methods with different sensitivities, we conducted a cross-sectional study in two rural communities located along the border between China's Yunnan Province and Myanmar's Shan and Kachin States, to estimate the prevalence of asymptomatic and symptomatic malaria. In Mong Pawk, all infections detected were asymptomatic, and the prevalence of Plasmodium falciparum was 0.3%, 4.3%, 4.0%, and 7.8% by light microscopy, rapid diagnostic test (RDT), conventional polymerase chain reaction (cPCR), and multiplexed real-time PCR (RT-PCR), respectively, and Plasmodium vivax prevalence was 0% by all detection methods. In Laiza, of 385 asymptomatic participants, 2.3%, 4.4%, and 12.2% were positive for P. vivax by microscopy, cPCR, and RT-PCR, respectively, and 2.3% were P. falciparum -positive only by RT-PCR. Of 34 symptomatic participants in Laiza, 32.4% were P. vivax- positive by all detection methods. Factors associated with infection included gender (males higher than females, P = 0.014), and young age group (5-17 age group compared with others, P = 0.0024). Although the sensitivity of microscopy was adequate to detect symptomatic infections, it missed the vast majority (86.5%) of asymptomatic infections. Although molecular detection methods had no advantage over standard microscopy or RDT diagnosis for clinically apparent infections, malaria elimination along the Myanmar-China border will likely require highly sensitive surveillance tools to identify asymptomatic infections and guide targeted screen-and-treat interventions.

Background: Low-density asymptomatic infections of Plasmodium spp. are common in low endemicity areas worldwide, but outside Africa, their contribution to malaria transmission is poorly understood. Community-based studies with highly sensitive molecular diagnostics are needed to quantify the asymptomatic reservoir of Plasmodium falciparum and P. vivax infections in Thai communities., Methods: A cross-sectional survey of 4309 participants was conducted in three endemic areas in Kanchanaburi and Ratchaburi provinces of Thailand in 2012. The presence of P. falciparum and P. vivax parasites was determined using 18S rRNA qPCR. Gametocytes were also detected by pfs25 / pvs25 qRT-PCRs., Results: A total of 133 individuals were found infected with P. vivax (3.09%), 37 with P. falciparum (0.86%), and 11 with mixed P. vivax/ P. falciparum (0.26%). The clear majority of both P. vivax (91.7%) and P. falciparum (89.8%) infections were not accompanied by any febrile symptoms. Infections with either species were most common in adolescent and adult males. Recent travel to Myanmar was highly associated with P. falciparum (OR = 9.0, P = 0.001) but not P. vivax infections (P = 0.13). A large number of P. vivax (71.5%) and P. falciparum (72.0%) infections were gametocyte positive by pvs25/pfs25 qRT-PCR. Detection of gametocyte-specific pvs25 and pfs25 transcripts was strongly dependent on parasite density. pvs25 transcript numbers, a measure of gametocyte density, were also highly correlated with parasite density (r 2  = 0.82, P < 0.001)., Conclusions: Asymptomatic infections with Plasmodium spp. were common in western Thai communities in 2012. The high prevalence of gametocytes indicates that these infections may contribute substantially to the maintenance of local malaria transmission.

Reporting cases of malaria to the Federal Office of Public Health has been mandatory in Switzerland since 1974. We analysed notifications of imported confirmed malaria cases between 2005 and 2015 in Switzerland or Liechtenstein. Data for previously visited countries, nationality and reason for travelling were analysed. In contrast with the impressive drop of malaria cases reported worldwide since 2000, we found that the number of malaria cases imported yearly in Switzerland doubled in 2014 and 2015 compared to the average for the preceding decade. Since 2014, Plasmodium vivax infection represented 36% of all diagnosed malaria cases in Switzerland, compared to 11% in the decade leading to 2013. Most of the vivax malaria patients originated from the Horn of Africa, especially from Eritrea. This rise in cases was a consequence not only of an increase in the number of Eritrean refugees, but also their vivax malaria incidence rate, which jumped from 1-3‰ previously to 12‰ in 2014. This is a trend that is not matched by national statistics in Eritrea. An unreported increased incidence in the country of origin (Eritrea) might be the cause of the rise of Pv cases imported into Switzerland, but infections are also likely to occur along the harsh and long migration journey. This epidemiology highlights the need to register and use primaquine for the treatment of latent-phase P. vivax malaria in Switzerland, a medicine currently neither marketed nor systematically reimbursed. Moreover, general practitioners should be aware of this specific epidemiological situation in order to avoid misdiagnosis of febrile Eritreans even months after they reach Switzerland.

Malaria is a widespread and life-threatening disease in tropical and subtropical regions. In patients with typical clinical symptoms, malaria is considered as a preliminary diagnosis if there is a travel history to malaria-endemic areas. The basis of the laboratory diagnosis of malaria is the microscopic examination of Giemsa stained smears. On the other hand, the diagnosis and differentiation of Plasmodium species with microscopic examination may have some difficulties. In the first case, adifferent appearance from the classical Plasmodium vivax erythrocytic forms in infected erythrocytes were detected in 1% of all erythrocytes in thin smear blood preparations of a 26-year-old male with complaints of fever and chills and a story of travel to Nigeria. It was observed that parasitic nuclei were not prominent, and were located in the cytoplasm irregularly as chromatin or dye particles, nucleus fragments similar to Schüffner's granules in the form of scattered and granular spots were present in some erythrocytes, the cytoplasm of some Plasmodium erythrocytic forms were irregular and nuclei were not seen. There were no Schüffner's granules in any of the infected erythrocytes. P.vivax was detected by the rapid diagnostic test (OptiMAL, DiaMed GmbH, Switzerland), which searches for the antigens of Plasmodium species, in the peripheral blood sample of the patients. The P.vivax 18S rRNA gene was also detected by the multiplex real-time polymerase chain reaction. Antibodies against Plasmodium species were searched by using the Pan Malaria Antibody CELISA (CeLLabs Pty Ltd, Brookvale, Australia) kit in the patient's serum sample and the optical density (OD) value of the patient sample was measured five times the OD value of the positive control. In the second case, adifferent appearance from the classical P.falciparum erythrocytic forms in infected erythrocytes were detected in 12% of all erythrocytes in thin smear blood preparations of a 31-year-old male who has been suffering from persistent fever, severe headache, pain in the eyes and was known to be working in Nigeria. It was observed that some Plasmodium trophozoites have 1/3 of the size of erythrocytes such as P.vivax and have non-granular cytoplasm, some erythrocytic forms were round and the nucleus and cytoplasm were hardly distinguished, some of them were seen as crescent and close to the nucleus of the cytoplasm and some erythrocytic forms had characteristically a single nucleus and a scattered cytoplasm, similar to mature trophozoites of P.vivax. Although the Plasmodium young trophozoites were similar to P.vivax in means of magnitude, the forms in which the nuclei adhered to the erythrocyte wall were common. There were no P.falciparum gametocyte forms. P.falciparum like young trophozoite was observedonly in one of the four smears. P.falciparum was detected by the commercial rapid diagnostic test and P.falciparum 18S rRNA gene was also detected by the multiplex real-time polymerase chain reaction. Antibody formation against Plasmodium species was not detected in the ELISA test. In these case reports, the importance of the support of rapid diagnostic tests, serological and molecular methods to microscopic diagnosis and species determination of two imported malaria cases were demonstrated.

Background: A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer., Methods: We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species., Results: Our LAMP method allowed amplification of all targeted 18S-rRNA genes of the reference plasmids with detection limits of 10-100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR., Conclusions: Our diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

Plasmodium vivax is the second most prevalent cause of malaria worldwide and the leading cause of malaria outside of Africa. Although infections are seldom fatal clinical disease can be debilitating and imposes significant health and economic impacts on affected populations. Estimates of transmission and prevalence intensity can be problematic because many episodes of vivax originate from hypnozoite stages in the liver that have remained dormant from previous infections by an unknown mechanism. Lack of treatment options to clear hypnozoites and the ability to infect mosquitoes before disease symptoms present represent major challenges for control and eradication of vivax malaria. Compounding these challenges is the unique biology of P. vivax and limited progress in development of experimental research tools, thereby hindering development of new drugs and vaccines. Renewed emphasis on vivax malaria research is beginning to make progress in overcoming some of these challenges., (Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.)

Plasmodium vivax malaria is an essential yet elusive target of tropical disease eradication efforts, and is the focus of this literature review. This review will reacquaint Special Operations Forces (SOF) Medics with the basic principles of malaria as context for understanding the several confounding issues particular to P. vivax infections. The review concludes with current malaria guidelines and malaria mitigation strategies., (2017.)

Introduction: Acute kidney injury (AKI) is reported to occur in patients with falciparum malaria but not uncommon with vivax malaria. AKI, anemia, thrombocytopenia and jaundice is a recurrent finding in severe malaria and can mimic as thrombotic microangiopathy (TMA). Relationship of malaria with TMA is unclear till date however evidences suggest their association., Methods & Material: We reviewed our electronic database to evaluate relationship of malaria with TMA, of cases of malaria, jaundice and AKI., Results: 4 patients found to have P. vivax malaria and histopathologically confirmed TMA. All had fever, oliguria, jaundice at presentation. The time between onset of symptoms and admission ranged from 7 to 14 days. All had parasitemia at presentation so were treated with Artesuanate. Hemodialysis and Plasmapheresis was done in all patients. On follow-up all patients recovered and asymptomatic urinary abnormality persisting in one patient., Conclusion: High index of suspicion should be kept for TMA in a patient who has nonrecovering AKI with persistent anemia and thrombocytopenia even after clinical and laboratory evidences of recovery from malaria, as response to plasmapheresis seems excellent in this subset of malarial AKI. There could be a pathogenetic link between P.vivax and TMA though yet to be confirmed in larger studies.

Background: Chloroquine (CQ) is the main anti-schizontocidal drug used in the treatment of uncomplicated malaria caused by Plasmodium vivax. Chloroquine resistant P. vivax (PvCR) malaria in the Western Pacific region, Asia and in the Americas indicates a need for biomarkers of resistance to improve therapy and enhance understanding of the mechanisms associated with PvCR. In this study, we compared plasma metabolic profiles of P. vivax malaria patients with PvCR and chloroquine sensitive parasites before treatment to identify potential molecular markers of chloroquine resistance., Methods: An untargeted high-resolution metabolomics analysis was performed on plasma samples collected in a malaria clinic in Manaus, Brazil. Male and female patients with Plasmodium vivax were included (n = 46); samples were collected before CQ treatment and followed for 28 days to determine PvCR, defined as the recurrence of parasitemia with detectable plasma concentrations of CQ ≥100 ng/dL. Differentially expressed metabolic features between CQ-Resistant (CQ-R) and CQ-Sensitive (CQ-S) patients were identified using partial least squares discriminant analysis and linear regression after adjusting for covariates and multiple testing correction. Pathway enrichment analysis was performed using Mummichog., Results: Linear regression and PLS-DA methods yielded 69 discriminatory features between CQ-R and CQ-S groups, with 10-fold cross-validation classification accuracy of 89.6% using a SVM classifier. Pathway enrichment analysis showed significant enrichment (p<0.05) of glycerophospholipid metabolism, glycosphingolipid metabolism, aspartate and asparagine metabolism, purine and pyrimidine metabolism, and xenobiotics metabolism. Glycerophosphocholines levels were significantly lower in the CQ-R group as compared to CQ-S patients and also to independent control samples., Conclusions: The results show differences in lipid, amino acids, and nucleotide metabolism pathways in the plasma of CQ-R versus CQ-S patients prior to antimalarial treatment. Metabolomics phenotyping of P. vivax samples from patients with well-defined clinical CQ-resistance is promising for the development of new tools to understand the biological process and to identify potential biomarkers of PvCR.

Background: Primaquine is essential for the radical cure of vivax malaria, however its broad application is hindered by the risk of drug-induced haemolysis in individuals with glucose-6-phosphate-dehydrogenase (G6PD) deficiency. Rapid diagnostic tests capable of diagnosing G6PD deficiency are now available, but these are not used widely., Methods: A series of qualitative interviews were conducted with policy makers and healthcare providers in four vivax-endemic countries. Routine G6PD testing is not part of current policy in Bangladesh, Cambodia or China, but it is in Malaysia. The interviews were analysed with regard to respondents perceptions of vivax malaria, -primaquine based treatment for malaria and the complexities of G6PD deficiency., Results: Three barriers to the roll-out of routine G6PD testing were identified in all sites: (a) a perceived low risk of drug-induced haemolysis; (b) the perception that vivax malaria was benign and accordingly treatment with primaquine was not regarded as a priority; and, (c) the additional costs of introducing routine testing. In Malaysia, respondents considered the current test and treat algorithm suitable and the need for an alternative approach was only considered relevant in highly mobile and hard to reach populations., Conclusions: Greater efforts are needed to increase awareness of the benefits of the radical cure of Plasmodium vivax and this should be supported by economic analyses exploring the cost effectiveness of routine G6PD testing.

Background: Malaria is one of the killer diseases in Ethiopia and it is still the first leading cause of death in health facilities. However, there is no information yet regarding the trends of malaria prevalence at health institution and particularly at Felegehiwot referral Hospital. Hence , knowing the trends of malaria prevalence at each health facilities is essential to design appropriate interventions. Therefore, the present study addressed the above gaps., Results: Overall, 14,750 blood films were diagnosed for malaria. Of these, 740 (5%) were confirmed with microscope. Plasmodium falciparum and Plasmodium vivax accounted for 397 (53%) and 331 (45%), respectively. Age groups >20 year (p < 0.02) and males (p < 0.025) were significantly affected., Conclusions: In conclusion, P. falciparum was predominant as compared to P. vivax. Hence, it needs close monitoring and intervention measures for control activities.

India is endemic for malaria with unstable transmission inhibiting the development of immunity and predisposing all age groups to the disease. Children under five are the greatest sufferers with maximum mortality. P. falciparum and P. vivax cause majority of cases. Fever is the cardinal symptom, though no set of signs and symptoms reliably distinguishes malaria from other causes of fever. In all suspected cases parasitological diagnosis should be confirmed before starting the treatment. Microscopy of blood smears is the gold standard for diagnosis. Rapid diagnostic tests are to be used where microscopy results are not available within 24 h. In complicated malaria and high risk patients like HIV, treatment can be commenced before confirmation, though all efforts to establish the diagnosis should be made. Chloroquine is used for uncomplicated vivax malaria while artemisinin based combination therapy (ACTs) is used for uncomplicated falciparum malaria. For complicated malaria, IV artesunate is the drug of choice irrespective of the Plasmodium species. It is important to follow recommendations diligently to decrease morbidity and mortality due to malaria and to avoid the problem of drug resistance. The gains of the past decade should be scaled up to make malaria elimination and eradication a reality.

Sensitive detection of specific antibodies by biosensors has become of major importance for monitoring and controlling epidemics. Here we report a development of a biosensor able to specifically measure antibodies in a drop of unmodified blood serum. Within minutes, the detection system measures presence of antibodies against Plasmodium vivax, a causing agent for malaria. The biosensor consists of a layer of carbon nanotubes (CNTs) which were casted on a carbon working electrode area of a three-electrode system and oxidized. An amine layer was produced next by modifying the surface with EDAC/NHS followed by reaction with a diamine compound. Finally, the protein fragments derived from P. vivax containing well-known antigen sequences were casted on this layer and bound through electrostatic interactions, involving hydrogen and ionic bonding. All these chemical changes occurring at the carbon surface along the biosensor assembly were followed and confirmed by Fourier Transformed Infrared s pectrometry (FTIR) and Raman spectroscopy. The presence of antibodies in serum was detected by monitoring the electrical properties of the layer, making use of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), against a standard iron probe. Overall, the charge-transfer resistance decreased after antibody binding, because there was an additional amount of protein bound to the surface. This hindered the access of the iron redox probe to the conductive support at the electrode surface. Electrical changes could be measured at antibody concentration as low as ~6-50pg/L (concentrations in the range of 10-15M) and as high as ~70μg/L. Specific measurement with low background was even possible in undiluted serum. Hence, this novel biosensor allows assessing serum antibody levels in real time and in un-manipulated serum samples on-site where needed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

AbstractPostpartum women may have an altered susceptibility to Plasmodium falciparum and Plasmodium vivax . The relationship between naturally acquired malarial immunity and susceptibility to malaria postpartum is yet to be determined. IgG levels were measured against P. falciparum and P. vivax antigens from delivery in 201 postpartum and 201 nonpregnant controls over 12 weeks. Associations between time-varying antibody levels and time to first microscopically confirmed species-specific infection were determined by Cox regression. Associations between antibody levels and prospective risk of Plasmodium infection were similar in postpartum and control women. A 2-fold increase in P. falciparum antibody levels was associated with increased prospective risk of P. falciparum infection (hazard ratio [HR] range = 1.37-1.94). Antibody levels against most P. vivax antigens displayed no association with prospective risk of P. vivax infection (HR range = 1.02-1.05) with the exception of Pv MSP1 19 antibodies that were weakly associated with prospective risk of P. vivax infection (HR = 1.14 (95% confidence interval = 1.02, 1.28) per 2-fold increase in levels). Associations between antibody levels and prospective risk of infection attenuated when adjusted for documented retrospective exposure. Serology may be a useful tool to predict and monitor women at increased risk of P. falciparum infection postpartum, particularly in the absence of a detailed history of retrospective infections.

Objective: To access a new step using PCR to amplify the targeted mtDNA sequence for detecting specifically Plasmodium vivax and its co-infections, false positive and false negative results with Plasmodium falciparum., Methods: In this study we have standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) was amplified by using a PCR technique as a tool to detect Plasmodium spp. Species specific primers were designed to hybridize with cytochrome c oxidase gene of P. vivax (cox I) and P. falciparum (cox III). Two hundred blood samples were collected on the basis of clinical symptoms which were initially examined through microscopic analysis after preparing Giemsa stained thick and thin blood smears. Afterwards genomic DNA was extracted from all samples and was then subjected to PCR amplification by using species specific primers and amplified segments were sequenced for confirmation of results., Results: One-hundred and thirty-two blood samples were detected as positive for malaria by PCR, out of which 64 were found to be positive by PCR and 53 by both microscopy and PCR for P.vivax infection. Nine samples were found to be false negative, one P.vivax mono infection was declared as co infection by PCR and 3 samples identified as having P.falciparum gametes were confirmed as P.vivax by PCR amplification. Sensitivity and specificity were found to be 85% and 92% respectively., Conclusions: Results obtained through PCR method were comparatively better and reliable than microscopy.

Background: The global decline of malaria burden and goals for elimination has led to an increased interest in the fine-scale epidemiology of malaria. Micro-geographic heterogeneity of malaria infection could have implications for designing targeted small-area interventions., Methods: Two-year longitudinal cohort study data were used to explore the spatial and spatio-temporal distribution of malaria episodes in 2040 children aged < 10 years in 16 villages near the Gilgel-Gibe hydropower dam in Southwest Ethiopia. All selected households (HHs) were geo-referenced, and children were followed up through weekly house-to-house visits for two consecutive years to identify febrile episodes of P. falciparum and P. vivax infections. After confirming the spatial dependence of malaria episodes with Ripley's K function, SatScan TM was used to identify purely spatial and space-time clusters (hotspots) of annual malaria incidence for 2 years follow-up: year 1 (July 2008-June 2009) and year 2 (July 2009-June 2010)., Results: In total, 685 P. falciparum episodes (in 492 HHs) and 385 P. vivax episodes (in 290 HHs) were identified, representing respectively incidence rates of 14.6 (95% CI: 13.4-15.6) and 8.2 (95% CI: 7.3-9.1) per 1000 child-months at risk. In year 1, the most likely (128 HHs with 63 episodes, RR = 2.1) and secondary (15 HHs with 12 episodes, RR = 5.31) clusters of P. vivax incidence were found respectively in southern and north-western villages; while in year 2, the most likely cluster was located only in north-western villages (85 HHs with 16 episodes, RR = 4.4). Instead, most likely spatial clusters of P. falciparum incidence were consistently located in villages south of the dam in both years: year 1 (167 HHs with 81 episodes, RR = 1.8) and year 2 (133 HHs with 67 episodes, RR = 2.2). Space-time clusters in southern villages for P. vivax were found in August-November 2008 in year 1 and between November 2009 and February 2010 in year 2; while for P. falciparum, they were found in September-November 2008 in year 1 and October-November 2009 in year 2., Conclusion: Hotspots of P. falciparum incidence in children were more stable at the geographical level and over time compared to those of P. vivax incidence during the study period.

Droplet digital polymerase chain reaction (ddPCR) is a partial PCR based on water-oil emulsion droplet technology. It is a highly sensitive method for detecting and delineating minor alleles from complex backgrounds and provides absolute quantification of DNA targets. The ddPCR technology has been applied for detection of many pathogens. Here the sensitive assay utilizing ddPCR for detection and quantification of Plasmodium species was investigated. The assay was developed for two levels of detection, genus specific for all Plasmodium species and for specific Plasmodium species detection. The ddPCR assay was developed based on primers and probes specific to the Plasmodium genus 18S rRNA gene. Using ddPCR for ultra-sensitive P. falciparum assessment, the lower level of detection from concentrated DNA obtained from a high volume (1 mL) blood sample was 11 parasites/mL. For species identification, in particular for samples with mixed infections, a duplex reaction was developed for detection and quantification P. falciparum/ P. vivax and P. malariae/ P. ovale. Amplification of each Plasmodium species in the duplex reaction showed equal sensitivity to singleplex single species detection. The duplex ddPCR assay had higher sensitivity to identify minor species in 32 subpatent parasitaemia samples from Cambodia, and performed better than real-time PCR. The ddPCR assay shows high sensitivity to assess very low parasitaemia of all human Plasmodium species. This provides a useful research tool for studying the role of the asymptomatic parasite reservoir for transmission in regions aiming for malaria elimination.