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Background: In patients with severe neutropenia, infections can rapidly become serious and life-threatening. It is essential to understand whether pregnancy induces changes in neutrophil levels thereby posing an increased threat to the health of gravidae., Methodology: This cross-sectional study was conducted in San Health District (Mali) and involved pregnant women infected or not by malaria parasites and non-pregnant healthy volunteers. Subjects were categorized as having neutropenia, normal neutrophil levels, and neutrophilia regarding their neutrophil levels. A logistic regression analysis was performed to determine factors associated with neutrophil level variation in pregnant women., Results: Whether or not the pregnant women were infected with malaria, 98 of the 202 cases (48.5%) showed neutrophilia. Surprisingly, 67 of the 71 cases of neutropenia (94.4%) observed in this study concerned healthy people who were not pregnant. The mean percentage of neutrophil levels was significantly (p < 0.001) lower (49.9%) in the first trimester compared to the second trimester of pregnancy (62.0%). A logistic regression model showed that compared to early pregnancy, the second (OR = 12.9, 95% CI 2.2-248.1, p = 0.018) and the third trimesters (OR = 13.7, 95% CI 2.3-257.5, p = 0.016) were strongly associated with the increase of neutrophil levels., Conclusions: Pregnancy can induce the production of mature neutrophils that are continually released into circulation. Neutrophil levels were lower during the first trimester of the pregnancy compared to the second and third trimesters, but not affected by the presence or absence of malaria infection., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Moussa Djimde, Charles Arama, Bouréma Koné, Hamadoun Diakité, Mohamed Keita, Mamadou D Samaké, Bréhima Tembely, Balla Bagayoko, Mohamed B Traoré, Japhet K Tshiongo, Hypolite M Mavoko, Alassane Dicko, Michel Vaillant, Petra F Mens, Henk DFH Schallig, Kassoum Kayentao.)

Introduction: Polymorphonuclear neutrophils (PMN) are involved in pathogen clearance by phagocytosis. However, the role of PMNs in the efficacy of artemisinin-based combination therapy (ACT) is poorly understood., Methodology: In a prospective longitudinal in vivo study, neutrophil rates were compared with malaria carriage after treatment with different ACTs: Artemether - lumefantrine (AL), Artesunate - amodiaquine (ASAQ), Dihydroartemisinin - piperaquine (DP) or Pyronaridine artesunate (PA). The study cases were classified as having neutropenia, normal neutrophil levels or neutrophilia depending on the level of neutrophils in the blood. This study included 3148 patients and was analyzed using R., Results: On day 7, only four patients in the neutropenia group and treated with AL had a malaria positive blood smear based on microscopy. On day 28, the rate of recurrent parasitemia in the AL arm was significantly higher in neutropenia patients (50.9%) than in patients with normal rates of neutrophils (43.1%) or in those with neutrophilia (6.0%) (p < 0.001). In ASAQ arm, the rate of recurrent Plasmodium falciparum parasitemia was 58.8% in the neutropenia group versus 29.4% in patients with normal rates of neutrophils and 11.8% in patients with neutrophilia (p < 0.001). No patient treated with DP with normal neutrophil counts or neutrophilia was carrying malaria parasites on day 28. Among the 15 patients with parasitemia on day 28 in the PA arm, 11 (73.33%) had neutropenia while 4 (26.67%) had a normal neutrophil count (p < 0.001)., Conclusions: Patients with neutropenia had higher rates of recurrent P. falciparum parasitemia after ACT., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Moussa Djimde, Kassoum Kayentao, Japhet Kabalu Tshiongo, Bakary Fofana, Charles Arama, Sodiomon B Sirima, Jean Bosco Ouedraogo, Abdoul Habib Beavogui, Issaka Sagara, Alassane Dicko, Petra F Mens, Henk DFH Schallig, Abdoulaye Djimde.)

This review is an update of an earlier narrative review published in 2015 on developments in the clinical management of cutaneous leishmaniasis (CL) including diagnosis, treatment, prevention and control measurements. CL is a vector-borne infection caused by the protozoan parasite Leishmania. The vector is the female sandfly. Globally, CL affects 12 million cases and annually 2 million new cases occur. CL is endemic in almost 100 countries and the total risk population is approximately 350 million people. WHO lists CL an emerging and uncontrolled disease and a neglected tropical disease. Local experience-based evidence remains the mainstay for the management of CL. Whereas intralesional therapeutic options are the first treatment option for most CL patients, those with mucocutaneous and disseminated involvement require a systemic therapeutic approach. Moreover, different Leishmania species can vary in their treatment outcomes. Therefore, species determination is critical for optimal CL clinical management. New DNA techniques allow for relatively easy Leishmania species determination, yet they are not easily implemented in resource-limited settings. There is a desperate need for novel, less toxic, and less painful treatment options, especially for children with CL. Yet, the large and well conducted studies required to provide the necessary evidence are lacking. To further control and potentially eliminate CL, we urgently need to improve vector control, and diagnostics, and we require efficient and safe vaccines. Alas, since CL primarily affects poor people, biotechnical companies dedicate little investment into the research programs that could lead to diagnostic, pharmaceutical, and vaccine innovations., (© 2022. The Author(s).)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SB declared a past co-authorship with the author DW to the handling editor.

Background: Management of uncomplicated Plasmodium falciparum malaria relies on artemisinin-based combination therapies (ACTs). These highly effective regimens have contributed to reductions in malaria morbidity and mortality. However, artemisinin resistance in Asia and changing parasite susceptibility to ACT in Africa have now been well documented. Strategies that retain current ACT as efficacious treatments are urgently needed., Methods: We present an open-label, randomised three-arm clinical trial protocol in three African settings representative of varying malaria epidemiology to investigate whether prolonged ACT-based regimens using currently available formulations can eliminate potentially resistant parasites. The protocol investigates whether a sequential course of two licensed ACT in 1080 children aged 6-120 months exhibits superior efficacy against acute P. falciparum malaria and non-inferior safety compared with standard single-course ACT given to 540 children. The primary endpoint is PCR-corrected clinical and parasitological response at day 42 or day 63 of follow-up. Persistence of PCR-detectable parasitaemia at day 3 is analysed as a key covariate. Secondary endpoints include gametocytaemia, occurrence of treatment-related adverse events in the double-ACT versus single-ACT arms, carriage of molecular markers of drug resistance, drug kinetics and patient adherence to treatment., Discussion: This protocol addresses efficacy and safety of sequential ACT regimens in P. falciparum malaria in Africa. The approach is designed to extend the useful life of this class of antimalarials with maximal impact and minimal delay, by deploying licensed medicines that could be swiftly implemented as sequential double ACT by National Malaria Control Programmes, before emerging drug resistance in Africa becomes a major threat to public health., Competing Interests: Competing interests: None declared.

Visceral leishmaniasis (VL) is a vector-borne disease widespread in the Mediterranean basin, including Cyprus. During the last decades no cases were notified from northern Cyprus, but herein three cases of VL (female: 2, male: 1, median age: 24.6 months) diagnosed during their hospital admission between January 2011 and December 2012 are reported. Diagnosis was based on clinical findings; 1 ≥ 1/64 titer positivity of immunofluorescence antibodies, Leishmania amastigotes in Giemsa-stained slides of bone marrow, as well as molecular identification confirmed that in all three the infecting pathogen was Leishmania infantum Fever, splenomegaly, and hepatomegaly were the typical clinical findings. First-line treatment with liposomal amphotericin B (AmBisome ® ; intravenous, 3 mg/kg) on days 1-5, followed by the same on days 10 and 21 yielded a successful outcome with no relapse in all cases. These confirmed VL cases found within 2 years demonstrate the presence of VL on the island., (© The American Society of Tropical Medicine and Hygiene.)

Objectives: To provide an overview of the most frequent aetiologies found in febrile episodes of children under 5 years from sub-Saharan Africa., Methods: MEDLINE and EMBASE were searched for publications in English and French on non-malaria fever episodes in African children under 5 years of age, which were published between January 1990 and July 2015. Case reports and conference abstracts were excluded., Results: In total, 3851 titles and abstracts were reviewed, and 153 were selected for full screening of which 18 were included in the present review. Bloodstream infection (BSI) was most commonly investigated (nine of 18) followed by urinary tract infection (UTI) (four of 18) and respiratory tract infection (RTI) (two of 18). Few studies investigated BSI and UTI in the same children (two of 18), or BSI and gastrointestinal infection (GII) (one of 18). As for BSI, the most frequently isolated bacteria were E. coli (four of 12), Streptococcus pneumonia (four of 12), Salmonella spp (three of 12) and Staphylococcus aureus (two of 12) with a positive identification rate of 19.7-33.3%, 5.2-27.6%, 11.7-65.4% and 23.5-42.0%, respectively. As for UTI, the main bacteria isolated were E. coli (six of six) and Klebsiella spp (six of six) with a positive rate of 20.0-72.3% and 10.0-28.5%, respectively. No bacterium was isolated in RTI group, but Human influenzae A and B were frequently found, with the highest positive identification rate in Tanzania (75.3%). Dengue virus (two of 12) was the most frequently reported viral infection with a positive identification rate of 16.7-30.8%. Finally, only rotavirus/adenovirus (69.2% positive identification rate) was found in GII and no bacterium was isolated in this group., Conclusions: The high prevalence of treatable causes of non-malaria fever episodes requires a proper diagnosis of the origin of fever followed by an appropriate treatment, thereby reducing the under-5 mortality in sub-Saharan Africa and preventing the overprescription of antibiotics and thus circumventing the rise of antibiotic resistance., (© 2016 John Wiley & Sons Ltd.)

Background: Leishmania (Viannia) guyanensis is believed to be the principal cause of cutaneous leishmaniasis (CL) in Suriname. This disease is treated with pentamidine isethionate (PI), but treatment failure has increasingly been reported., Aim: To evaluate PI for its clinical efficacy, to compare parasite load, and to assess the possibility of treatment failure due to other infecting Leishmania species., Methods: Parasite load of patients with CL was determined in skin biopsies using real-time quantitative PCR before treatment and 6 and 12 weeks after treatment. Clinical responses were evaluated at week 12 and compared with parasite load. In parallel, molecular species differentiation was performed., Results: L. (V.) guyanensis was the main infecting species in 129 of 143 patients (about 90%). PI treatment led to a significant decrease (P < 0.001) in parasite counts, and cured about 75% of these patients. Treatment failure was attributable to infections with Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis and L. (V.) guyanensis (1/92, 1/92 and 22/92 evaluable cases, respectively). There was substantial agreement beyond chance between the parasite load at week 6 and the clinical outcome at week 12, as indicated by the κ value of 0.61., Conclusions: L. (V.) guyanensis is the main infecting species of CL in Suriname, followed by L. (V.) braziliensis and L. (L.) amazonensis. Furthermore, patient response to PI can be better anticipated based on the parasite load 6 weeks after the treatment rather than on parasite load before treatment., (© 2015 The Authors Clinical and Experimental Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists, North American Clinical Dermatologic Society and St Johns Dermatological Society.)

Viability estimation of the highly resistant oocysts of Cryptosporidium remains a key issue for the monitoring and control of this pathogen. We present here a simple 'one tube' quantitative PCR (qPCR) protocol for viability estimation using a DNA extraction protocol which preferentially solubilizes excysted sporozoites rather than oocysts. Parasite DNA released from excysted sporozoites was quantified by real-time qPCR using a ribosomal DNA marker. The qPCR signal was directly proportional to the number of oocysts excysted, and a power-law relationship was noted between oocyst age and the proportion excysting. Unexcysted oocysts released negligible amounts of DNA making the method suitable for estimating viability of as few as 10 oocysts.

Ethnopharmacological Relevance: Plant-based preparations are extensively used in Surinamese folk medicine for treating leishmaniasis, but often without a scientific rationale., Aim of the Study: To evaluate 25 Surinamese medicinal plants for their potential efficacy against leishmaniasis., Materials and Methods: Concentrated plant extracts were evaluated for their effect on the viability of L. (V.) guyanensis AMC, L. (L.) major NADIM5, and L. (L.) donovani GEDII promastigotes, as well as intracellular amastigotes of L. (L.) donovani BHU814 in infected THP-1 cells. Selectivity was assessed by cytotoxicity against THP-1 cells., Results: The only plant extract that showed potentially meaningful anti-leishmanial activity was that from Solanum lycocarpum that displayed mean IC50 values of about 51, 61, and <16 µg/mL against L. (V) guyanensis, L. (L) major, and L. (L) donovani promastigotes, respectively; about 374 µg/mL against L. (L) donovani amastigotes; and >500 µg/mL against THP-1 cells. The Bryophyllum pinnatum, Inga alba, and Quassia amara extracts displayed moderate to high IC50 values against promastigotes (about 51 to >500 µg/mL) and/or amastigotes (about 224 to >500 µg/mL) but were relatively toxic to THP-1 cells (IC50 values <16 to about 42 µg/mL). The remaining plant extracts exhibited in many cases IC50 values close to, around, or above 500µg/mL against promastigotes, amastigotes, and THP-1 cells., Conclusions: The S. lycocarpum preparation may be useful against leishmaniasis and may have a good safety index, warranting further investigations into its active constituents and mechanism(s) of action., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Health-related stigma and its dramatic consequences for those stigmatized have long been a crucial concern for public health authorities globally. However, before concluding that stigma spoils the lives of people with a particular disease or disability and is a major obstacle to obtaining/providing adequate health care, it is necessary to first determine whether there is actual stigmatization related to the condition concerned. The purpose of this article is to nuance the concept of stigma through a detailed ethnographic exploration of the experiences and views of patients and others affected by the parasitic skin disease cutaneous leishmaniasis (CL) in Suriname, South America. Qualitative data on the perceptions, treatment and illness experiences of CL in Suriname was collected in 2009 and 2010 among 205 CL patients at the Dermatology Service in the capital city Paramaribo, and among 321 people in different rural hinterland villages. The exploration reveals the complex and sometimes confusing statements of patients and observers of social reactions to the disease. The authors conclude that--in contrast to other societies--CL is not generally a stigmatized disease in Suriname (though this is not to deny that stigmatization may occur occasionally). Over the past decades, the concepts of stigma and stigmatization have been abundantly theorized. But when theory drifts away from ethnographic evidence, it may turn into imprecise popular speech. In this article, we warn against inflation of the term stigma and show, through an in-depth qualitative description of reactions to symptoms of CL in Suriname, why negative reactions may not necessarily entail stigma., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Functional impairment of dendritic cells (DCs) is part of a survival strategy evolved by Leishmania and Plasmodium parasites to evade host immune responses. Here, the effects of co-exposing human monocyte-derived DCs to Leishmania donovani promastigotes and Plasmodium falciparum-infected erythrocytes were investigated. Co-stimulation resulted in a dual, dose-dependent effect on DC differentiation which ranged from semi-mature cells, secreting low interleukin(-12p70 levels to a complete lack of phenotypic maturation in the presence of high parasite amounts. The effect was mainly triggered by the Leishmania parasites, as illustrated by their ability to induce semi-mature, interleukin-10-producing DCs, that poorly responded to lipopolysaccharide stimulation. Conversely, P. falciparum blood-stage forms failed to activate DCs and only slightly interfered with lipopolysaccharide effects. Stimulation with high L. donovani concentrations triggered phosphatidylserine translocation, whose onset presented after initiating the maturation impairment process. When added in combination, the two parasites could co-localize in the same DCs, confirming that the leading effects of Leishmania over Plasmodium may not be due to mutual exclusion. Altogether, these results suggest that in the presence of visceral leishmaniasis-malaria co-infections, Leishmania-driven effects may overrule the more silent response elicited by P. falciparum, shaping host immunity towards a regulatory pattern and possibly delaying disease resolution., (© 2015 John Wiley & Sons Ltd.)

Visceral leishmaniasis (VL) is a serious chronic disease with a lethality rate of up to 10% in humans. In urban areas of Brazil, dogs are the main reservoirs of the etiological agent (Leishmania infantum) of VL, and the Brazilian Ministry of Health recommends the euthanasia of animals that are seropositive in both the immunochromatographic dual path platform rapid test (DPP(®); Bio-Manguinhos) and the enzyme-linked immunosorbent assay (ELISA) with an L. major-like antigen (Bio-Manguinhos). Vaccination is an additional tool in the control of canine VL, but the use of Leishmune(®) (Zoetis Indústria de Produtos Veterinários, São Paulo, SP, Brazil), which contains the fucose mannose ligand (FML) isolated from L. donovani, is not currently recommended by the Brazilian Ministry of Health because vaccinated animals may exhibit positive serology and there are reservations regarding the efficacy of the vaccine. The aims of the present study were: (i) to verify the abilities of the fast agglutination screening test (FAST), the direct agglutination test (DAT), the indirect fluorescent-antibody test (IFAT), the DPP rapid test, and ELISA tests with L. major-like and FML antigens to differentiate between L. infantum-infected and Leishmune(®)-vaccinated dogs, and (ii) to analyze the sensitivities and specificities of the different methods. The reactivities to these tests of Leishmune(®)-vaccinated dogs (n = 71), asymptomatic (n = 20) and symptomatic (n = 20) naturally infected dogs, and unvaccinated healthy control dogs (n = 5) were compared. None of the Leishmune(®)-vaccinated dogs tested seropositive in FAST and DAT, although one dog was reactive to DPP and four dogs to ELISA/L. major-like and IFAT tests. While 69 (97%) of vaccinated dogs reacted to ELISA/FML, only one was seropositive in both ELISA/L. major-like and IFAT tests. Individually, all immunodiagnostic tests presented high specificities and positive likelihood ratios (LR+), and high specificity values were obtained when the tests were considered in pairs. However, sensitivity and LR- values were low for ELISA/L. major-like and IFAT tests individually, and for all pair combinations of tests except for FAST with DPP.

This review focuses on recent developments in the diagnosis, treatment, management, and strategies for the prevention and control of cutaneous leishmaniasis (CL) caused by both Old and New World Leishmania species. CL is caused by the vector-borne protozoan parasite Leishmania and is transmitted via infected female sandflies. The disease is endemic in more than 98 countries and an estimated 350 million people are at risk. The overall prevalence is 12 million cases and the annual incidence is 2-2.5 million. The World Health Organization considers CL a severely neglected disease and a category 1 emerging and uncontrolled disease. The management of CL differs from region to region and is primarily based on local experience-based evidence. Most CL patients can be treated with topical treatments, but some Leishmania species can cause mucocutaneous involvement requiring a systemic therapeutic approach. Moreover, Leishmania species can vary in their sensitivity to available therapeutic options. This makes species determination critical for the choice of treatment and the clinical outcome of CL. Identification of the infecting parasite used to be laborious, but now the Leishmania species can be identified relatively easy with new DNA techniques that enable a more rational therapy choice. Current treatment guidelines for CL are based on poorly designed and reported trials. There is a lack of evidence for potentially beneficial treatments, a desperate need for large well-conducted studies, and standardization of future trials. Moreover, intensified research programs to improve vector control, diagnostics, and the therapeutic arsenal to contain further incidence and morbidity are needed.

The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Malaria still causes high morbidity and mortality around the world, mainly in sub-Saharan Africa. Community case management of malaria (CCMm) by community health workers (CHWs) is one of the strategies to combat the disease by increasing access to malaria treatment. Currently, the World Health Organization recommends to treat only confirmed malaria cases, rather than to give presumptive treatment., Objectives: This systematic review aims to provide a comprehensive overview of the success or failure of critical steps in CCMm with rapid diagnostic tests (RDTs)., Methods: The databases of Medline, Embase, the Cochrane Library, the library of the 'Malaria in Pregnancy' consortium, and Web of Science were used to find studies on CCMm with RDTs in SSA. Studies were selected according to inclusion and exclusion criteria, subsequently risk of bias was assessed and data extracted., Results: 27 articles were included. CHWs were able to correctly perform RDTs, although specificity levels were variable. CHWs showed high adherence to test results, but in some studies a substantial group of RDT negatives received treatment. High risk of bias was found for morbidity and mortality studies, therefore, effects on morbidity and mortality could not be estimated. Uptake and acceptance by the community was high, however negative-tested patients did not always follow up referral advice. Drug or RDT stock-outs and limited information on CHW motivation are bottlenecks for sustainable implementation. RDT-based CCMm was found to be cost effective for the correct treatment of malaria in areas with low to medium malaria prevalence, but study designs were not optimal., Discussion: Trained CHWs can deliver high quality care for malaria using RDTs. However, lower RDT specificity could lead to missed diagnoses of non-malarial causes of fever. Other threats for CCMm are non-adherence to negative test results and low referral completion. Integrated CCM may solve some of these issues. Unfortunately, morbidity and mortality are not adequately investigated. More information is needed about influencing sociocultural aspects, CHW motivation and stock supply., Conclusion: CCMm is generally well executed by CHWs, but there are several barriers for its success. Integrated CCM may overcome some of these barriers.

Background: The immune system plays a critical role in the development of co-infections, promoting or preventing establishment of multiple infections and shaping the outcome of pathogen-host interactions. Its ability to mediate the interplay between visceral leishmaniasis (VL) and malaria has been suggested, but poorly documented. The present study investigated whether concomitant infection with Leishmania donovani complex and Plasmodium falciparum in naturally co-infected patients altered the immunological response elicited by the two pathogens individually., Results: Circulating levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, IL-17A and tumor necrosis factor (TNF) were assessed in sera of patients infected with active VL and/or malaria and healthy individuals from Gedarif State, Sudan. Comparative analysis of cytokine profiles from co- and mono-infected patients highlighted significant differences in the immune response mounted upon co-infection, confirming the ability of L. donovani and P. falciparum to mutually interact at the immunological level. Progressive polarization towards type-1 and pro-inflammatory cytokine patterns characterized the co-infected patients, whose response partly reflected the effect elicited by VL (IFN-γ, TNF) and malaria (IL-2, IL-13), and partly resulted from a synergistic interaction of the two diseases upon each other (IL-17A). Significantly reduced levels of P. falciparum parasitaemia (P <0.01) were detected in the co-infected group as opposed to the malaria-only patients, suggesting either a protective or a non-detrimental effect of the co-infection against P. falciparum infection., Conclusions: These findings suggest that a new immunological scenario may occur when L. donovani and P. falciparum co-infect the same patient, with potential implications on the course and resolution of these diseases.

Background: Canine leishmaniosis (CanL) due to Leishmania infantum is a global zoonosis endemic in more than 70 countries in Europe, North Africa, Asia and America; however, data on this infection is scarce from southern Africa. The aim of this study was to survey dogs in Luanda, Angola, for Leishmania infection., Findings: One hundred-and-three dogs presented to a veterinary medical centre in Luanda were serologically and molecularly assessed for Leishmania with the direct agglutination test (DAT) and polymerase chain reaction (PCR). Two dogs were seropositive, with DAT titres of 800 and ≥6400; the latter was also found to be PCR-positive and confirmed to be infected with L. infantum by DNA sequence analysis. No other dog was found to be PCR-positive. The first dog had been imported from Portugal, but the latter had never left Angola (neither had its parents), strongly suggesting an autochthonous infection., Conclusions: Although other cases of CanL have previously been described in the country, this is the first reported study of canine Leishmania infection at the population level, as well as the first report on the molecular characterization of L. infantum in dogs from Angola.

The first case of cutaneous leishmaniosis in a horse from the north of Portugal, with a 1.5 cm in diameter ulcerated nodular lesion on the left face, is reported. The skin nodule was surgically excised and assessed by histopathology, including an immunohistochemistry method applied for the first time to equine tissues which clearly demonstrated leishmanial amastigote forms. Two serological determinations with the direct agglutination test performed 13 months apart showed seroconversion specific for Leishmania from a <25 to a 200 antibody titre. Polymerase chain reaction followed by kinetoplast DNA sequencing provided a 116-bp sequence with 98% identity to Leishmania infantum closest sequence deposited in GenBank. No recurrence was observed after complete surgical excision. Leishmaniosis should be included in the differential diagnosis of cutaneous nodular or papular lesions in the equine species in Portugal., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Diagnosis of human African trypanosomiasis (HAT) using molecular tests should ideally achieve high sensitivity without compromising specificity. This study compared 2 simplified tests, nucleic acid sequence-based amplification (NASBA) combined with oligochromatography (OC) and loop-mediated isothermal amplification (LAMP), executed on 181 blood samples from 65 Trypanosoma brucei gambiense HAT patients, 86 controls, and 30 serological suspects from Uganda. Basing on the composite reference standard, the diagnostic sensitivity and specificity of NASBA were 93.9% (95% confidence interval [CI] = 84.9-98.3%) and 100% (95% CI = 94.9-100%), respectively. The same parameters for LAMP were 76.9% (95% CI = 64.8-86.5%) and 100% (95% CI = 91.6-100%), respectively. The level of agreement between LAMP and microscopy was good with a kappa (κ) value of 79.2% (95% CI = 69.4-88.9%), while that of NASBA-OC/microscopy was very good (κ value 94.6%; 95% CI = 89.3-99.8%). The sensitivity of NASBA-OC was significantly higher than that of LAMP (Z = 2.723; P = 0.007). These tests have potential application to HAT surveillance., (© 2013.)

Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.

Background: Parasite clearance time after artemisinin-based combination therapy (ACT) may be increasing in Asian and African settings. The association between parasite clearance following ACT and transmissibility is currently unknown., Methods: We determined parasite clearance dynamics by duplex quantitative polymerase chain reaction (qPCR) in samples collected in the first 3 days after treatment of uncomplicated malaria with ACT. Gametocyte carriage was determined by Pfs25 quantitative nucleic acid sequence-based amplification assays; infectiousness to mosquitoes by membrane-feeding assays on day 7 after treatment., Results: Residual parasitemia was detected by qPCR in 31.8% (95% confidence interval [CI], 24.6-39.8) of the children on day 3 after initiation of treatment. Residual parasitemia was associated with a 2-fold longer duration of gametocyte carriage (P = .0007), a higher likelihood of infecting mosquitoes (relative risk, 1.95; 95% CI, 1.17-3.24; P = .015), and a higher parasite burden in mosquitoes (incidence rate ratio, 2.92; 95% CI, 1.61-5.31; P < .001). Children with residual parasitemia were also significantly more likely to experience microscopically detectable parasitemia during follow-up (relative risk, 11.25; 95% CI, 4.08-31.01; P < .001)., Conclusions: Residual submicroscopic parasitemia is common after ACT and is associated with a higher transmission potential. Residual parasitemia may also have consequences for individual patients because of its higher risk of recurrent parasitemia.

Currently available drugs for treatment of Leishmania infections are highly toxic and drug resistance to first line therapies has been observed. New, safer and more effective drugs are urgently needed to improve clinical resolution of the disease and reduce the risks associated with it. High-throughput screening of new compounds against cultured promastigotes is easy to perform, but the results are poorly predictive of in vivo efficacy. Intra-macrophage amastigote models provide a better proxy of the clinically relevant stage of disease and should be routinely implemented in the search for new anti-leishmanial agents, despite being labor intensive. This study describes the use of a duplex quantitative Reverse-Transcriptase PCR (qRT-PCR) for assessment of drug activity against Leishmania intracellular amastigotes and their host cells. The assay simultaneously quantifies Leishmania 18S ribosomal RNA and the human β2-microglobulin (β-2M) mRNA, used for monitoring drug cytotoxicity and test performance. Accurate determination of parasite viability by the newly developed qRT-PCR was confirmed by parallel assessment of compound performance against standard microscopy. Highly reproducible anti-leishmanial activities were obtained with a set of structurally- and pharmacologically-diverse compounds, whose toxicity against host cells correlated with a low β-2M amplification. Sensitive and versatile, this duplex qRT-PCR offers a valuable tool for assessment of drug activities against Leishmania amastigotes and their host cells.

Background: Sand flies (Diptera: Psychodidae) are the vectors of Leishmania parasites, the causative agents of leishmaniasis. Cutaneous leishmaniasis is an increasing public health problem in the Republic of Suriname and is mainly caused by Leishmania (Vianna) guyanensis, but L. (V.) braziliensis, L. (L.) amazonensis, and L. (V.) naiffi also infect humans. Transmission occurs predominantly in the forested hinterland of the country. Information regarding the potential vectors of leishmaniasis in Suriname is limited. This study aims to broaden the knowledge about vectors involved in the transmission of cutaneous leishmaniasis in Suriname. For this purpose, sand flies were characterized in various foci of cutaneous leishmaniasis in the country, the districts of Para, Brokopondo, and Sipaliwini., Methods: Sand flies were collected in areas around mining plots and villages using CDC light traps in the period between February 2011 and March 2013. They were categorized by examination of the spermathecea (females) and the external genitalia (males)., Results: A total of 2,743 sand fly specimens belonging to 34 different species were captured, including four species (Lutzomyia aragaoi, Lu. ayrozai, Lu. damascenoi, and Lu. sordellii) that had never before been described for Suriname. Five percent of the catch comprised Lu. squamiventris sensu lato, one female of which was positive with L. (V.) braziliensis and was captured in a gold mining area in Brokopondo. Other sand fly species found positive for Leishmania parasites were Lu. trichopyga, Lu. ininii, and Lu. umbratilis, comprising 32, 8, and 4%, respectively, of the catch. These were captured at gold mining areas in Brokopondo and Sipaliwini, but the Leishmania parasites they had ingested could not be identified due to insufficient amounts of DNA., Conclusions: The sand fly fauna in Suriname is highly diverse and comprises Lutzomyia species capable of transmitting Leishmania parasites. Four new Lutzomyia species have been found, and four species - Lu. squamiventris (s.l.), Lu. trichopyga, Lu. ininii, and Lu. umbratilis - have been found to harbor Leishmania parasites. The latter were among the most abundant species captured. These observations may contribute to the understanding of leishmaniasis transmission and the development of control programs in Suriname.

Background: Leishmania infantum and Toxoplasma gondii are protozoa with zoonotic and economic importance. Prevalences of antibodies to these agents were assessed in 173 horses from the north of Portugal., Findings: Antibodies to L. infantum were detected by the direct agglutination test (DAT); seven (4.0%) horses were seropositive with DAT titres of 200 (n = 5), 800 (n = 1) and ≥ 1600 (n = 1). Antibodies to T. gondii were assayed by the modified agglutination test (MAT); 23 (13.3%) horses were seropositive with MAT titres of 20 (n = 13), 40 (n = 5), 80 (n = 3) and ≥ 160 (n = 2). No statistical differences were found among equine categories of gender (female, male and gelding), age (1.5-6, 7-12 and 13-30 years), type of housing (indoors and mixed/outdoors), ability (recreation, farming and sports) and clinical status (apparently healthy and sick) for both agents., Conclusions: Horses are exposed to and may be infected with L. infantum and T. gondii in the north of Portugal.

Background: Artemisinin-based combination therapy (ACT) reduces the potential for malaria transmission, compared with non-ACTs. It is unclear whether this effect differs between ACTs., Methods: A total of 298 children (age, 6 months to 10 years) with uncomplicated falciparum malaria were randomized to artemether-lumefantrine (AL; n = 153) or dihydroartemisinin-piperaquine (DP; n = 145) in Mbita, a community in western Kenya. Gametocyte carriage was determined by molecular methods on days 0, 1, 2, 3, 7, 14, 28, and 42 after treatment initiation. The gametocyte infectiousness to mosquitoes was determined by mosquito-feeding assays on day 7 after beginning therapy., Results: The cumulative risk of recurrent parasitemia on day 42 after initiation of treatment, unadjusted by polymerase chain reaction findings, was 20.7% (95% confidence interval [CI], 14.4-28.2) for AL, compared with 3.7% (95% CI, 1.2-8.5) for DP (P < .001). The mean duration of gametocyte carriage was 5.5 days (95% CI, 3.6-8.5) for AL and 15.3 days (95% CI, 9.7-24.2) for DP (P = .001). The proportion of mosquitoes that became infected after feeding on blood from AL-treated children was 1.88% (43 of 2293), compared with 3.50% (83 of 2371) for those that fed on blood from DP-treated children (P = .06); the oocyst burden among mosquitoes was lower among those that fed on blood from AL-treated children (P = .005) CONCLUSIONS: While DP was associated with a longer prophylactic time after treatment, gametocyte carriage and malaria transmission to mosquitoes was lower after AL treatment., Clinical Trials Registration: NCT00868465.

Background: Canine leishmaniosis (CanL) is an endemic zoonosis in the southern regions of Europe. This paper reports the trend in CanL seroprevalence in the municipality of Évora (southern Portugal), where the disease is endemic, over a period of 20 years. The work comprises three different studies that were conducted in the years of 1990 (n = 3,614), 1999 (n = 3,563) and 2010 (n = 1,485 dogs). Blood samples were collected during the anti-rabies vaccination campaigns. Anti-Leishmania antibodies were detected with the direct agglutination test (DAT)., Findings: The total percentages of DAT seropositive dogs were 3.9% (in 1990), 9.4% (in 1999) and 5.6% (in 2010). The overall seroprevalence was significantly higher in 1999 compared to 1990, but in 2010 a significant decrease was found in comparison with 1999. However, compared to 1990 the overall seroprevalence was still significantly higher in 2010. From 1990 to 2010 seroprevalence has switched from significantly lower to higher in the rural areas. Relatively few dogs showed clinical signs of overt disease (0.8% to 2.0%) with lymphadenopathy, onychogryphosis and skin involvement as most frequently observed. Gender associated differences in seroprevalence were not found, and most commonly seropositive dogs were working or stray animals. The mean age of seropositive dogs was significantly higher than seronegative dogs in all three sampling rounds., Conclusions: A high proportion of dogs, which are apparently healthy, yet seropositive, may remain an important factor in limiting the outcome of zoonotic leishmaniosis control efforts.

Background: In areas where visceral leishmaniasis (VL) and malaria are co-endemic, co-infections are common. Clinical implications range from potential diagnostic delay to increased disease-related morbidity, as compared to VL patients. Nevertheless, public awareness of the disease remains limited. In VL-endemic areas with unstable and seasonal malaria, vulnerability to the disease persists through all age-groups, suggesting that in these populations, malaria may easily co-occur with VL, with potentially severe clinical effects., Methods: A retrospective case-control study was performed using medical records of VL patients admitted to Tabarakallah and Gedarif Teaching Hospitals (Gedarif State) and Al`Azaza kala-azar Clinic (Sennar State), Sudan (2005-2010). Patients positively diagnosed with VL and malaria were identified as cases, and VL patients without microscopy-detectable malaria as controls. Associations between patient characteristics and the occurrence of the co-infection were investigated using logistic regression analysis. Confirmation of epidemiological outcomes was obtained with an independently collected dataset, composed by Médecins Sans Frontières (MSF) at Um-el-Kher and Kassab Hospitals, Gedarif State (1998)., Results: The prevalence of malaria co-infection among VL surveyed patients ranged from 3.8 to 60.8%, with a median of 26.2%. Co-infected patients presented at hospital with deteriorated clinical pictures. Emaciation (Odds Ratio (OR): 2.46; 95% Confidence Interval (95% CI): 1.72-3.50), jaundice (OR: 2.52; 95% CI: 1.04-6.09) and moderate anemia (OR: 1.58; 95% CI: 1.10-2.28) were found to be positively associated with the co-infection, while severity of splenomegaly (OR: 0.53; 95% CI: 0.35-0.81) and, to a less extent, hepatomegaly (OR: 0.52; 95% CI: 0.27-1.01) appeared to be reduced by concomitant VL and malaria. The in-hospital case-fatality rates did not significantly differ between co- and mono-infected patients (OR: 1.13; 95% CI: 0.59-2.17). Conversely, a significantly increased mortality rate (OR: 4.38; 95% CI: 1.83-10.48) was observed by MSF amongst co-infected patients enrolled at Um-el-Kher and Kassab Hospitals, who also suffered an enhanced risk of severe anemia (OR: 3.44; 95% CI: 1.68-7.02) compared to VL mono-infections., Conclusions: In endemic VL areas with unstable seasonal malaria, like eastern Sudan, VL patients are highly exposed to the risk of developing concomitant malaria. Prompt diagnosis and effective treatment of malaria are essential to ensure that its co-infection does not result into poor prognoses.

Declining malaria transmission and known difficulties with current diagnostic tools for malaria, such as microscopy and rapid diagnostic tests (RDTs) in particular at low parasite densities, still warrant the search for sensitive diagnostic tests. Molecular tests need substantial simplification before implementation in clinical settings in countries where malaria is endemic. Direct blood PCR (db-PCR), circumventing DNA extraction, to detect Plasmodium was developed and adapted to be visualized by nucleic acid lateral flow immunoassay (NALFIA). The assay was evaluated in the laboratory against samples from confirmed Sudanese patients (n = 51), returning travelers (n = 214), samples from the Dutch Blood Bank (n = 100), and in the field in Burkina Faso (n = 283) and Thailand (n = 381) on suspected malaria cases and compared to RDT and microscopy. The sensitivity and specificity of db-PCR-NALFIA compared to the initial diagnosis in the laboratory were 94.4% (95% confidence interval [CI] = 0.909 to 0.969) and 97.4% (95% CI = 0.909 to 0.969), respectively. In Burkina Faso, the sensitivity was 94.8% (95% CI = 0.88.7 to 97.9%), and the specificity was 82.4% (95% CI = 75.4 to 87.7%) compared to microscopy and 93.3% (95% CI = 87.4 to 96.7%) and 91.4% (95% CI = 85.2 to 95.3%) compared to RDT. In Thailand, the sensitivity and specificity were 93.4% (CI = 86.4 to 97.1%) and 90.9 (95% CI = 86.7 to 93.9%), respectively, compared to microscopy and 95.6% (95% CI = 88.5 to 98.6%) and 87.1% (95% CI = 82.5 to 90.6) compared to RDT. db-PCR-NALFIA is highly sensitive and specific for easy and rapid detection of Plasmodium parasites and can be easily used in countries where malaria is endemic. The inability of the device to discriminate Plasmodium species requires further investigation.

Background: Molecular tools are very sensitive and specific and could be an alternative for the diagnosis of malaria. The complexity and need for expensive equipment may hamper implementation and, therefore, simplifications to current protocols are warranted., Methods: A PCR detecting the different Plasmodium species and differentiating between Plasmodium falciparum and Plasmodium vivax was developed and combined with a nucleic acid lateral flow immuno-assay (PCR-NALFIA) for amplicon detection. The assay was thoroughly evaluated for the analytical sensitivity and specificity in the laboratory, the robustness and reproducibility in a ring trial and accuracy and predictive value in a field trial., Results: The analytical sensitivity and specificity were 0.978 (95% CI: 0.932-0.994) and 0.980 (95% CI: 0.924-0.997), respectively, and were slightly less sensitive for the detection of P. vivax than for P. falciparum. The reproducibility tested in three laboratories was very good (k = 0.83). This evaluation showed that the PCR machine used could influence the results. Accuracy was evaluated in Thailand and compared to expert microscopy and rapid diagnostic tests (RDTs). The overall and P. falciparum-specific sensitivity and specificity was good ranging from 0.86-1 and 0.95-0.98 respectively, compared to microscopy. Plasmodium vivax detection was better than the sensitivity of RDT, but slightly less than microscopy performed in this study., Conclusion: PCR-NALFIA is a sensitive, specific and robust assay able to identify Plasmodium species with good accuracy. Extensive testing including a ring trial can identify possible bottlenecks before implementation and is therefore essential to perform in additon to other evaluations.

Rapid diagnostics tests (RDTs) detect malaria specific antigen(s) in the circulation, even when parasites are sequestered in the placenta and not visible by microscopy. However, research on their diagnostic accuracy during pregnancy is limited. Pregnant women (n = 418) were screened for malaria during routine antenatal care by using two RDTs that detect histidine-rich protein 2 (HRP2) or Plasmodium lactate dehydrogenase, and enzyme-linked immunosorbent assays with antibodies that detect dihydrofolate reductase-thymidylate synthase or heme-detoxification protein, and compared with real-time polymerase chain reaction (RT-PCR) and microscopy for evaluation of their diagnostic accuracy. Prevalence of malaria infection was high (53% by PCR). The RT-PCR and the HRP2 RDT detected most cases of malaria during pregnancy, whereas microscopy, the Plasmodium lactate dehydrogenase RDT, and enzyme-linked immunosorbent assays for dihydrofolate reductase-thymidylate synthase and heme-detoxification protein antibodies did not detect several low-density infections. Therefore, the HRP2 RDT could be a useful tool in high-transmission areas for diagnosis of malaria in asymptomatic pregnant women.

Objectives: To evaluate persistence of several Plasmodium antigens in pregnant women after treatment and compare diagnostics during treatment follow-up., Methods: Thirty-two pregnant women (N = 32) with confirmed malaria infection by a histidine-rich protein 2 (HRP2)-based rapid diagnostic test (RDT) and microscopy were followed for 28 days after artemisinin-based combination therapy (ACT). A Plasmodium lactate dehydrogenase (pLDH)-based RDT and two ELISAs based on the detection of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and haeme detoxification protein (HDP) were compared with each other and to RT-PCR at each visit., Results: The mean visit number (95% confidence interval) on which the HRP2-based RDT was still positive after treatment was 3.4 (2.7-4.1) visits with some patients still positive at day 28. This is significantly later than the pLDH-based RDT [0.84 (0.55-1.1)], microscopy (median 1, range 1-3), DHFR-TS-ELISA [1.7 (1.1-2.3)] and RT-PCR (median 2, range 1-5) (P < 0.05), but not significantly later than HDP-ELISA [2.1 (1.6-2.7)]. Lower gravidity and higher parasite density at day 0 resulted in significantly longer positive results with most tests (P < 0.05)., Conclusions: HRP2 can persist up to 28 days after ACT treatment; therefore, this test is not suitable for treatment follow-up in pregnant women and can generate problems when using this test during intermittent preventive treatment (IPTp). DHFR-TS is less persistent than HRP2, making it a potentially interesting target for diagnosis., (© 2012 Blackwell Publishing Ltd.)

The main causative agent of cutaneous leishmaniasis (CL) in Suriname is Leishmania (Viannia) guyanensis. This case report presents a patient infected with Leishmania (Viannia) braziliensis, a species never reported before in Suriname. This finding has clinical implications, because L. braziliensis has a distinct clinical phenotype characterized by mucocutaneous leishmaniasis, a more extensive and destructive form of CL that requires different treatment. Clinicians should be aware that chronic cutaneous ulcers in patients from the Guyana region could be caused by L. braziliensis.

Background and Methodology: Due to geographic overlap of malaria and visceral leishmaniasis (VL), co-infections may exist but have been poorly investigated. To describe prevalence, features and risk factors for VL-malaria co-infections, a case-control analysis was conducted on data collected at Amudat Hospital, Uganda (2000-2006) by Médecins sans Frontières. Cases were identified as patients with laboratory-confirmed VL and malaria at hospital admission or during hospitalization; controls were VL patients with negative malaria smears. A logistic regression analysis was performed to study the association between patients' characteristics and the occurrence of the co-infection., Results: Of 2414 patients with confirmed VL, 450 (19%) were positively diagnosed with concomitant malaria. Most co-infected patients were males, residing in Kenya (69%). While young age was identified by multivariate analysis as a risk factor for concurrent VL and malaria, particularly the age groups 0-4 (odds ratio (OR): 2.44; 95% confidence interval (CI): 1.52-3.92) and 5-9 years (OR: 2.23, 95% CI: 1.45-3-45), mild (OR: 0.53; 95% CI: 0.32-0.88) and moderate (OR: 0.45; 95% CI: 0.27-0.77) anemia negatively correlated with the co-morbidity. VL patients harboring skin infections were nearly three times less likely to have the co-infection (OR: 0.35; 95% CI: 0.17-0.72), as highlighted by the multivariate model. Anorexia was slightly more frequent among co-infected patients (OR: 1.71; 95% CI: 0.96-3.03). The in-hospital case-fatality rate did not significantly differ between cases and controls, being 2.7% and 3.1% respectively (OR: 0.87; 95% CI: 0.46-1.63)., Conclusions: Concurrent malaria represents a common condition among young VL patients living in the Pokot region of Kenya and Uganda. Although these co-morbidities did not result in a poorer prognosis, possibly due to early detection of malaria, a positive trend towards more severe symptoms was identified, indicating that routine screening of VL patients living in malaria endemic-areas and close monitoring of co-infected patients should be implemented.

Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, KD) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have KD values of 0.10 nM ± 0.014 (D5) and 0.068 ± 0.015 nM (D6) for DHFR-TS mAbs, 0.10 ± 0.022 nM (H16) and 0.21 ± 0.022 nM (H18) for HDP mAbs and 0.11 ± 0.028 nM (G23) and 0.33 ± 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (KD of 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1-13), making them promising reagents for further test development.

Background: A range of molecular amplification techniques have been developed for the diagnosis of Human African Trypanosomiasis (HAT); however, careful evaluation of these tests must precede implementation to ensure their high clinical accuracy. Here, we investigated the diagnostic accuracy of molecular amplification tests for HAT, the quality of articles and reasons for variation in accuracy., Methodology: Data from studies assessing diagnostic molecular amplification tests were extracted and pooled to calculate accuracy. Articles were included if they reported sensitivity and specificity or data whereby values could be calculated. Study quality was assessed using QUADAS and selected studies were analysed using the bivariate random effects model., Results: 16 articles evaluating molecular amplification tests fulfilled the inclusion criteria: PCR (n = 12), NASBA (n = 2), LAMP (n = 1) and a study comparing PCR and NASBA (n = 1). Fourteen articles, including 19 different studies were included in the meta-analysis. Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to 99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3). Differences in study design and readout method did not significantly change estimates although use of satellite DNA as a target significantly lowers specificity. Sensitivity and specificity of PCR on CSF for staging varied from 87.6% to 100%, and 55.6% to 82.9% respectively., Conclusion: Here, PCR seems to have sufficient accuracy to replace microscopy where facilities allow, although this conclusion is based on multiple reference standards and a patient population that was not always representative. Future studies should, therefore, include patients for which PCR may become the test of choice and consider well designed diagnostic accuracy studies to provide extra evidence on the value of PCR in practice. Another use of PCR for control of disease could be to screen samples collected from rural areas and test in reference laboratories, to spot epidemics quickly and direct resources appropriately.

Background: During pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed., Methods: MEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women., Results: The results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy., Conclusion: The findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations concerning the use of these tests. Nevertheless, more studies with placental histology as reference test are urgently required to reliably determine the accuracy of RDTs and PCR for the diagnosis of placental malaria. P. vivax-infections have been neglected in diagnostic test accuracy studies of malaria in pregnancy.

Human African trypanosomiasis (HAT) is caused by sub-species of the parasitic protozoan Trypanosoma brucei and is transmitted by tsetse flies, both of which are endemic only to sub-Saharan Africa. Several cases have been reported in non-endemic areas, such as North America and Europe, due to travelers, ex-patriots or military personnel returning from abroad or due to immigrants from endemic areas. In this paper, non-endemic cases reported over the past 20 years are reviewed; a total of 68 cases are reported, 19 cases of Trypanosoma brucei gambiense HAT and 49 cases of Trypanosoma brucei rhodesiense HAT. Patients ranged in age from 19 months to 72 years and all but two patients survived. Physicians in non-endemic areas should be aware of the signs and symptoms of this disease, as well as methods of diagnosis and treatment, especially as travel to HAT endemic areas increases. We recommend extension of the current surveillance systems such as TropNetEurop and maintaining and promotion of existing reference centers of diagnostics and expertise. Important contact information is also included, should physicians require assistance in diagnosing or treating HAT., (Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Leishmania (Kinetoplastida: Trypanosomatidae) are protozoan parasites of significant medical and veterinary importance. Over the last decade, visceral leishmaniasis (VL) has emerged as a major opportunistic infection associated with HIV/AIDS in North Western Ethiopia. This paper reports on serological evidence of possible Leishmania donovani (L. donovani) infection in dogs using two serological tests: direct agglutination test (DAT) and Kalazar detect rapid test (KDRT). Two hundred and seventeen asymptomatic local breed dogs were examined for L. donovani antibodies. Performance of the DAT and KDRT was assessed in 162 matching samples of blood collected on filter paper and serum, respectively. Using DAT and KDRT testing in parallel, the overall seroprevalence of L. donovani infection was 27.7% and 14.8%, respectively. The degree of agreement was found to be fair (68.8%, k = 0.234). Univariable logistic regression analysis of some risk factors for L. donovani infection in dogs using DAT indicates that place of residence, sex, age, dog keeping purpose and dog housing condition were not significantly associated with seropositivity. The high proportion of positive dogs suggests the exposure of these animals to L. donovani infection and needs further investigation. Isolation and typing of the parasite aiming at confirming the role of these animals in maintenance and transmission of kala-azar is advocated., (© 2011 Blackwell Verlag GmbH.)

Molecular tools continue to be important in the prevention and control of parasitic diseases. However, using these techniques directly in the field remains a major challenge. Therefore, the preservation of clinical samples collected from endemic field areas for later analysis remains an important preanalytical process. This study aimed at identifying a suitable protocol for stabilization and preservation of RNA and DNA in bioclinical specimens for Trypanosoma, Leishmania, and Plasmodium research. Both spiked and unspiked blood samples were preserved in 7 protocols (different media; storage temperatures). Samples were evaluated for possible degradation of DNA and RNA along the storage duration up to the 10th week. Nucleic acid targets were assessed as follows: (i) Trypanosoma and Plasmodium RNA analysis was done using real-time nucleic acid sequence-based amplification (RT-NASBA) for 18S rRNA and for stage-specific Pfs25 mRNA, respectively; (ii) Trypanosoma DNA assessment analysis was conducted by using a conventional PCR for 18S rDNA; (iii) Leishmania RNA analysis was performed with a quantitative NASBA for 18S rRNA and Leishmania DNA assessment with an RT-PCR for 18S rDNA. Findings suggested that a newly developed L3™ buffer proved to be reliable and suitable for both short- and long-term preservation of parasite nucleic acid material. This buffer is envisaged to be suitable for utilization in field situations where resources are limited., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cutaneous leishmaniasis caused by Leishmania major infection affected 172 (18.3%) of 938 Dutch military troops deployed in northern Afghanistan in 2005. The high attack rate was a result of initial insufficient availability of means of prevention and insufficient adherence to preventive measures. At presentation, the lymphatic system was involved in 24.8%. Treatment with intralesional injections of antimony with or without cryotherapy was satisfactory, but 19.5% of patients received secondary treatment with miltefosine. Six months after treatment, 128 (77.1%) of 166 treated patients were cured, 16 (9.6%) were lost to follow-up, and 22 (13.3%) already experienced cure at six weeks but were not seen at six months. Natural evolution played a role in this observational study, which showed cure of all patients seen at six months. In general, management of cutaneous leishmaniasis was feasible under field conditions.

Placental malaria is the placental sequestration of Plasmodium falciparum infected erythrocytes that accumulate in the intervillous space, resulting in pathological alterations. The intervillous space, the main compartment for exchange of nutrients and delivery of oxygen to the fetus, is of utmost importance for fetal development. Events leading to adverse outcomes of placental malaria can be summarized in four steps: (1) accumulation of P. falciparum infected erythrocytes; (2) infiltration of monocytes and macrophages; (3) alteration of the placental cytokine balance and (4) pathogenesis of adverse pregnancy outcomes. These events are triggered by chemokines and cytokines leading to impaired materno-fetal exchange and damage to the placenta. This review describes the events during placental malaria infection at molecular level and presents a simplified model describing all crucial steps leading to adverse pregnancy outcomes based on a review of recent literature (August 2009)., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: The Leishmania OligoC-TesT and NASBA-Oligochromatography (OC) were recently developed for simplified and standardised molecular detection of Leishmania parasites in clinical specimens. We here present the phase II evaluation of both tests for diagnosis of visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and post kala-azar dermal leishmaniasis (PKDL) in Sudan., Methodology: The diagnostic accuracy of the tests was evaluated on 90 confirmed and 90 suspected VL cases, 7 confirmed and 8 suspected CL cases, 2 confirmed PKDL cases and 50 healthy endemic controls from Gedarif state and Khartoum state in Sudan., Principal Findings: The OligoC-TesT as well as the NASBA-OC showed a sensitivity of 96.8% (95% CI: 83.8%-99.4%) on lymph node aspirates and of 96.2% (95% CI: 89.4%-98.7%) on blood from the confirmed VL cases. The sensitivity on bone marrow was 96.9% (95% CI: 89.3%-99.1%) and 95.3% (95% CI: 87.1%-98.4%) for the OligoC-TesT and NASBA-OC, respectively. All confirmed CL and PKDL cases were positive with both tests. On the suspected VL cases, we observed a positive OligoC-TesT and NASBA-OC result in 37.1% (95% CI: 23.2%-53.7%) and 34.3% (95% CI: 20.8%-50.9%) on lymph, in 72.7% (95% CI: 55.8%-84.9%) and 63.6% (95% CI: 46.6%-77.8%) on bone marrow and in 76.9% (95% CI: 49.7%-91.8%) and 69.2% (95% CI: 42.4%-87.3%) on blood. Seven out of 8 CL suspected cases were positive with both tests. The specificity on the healthy endemic controls was 90% (95% CI: 78.6%-95.7%) for the OligoC-TesT and 100% (95% CI: 92.9%-100.0%) for the NASBA-OC test., Conclusions: Both tests showed high sensitivity on lymph, blood and tissue scrapings for diagnosis of VL, CL and PKDL in Sudan, but the specificity for clinical VL was significantly higher with NASBA-OC.

Background: This study describes the laboratory evaluation of a novel diagnostic platform for malaria. The Magneto Optical Test (MOT) is based on the bio-physical detection of haemozoin in clinical samples. Having an assay time of around one minute, it offers the potential of high throughput screening., Methods: Blood samples of confirmed malaria patients from different regions of Africa, patients with other diseases and healthy non-endemic controls were used in the present study. The samples were analysed with two reference tests, i.e. an histidine rich protein-2 based rapid diagnostic test (RDT) and a conventional Pan-Plasmodium PCR, and the MOT as index test. Data were entered in 2 x 2 tables and analysed for sensitivity and specificity. The agreement between microscopy, RDT and PCR and the MOT assay was determined by calculating Kappa values with a 95% confidence interval., Results: The observed sensitivity/specificity of the MOT test in comparison with clinical description, RDT or PCR ranged from 77.2 - 78.8% (sensitivity) and from 72.5 - 74.6% (specificity). In general, the agreement between MOT and the other assays is around 0.5 indicating a moderate agreement between the reference and the index test. However, when RDT and PCR are compared to each other, an almost perfect agreement can be observed (k = 0.97) with a sensitivity and specificity of >95%., Conclusions: Although MOT sensitivity and specificity are currently not yet at a competing level compared to other diagnostic test, such as PCR and RDTs, it has a potential to rapidly screen patients for malaria in endemic as well as non-endemic countries.

Background: The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) have been recently modified by coupling to oligochromatography (OC) for easy and fast visualisation of products. In this study we evaluate the sensitivity and specificity of the PCR-OC and NASBA-OC for diagnosis of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense human African trypanosomiasis (HAT)., Methodology and Results: Both tests were evaluated in a case-control design on 143 HAT patients and 187 endemic controls from the Democratic Republic of Congo (DRC) and Uganda. The overall sensitivity of PCR-OC was 81.8% and the specificity was 96.8%. The PCR-OC showed a sensitivity and specificity of 82.4% and 99.2% on the specimens from DRC and 81.3% and 92.3% on those from Uganda. NASBA-OC yielded an overall sensitivity of 90.2%, and a specificity of 98.9%. The sensitivity and specificity of NASBA-OC on the specimens from DRC was 97.1% and 99.2%, respectively. On the specimens from Uganda we observed a sensitivity of 84.0% and a specificity of 98.5%., Conclusions/significance: The tests showed good sensitivity and specificity for the T. b. gambiense HAT in DRC but rather a low sensitivity for T. b. rhodesiense HAT in Uganda.

Objective: To evaluate the repeatability and reproducibility of four simplified molecular assays for the diagnosis of Trypanosoma brucei spp. or Leishmania ssp. in a multicentre ring trial with seven participating laboratories., Methods: The tests are based on PCR or NASBA amplification of the parasites nucleic acids followed by rapid read-out by oligochromatographic dipstick (PCR-OC and NASBA-OC)., Results: On purified nucleic acid specimens, the repeatability and reproducibility of the tests were Tryp-PRC-OC, 91.7% and 95.5%; Tryp-NASBA-OC, 95.8% and 100%; Leish-PCR-OC, 95.9% and 98.1%; Leish-NASBA-OC, 92.3% and 98.2%. On blood specimens spiked with parasites, the repeatability and reproducibility of the tests were Tryp-PRC-OC, 78.4% and 86.6%; Tryp-NASBA-OC, 81.5% and 89.0%; Leish-PCR-OC, 87.1% and 91.7%; Leish-NASBA-OC, 74.8% and 86.2%., Conclusion: As repeatability and reproducibility of the tests were satisfactory, further phase II and III evaluations in clinical and population specimens from disease endemic countries are justified.

Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved region of the 18S ribosomal RNA (rRNA) gene; amplification was visualized by the pre-amplification addition of fluorescent detection reagent (FDR) and a simple UV lamp. By using a reverse-transcriptase step, the system detected infections between 10 and 100 parasites per mL. The assay was tested on a range of nucleic acid extracts from Leishmania species, visceral leishmaniasis (VL) patients from Sudan, and cutaneous leishmaniasis (CL) patients from Suriname. The sensitivity of RT-LAMP from the blood of VL patients was 83% (N = 30) compared with microscopy of bone-marrow and lymph-node aspirates; for CL patients the observed sensitivity was 98% (N = 43). The potential to use LAMP as a diagnostic tool for leishmaniasis is discussed.

Background: Molecular methods to detect Leishmania parasites are considered specific and sensitive, but often not applied in endemic areas of developing countries due to technical complexity. In the present study isothermal, nucleic acid sequence based amplification (NASBA) was coupled to oligochromatography (OC) to develop a simplified detection method for the diagnosis of leishmaniasis. NASBA-OC, detecting Leishmania RNA, was evaluated using clinical samples from visceral leishmaniasis patients from East Africa (n = 30) and cutaneous leishmaniasis from South America (n = 70) and appropriate control samples., Results: Analytical sensitivity was 10 parasites/ml of spiked blood, and 1 parasite/ml of culture. Diagnostic sensitivity of NASBA-OC was 93.3% (95% CI: 76.5%-98.8%) and specificity was 100% (95% CI: 91.1%-100%) on blood samples, while sensitivity and specificity on skin biopsy samples was 98.6% (95% CI: 91.2%-99.9%) and 100% (95% CI: 46.3%-100%), respectively., Conclusion: The NASBA-OC format brings implementation of molecular diagnosis of leishmaniasis in resource poor countries one step closer.

Malaria is one of the most important infectious diseases in the world. Although most cases occur in the tropical regions of Africa, Asia, Central and South America, there is in Europe a significant increase in the number of imported cases in non-endemic countries, in particular due to the higher mobility in today's society. The prevalence of a possible asymptomatic infection with Plasmodium species was assessed using Nucleic Acid Sequence Based Amplification (NASBA) assays on clinical samples collected from 195 study cases with no clinical signs related to malaria and coming from sub-Saharan Africa. In addition, base-line demographic, clinical and socio-economic information was collected from study participants who also underwent a full clinical examination. Sixty-two study subjects (31.8%) were found positive for Plasmodium using a pan Plasmodium specific NASBA based on the small subunit 18S rRNA gene (18S NASBA). Twenty-four samples (38%) of the 62 positive study cases were found positive with a Pfs25 mRNA NASBA, which specifically detects gametocytes of Plasmodium falciparum. This study showed that a substantial proportion of people originating from malaria endemic countries harbour malaria parasites in their blood. If transmission conditions are available, they could be a reservoir.

In a retrospective, observational study involving 34 patients with Leishmania major infection, 31 of whom had experienced unsuccessful treatment with intralesional antimony (ilSb(v)), miltefosine proved effective. Thirty patients experienced cure after receipt of miltefosine, 3 after receipt of additional ilSb(v), and 1 after 28 daily intravenous injections of antimony. Temporary diminution of ejaculate volume was reported by 21 patients.