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The contribution of migrated people from once green Sahara (about 10,000-6000 years BC) towards Mediterranean area had probably a double effect: both genetic and cultural connections have been described between Western Europe and North Africa. Sudanese populations from different ethnicities have been studied for HLA-A, -B, -DRB1 and -DQB1 antigens by a standard microlymphotoxicity method. Results found show that Nubians are genetically related with African Sub-Saharan populations and distant from other Sudanese tribes, who are closer to Mediterranean populations than to Sub-Saharan ones. This is concordant with other authors and meta-analysis data. Our present work is, to our knowledge, the first and only one HLA research that studies Sudanese people according to different Sudan ethnic groups: samples were collected before Sudan partition between North and South. A prehistoric genetic and peoples exchange between Africa and the Mediterranean basin may be observed and is supported with the results obtained in this Sudanese HLA study. However, demic diffusion model of agriculture and other anthropological traits from Middle East to West Europe/Maghreb do not exist: a more detailed Sahel and North African countries ancient and recent admixture studies are also being carried out which may clearer explain pastoralists/agriculture innovations origins in Eurafrican Mediterranean and Atlantic façade., (© 2023. Springer Nature Limited.)

Background: Recombinant antigens rK39 (based on kinesin sequence) and rK28 (comprising kinesin and HASPB sequences) are a mainstay of serological diagnosis for visceral leishmaniasis (VL). However, their key epitopes and the significance of their structural conformation are not clearly defined, particularly in relation to reported cross-reactivity with sera from patients with malaria, schistosomiasis, and tuberculosis., Methods: To assess the effect of conformation on antigenicity with Sudanese VL sera, antigens rK39 and rK28 were heat-denatured at 95 °C for 10 min and then assayed by enzyme-linked immunosorbent assay (ELISA). Amino acid sequences of rK39 and rK28 were submitted to NCBI BLASTp to assess homology with Plasmodium, Schistosoma, and Mycobacterium., Results: Heat denaturation significantly diminished the antigenicity of rK39 compared to non-denatured antigen (P = 0.001), but not for rK28 (P = 0.275). In BLASTp searches, HASPB sequences from rK28 had similarities with sequences from Plasmodium, encompassing software-predicted B-cell epitopes., Conclusions: The antigenicity of rK39 appears to be dependent on structural conformation, whereas that of rK28 depends on linear sequence. HASPB sequence homology with Plasmodium may be responsible for the reported cross-reactivity of rK28 with malaria sera. Further work is warranted to refine the specificity of these antigens., (© 2022. The Author(s).)

Background: Persistent fever, defined as fever lasting for 7 days or more at first medical evaluation, has been hardly investigated as a separate clinical entity in the tropics. This study aimed at exploring the frequencies and diagnostic predictors of the ubiquitous priority (i.e., severe and treatable) infections causing persistent fever in the tropics., Methods: In six different health settings across four countries in Africa and Asia (Sudan, Democratic Republic of Congo [DRC], Nepal, and Cambodia), consecutive patients aged 5 years or older with persistent fever were prospectively recruited from January 2013 to October 2014. Participants underwent a reference diagnostic workup targeting a pre-established list of 12 epidemiologically relevant priority infections (i.e., malaria, tuberculosis, HIV, enteric fever, leptospirosis, rickettsiosis, brucellosis, melioidosis, relapsing fever, visceral leishmaniasis, human African trypanosomiasis, amebic liver abscess). The likelihood ratios (LRs) of clinical and basic laboratory features were determined by pooling all cases of each identified ubiquitous infection (i.e., found in all countries). In addition, we assessed the diagnostic accuracy of five antibody-based rapid diagnostic tests (RDTs): Typhidot Rapid IgM, Test-it TM Typhoid IgM Lateral Flow Assay, and SD Bioline Salmonella typhi IgG/IgM for Salmonella Typhi infection, and Test-it TM Leptospira IgM Lateral Flow Assay and SD Bioline Leptospira IgG/IgM for leptospirosis., Results: A total of 1922 patients (median age: 35 years; female: 51%) were enrolled (Sudan, n = 667; DRC, n = 300; Nepal, n = 577; Cambodia, n = 378). Ubiquitous priority infections were diagnosed in 452 (23.5%) participants and included malaria 8.0% (n = 154), tuberculosis 6.7% (n = 129), leptospirosis 4.0% (n = 77), rickettsiosis 2.3% (n = 44), enteric fever 1.8% (n = 34), and new HIV diagnosis 0.7% (n = 14). The other priority infections were limited to one or two countries. The only features with a positive LR ≥ 3 were diarrhea for enteric fever and elevated alanine aminotransferase level for enteric fever and rickettsiosis. Sensitivities ranged from 29 to 67% for the three RDTs targeting S. Typhi and were 9% and 16% for the two RDTs targeting leptospirosis. Specificities ranged from 86 to 99% for S. Typhi detecting RDTs and were 96% and 97% for leptospirosis RDTs., Conclusions: Leptospirosis, rickettsiosis, and enteric fever accounted each for a substantial proportion of the persistent fever caseload across all tropical areas, in addition to malaria, tuberculosis, and HIV. Very few discriminative features were however identified, and RDTs for leptospirosis and Salmonella Typhi infection performed poorly. Improved field diagnostics are urgently needed for these challenging infections., Trial Registration: NCT01766830 at ClinicalTrials.gov., (© 2022. The Author(s).)

Objectives: Community-level antibiotic use contributes to antimicrobial resistance, but is rarely monitored as part of efforts to optimize antibiotic use in low- and middle-income countries (LMICs). We investigated antibiotic use in the 4 weeks before study inclusion for persistent fever., Methods: The NIDIAG-Fever (Neglected Infectious diseases DIAGnosis-Fever) study investigated aetiologies of infections in patients ≥5 years old with fever ≥1 week in six healthcare facilities in Cambodia, the Democratic Republic of the Congo (DRC), Nepal, and Sudan. In the present nested cross-sectional study, we describe prevalence and choice of antibiotics before and at study inclusion, applying the Access/Watch/Reserve (AWaRe) classification of the WHO List of Essential Medicines. Factors associated with prior antibiotic use were analysed., Results: Of 1939 participants, 428 (22.1%) reported the prior use of one or more antibiotics, ranging from 6.3% (24/382, Cambodia) to 35.5% (207/583, Nepal). Of 545 reported antibiotics, the most frequent were Watch group antibiotics (351/545, 64.4%), ranging from 23.6% (DRC) to 82.1% (Nepal). Parenteral administration ranged from 5.9% to 69.6% between study sites. Antibiotic use was most frequent among young patients (5-17 years of age; risk ratio 1.42, 95%CI 1.19-1.71) and men (RR 1.29; 95%CI 1.09-1.53). No association was found with specific symptoms. Of 555 antibiotics started before study inclusion, 275 (49.5%) were discontinued at study inclusion., Conclusions: Watch antibiotics were frequently used, and discontinued upon study inclusion. The antibiotic use frequency and choice varied importantly between LMICs. Data on local antibiotic use are essential to guide efforts to optimize antibiotic use in LMICs, should not be restricted to hospitals, and need to take local healthcare utilization into account., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vancomycin-resistant enterococci (VRE) poses a formidable challenge to public health due to its inherent resistance to multiple antibiotics coupled with the ability to transfer genetic determinants to dangerous pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). The purpose of this study was to investigate the incidence of vancomycin resistance in enterococci among clinical isolates at a tertiary care military hospital in the eastern region of Saudi Arabia and to detect van genes using multiplex-PCR. Overall, 246 isolates of enterococci were collected from various clinical specimens. The isolates were identified, and antimicrobial susceptibility testing was done using the Vitek 2 system. Multiplex PCR was performed on the VRE isolates, thus identified to determine the van genes harbored. A total of 15 VRE were identified, of which 14 (93.3%) were Enterococcus faecium , and 1(6.7%) was Enterococcus casseliflavus with intrinsic vanC resistance. Of the 14 vancomycin-resistant Enterococcus faecium , 8 (57.1%) harbored vanB genes, while 6 (42.8%) harbored vanA genes. All the VRE were susceptible to linezolid and tigecycline. Our study detected a low prevalence (6.1%) of VRE among clinical isolates of enterococci and that the vanB gene predominates in such strains. Susceptibility profiles indicated that linezolid and tigecycline are still effective against these multidrug-resistant pathogens. Pus specimens yielded the highest percentage (53.3%) of isolates from which VRE was obtained, and this finding is novel among studies done in Saudi Arabia., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L. donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays. VL urine samples were incubated with immobilised anti-L. donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification. We identified six L. donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Local health personnel have drawn attention to an apparent increase in incidence and severity of cutaneous leishmaniasis (CL) in Sudan. The objective of this study was to investigate CL burden and surveillance., Methods: Surveillance data were compiled from the KalaCORE programme, Leishmania coordinators in Northern Kordofan and Southern Darfur, and Khartoum Dermatology Hospital. CL lesions were sampled from 14 suspected cases from Northern Kordofan and the Hospital for Tropical Diseases in Omdurman. PCR-restriction fragment length polymorphism analysis and multilocus sequencing were used to characterize the disease agent., Results: All sites reported substantial increases from 2014 to 2016/7, far exceeding World Health Organization case reports for 2014, consistent with a widespread outbreak. Single seasonal peak incidence was observed, except for two peaks in Southern Darfur. In Northern Kordofan, the odds ratio for CL in the 35-44 years age group was 2.6 times higher than in the >45 years age group (p<0.0001); in Southern Darfur, the OR was 2.38 greater in males than females (p<0.0001). Lesions included severe presentations, despite chemotherapy. Leishmania major was identified as the agent., Conclusions: Active surveillance is required to understand the extent of CL in Sudan, as well as training to standardize surveillance, diagnosis, reporting, and quality control. Point-of-care rapid diagnosis would be valuable. Genotyping and phenotyping are required to monitor the emergence of pathogenic strains, drug resistance, outbreaks, and changes in severity., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Visceral leishmaniasis (VL), caused by the Leishmania donovani complex, is a fatal, neglected tropical disease that is targeted for elimination in India, Nepal, and Bangladesh. Improved diagnostic tests are required for early case detection and for monitoring the outcomes of treatments. Previous investigations using Leishmania lysate antigen demonstrated that the immunoglobulin (Ig) G1 response is a potential indicator of a patient's clinical status after chemotherapy., Methods: IgG1 or IgG enzyme-linked immunosorbent assays (ELISAs) with rK39 or lysate antigens and novel IgG1 rK39 rapid diagnostic tests (RDTs) were assessed with Indian VL serum samples from the following clinical groups: paired pre- and postchemotherapy (deemed cured); relapsed; other infectious diseases; and endemic, healthy controls., Results: With paired pre- and post-treatment samples (n = 37 pairs), ELISAs with rK39- and IgG1-specific conjugates gave a far more discriminative decrease in post-treatment antibody responses when compared to IgG (P < .0001). Novel IgG1 rK39 RDTs provided strong evidence for decreased IgG1 responses in patients who had successful treatment (P < .0001). Furthermore, both IgG1 rK39 RDTs (n = 38) and ELISAs showed a highly significant difference in test outcomes between cured patients and those who relapsed (n = 23; P < .0001). RDTs were more sensitive than corresponding ELISAs., Conclusions: We present strong evidence for the use of IgG1 in monitoring treatment outcomes in VL, and the first use of an IgG1-based RDT using the rK39 antigen for the discrimination of post-treatment cure versus relapse in VL. Such an RDT may have a significant role in monitoring patients and in targeted control and elimination of this devastating disease., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a crude lysate antigen (CLA) as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs., Methodology: Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera., Results: Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60-97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive., Conclusion: We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL., Competing Interests: BCBH was and PM is employee of Coris BioConcept and they do not have any share in the company. Sergey Litvinov is an employee of Aptum Biologics Ltd. which is an official partner institution in the Consortium that produced this work. Aptum Biologics Ltd. has and provided to the Consortium the software for epitope prediction, and beyond this has no consultancy, employment or any other forms of financial benefits from this work, nor affiliation with whatever potential intellectual property that may arise from this particular work. All other authors have declared that no competing interests exist.

Background: There is a recognized need for an improved diagnostic test to assess post-chemotherapeutic treatment outcome in visceral leishmaniasis (VL) and to diagnose post kala-azar dermal leishmaniasis (PKDL). We previously demonstrated by ELISA and a prototype novel rapid diagnostic test (RDT), that high anti- Leishmania IgG1 is associated with post-treatment relapse versus cure in VL. Methodology: Here, we further evaluate this novel, low-cost RDT, named VL Sero K-SeT, and ELISA for monitoring IgG1 levels in VL patients after treatment. IgG1 levels against L. donovani lysate were determined. We applied these assays to Indian sera from cured VL at 6 months post treatment as well as to relapse and PKDL patients. Sudanese sera from pre- and post-treatment and relapse were also tested. Results: Of 104 paired Indian sera taken before and after treatment for VL, when deemed clinically cured, 81 (77.9%) were positive by VL Sero K-SeT before treatment; by 6 months, 68 of these 81 (84.0%) had a negative or reduced RDT test line intensity. ELISAs differed in positivity rate between pre- and post-treatment ( p = 0.0162). Twenty eight of 33 (84.8%) Indian samples taken at diagnosis of relapse were RDT positive. A comparison of Indian VL Sero K-SeT data from patients deemed cured and relapsed confirmed that there was a significant difference ( p < 0.0001) in positivity rate for the two groups using this RDT. Ten of 17 (58.8%) Sudanese sera went from positive to negative or decreased VL Sero K-SeT at the end of 11-30 days of treatment. Forty nine of 63 (77.8%) PKDL samples from India were positive by VL Sero K-SeT. Conclusion: We have further shown the relevance of IgG1 in determining clinical status in VL patients. A positive VL Sero K-SeT may also be helpful in supporting diagnosis of PKDL. With further refinement, such as the use of specific antigens, the VL Sero K-SeT and/or IgG1 ELISA may be adjuncts to current VL control programmes.

We describe the case of a 12-year-old boy from Sudan who presented with fever of 1-week duration, headache, cough, and vomiting. A set of diagnostic tests led to the diagnosis of three infectious diseases: visceral leishmaniasis (probable diagnosis based on positive direct agglutination test), enteric fever (blood culture grown with Salmonella Paratyphi), and brucellosis (blood culture grown with Brucella melitensis ). The patient received specific treatment of the three infections and recovered. This case illustrates the occurrence and possible implications of coinfections in patients with persistent fever, including conditions that are hard to diagnose in field settings, such as brucellosis and enteric fever.

Introduction: Cutaneous leishmaniasis (CL) is the most frequent form of leishmaniasis, with 0.7 to 1.2 million cases per year globally. However, the burden of CL is poorly documented in some regions. We carried out this review to synthesize knowledge on the epidemiological burden of CL in sub-Saharan Africa., Methods: We systematically searched PubMed, CABI Global health, Africa Index Medicus databases for publications on CL and its burden. There were no restrictions on language/publication date. Case series with less than ten patients, species identification studies, reviews, non-human, and non-CL focused studies were excluded. Findings were extracted and described. The review was conducted following PRISMA guidelines; the protocol was registered in PROSPERO (42016036272)., Results: From 289 identified records, 54 met eligibility criteria and were included in the synthesis. CL was reported from 13 of the 48 sub-Saharan African countries (3 eastern, nine western and one from southern Africa). More than half of the records (30/54; 56%) were from western Africa, notably Senegal, Burkina Faso and Mali. All studies were observational: 29 were descriptive case series (total 13,257 cases), and 24 followed a cross-sectional design. The majority (78%) of the studies were carried out before the year 2000. Forty-two studies mentioned the parasite species, but was either assumed or attributed on the historical account. Regional differences in clinical manifestations were reported. We found high variability across methodologies, leading to difficulties to compare or combine data. The prevalence in hospital settings among suspected cases ranged between 0.1 and 14.2%. At the community level, CL prevalence varied widely between studies. Outbreaks of thousands of cases occurred in Ethiopia, Ghana, and Sudan. Polymorphism of CL in HIV-infected people is a concern. Key information gaps in CL burden here include population-based CL prevalence/incidence, risk factors, and its socio-economic burden., Conclusion: The evidence on CL epidemiology in sub-Saharan Africa is scanty. The CL frequency and severity are poorly identified. There is a need for population-based studies to define the CL burden better. Endemic countries should consider research and action to improve burden estimation and essential control measures including diagnosis and treatment capacity., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: The authors have declared that no competing interests exist.

In resource-limited settings, the scarcity of skilled personnel and adequate laboratory facilities makes the differential diagnosis of fevers complex [1-5]. Febrile illnesses are diagnosed clinically in most rural centers, and both Rapid Diagnostic Tests (RDTs) and clinical algorithms can be valuable aids to health workers and facilitate therapeutic decisions [6,7]. The persistent fever syndrome targeted by NIDIAG is defined as presence of fever for at least one week. The NIDIAG clinical research consortium focused on potentially severe and treatable infections and therefore targeted the following conditions as differential diagnosis of persistent fever: visceral leishmaniasis (VL), human African trypanosomiasis (HAT), enteric (typhoid and paratyphoid) fever, brucellosis, melioidosis, leptospirosis, malaria, tuberculosis, amoebic liver abscess, relapsing fever, HIV/AIDS, rickettsiosis, and other infectious diseases (e.g., pneumonia). From January 2013 to October 2014, a prospective clinical phase III diagnostic accuracy study was conducted in one site in Cambodia, two sites in Nepal, two sites in Democratic Republic of the Congo (DRC), and one site in Sudan (clinicaltrials.gov no. NCT01766830). The study objectives were to (1) determine the prevalence of the target diseases in patients presenting with persistent fever, (2) assess the predictive value of clinical and first-line laboratory features, and (3) assess the diagnostic accuracy of several RDTs for the diagnosis of the different target conditions., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: The authors have declared that no competing interests exist.

Background: Visceral leishmaniasis (VL), caused by protozoa of the Leishmania donovani complex, is a widespread parasitic disease of great public health importance; without effective chemotherapy symptomatic VL is usually fatal. Distinction of asymptomatic carriage from progressive disease and the prediction of relapse following treatment are hampered by the lack of prognostic biomarkers for use at point of care., Methodology/principal Findings: All IgG subclass and IgG isotype antibody levels were determined using unpaired serum samples from Indian and Sudanese patients with differing clinical status of VL, which included pre-treatment active VL, post-treatment cured, post-treatment relapsed, and post kala-azar dermal leishmaniasis (PKDL), as well as seropositive (DAT and/or rK39) endemic healthy controls (EHCs) and seronegative EHCs. L. donovani antigen-specific IgG1 levels were significantly elevated in relapsed versus cured VL patients (p<0.0001). Using paired Indian VL sera, consistent with the known IgG1 half-life, IgG1 levels had not decreased significantly at day 30 after the start of treatment (p = 0.8304), but were dramatically decreased by 6 months compared to day 0 (p = 0.0032) or day 15 (p<0.0001) after start of treatment. Similarly, Sudanese sera taken soon after treatment did not show a significant change in the IgG1 levels (p = 0.3939). Two prototype lateral flow immunochromatographic rapid diagnostic tests (RDTs) were developed to detect IgG1 levels following VL treatment: more than 80% of the relapsed VL patients were IgG1 positive; at least 80% of the cured VL patients were IgG1 negative (p<0.0001)., Conclusions/significance: Six months after treatment of active VL, elevated levels of specific IgG1 were associated with treatment failure and relapse, whereas no IgG1 or low levels were detected in cured VL patients. A lateral flow RDT was successfully developed to detect anti-Leishmania IgG1 as a potential biomarker of post-chemotherapeutic relapse.

In the diagnosis of leishmaniasis, identification of the causative Leishmania species is relevant for treatment, prognosis, and epidemiology. Three new hsp70-based PCR variants were developed and recently validated on clinical samples from Peru, without the need for culturing. We evaluated their performance on 133 clinical samples (bone marrow, blood, buffy coat, lymph node aspirates, lesion biopsies) from 42 cutaneous and 56 visceral leishmaniasis patients and 35 negative cases, all from Old World countries (Italy, Sudan, Israel, and Tunisia). The 3 new PCRs were significantly more sensitive than those previously described for hsp70, and their respective restriction fragment analyses were more efficient for species identification. In 79% of the parasitologically confirmed positive samples, the species could be identified directly from sample DNA. This evaluation demonstrated that these new tools are globally applicable in different geographical, clinical, and sampling contexts, and they could become the reference method for identification of Leishmania species in clinical specimens., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Visceral leishmaniasis (VL), a widely distributed systemic disease caused by infection with the Leishmania donovani complex (L. donovani and L. infantum), is almost always fatal if symptomatic and untreated. A rapid point-of-care diagnostic test for anti-Leishmania antibodies, the rK39-immunochromatographic test (rK39-ICT), has high sensitivity and specificity in South Asia but is less sensitive in East Africa. One of the underlying reasons may be continent-specific molecular diversity in the rK39 antigen within the L. donovani complex. However, a second reason may be differences in specific IgG anti-Leishmania levels in patients from different geographical regions, either due to variable antigenicity or immunological response., Methodology/principal Findings: We determined IgG titres of Indian and Sudanese VL patients against whole cell lysates of Indian and Sudanese L. donovani strains. Indian VL patients had significantly higher IgG titres against both L. donovani strains compared to Sudanese VL patients (p<0.0001). Mean reciprocal log10 50% end-point titres (1/log10t50) were i) 3.80 and 3.88 for Indian plasma and ii) 2.13 and 2.09 for Sudanese plasma against Indian and Sudanese antigen respectively (p<0.0001). Overall, the Indian VL patients therefore showed a 46.8-61.7 -fold higher mean ELISA titre than the Sudanese VL patients. The higher IgG titres occurred in children (<16 years old) and adults of either sex from India (mean 1/log10t50: 3.60-4.15) versus Sudan (mean 1/log10t50: 1.88-2.54). The greatest difference in IgG responses was between male Indian and Sudanese VL patients of ≥ 16 years old (mean 1/log10t50: 4.15 versus 1.99 = 144-fold (p<0.0001)., Conclusions/significance: Anti-Leishmania IgG responses among VL patients in Sudan were significantly lower than in India; this may be due to chronic malnutrition with Zn(2+) deficiency, or variable antigenicity and capacity to generate IgG responses to Leishmania antigens. Such differential anti-Leishmania IgG levels may contribute to lower sensitivity of the rK39-ICT in East Africa.

Background: Poor access to diagnosis stymies control of visceral leishmaniasis (VL). Antibody-detecting rapid diagnostic tests (RDTs) can be performed in peripheral health settings. However, there are many brands available and published reports of variable accuracy., Methods: Commercial VL RDTs containing bound rK39 or rKE16 antigen were evaluated using archived human sera from confirmed VL cases (n = 750) and endemic non-VL controls (n = 754) in the Indian subcontinent (ISC), Brazil, and East Africa to assess sensitivity and specificity with 95% confidence intervals. A subset of RDTs were also evaluated after 60 days' heat incubation (37°C, 45°C). Interlot and interobserver variability was assessed., Results: All test brands performed well against ISC panels (sensitivity range, 92.8%-100%; specificity range, 96%-100%); however, sensitivity was lower against Brazil and East African panels (61.5%-91% and 36.8%-87.2%, respectively). Specificity was consistently > 95% in Brazil and ranged between 90.8% and 98% in East Africa. Performance of some products was adversely affected by high temperatures. Agreement between lots and readers was good to excellent (κ > 0.73-0.99)., Conclusions: Diagnostic accuracy of VL RDTs varies between the major endemic regions. Many tests performed well and showed good heat stability in the ISC; however, reduced sensitivity against Brazilian and East African panels suggests that in these regions, used alone, several RDTs are inadequate for excluding a VL diagnosis. More research is needed to assess ease of use and to compare performance using whole blood instead of serum and in patients coinfected with human immunodeficiency virus.

For the epidemiological monitoring and clinical case management of leishmaniasis, determination of the causative Leishmania species gains importance. Current assays for the Old World often suffer from drawbacks in terms of validation on a geographically representative sample set and the ability to recognize all species complexes. We want to contribute to standardized species typing for Old World leishmaniasis. We determined the ribosomal DNA internal transcribed spacer 1 sequence of 24 strains or isolates, and validated four species-specific polymerase chain reactions (PCRs) amplifying this target. They discriminate L. aethiopica, L. tropica, L. major, and the L. donovani complex, use the same cycling conditions, and include an internal amplification control. Our PCRs amplify 0.1 pg of Leishmania DNA, while being 100% specific for species identification on an extensive panel of geographically representative strains and isolates. Similar results were obtained in an endemic reference laboratory in Kenya. Species could also be identified in clinical specimens. The presented PCRs require only agarose gel detection, and have several other advantages over many existing assays. We outline potential problems, suggest concrete solutions for transferring the technique to other settings, and deliver the proof-of-principle for analyzing clinical samples.

Background: Visceral leishmaniasis (VL) is diagnosed by microscopic confirmation of the parasite in bone marrow, spleen or lymph node aspirates. These procedures are unsuitable for rapid diagnosis of VL in field settings. The development of rK39-based rapid diagnostic tests (RDT) revolutionized diagnosis of VL by offering high sensitivity and specificity in detecting disease in the Indian subcontinent; however, these tests have been less reliable in the African subcontinent (sensitivity range of 75-85%, specificity of 70-92%). We have addressed limitations of the rK39 with a new synthetic polyprotein, rK28, followed by development and evaluation of two new rK28-based RDT prototype platforms., Methodology/principal Findings: Evaluation of 62 VL-confirmed sera from Sudan provided sensitivities of 96.8% and 93.6% (95% CI = K28: 88.83-99.61%; K39: 84.30-98.21%) and specificities of 96.2% and 92.4% (95% CI = K28: 90.53-98.95%; K39: 85.54-96.65%) for rK28 and rK39, respectively. Of greater interest was the observation that individual VL sera with low rK39 reactivity often had much higher rK28 reactivity. This characteristic of the fusion protein was exploited in the development of rK28 rapid tests, which may prove to be crucial in detecting VL among patients with low rK39 antibody levels. Evaluation of two prototype lateral flow-based rK28 rapid tests on 53 VL patients in Sudan and 73 VL patients in Bangladesh provided promisingly high sensitivities (95.9% [95% CI = 88.46-99.1 in Sudan and 98.1% [95% CI = 89.93-99.95%] in Bangladesh) compared to the rK39 RDT (sensitivities of 86.3% [95% CI = 76.25-93.23%] in Sudan and 88.7% [95% CI = 76.97-95.73%] in Bangladesh)., Conclusions/significance: Our study compares the diagnostic accuracy of rK39 and rK28 in detecting active VL cases and our findings indicate that rK28 polyprotein has great potential as a serodiagnostic tool. A new rK28-based RDT will prove to be a valuable asset in simplifying VL disease confirmation at the point-of-care.

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.

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.

Objective: To estimate the sensitivity and specificity of the OligoC-TesT and nucleic acid sequence-based amplification coupled to oligochromatography (NASBA-OC) for molecular detection of Leishmania in blood from patients with confirmed visceral leishmaniasis (VL) and healthy endemic controls from Kenya., Methods: Blood specimens of 84 patients with confirmed VL and 98 endemic healthy controls from Baringo district in Kenya were submitted to both assays., Results: The Leishmania OligoC-TesT showed a sensitivity of 96.4% (95% confidence interval [CI]: 90-98.8%) and a specificity of 88.8% (95% CI: 81-93.6%), while the sensitivity and specificity of the NASBA-OC were 79.8% (95% CI: 67-87%) and 100% (95% CI: 96.3-100%), respectively., Conclusion: Our findings indicate high sensitivity of the Leishmania OligoC-TesT on blood while the NASBA-OC is a better marker for active disease.

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.

Background: Definite diagnosis of Leishmania infections is based on demonstration of the parasite by microscopic analysis of tissue biopsy specimens or aspirate samples. However, microscopy generally shows low sensitivity and requires invasive sampling., Methods: We describe here the development of a simple and rapid test for the detection of polymerase chain reaction-amplified Leishmania DNA. A phase 1 evaluation of the text was conducted in clinical samples from 60 nonendemic and 45 endemic control subjects and from 44 patients with confirmed cutaneous leishmaniasis (CL), 12 with mucocutaneous leishmaniasis (MCL), and 43 with visceral leishmaniasis (VL) from Peru, Kenya, and Sudan., Results: The lower detection limits of the assay are 10 fg of Leishmania DNA and 1 parasite in 180 microL of blood. The specificity was 98.3% (95% confidence interval [CI], 91.1%-99.7%) and 95.6% (95% CI, 85.2%-98.8%) for nonendemic and endemic control samples, respectively, and the sensitivity was 93.2% (95% CI, 81.8%-97.7%), 91.7% (95% CI, 64.6%-98.5%), and 86% (95% CI, 72.7%-93.4%) for lesions from patients with CL or MCL and blood from patients with VL, respectively., Conclusions: The Leishmania OligoC-TesT showed high specificity and sensitivity in clinical samples and was able to detect the parasite in samples obtained by less invasive means, such as blood, lymph, and lesion scrapings. The assay is a promising new tool for simplified and standardized molecular detection of Leishmania parasites.

Three diagnostic tests for visceral leishmaniasis (VL), the freeze-dried direct agglutination test (FD-DAT), the rK39 dipstick and a urine latex antigen test (KAtex), were evaluated for use in primary care in East Africa and the Indian subcontinent. Clinical suspects were prospectively recruited and tissue, blood and urine samples were taken. Direct microscopic examination of tissue smear, and FD-DAT, rK39 and KAtex were performed. Sensitivity and specificity with 95% credible intervals were estimated using Bayesian latent class analysis. On the Indian subcontinent both the FD-DAT and the rK39 strip test exceeded the 95% sensitivity and 90% specificity target, but not so in East Africa. Sensitivity of the FD-DAT was high in Ethiopia and Kenya but lower in Sudan, while its specificity was below 90% in Kenya. Sensitivity of the rK39 was below 80% in the three countries, and its specificity was only 70% in Ethiopia. KAtex showed moderate to very low sensitivity in all countries. FD-DAT and rK39 can be recommended for clinical practice on the Indian subcontinent. In East Africa, their clinical use should be carefully monitored. More work is needed to improve existing formats, and to develop better VL diagnostics.

Kala azar (KA) is a lethal disease caused by Leishmania parasites (Leishmania donovani s.l.) that multiply in large numbers in deep organs such as spleen and liver. The host immunological response to these organisms is complex and experimental studies in animals have detected a large number of genetic loci involved in the control of infection and disease. We report here on a study in a human population of Sudan carried out during an outbreak of KA. The following conclusions are presented: (1) environmental factors that could have affected the distribution of the insect vector, influenced progression of KA in the initial phase of the epidemics - but they became less important later at the peak of transmission, probably after infected phlebotomies had spread to all parts of the village -; (2) Leishmania population during the epidemics was heterogeneous, suggesting a possible parasite evolution during the outbreak; (3) the incidence of KA varied markedly among age groups, families and ethnic groups. Susceptibility to KA was shown to depend on a locus on chromosomes 22q12 and on NRAMP1 on chromosome 2q35; the data also suggested a third locus in the region 2q23-q24. Overall, this study indicates complex interactions between host genes and environment in the spreading of KA in that population. It is also suspected that the large parasite diversity observed in the outbreak has contributed to disease spreading across host genetic barriers.

Kala-azar (KA) is a life-threatening protozoal disease caused by Leishmania parasites (L. donovani, L. chagasi, and L. infantum). The disease, which is also called "visceral leishmaniasis," is prevalent in Africa, South America, Asia, and the Mediterranean basin. Epidemics occur periodically, killing a large number of infected individuals. Factors determining whether a patient remains asymptomatic or develops KA are still largely unknown. In a previous study that was performed during an outbreak of KA in a village on the Ethiopian-Sudanese border, we showed that KA was more frequent in certain families and ethnic groups, thereby suggesting that host genetic factors play an important role in the development of the disease. Here, we report the results of a genomewide linkage study performed on 63 Sudanese families selected from the most affected ethnic group and including 169 children with KA. Significant linkage (LOD score 3.50 [P=3x10-5] in all patients; LOD score 3.90 [P=10-5] in patients who were affected early in the outbreak) was obtained with markers on chromosome 22q12. These results are the first evidence of a major genetic effect on the development of human KA. They may lead to identification of genes critical in the pathogenesis of this disease and to new therapeutic interventions against this parasite, which is developing resistance to available drugs.

This paper describes a new latex agglutination test ('KATEX') for the detection of leishmanial antigen in the urine of patients with visceral leishmaniasis. In preliminary laboratory trials, using urine collected from well-defined cases and controls from Brazil, Yemen and Nepal, the test had 100% specificity and a sensitivity between 68 and 100%. When used in a time-course experiment in cotton rats infected with Leishmania donovani, the test became positive 1 week after inoculation and antigen levels in urine declined rapidly after chemotherapy (the test was negative before the end of the course of treatment). Finally, in an integrated study performed in Sudan, KATEX was compared to microscopy and four different serological tests in a group of 73 patients having presented with clinical manifestations suggestive of visceral leishmaniasis. Compared to microscopy, KATEX performed better than any single serological test in predicting positivity and a particularly good result was obtained by combining KATEX and the direct agglutination test (DAT).

Background: Substantial uncertainty surrounds the specificity of the Direct Agglutination Test (DAT) for visceral leishmaniasis (VL) in clinical suspects, since no good gold standard exists for unequivocally identifying diseased subjects. We explored the Latent Class Analysis (LCA) modelling technique to circumvent this problem., Patients and Methods: Data on 149 clinical suspects recruited in 1993-96 during a multicentre study in Sudan were re-examined. Clinical data, lymph node and bone marrow aspirate and DAT results were available. IFAT was performed in 1997 on stored filter paper blood of 80 individuals. Classical Validity Analysis (CVA) in a 2 x 2 contingency table with parasitology as a gold standard was compared with the parameter estimates produced by the best fitting LCA model., Results: The sensitivity estimates of DAT produced by CVA (98% (89%-100%)) were almost exactly reproduced by LCA. The specificity estimates by LCA were substantially higher than those obtained in CVA. Specificity of DAT depended, however, on whether the subject was treated for VL before. In subjects without prior treatment, CVA estimated DAT specificity at 68% (56%-79%), whereas LCA estimated it at 85% (63%-100%)., Conclusion: LCA modelling proved a useful tool, as it gave consistent estimates of test characteristics and allowed for control of confounding factors and interaction effects. Since VL is a life-threatening disease for which expensive but effective and safe treatment exists, a clinical suspect in an endemic area should be treated on the basis of a positive DAT result.

The validity of the direct agglutination test (DAT) for visceral leishmaniasis (VL) was studied with a standardized field kit on 148 clinically suspected persons and 176 healthy controls recruited between 1993 and 1994 from an endemic area in Gedaref State, Sudan. A sensitivity of 95.9% and a specificity of 99.4% were found at a 1: 8,000 cut-off titer when parasitologically confirmed cases were compared with healthy controls. While corroborating previously reported sensitivity and specificity estimates of this serodiagnostic test, this study examined the bias generated by commonly used test validation procedures. The fundamental methodologic problem in VL test validation is the absence of a reliable gold standard. Moreover, any operational guideline on DAT use has to consider the critical dependency of the predictive values of the test on VL prevalence rates. The DAT diagnostic cut-off titer depends upon many external factors, among which the prevalence of disease in the area and the case mix seem the most important.

Objective: To evaluate the repeatability and reproducibility of the serological direct agglutination test (DAT) for visceral leishmaniasis (VL) with aqueous antigen in a multi-centre study in VL-endemic areas in Sudan, Kenya and Nepal., Methods: Repeatability within each centre and reproducibility between the centres' results and an external reference laboratory (Belgium) was assessed on 1596 triplicate plain blood samples collected on filter paper., Results: High kappa values (range 0.86-0.97) indicated excellent DAT repeatability within the centres. The means of the titre differences between the reference laboratory and the centres in Sudan, Kenya and Nepal (2.3, 2.4 and 1.1, respectively, all significantly different from 0) showed weak reproducibility across centres. 95% of the titre differences between the reference laboratory and the respective centres were accounted for by large intervals: 0.6-9 fold titre variation for Sudan, 0.7-8 fold for Kenya and 0.26-4 fold for Nepal., Conclusion: High repeatability of DAT confirms its potential, but reproducibility problems remain an obstacle to its routine use in the field. Reproducibility was hindered by alteration of the antigen through temperature and shaking, especially in Kenya and Sudan, and by nonstandardization of the test reading. DAT handling procedures and antigen quality must be carefully standardized and monitored when introducing this test into routine practice.

Although polyomavirus JC (JCV) is the proven pathogen of progressive multifocal leukoencephalopathy, the fatal demyelinating disease, this virus is ubiquitous as a usually harmless symbiote among human beings. JCV propagates in the adult kidney and excretes its progeny in urine, from which JCV DNA can readily be recovered. The main mode of transmission of JCV is from parents to children through long cohabitation. In this study, we collected a substantial number of urine samples from native inhabitants of 34 countries in Europe, Africa, and Asia. A 610-bp segment of JCV DNA was amplified from each urine sample, and its DNA sequence was determined. A worldwide phylogenetic tree subsequently constructed revealed the presence of nine subtypes including minor ones. Five subtypes (EU, Af2, B1, SC, and CY) occupied rather large territories that overlapped with each other at their boundaries. The entire Europe, northern Africa, and western Asia were the domain of EU, whereas the domain of Af2 included nearly all of Africa and southwestern Asia all the way to the northeastern edge of India. Partially overlapping domains in Asia were occupied by subtypes B1, SC, and CY. Of particular interest was the recovery of JCV subtypes in a pocket or pockets that were separated by great geographic distances from the main domains of those subtypes. Certain of these pockets can readily be explained by recent migrations of human populations carrying these subtypes. Overall, it appears that JCV genotyping promises to reveal previously unknown human migration routes: ancient as well as recent.

Trypsin treatment of Leishmania promastigote antigen has proved to be indispensible in the direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). In the present study four antigen batches were prepared with pronase (400 micrograms/ml), lipase (0.45% [wt/vol]), pancreatin (0.3% [wt/vol]), or 2-mercaptoethanol (2-ME) (1.2% [vol/vol]) at a ratio of 20:1 versus promastigote packed cell volume or a density of 10(8)/ml. Batches prepared in this way performed satisfactorily when compared with the performance of the initial trypsinated antigen. Even higher was the sensitivity and specificity of the 2-ME-processed antigen, scoring a minimum DAT titer of 1:102,400 in the VL and CVL group and a maximum of 1:400 in the negative control group. Corresponding titers ranging from 1:6,400 to 1:12,800 and 1:800 to 1:1,600 were obtained with the antigen variants processed with pronase, lipase, pancreatin, or trypsin. By combining the use of indigenous Leishmania donovani subspecies from Sudan, Bangladesh, or Morocco and incorporating 2-ME instead of trypsin in the antigen processing step, a threefold increase in titer was attained in sera from the respective areas where VL is endemic. 2-ME-processed antigen suspensions maintained stability at 4 degrees C for up to 9 months, as evidenced by the absence of autoagglutination and the reproducibility of DAT readings with standard sera. The specificity of DAT was further improved by supplementation of the sample diluent with 0.03 M urea and incubation of the test plates at 37 degrees C for 1 h. Titers ranging from 1:200 to 1:12,800 in the sera of patients and laboratory animals infected with various trypanosoma species were significantly reduce (/=1:51,200) against 2-ME-processed antigen, despite the incorporation of urea into the DAT.