Etiological spectrum of persistent fever in the tropics and predictors of ubiquitous infections: a prospective four-country study with pooled analysis.

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-itTM Typhoid IgM Lateral Flow Assay, and SD Bioline Salmonella typhi IgG/IgM for Salmonella Typhi infection, and Test-itTM 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.

Detection of Immunoglobulin G1 Against rK39 Improves Monitoring of Treatment Outcomes in Visceral Leishmaniasis.

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.

Exome Sequencing Identifies Two Variants of the Alkylglycerol Monooxygenase Gene as a Cause of Relapses in Visceral Leishmaniasis in Children, in Sudan.

Background: Visceral leishmaniasis (kala-azar, KA) is the most severe form of leishmaniasis, characterized by fever, weight loss, hepatosplenomegaly, and lymphadenopathy. During an outbreak of KA in Babar El Fugara (Sudan), 5.7% of cured patients displayed relapses, with familial clustering in half the cases. Methods: We performed whole-exome sequencing on 10 relapsing individuals and 11 controls from 5 nuclear families. Results: Rare homozygous and compound-heterozygous nonsense (c.1213C > T, rs139309795, p.Arg405*) and missense (c.701A > G, rs143439626, p.Lys234Arg) mutations of the alkylglycerol monooxygenase (AGMO) gene were associated with KA relapse in 3 families. Sequencing in additional family members confirmed the segregation of these mutations with relapse and revealed an autosomal dominant mode of transmission. These mutations were detected heterozygous in 2 subjects among 100 unrelated individuals with KA who never relapsed after cure, suggesting incomplete penetrance of AGMO deficiency. AGMO is expressed in hematopoietic cells, and is strongly expressed in the liver. AGMO modulates PAF production by mouse macrophages, suggesting that it may act through the PAF/PAF receptor pathway previously shown to have anti-Leishmania activity. Conclusions: This is the first demonstration that relapses after a first episode of KA are due to differences in human genetic susceptibility and not to modifications of parasite pathogenicity.

HLA alleles and haplotypes in Sudanese population and their relationship with Mediterraneans.

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.

A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis.

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.

Linear and conformational determinants of visceral leishmaniasis diagnostic antigens rK28 and rK39.

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.

Antibiotic use prior to seeking medical care in patients with persistent fever: a cross-sectional study in four low- and middle-income countries.

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.

Sensitivity and specificity of the Leishmania OligoC-TesT and NASBA-oligochromatography for diagnosis of visceral leishmaniasis in Kenya.

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.

Accordance and concordance of PCR and NASBA followed by oligochromatography for the molecular diagnosis of Trypanosoma brucei and Leishmania.

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.

Isolation and characterisation of Leishmania donovani protein antigens from urine of visceral leishmaniasis patients.

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.

Epidemiological and molecular investigation of resurgent cutaneous leishmaniasis in Sudan.

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.

Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis.

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.

Uncharted territory of the epidemiological burden of cutaneous leishmaniasis in sub-Saharan Africa-A systematic review.

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.

Case Report: Visceral Leishmaniasis with Salmonella Paratyphi and Brucella melitensis Coinfection as a Cause of Persistent Fever in a Patient from Sudan.

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.

Visceral Leishmaniasis IgG1 Rapid Monitoring of Cure vs. Relapse, and Potential for Diagnosis of Post Kala-Azar Dermal Leishmaniasis.

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.

Genes and environment in susceptibility to visceral leishmaniasis.

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.

Diagnosis of Persistent Fever in the Tropics: Set of Standard Operating Procedures Used in the NIDIAG Febrile Syndrome Study.

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.

Epidemiology of visceral leishmaniasis in Atbara River area, eastern Sudan: the outbreak of Barbar El Fugara village (1996-1997).

An outbreak of visceral leishmaniasis (VL) started in 1995 in the Atbara River area in eastern Sudan. This article reports on this outbreak and on the clinical and immunological studies that were carried out in a village, with the highest incidence of VL cases, from 1996 to 1997. A significant increase in VL incidence was recorded in a dozen villages in this area; one village, Barbar El Fugara accounted for half of the total number of cases recorded at the regional hospital. A total of 152 VL and 61 post kala-azar dermal lesion (PKDL) cases were diagnosed and treated in Barbar. Household (n = 671) and school (n = 276) surveys were performed using the leishmanin skin test (LST) and the direct agglutination test (DAT). LST positivity was 23.1 and 15.7%, whereas DAT positivity was 8.9 and 26.4% in both surveys, respectively. No gender differences were observed in either test. Unlike DAT, LST positivity was predominant in the higher age groups that also exhibited lower prevalence of VL. Few individuals were positive by both tests (1.3%, 5.2%) while the majority (68.8%, 64.8%) had no evidence of acquired immune response, suggesting either a role of innate immunity in preventing parasite establishment or, unexpectedly, lack of exposure to Leishmania. Subclinical parasitism was also demonstrated, as evidence of both acquired humoral and cellular immune responses was observed in individuals with no past history of the disease. The wide spectrum of L. donovani/human interactions may be explained by differential exposure to environmental risk factors, parasite strain polymorphisms or host genetic makeup.

The interplay between environmental and host factors during an outbreak of visceral leishmaniasis in eastern Sudan.

Parasitic diseases, including human visceral leishmaniasis, are multifactorial. Factors that are expected to play an important role in the parasite-human interaction are exposure, parasite "virulence" and host resistance factors. In populations exposed to Leishmania donovani most subjects do not allow the parasites to establish themselves or remain asymptomatic. Some individuals, however, fail to control parasite expansion and dissemination and develop a visceral disease. We report here the results of a longitudinal survey whose aims were to identify risk factors underlying visceral leishmaniasis (VL) susceptibility during an outbreak that occurred in a Sudanese village between 1995 and 1999. Most of the 660 subjects (90%) living in the central district were exposed to Leishmania and 20.9% (n = 138), mostly teenagers, developed VL. VL cases increased markedly in adults late in the outbreak, suggesting some changes in adult resistance status or in Leishmania "virulence" during the epidemic. Age and ethnic origin of the patients were the most important critical risk factors to account for the distribution of the VL cases that were recorded during the whole epidemic. This and the high frequency of VL in certain families suggest that host genetic factors played an important role in shaping the outbreak in this village. However, environmental factors (the presence of cows and neems in the households) that increase/decrease exposure to the parasite had significant effects on the distribution of VL cases in the village in the first phase of the outbreak.