Detection Of Visceral Leishmaniasis Research Articles

Demographic and Clinical Features of Visceral Leishmaniasis in Chad: A Prospective Cross-Sectional Survey.

Visceral leishmaniasis (VL), caused by the protozoan Leishmania donovani complex, is endemic in many parts of the world. Little known in Chad, VL has been recently documented from previously nonendemic areas. We report an epidemiological investigation of VL in the Léré district hospital in southwestern Chad. After informed consent, 40 VL patients were enrolled in the study. Diagnosis made using the formalin serological test was confirmed by polymerase chain reaction on blood samples. Clinical parameters were obtained from the physician or nurse caregiver, and from patients. Of a total of 40 serology positive patients, L. donovani DNA was found in 33 (82.5%), with 55% being male patients. The most affected age groups were 15-29 (47.5%) and 0-14 (32.5%) years. Fever, weight loss, and pallor were frequent symptoms. Notably, splenomegaly and hepatomegaly were uncommon clinical signs. Common comorbidities included malaria (25%) and hepatitis B (15%), followed by gastric ulcer (10%) and tuberculosis (7.5%). These comorbidities were concurrent with VL and were diagnosed microscopically in blood and serum for malaria and tuberculosis, respectively, and by the rapid diagnostic test using serum for hepatitis B and gastric ulcer. Thirty-five percent of cases were treated with meglumine antimoniate, and three patients (7.5%), all with comorbidities, died. Sixty percent of patients lived close to the main town. Our data demonstrate that VL is endemic in the health district of Léré. Improving health education regarding L. donovani infection in endemic areas of Chad and providing training of health workers on early detection and management of VL are needed to help save lives.

Comparison of SensPERT® Leishmania rapid test with two other immunochromatographic tests for the diagnosis of canine visceral leishmaniasis in Brazil.

In urban environments, dogs serve as the primary reservoir for visceral leishmaniasis (VL). Rapidly diagnosing canine VL through tests enables early treatment and a favorable prognosis. This study aimed to assess the diagnostic performance of the SensPERT® Leishmania test kit (Dechra®), Alere® Leishmaniasis Ac test kit, and the rapid test dual path platform (TR-DPP®) Bio-Manguinhos in detecting VL. 30 serum samples from reactive VL dogs and 30 serum samples from healthy dogs were employed for assessing the sensitivity and specificity variation between SensPERT® Leishmania test kit, Alere® Leishmaniasis Ac test kit, and rapid test dual platform - TR-DPP®. The SensPERT® Leishmania test outperformed Alere® and TR-DPP® in terms of sensitivity, specificity, positive and negative predictive values and demonstrated near-perfect concordance with Alere® and substantial concurrence with TR-DPP®. The SensPERT® Leishmania rapid test proved to be a promising test in the detection of VL in dogs.

Real Time PCR-based diagnosis of human visceral leishmaniasis using urine samples.

Diagnosis of visceral leishmaniasis (VL) through the detection of its causative agents namely Leishmania donovani and L. infantum is traditionally based on immunochromatographic tests, microscopy of bone marrow, spleen aspirates, liver or lymph node and differential diagnosis. While the first process has low specificity, the later one carries the risk of fatal hemorrhage. Over the last decade, multiple Polymerase Chain Reaction (PCR) based diagnosis has been developed using blood and urine sample with a varying degree of sensitivity and specificity, an issue worth improving for precision diagnosis. Earlier, we reported a PCR-based diagnosis of L. donovani in peripheral blood using a novel set of PCR primers with absolute specificity. Using the same set of primers and PCR conditions, here we describe diagnosis of L. donovani from urine, for a non-invasive, rapid and safe diagnosis. Diagnosis of VL was carried out using urine samples collected from clinically diagnosed VL patients (n = 23) of Bangladesh in Real Time PCR. Test results were validated by comparing blood samples from the same set of patients. Sensitivity and specificity of this diagnosis was analyzed using retrospective bone marrow samples, collected earlier from confirmed VL patients (n = 19). The method showed 100% sensitivity in detecting L. donovani in urine and corresponding blood and retrospective bone marrow samples, as well as 100% specificity in control groups. A Real Time PCR-based molecular detection system using urine sample is hereafter presented what could be a, non-invasive approach for VL detection with precision and perfection.

Serological studies on rK39 negative Visceral Leishmaniasis in an endemic focus of Leishmania donovani induced Cutaneous Leishmaniasis

Sri Lanka reports a focus of L. donovani induced cutaneous leishmaniasis (CL). Our more recent parasite and clinical studies and historical evidence point towards long term existence of Leishmania in the country, indicating a possible evolution leading to antigenic heterogenicity as well. In-house enzyme-linked immunosorbent assay (ELISA) that was developed during phase 1 study indicated >80% sero-positivity in local CL, while visceral leishmaniasis (VL) remained very rare with majority being negative when tested with rK39 assay. A novel serological tool was developed and sero-positivity of VL was assessed for the first time. The assay showed 100.0% sensitivity and 98.3% specificity for detection of VL. Samples were showed less positivity with established direct agglutination test (DAT) and rK39 strip test. The assay was less expensive than that of established rapid diagnostic tests (RDTs), culture and PCR assays. This assay may be useful in diagnosing clinical VL infections, detection of light microscopy (LM) negative patients, tracking post treatment stages, field screening of asymptomatic cases and in further serological studies.

Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended.

Genetic diversity analysis of Chinese Leishmania isolates and development of L. donovani complex-specific markers by RAPD

BackgroundLeishmaniasis is one of the most neglected tropical diseases in the world and remains endemic in some underdeveloped regions, including western China. The phylogeny and classification of Chinese Leishmania has not been completely clarified to date, especially within the Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers have been performed. More analytic methods and data are still needed. Random amplified polymorphic DNA (RAPD) technology can sensitively identify slight intraspecific differences, and it is a powerful tool to seek species-specific markers. This work attempted to identify Chinese Leishmania isolates from diverse geographic regions at the genomic level. Meanwhile, specific markers of the L. donovani complex were also developed by RAPD.MethodsRAPD was applied to 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into a data matrix, based on which genetic similarity was calculated, and a UPGMA dendrogram was constructed to analyse the genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of the L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified region (SCAR) markers, which were validated preliminarily in 17 available Leishmania strains in this study and analysed by bioinformatics.ResultsThe cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, the strains of the L. donovani complex clearly divided into two clades, and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of the L. donovani complex, i.e., 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on 17 available Leishmania strains in this study. Through bioinformatic analyses, Marker 1-AD17 may have more specificity for PCR detection of VL, and Marker 3-O13 has the potential to encode a protein.ConclusionsThe RAPD results verified that the undescribed Leishmania species causing visceral leishmaniasis (VL) in China was a unique clade distinguished from L. donovani and revealed that there was genetic differentiation among Chinese L. donovani. The identification of L. donovani-specific markers may help to provide a foundation for future research attempting to develop new specific diagnostic markers of VL and identify specific gene functions.

Basic knowledge about visceral leishmaniasis before and after educational intervention among primary health care professionals in Midwestern Brazil.

ABSTRACTHealth education and training of primary health care (PHC) professionals are highly recommended to reduce the occurrence and lethality of visceral leishmaniasis (VL). This study assessed the impact of an educational intervention on the basic knowledge about visceral leishmaniasis (VL) among PHC professionals from the Brazilian municipality of Rondonopolis, an important endemic area for VL. Responses provided by physicians, nurses, nursing technicians and community health agents from 12 PHC facilities were recorded through the application of self-completed and semi-structured questionnaires before (n=92) and after (n=64) an in-person health training course covering various aspects of VL. Closed- and open-ended responses were compared by the chi-square test and analyses of word clouds, respectively. The proportion of professionals aware of the correct etiological agent (p<0.001) and transmission route (p<0.001) of VL increased post-intervention. In addition, they increased their ability to recognize fever (p<0.001), weakness (p<0.001), weight loss (p<0.001), pallor (p<0.001) and abdominal distention (p=0.013) as clinical manifestations of human VL, and weakness (p<0.001), alopecia (p<0.001) and weight loss (p=0.019) as signs of canine VL. Analyses of word clouds suggested that the participants became more aware of the role of dogs in VL transmission and the role of environmental management in the prevention of VL. In conclusion, the intervention positively impacted the baseline knowledge concerning VL among the professionals. This can support the planning of educational activities for the PHC team regarding early case detection, prevention and control of VL in endemic areas.

Serodiagnosis of Visceral Leishmaniasis in Northeastern Italy: Evaluation of Seven Serological Tests.

This study compares the performance of seven assays, including two ELISA (Leishmania ELISA IgG + IgM, Vircell Microbiologists; Leishmania infantum IgG ELISA, NovaTec), three rK39-based immunochromatographic tests (rK39-ICTs) (Leishmania Dipstick Rapydtest, Apacor; On Site Leishmania IgG/IgM Combo Rapid Test, CTK Biotech; LEISHMANIA Strip quick Test, Cypress Diagnostic), one indirect immunofluorescent antibody test (IFAT) (Leishmania-Spot IF, BioMérieux), and one western blot (WB) (Leishmania WESTERN BLOT IgG, LDBio Diagnostics) for serodiagnosis of visceral leishmaniasis (VL). Serum samples from 27 VL patients living in northeastern Italy were analyzed, as well as the serum samples from 50 individuals in whom VL diagnosis was excluded. The WB and the IFAT had 96% sensitivity, followed by the ELISA (63% and 74%, respectively). The rK39-ICT exhibited the worst performance among the serological tests, with sensitivities ranging from 52% to 70%. By combining selected ELISA/ICT, the sensitivity of VL detection reached 89%. IFAT and WB outperformed ELISA and rK39-ICT by possessing optimal sensitivity, but their high cost and complexity of execution would not allow their employment as screening tests. In conclusion, the combination of easy-to-perform tests, such as ICT and ELISA, could improve sensitivity in the serodiagnosis of Mediterranean VL.

Modifications in a Reference Freeze-Dried Direct Agglutination Test to Improve Visceral Leishmaniasis Detection.

Currently, a significantly lower temperature (35°C) than initially established (56°C) is indicated as the maximum temperature storage for the commercial reference visceral leishmaniasis (VL) freeze-dried direct agglutination test (FD-DAT). Despite an approximately 50% loss in the number of promastigotes in an FD-DAT batch that expired 7 years earlier, the promastigotes maintained a similar morphology to the equivalent valid batch implying most likely that auto-agglutination, rather than aging, is the main reason for expiry. The substitution of normal saline which was initially recommended for reconstitution, by citrate-saline/formaldehyde (CSF) as an anti-clumping/preservative agent resulted in restoration of validity comparable with that of the freeze-dried original or the liquid direct agglutination test (LQ-DAT) version (Friedman ANOVA test = 1.0588; P = 0.5890). Following a similar reconstitution procedure as for the 7-year expired antigen, using significantly lower promastigote concentration (1.4 × 107/mL) than in the non-expired (9.0 × 107/mL), good reliability for VL detection and stability at 4°C (> 12 months) were achieved. In comparison with the original version using normal saline ($32.0/vial), the cost-effectiveness of the FD-DAT was appreciably improved by the CSF incorporation and lowering of promastigote concentration per unit suspension medium ($12.8/vial). With diagnostic reliability comparable with the full-out titration used, FD-DAT procedure based on single sample dilution at the VL cutoff (1:3,200) permitted the use of significantly smaller antigen volumes (0.1 mL vs. > 1.5 mL), therefore contributing to a further reduction in the application cost. The successful replacement of β-mercaptoethanol (β-ME) by urea (T = 21.00; P = 0.0868) provided the required safety for the test procedure similar to the widely applied LQ-DAT.

Molecular Characterization of Visceral Leishmaniasis in Asymptomatic Dogs in North Khorasan, Northeastern Iran

Background: Leishmania infantum parasites are the main causative agents of visceral leishmaniasis that threaten a wide range of humans and canines in Iran. Objectives: Our aim was to survey Leishmania parasite species and simultaneous comparison of canine organs in endemic areas for the diagnosis of visceral leishmaniasis using the ITS-rDNA gene, sequencing, and phylogenetic analysis. Methods: In this study, sampling was done with vacuum tubes containing EDTA from blood and sterile swabs from the snout and conjunctiva of asymptomatic sheepdogs (n = 37) using a non-invasive method in north Khorasan, northeastern Iran, from 28 July to 4 August 2018. The DNA of collected samples was extracted, amplified, and sequenced by targeting the ITS-rDNA gene. To demonstrate the taxonomic status of Leishmania spp., sequences were subjected to phylogenetic analysis based on the maximum likelihood method. Results: We obtained 37 samples from asymptomatic dogs of which, 10 dogs were definitely diagnosed with L. infantum and one dog infected with L. tropica. The blood (n = 8) and right conjunctiva (n = 6) samples were the most infected samples. The highest number of infections in dogs was in the age group of 5 - 10 years indicating that this group is more sensitive to visceral leishmaniasis in this region. Conclusions: The current findings indicate that non-invasive sampling and molecular methods are reliable and suitable in the detection of visceral leishmaniasis. This is the first report of the visceral involvement of a shepherd dog with L. tropica in northeastern Iran. The remarkable occurrence of visceral leishmaniasis (29.7%) in asymptomatic sheepdogs reflects a health alert to conduct the surveillance and monitoring of susceptible individuals/reservoirs in the region.

Are We Now Well Prepared for Another Major Visceral Leishmaniasis Epidemic in Sudan?

To minimize the chance for future visceral leishmaniasis (VL) epidemics such as the 1988–1991 epidemic in Sudan, several VL detection tools have been introduced. There are many VL diagnostics with excellent sensitivities, specificities, and ease of use reported. However, additional test characteristics should be considered for use in the detection of future VL epidemics. The potential for local production or uninterrupted availability, low production and application costs, and stability at ≥45°C are of the utmost importance. Of the antibody-, antigen-, or DNA-based methods introduced, only a liquid direct agglutination test (LQ-DAT) remains in routine use. The LQ-DAT test may be the ideal diagnostic for detection of VL epidemics due to its low cost ($0.50/patient), stability under frequent and long-duration electric failures, and high level of reproducibility. The improved reliability for VL detection achieved locally through incorporating autochthonous L. donovani strains in antigen processing and precluding toxicants in test execution provides optimal sensitivity and safety for routine and mass application.

User and environment friendly direct agglutination test for the sero-diagnosis of visceral leishmaniasis: exclusion of formaldehyde and β-mercaptoethanol in test execution.

Based on world-wide evaluation, the direct agglutination test (DAT) is now generally acknowledged as one of the leading diagnostics for visceral leishmaniasis (VL). To enhance more routine and mass application, but simultaneously ensure safety to both user and environment, further improvements need to be introduced. In the current format, a two-sixfold titre decrease was observed due to using formaldehyde as an antigen preservative in DAT. Successful formaldehyde preservative exclusion was achieved by increasing its concentration to 3 % (wt/vol) for conserving promastigote status after β-mercaptoethanol (β-ME) treatment and repeating exposure of the parasite to the fixative after Coomassie Brilliant Blue staining. Microbial contamination was not observed in any of the antigen aliquots preserved in 0.05 % (wt/vol) sodium dichloroisocyanurate (chlorine) instead of formaldehyde for 6 months or longer. By excluding formaldehyde, restoring the normal antibody level, prior to treatment of sera with β-ME only minimally influenced the test outcome. A comparable successful reduction in non-specific agglutination, as with β-ME, was achieved by incorporating urea (0.3 % wt/vol) in the improved DAT procedure (P=0.646; T=23.0). As with the current procedure, the improved equivalent (formaldehyde and β-ME free) showed good reliability for VL detection (VL - Fr=52.39, W=0.70, P<0.001; and non-VL - Fr=65.97, W=0.83, P<0.001). A much lower cut-off (titre 1 : 400 versus 1 : 3200) for VL diagnosis can be adopted if urea is integrated in the improved procedure. By introducing the modifications mentioned, we think we have succeeded to a reasonable degree in increasing the DAT potential for VL control.

Epidemiological and clinical features of visceral leishmaniasis in children in Alicante Province, Spain

Background: Visceral leishmaniasis (VL) is endemic to the Mediterranean basin. In children, VL often presents with non-specific symptoms and can be life-threatening without proper treatment.Aim: To describe the epidemiological and clinical features of pediatric VL in children in Alicante, Spain.Methods: The study included all paediatric (<15 years) cases admitted to three hospitals in the province of Alicante from May 1992 to May 2015 with diagnosis of VL (detection was either by anti-Leishmania antibodies in serology or Leishmania in blood and/or bone marrow aspirates).Results: There were 38 cases of pediatric VL (18 aged <24 months, 15 aged 24–59 months and 5 aged ≥5 years). The main symptoms were fever (97.4%), followed by pallor (75.0%) and loss of appetite (46.4%). Eighty-seven per cent of patients were anaemic (haemoglobin < 9 g/dL), 73.7% had neutropenia and 68.4% had thrombocytopenia. Before 2004, 92.3% of patients were treated with meglumine antimoniate (MA) and 7.7% with liposomal amphotericin B (LAmB); after 2004, 84% were treated with LAmB and just one (16%) with MA (p < 0.001). LAmB performed better than MA in terms of mean treatment length (7.4 days vs 25.9 days, p < 0.001), time to becoming afebrile (1.7 vs 13.7 days, p < 0.001), and length of hospital stay (10.9 vs 19.4 days, p = 0.001).Conclusion: Paediatric VL in Alicante mainly affects children under five. Children aged ≤24 months present with a lower haemoglobin and white blood cell count. Treatment with LAmB reduces treatment length, time to becoming afebrile and length of hospital stay.

Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis.

Current strategies for the control of zoonotic visceral leishmaniasis (VL) rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay) with sera from infected humans and dogs. None of those was valid for the detection of human VL (26–52% sensitivity) although their performance was increased in the canine sera (48–91% sensitivity), with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This “Mix” resulted in similar levels of sensitivity for both human (84%) and canine (88%) sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis.

Impact of ASHA training on active case detection of visceral leishmaniasis in Bihar, India.

BackgroundOne of the major challenges for management of visceral leishmaniasis (VL) is early diagnosis of cases to improve treatment outcome and reduce transmission. We have therefore investigated active case detection of VL with the help of accredited social health activists (ASHA). ASHAs are women who live in the community and receive performance-based incentives for overseeing maternal and other health-related issues in their village.Methods and Principal FindingThrough conducting interviews with 400 randomly selected ASHAs from four primary health care centers (PHCs), it was observed that their level of knowledge about visceral leishmaniasis (VL) regarding transmission, diagnosis, and treatment was limited. The baseline data indicated that less than 10% of VL cases seeking treatment at the PHCs were referred by ASHAs. To increase the knowledge and the referral rate of VL cases by ASHAs, training sessions were carried out during the monthly ASHA meetings at their respective PHCs. Following a single training session, the referral rate increased from less than 10% to over 27% and the overall knowledge about VL substantially improved. It was not possible, however, to demonstrate that ASHA training reduced the time that individuals had fever before treatment at the PHC.ConclusionsTraining ASHAs to identify VL cases in villages for early diagnosis and treatment at the local PHC is feasible and should be undertaken routinely to improve knowledge about VL.

Performance of a rapid diagnostic test for the detection of visceral leishmaniasis in a large urban setting

Rapid diagnostic tests (RDTs) may improve the early detection of visceral leishmaniasis (VL), but their real-world performance requires additional study. Therefore, we evaluated the performance of an rK39-based RDT (Kalazar Detect™) for the detection of VL in an endemic, large urban area. Data were collected from a registry of rK39 RDT performed at 11 emergency care units in Belo Horizonte, Brazil, and from a national database of reportable communicable diseases of the Sistema de Informação de Agravos de Notificação (SINAN). The rapid rK39 test was performed in 476 patients, with 114 (23.9%) positive results. The analysis of rK39 RDT performance was based on 381 (80%) cases reported to the SINAN database, of which 145 (38.1%) were confirmed cases. Estimates for sensitivity and specificity were 72.4% (95% CI: 64.6-79%) and 99.6% (95%CI: 97.6-99.9%), respectively. Positive and negative predictive values were estimated at 99.1% (95%CI: 94.9-99.8%) and 85.5% (95%CI: 80.8-89.1%), respectively. In addition, close agreement between the rK39 RDT and indirect immunofluorescence was observed. In summary, the rK39 RDT showed a high specificity but only moderate sensitivity. In endemic areas for VL, treatment may be considered in cases with clinical manifestations and a positive rK39 RDT, but those with a negative test should be subjected to further investigation.

Epidemiological Status of Visceral Leishmaniasis in Iran: Experiences and Review of Literature

Background: The Mediterranean form of visceral leishmaniasis (VL), or kala-azar, is a potentially fatal vector-borne zoonotic disease. At the present time, VL caused by Leishmania infantum is recognized as an important parasitic disease in Iran. Objective: The aim of the study was to review the epidemiological aspects of VL in Iran during 1996-2010. Methods: Twenty seven eligible journal articles on VL epidemiological aspects were indexed in electronic databases (ISI and MEDLINE) from 1996 to 2010 and selected for this review accompanied by my personal experiences over two decades. For the detection of VL in humans and animal reservoir hosts, anti-Leishmania antibodies were detected using direct agglutination test (DAT). Parasitological examinations were performed on suspected VL patients and all captured wild canines and rodents. Different molecular methods were used for identification of Leishmania species isolated from infected humans, animal reservoirs, and vectors. Results: More than 3000 cases of symptomatic VL were detected in 31 provinces of Iran from 1996 to 2010. The majority of VL cases (92.8%) were found among children up to 12 years old. DAT surveillance could help decrease mortality and morbidity in VL endemic areas of Iran. The principal animal reservoir hosts of the infection were domestic and wild canines. Infections of man and desert rodents were accidental. Phlebotomus kandelakii; P. perfiliewi in northwest Iran; and P. major, P. keshishiani, and P. alexandri in southern parts of Iran were parasitologically or molecularly shown to be the principal VL vectors. Conclusion: The Mediterranean form of VL is a serious zoonotic disease that occurs sporadically in all geographical zones of Iran except northwestern and southern Iran, where the disease is endemic

Leishmaniasis in Sri Lanka: a decade old story

Visceral leishmaniasis is a vector-borne protozoan disease targeted for elimination from the Indian subcontinent by year 2020. Sri Lanka is a new focus of human leishmaniasis caused by a genetically distinct variant of Leishmania donovani , which is the species more widely known to cause visceral leishmaniasis (VL). The clinical entity that is most frequent in Sri Lanka is cutaneous leishmaniasis (CL), though a few cases of muco-cutaneous (MCL) and VL have also been reported in the recent past. In CL, papules, nodules, ulcerating nodules and ulcers occur mainly as single lesions on exposed body areas of affected individuals. A wide age range is affected including both sexes. The potential for visceralization in the cutaneous variant of L. donovani in Sri Lanka is not known. There is no evidence for a serological response in CL patients who have demonstrated negative for rK 39 antibodies in sera, (rk39 test being the recommended test for VL detection), while MCL and VL cases have been rK39 positive. Phlebotomus argentipes , the vector of L. donovani in other parts of the world is a widely prevalent insect in almost all parts of Sri Lanka. Studies are underway for vector identification. L. donovani is transmitted between this vector and the human host, the only known host for this species. However, domestic dogs have shown the presence of Leishmania parasites and also the presence of anti - L. donovani antibodies in their sera, providing primary evidence for the likely presence of an animal reservoir in Sri Lanka. Field-based risk factor studies have shown that there is peri-domestic as well as zoonotic/outdoor transmission cycles in different parts of the country. At present patients are detected mainly passively based on self referrals. Only a proportion of these patients proceed for pre-treatment laboratory confirmation due to the lack of freely available investigation facilities. Leishmaniasis was made a notifiable disease in Sri Lanka in 2008. Action plan for its control was drawn up with primary involvement of the Ministry of Health and other stake holders in 2008. Preventive and control activities are required to be put in place sooner rather than later. Enhanced case detection and active treatment are of prime value in controlling L. donovani infections. Availability of cost effective and field friendly diagnostic services in a decentralized manner, timely case management and vector control using appropriate protocols are necessary. To achieve this, a considerable amount of information is already available, and further research is needed to fill in the essential gaps. DOI: http://dx.doi.org/10.4038/sljid.v2i2.4420 Sri Lankan Journal of Infectious Diseases Vol.2(2) 2012:2-12

Diagnosis of visceral leishmaniasis: developments over the last decade

Diagnostic parameters for visceral leishmaniasis (VL), a potentially fatal parasitic disease caused by Leishmania donovani, have been redefined in the last decade with the development of serological and molecular tests, though a definitive diagnosis still banks on the century-old parasitological methods in many areas. Recombinant antigens have improved performance of serodiagnostic methods. Serology-based tests, rk39 antigen dipstick, and direct agglutination test commonly employed in the field are highly sensitive methods, however, fail to distinguish past infections. Molecular approaches have become increasingly relevant due to remarkable sensitivity, specificity, and flexibility in choice of samples. Quantitative polymerase chain reaction is a highly sensitive and specific tool used in referral labs for detection/assessment of parasite load in VL patients and subsequently in monitoring treatment response to antileishmanial agents. The method displays potential to provide threshold for distinguishing asymptomatics in endemic areas. Currently, improvement in VL diagnostics is required for successful decentralized (point-of-care) testing in field conditions and to detect VL-HIV co-infection. Techniques such as loop-mediated isothermal amplification offer a reliable molecular diagnostic method for field application. The diagnosis based on bioanalytics/biosensors promise frontiers for point-of-care VL detection after adequate standardization. This review summarizes the recent developments in VL diagnostics, drawing attention towards the need for standardization of the diagnostics across the affected regions.

Asymptomatic infection of visceral leishmaniasis in hyperendemic areas of Vaishali district, Bihar, India: a challenge to kala-azar elimination programmes

A cohort of 91 asymptomatic individuals with visceral leishmaniasis (VL) were identified during base line screening using recombinant 39-aminoacid antigen (rk-39) and polymerase chain reaction (PCR) conducted from December 2005 to June 2006 involving 997 individuals of two highly endemic villages of Vaishali district, Bihar. The point prevalence of asymptomatic infection was 98 per 1000 persons at baseline. There was no statistically significant difference between rk-39 and PCR positivity rate (P>0.05), even though PCR positivity alone was found significantly higher (4.2%) than rk-39 positivity alone (2.6%). The monthly follow-up of the asymptomatic cohort revealed a disease conversion rate of 23.1 per 100 persons within a year. There was a statistically significant difference in conversion of disease when individuals were positive by both tests as compared to single tests by rk-39 and PCR (P<0.01). Disease conversion rate in the subjects residing in households with a history of VL (62%, 13/21) was higher than those residing in the households without a history of VL (38%, 8/21). Most of the identified asymptomatic individuals were from low socio-economic strata similar to that of VL cases in general. Apart from rk-39, PCR may be considered for screening of asymptomatic Leishmania donovani infection in large-scale epidemiological studies. Screening of asymptomatic cases and their close follow-up to ascertain early detection and treatment of VL may be considered in addition to the existing VL control strategies.