rK39 ELISA Research Articles

Monitoring of Leishmania transmission in the postelimination phase: The potential of serological surveys

ObjectivesMonitoring of Leishmania transmission is considered a strategic priority for sustaining elimination of visceral leishmaniasis as a public health problem in the Indian subcontinent. The objective of this study was to evaluate whether serological surveys can distinguish between communities with and without Leishmania transmission, and to assess which serological marker performs best. MethodsSeven villages were selected from Bihar and Uttar Pradesh state, India, and categorized as either currently endemic (CE), previously endemic (PE) or nonendemic (NE). Blood samples were analyzed with the rK39 RDT, direct agglutination test (DAT), and rK39 ELISA. ResultsContrary to the rK39 RDT and DAT, the rK39 ELISA showed a significant difference between all three categories of endemicity, with a seroprevalence of 5.21% in CE villages, 1.55% in PE villages, and 0.13% in NE villages. Even when only looking at the seroprevalence among children aged <10 years, the rK39 ELISA was still able to differentiate between villages with and without ongoing transmission. ConclusionOur findings suggest the rK39 ELISA to be the most promising marker for monitoring of Leishmania transmission. Further validation is required, and practical, context-adapted recommendations need to be formulated in order to guide policymakers toward meaningful and sustainable surveillance strategies in the post-elimination phase.

Dried Blood Spots (DBS): A suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era.

Serum or whole blood collection, processing, transport and storage still present significant challenges in low resource settings where mass surveillance is required to sustain disease elimination. Therefore, in this study, we explored the diagnostic efficacy of dried blood spots (DBS) as a minimally invasive and potentially cost-effective alternative sampling technique to whole blood sampling procedures for subsequent detection of Leishmania donovani antibodies or DNA. Archived serum, DNA samples from whole blood of visceral leishmaniasis (VL) cases and healthy controls, and DBS from corresponding cases and controls, were used. Both molecular and serological assays were optimized to detect L. donovani antibodies or DNA in DBS elute and results were compared against those obtained with whole blood. Serological assays (both rK28 ELISA and rK39 ELISA) of DBS samples showed sensitivity and specificity of 100% and had excellent agreement with results from whole blood samples (kappa value ranged from 0.98-1). Bland-Altman analysis of OD values from rK28-ELISA with DBS elute and patients' serum showed an excellent agreement (ICC = 0.9) whereas a good agreement (ICC = 0.8) was observed in the case of rK39-ELISA. However, qPCR and RPA of DBS samples had a diminished sensitivity of 76% and 68%, respectively, and poor agreement was observed with the whole blood samples. Our results demonstrate that DBS offer excellent diagnostic efficiency for serological assays and represent a viable alternative to whole blood sampling procedures.

Detection of asymptomatic Leishmania infection in blood donors at two blood banks in Ethiopia.

Visceral leishmaniasis (VL) is a disease caused by Leishmania parasites. While predominantly transmitted by sandflies, cases of VL transmitted through blood transfusion have been reported, particularly in immunocompromised recipients. Although Leishmania parasites have been found in blood donors in some VL endemic areas, this has never been studied in East-Africa, where HIV prevalence is relatively high. We established the prevalence of asymptomatic Leishmania infection and associated socio-demographic factors among blood donors presenting at two blood bank sites (Metema and Gondar) in northwest Ethiopia between June and December 2020. Metema is located in a VL-endemic area; Gondar has historically been considered VL non-endemic but as an outbreak of VL has occurred around Gondar, it was defined as previously VL non-endemic. Blood samples were tested by the rK39 rapid diagnostic test (RDT), rK39 ELISA, direct agglutination test (DAT) and qPCR targeting kinetoplast DNA (kDNA). Asymptomatic infection was defined as positive by any of these tests in a healthy person. A total of 426 voluntary blood donors were included. The median age was 22 years (IQR, 19-28 years); 59% were male and 81% resided in urban areas. Only one participant had a history of VL and three had a family history of VL. Asymptomatic infection was detected in 15.0% (n = 32/213) in Metema and 4.2% (n = 9/213) in Gondar. The rK39 ELISA was positive in 5.4% (n = 23/426), the rK39 RDT in 2.6% (11/426), PCR in 2.6% (11/420) and DAT in 0.5% (2/426). There were six individuals with two positive tests: one positive on rK39 RDT and PCR and five positive on rK39 RDT and ELISA. The prevalence of asymptomatic infection was higher in Metema (VL-endemic) and males but was not associated with age, a history of VL amongst family members or living in a rural area. Antibodies against Leishmania and parasite DNA was detected in a substantial number of blood donors. Future research should be directed at better defining the risk to recipients, including parasite viability studies and longitudinal studies amongst recipients.

Assessment of Risk of Exposure to Leishmania Parasites among Renal Disease Patients from a Renal Unit in a Sri Lankan Endemic Leishmaniasis Focus.

Leishmania donovani causes both cutaneous and visceral leishmaniasis (CL and VL) in Sri Lanka, where chronic kidney disease (CKD) and kidney transplant recipients' (KTR) geographical areas overlap. This study aimed to determine the risk of exposure to Leishmania infection among renal patients. This cross-sectional study in a renal unit assessed clinical symptoms and signs of CL and VL in recipients of blood/kidney or immunosuppressives. Sera were tested with Leishmania-specific DAT and rK-39 ELISA. There were 170 participants. A total of 84.1% (n = 143) were males (CKD: 101, KTR; 42, mean age 45) and 27 were females (females: CKD: 23, KTR: 4, mean age 39 years). Recipients of blood transfusion/s within last 2 years: 75.9% (CKD: 115, KTR: 14), on immunosuppressive therapy: 34.1% (CKD: 13, KTR: 45). Two CKD patients repeatedly showed clear positive titres (1: 12,800 and 1: 3200) with Leishmania-DAT and another two (CKD) became marginally positive with rK39-ELISA. Prevalence of anti-Leishmania antibodies: 2.4% (4/170). All four patients were clinically asymptomatic and were recipients of recent blood transfusions. Attributable risk of exposure to Leishmania infection through blood transfusions was 0.032, OR 2.99 (95% CI = 0.16 to 56.45, p = 0.47). Therefore, routine screening of kidney/blood donors and CKD and KTR patients in Sri Lanka may not be necessary.

Assessing L. donovani Skin Parasite Load: A Proof of Concept Study of a Microbiopsy Device in an Indian Setting.

BackgroundIn the endgame of the elimination initiative of visceral leishmaniasis (VL) on the Indian subcontinent, one of the main questions remaining is whether asymptomatically infected individuals also contribute to transmission. We piloted a minimally invasive microbiopsy device that could help answer this question. While the potential of this device has been previously illustrated in Ethiopia, no such information is available for the setting of the Indian subcontinent. In this proof of concept study we aimed to assess 1) to what extent skin parasite load obtained with the new microbiopsy device correlates with disease status, 2) to what extent skin parasite load correlates with blood parasite load in the same subject, and 3) to what extent the skin parasite load obtained from different sampling sites on the body correlates with one another.MethodsWe performed a pilot study in Bihar, India, including 29 VL patients, 28 PKDL patients, 94 asymptomatically infected individuals, 22 endemic controls (EC), and 28 non-endemic controls (NEC). Presence of infection with L. donovani in the blood was assessed using Direct Agglutination Test, rK39 ELISA, Whole Blood Analysis measuring IFN-γ and qPCR. A skin sample was collected with the microbiopsy device on two different locations on the body. PKDL patients provided a third skin sample from the edge of a PKDL lesion. Parasite load in the skin was measured by qPCR.FindingsWe found a clear correlation between the skin parasite load obtained with the microbiopsy device and disease status, with both higher skin parasite loads and higher proportions of positive skin samples in VL and PKDL patients compared to asymptomatics, EC, and NEC. No clear correlation between skin parasite load and blood parasite load was found, but a moderate correlation was present between the skin parasite load in arm and neck samples. In addition, we found four positive skin samples among asymptomatic individuals, and 85% of PKDL lesions tested positive using this microbiopsy device.ConclusionsIn line with previous pilot studies, our results from an Indian setting suggest that the microbiopsy device provides a promising tool to measure skin parasite load, and – if validated by xenodiagnosis studies – could facilitate much needed larger scale studies on infectiousness of human subgroups. In addition, we advocate further evaluation of this device as a diagnostic tool for PKDL.

Evaluation of a new multi-epitope sequence of eight known Leishmania infantum antigens for HVL diagnosis by ELISA and Western blot.

Leishmaniasis, known as a disease with high prevalence proportion throughout the world, is caused by protozoan parasites. Visceral leishmaniasis is the most severe form of this condition reported sporadically from all regions in Iran. Between different diagnostic tests, serodiagnosis of this infection is of utmost importance in both humans and dogs. Although rK39 ELISA test has been extensively validated in endemic areas, there are currently challenges regarding a more appropriate serodiagnostic test. A novel multi-epitope construct was designed consisting of highexposedB cell epitopes using eight important antigens of Leishmania infantum (Gp63, KMP-11, HSP70, CPA, H2A, H3, LACK and TRYP). Our artificial sequence, a Multi-epitope Recombinant Protein (MRP), was consequently produced and purified. Then, immunoreactivity was investigated by ELISA test and western blotting as well. In the present study, the cutoff value (1.052) for the new MRP-ELISA was determined by receiver operator characteristic (ROC) curve analysis using 35 known positive and 20 known negative HVL sera previously tested for antibodies to L. infantum by DAT, showing a sensitivity of 93.1% and a specificity of 77.4%. The blotting test also showed a favorable band to detect visceral leishmaniasis. According to the results, this new antigen had acceptable potential in detecting VL positive cases once western blotting was utilized, but the ELISA test did not proceed as expected for detecting true negative cases, probably due to some optimization issues.The present study is a promising start.

Diagnostic accuracy of direct agglutination test, rK39 ELISA and six rapid diagnostic tests among visceral leishmaniasis patients with and without HIV coinfection in Ethiopia.

Diagnosis of a first-time visceral leishmaniasis (VL) infection in Ethiopia is established by use of a rapid diagnostic test (RDT) detecting antibodies against rK39, direct agglutination test (DAT) and microscopy according to the national algorithm. The performance of individual tests and algorithm is variable and depends on several factors, one being HIV status. Limited data are available on the performance of tests in VL-HIV coinfected patients. Assessment of the performance of DAT (ITM-A), rK39 ELISA (Serion) and six RDT (Onsite Leishmania Ab CTK, Antigen ICT Xinjier, IT Leish Biorad, Kalazar Detect Inbios, rK39 IgG1 Coris, rk28 IgG1 Coris) for the diagnosis of VL was done on a panel of 91 stored serum and plasma samples of 'first-episode' suspected VL patients, with HIV coinfection (n = 51) and without (n = 40). A combined reference standard was used: either positive microscopy on tissue aspirates, or in case of negative microscopy, positive PCR results on the aspirate slide. Additionally, endemic healthy controls (n = 20), non-endemic controls (n = 10) and patients with confirmed malaria infection (n = 10) were tested for specificity evaluation. Sensitivities ranged from 69.2% for DAT (applied cut-off ≥ 1/3200) to 92.2% for the Onsite RDT, whereas specificities ranged from 20.0% for Kalazar Antigen ICT to 100% for IT Leish and rK39 IgG1. Sensitivities from all assays decreased upon stratification according to HIV status but was only significantly different for rK39 Serion ELISA (p-value 0.0084) and the Onsite RDT (p-value 0.0159). In conclusion, performance of commercially available assays for VL on samples from Northern-Ethiopian patients varied widely with a substantial decrease in sensitivity in the VL-HIV coinfected group. Clear guidelines on minimal performance criteria of individual tests and algorithms are needed, as well as which reference standard should be used to determine the performance.

Evidence-based diagnostic algorithm for visceral leishmaniasis in Bangladesh

Evidence-based diagnostic algorithm is highly recommended for the visceral leishmaniasis (VL). This cross-sectional study was performed in Bangladesh to evaluate VL diagnostic tools including serology, buffy coat smear microscopy for LD body and various DNA-based techniques using buffy coat in 100 confirmed VL cases and 100 controls. The performance of tools against spleen smear (gold standard) was evaluated using kappa coefficient. Diagnostic precision and other inherent indicators were considered for index scoring (IS) of performance of tools using factor analysis. A diagnostic algorithm was formulated based on the IS and availability of the tools at different health care facilities of Bangladesh. A high level of agreement (kappa≥ 0.80) was observed for all the diagnostic tools. The highest kappa coefficients were found for rK39 RDT and rK39 ELISA (0.95), followed by ssuRNA-PCR (0.94), Buffy coat smear (0.93), rK28 ELISA (0.92), rK28 RDT (0.89), LAMP (0.89), Mini-exon PCR (0.86), ITS1 (0.85), and ITS2 PCR (0.80). rK39 RDT was found to be the best diagnostic test (IS: 1.7) followed by rK28 RDT (IS: 1.5), buffy coat smear microscopy (IS: 0.5), rK39 & rK28 ELISA (IS: 0.3), ssuRNA-PCR (IS: -0.7) and LAMP, Mini-exon, ITS1, & ITS2 PCR (IS: -0.9). rK39 RDT has been proposed as the best option for primary health care facilities, while buffy coat smear microscopy was found to be a good adjunct for confirmation of serology-positive cases and proposed for secondary and tertiary facilities. ssuRNA-PCR or LAMP can be an alternate confirmation tool only applicable to the tertiary facilities.

Determinants for progression from asymptomatic infection to symptomatic visceral leishmaniasis: A cohort study.

BackgroundAsymptomatic Leishmania donovani infections outnumber clinical presentations, however the predictors for development of active disease are not well known. We aimed to identify serological, immunological and genetic markers for progression from L. donovani infection to clinical Visceral Leishmaniasis (VL).MethodsWe enrolled all residents >2 years of age in 27 VL endemic villages in Bihar (India). Blood samples collected on filter paper on two occasions 6–12 months apart, were tested for antibodies against L. donovani with rK39-ELISA and DAT. Sero converters, (negative for both tests in the first round but positive on either of the two during the second round) and controls (negative on both tests on both occasions) were followed for three years. At the start of follow-up venous blood was collected for the following tests: DAT, rK39- ELISA, Quantiferon assay, SNP/HLA genotyping and L.donovani specific quantitative PCR.ResultsAmong 1,606 subjects enrolled,17 (8/476 seroconverters and 9/1,130 controls) developed VL (OR 3.1; 95% CI 1.1–8.3). High DAT and rK39 ELISA antibody titers as well as positive qPCR were strongly and significantly associated with progression from seroconversion to VL with odds ratios of 19.1, 30.3 and 20.9 respectively. Most VL cases arose early (median 5 months) during follow-up.ConclusionWe confirmed the strong association between high DAT and/or rK39 titers and progression to disease among asymptomatic subjects and identified qPCR as an additional predictor. Low predictive values do not warrant prophylactic treatment but as most progressed to VL early during follow-up, careful oberservation of these subjects for at least 6 months is indicated.

Long-lasting insecticidal nets to prevent visceral leishmaniasis in the Indian subcontinent; methodological lessons learned from a cluster randomised controlled trial.

Long-lasting insecticidal nets to prevent visceral leishmaniasis in the Indian subcontinent; methodological lessons learned from a cluster randomised controlled trial.

Higher parasite burden In asymptomatic individuals In endemic areas of visceral leishmaniasis

Background: In an area endemic for Visceral Leishmaniasis (VL), subclinical or asymptomatic infections play a crucial role in progression of disease. We determined the parasite load by quantitative PCR (qPCR) in healthy infected population living in an area endemic for Visceral Leishmaniasis. We enrolled 13366 persons from 11 villages of highly endemic region of Bihar, India. Methods & Materials: We conducted two sero-surveys by rK-39 ELISA and DAT with 1 year time interval for identification of incident infected healthy (Asymptomatic) individuals. Parasite load by using TaqMan based qPCR were done on peripheral blood using kDNA specific primers and probe on these sero-converted individuals and its matched control populations. Individuals having parasite load greater than 1 genome/ml of blood was considered as positive by qPCR. Follow-up visit to the homes of each individual were made to monitor the disease conversion in this cohort. Results: Between seroconversion and qPCR were accessed by kappa value. Total 235 persons were converted their serology within 12 month intervals. Of these 235 sero-converters 105 (44.6%) individuals were also positive by qPCR. However, similar number of controls groups (87/237, 37%) also showed positivity by qPCR. The agreement between sero-converter and qPCR was poor (k = 0.12). And among all individuals only one was progress to disease. Conclusion: So there is no major difference in qPCR result between sero-negatives and sero-converters and among all individuals only one were converted into disease that has parasite load 146 parasite genome/ml of blood. These findings suggest the usefulness of parasite load in healthy individuals living in an endemic area of Bihar and contribute as a good tool for VL elimination programme.

Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar, India

Recent epidemiological reports indicate that asymptomatic human infections with Leishmania donovani, the causative agent of visceral leishmaniasis or Kala-azar (KA), occur frequently in India. We explored markers of infection. Blood samples were collected from 286 healthy subjects from 16 villages in the Muzaffarpur district of Bihar. These individuals were classified into three groups: (i) persons with no history of KA and living in a house where no KA cases were previously reported, (ii) persons with no history of KA but living in a house where KA cases were diagnosed at the time of sampling or in the past, and (iii) successfully treated KA patients. Each sample was tested using a Leishmania-specific PCR to detect Leishmania DNA, and two serological tests to demonstrate anti-Leishmania antibodies: the Direct Agglutination Test and rK39 ELISA. PCR positivity was similar among the three groups (20-25%). In contrast, among treated patients, the percentage of serologically positive individuals was roughly five times that of healthy individuals with no KA history, as measured with either test. Living in a house where KA had been reported did not affect seropositivity. A significant proportion of asymptomatic infections of Leishmania exist in endemic regions. Using a combination of molecular and serological tests increases the capacity to detect infections at different stages. Further work is required to understand the kinetics of the markers.

Latent Infection with Leishmania donovani in Highly Endemic Villages in Bihar, India

IntroductionAsymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4–10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.MethodsWe selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.ResultsWe enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30). Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years).DiscussionAlthough infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.

Comparative evaluation of rK39 and soluble antigen ELISA, IFAT and parasitological test for the diagnosis of Canine Visceral Leishmaniasis.

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Systematic Review into Diagnostics for Post-Kala-Azar Dermal Leishmaniasis (PKDL)

Identification of post-kala-azar dermal leishmaniasis (PKDL) is important due to the long and toxic treatment and the fact that PKDL patients may serve as a reservoir for visceral leishmaniasis (VL). We summarized the published literature about the accuracy of diagnostic tests for PKDL. We searched Medline for eligible studies investigating the diagnostic accuracy of any test for PKDL. Study quality was assessed using QUADAS-2. Data were extracted from 21 articles including 43 separate studies. Twenty-seven studies evaluated serological tests (rK39 dipstick, ELISA, DAT, and leishmanin tests), six studies molecular tests, eight microscopy, and two cultures. Only a few of these studies reported a valid estimate of diagnostic accuracy, as most were case-control designs or used a reference standard with low sensitivity. The included studies were very heterogeneous, for example, due to a large variety of reference standards used. Hence, no summary estimates of sensitivity or specificity could be made. We recommend well-designed diagnostic accuracy trials that evaluate, side-by-side, all currently available diagnostics, including clinical symptoms, serological, antigen, molecular, and parasitological tests and possible use of statistical modelling to evaluate diagnostics when there is no suitable gold standard.

Comparative Study of rK39 Leishmania Antigen for Serodiagnosis of Visceral Leishmaniasis: Systematic Review with Meta-Analysis

BackgroundThe rK39 recombinant protein is derived from a specific antigen produced by the Leishmania donovani complex, and has been used in the last two decades for the serodiagnosis of visceral leishmaniasis. We present here a systematic review and meta-analysis of studies evaluating serologic assays to diagnose visceral leishmaniasis to determine the accuracy of rK39 antigen in comparison to the use of other antigen preparations.Methodology/Principal FindingsA systematic review with meta-analysis of the literature was performed to compare the rK39 strip-test and ELISA formats against serological tests using promastigote antigens derived from whole or soluble parasites for Direct Aglutination Test (DAT), Indirect Immunofluorescence test (IFAT) and ELISA with a promastigote antigen preparation (p-ELISA). Gold standard diagnosis was defined by the demonstration of amastigotes on hematological specimens. A database search was performed on Medline, Lilacs, Scopus, Isi Web of Science, and Cochrane Library. Quality of data was assessed using the QUADAS questionnaire. A search of the electronic databases found 352 papers of which only 14 fulfilled the selection criteria. Three evaluated the rK39 ELISA, while 13 evaluated the rK39 immunochromatographic strip test. The summarized sensitivity for the rK39-ELISA was 92% followed by IFAT 88% and p-ELISA 87%. The summarized specificity for the three diagnostic tests was 81%, 90%, and 77%. Studies comparing the rK39 strip test with DAT found a similar sensitivity of 94%, although the DAT had a slightly higher specificity. The rK39 strip test was more sensitive and specific than the IFAT and p-ELISA. We did not detect any difference in the sensitivity and specificity between strips produced by different manufacturers.ConclusionsThe rK39 protein used either in a strip test or in an ELISA, and the DAT are the best choices for implementation of rapid, easy and efficient test for serodiagnosis of VL.

Evaluation of rK28 antigen for serodiagnosis of visceral Leishmaniasis in India

Antibody detection is a safely applied method at the wide scale in diagnosis of visceral Leishmaniasis (VL). In order to further advance serodiagnosis, the rK28 antigen has been recently introduced as a candidate for diagnosis of VL. We evaluated the sensitivity and specificity of the rK28 antigen in a micro-ELISA format in comparison to the rk39 antigen. The test was conducted on 252 parasitologically confirmed VL cases, 103 endemic healthy controls, 95 non-endemic healthy controls, 88 other infectious disease and 53 follow-up cases. Of 252 parasitologically confirmed VL cases, 251 cases were reported positive by rK28 antigen, yielding 99.6% sensitivity (95% CI, 0.97–0.99), which was similar to the sensitivity of rK39 ELISA (99.6%) (95% CI, 0.97–0.99). Specificity of the rK28 antigen in non-endemic and endemic healthy controls was 100% (95% CI 0.96–1) and 94.17% (95% CI, 0.88–0.97), respectively. In 88 different diseases, specificity was 95.45% (95% CI, 0.84–0.96). With the rK39 antigen, specificity of non-endemic and endemic controls and different diseases was 100% (95% CI 0.96–1), 92.23% (95% CI 0.85–0.96) and 96.59% (95% CI 0.90–0.98), respectively. Our results show that rK39 and rK28 antigens have similar sensitivity and specificity and rK28 can also be used as a serodiagnostic tool in the endemic population of Bihar.

Herramientas no invasivas en Venezuela: comparación entre las pruebas inmunoserológicas DAT, rK26 y rK39 en el diagnóstico de leishmaniasis visceral

Human visceral leishmaniasis is a serious public health problem in endemic countries because of its high potential lethality, particularly in children. Rapid diagnosis is essential to early treatment and control of visceral leishmaniasis. The aim was to compare three serodiagnostic tools for human visceral leishmaniasis. Three methods were compared: the rK39 dipstick (Kalazar detection test, Inbios International Inc.), ELISA rK26 and direct agglutination test (DAT) (KIT Biomedical Research). Fifty serum samples from patients positive for rK39 ELISA were compared from four endemic provinces in Venezuela: Nueva Esparta (Margarita island), Lara, Anzoátegui and Trujillo. Additional serum samples from 17 healthy volunteers and 25 patients with other diseases were included. The rK39 ELISA served as the baseline standard method. Sensitivity, specificity, positive predictive value, negative predictive value and likelihood ratio were calculated for each test. All methods had a positive correlation with rK39 ELISA (p<0.0001). They showed high sensitivity and specificity. The direct agglutination test and the rK39 dipstick showed high sensitivity values, 89.7% (95% CI: 81.34.0-98.2%) and 94.2% (95% CI: 87.7-100%), respectively, and high specificity, 81.0% (95% CI: 80.0-99.5%) and 100%. The rK26 ELISA showed good specificity, 99% (95% CI: 95.2-100%), but a very low sensitivity, 37% (95% CI: 23.4-50.2%). Overall results indicated that DAT and rK39 dipstick have the highest specificity and sensitivity. Both are simple, cost-effective and field applicable tests. Therefore, they are recommended for early and accurate diagnosis of visceral leishmaniasis.

Positive rK39 Serologic Assay Results in US Servicemen with Cutaneous Leishmaniasis

The rK39 test is a serologic assay for the rapid diagnosis of visceral leishmaniasis (VL). Serum from a North American cohort of 59 otherwise asymptomatic soldiers with cutaneous leishmaniasis (CL) was tested with the rK39 dipstick and ELISA assays, and 10.2% and 28.8% had positive results, respectively. CL is associated with a reactive rK39 assay result in some patients without clinical evidence of VL.

Detection of Leishmania kDNA in human serum samples for the diagnosis of visceral leishmaniasis

The performance of PCR to detect Leishmania kDNA in serum for the diagnosis of visceral leishmaniasis (VL) was assessed in serum samples from 65 patients with VL, 17 non-infected individuals and 17 patients with other febrile hepatosplenic diseases. Serum PCR showed a sensitivity of 85%, specificity of 100% and efficiency of 90%. The sensitivity values obtained for blood PCR (97%) and rK39 ELISA (95%) were significantly higher (P=0.01) than the values observed for L. chagasi ELISA (88%) and serum PCR (85%), whilst no difference was observed among the specificity rates obtained with rK39 ELISA (94%; P=0.47) and L. chagasi ELISA (85%; P=0.06). This work suggests that the use of serum samples may be an alternative for the diagnosis of VL when peripheral blood samples are not available or require significant operational efforts.