AmBisome Monotherapy and Combination AmBisome-Miltefosine Therapy for the Treatment of Visceral Leishmaniasis in Patients Coinfected With Human Immunodeficiency Virus in India: A Randomized Open-Label, Parallel-Arm, Phase 3 Trial.

BACKGROUND: Visceral leishmaniasis (VL) in patients with human immunodeficiency virus (HIV) presents an increasingly important patient cohort in areas where both infections are endemic. Evidence for treatment is sparce, with no high-quality studies from the Indian subcontinent. METHODS: This is a randomized, open-label, parallel-arm, phase 3 trial conducted within a single hospital in Patna, India. One hundred and fifty patients aged ≥18 years with serologically confirmed HIV and parasitologically confirmed VL were randomly allocated to 1 of 2 treatment arms, either a total 40 mg/kg intravenous liposomal amphotericin B (AmBisome; Gilead Pharmaceuticals) administered in 8 equal doses over 24 days or a total 30 mg/kg intravenous AmBisome administered in 6 equal doses given concomitantly with a total 1.4 g oral miltefosine administered through 2 daily doses of 50 mg over 14 days. The primary outcome was intention-to-treat relapse-free survival at day 210, defined as absence of signs and symptoms of VL or, if symptomatic, negative parasitological investigations. RESULTS: Among 243 patients assessed for eligibility, 150 were recruited between 2 January 2017 and 5 April 2018, with no loss to follow-up. Relapse-free survival at day 210 was 85% (64/75; 95% CI, 77-100%) in the monotherapy arm, and 96%, (72/75; 90-100%) in the combination arm. Nineteen percent (28/150) were infected with concurrent tuberculosis, divided equally between arms. Excluding those with concurrent tuberculosis, relapse-free survival at day 210 was 90% (55/61; 82-100%) in the monotherapy and 97% (59/61; 91-100%) in the combination therapy arm. Serious adverse events were uncommon and similar in each arm. CONCLUSIONS: Combination therapy appears to be safe, well tolerated, and effective, and halves treatment duration of current recommendations. CLINICAL TRIALS REGISTRATION: Clinical Trial Registry India (CTRI/2015/05/005807; the protocol is available online at https://osf.io/avz7r).

A Novel Antigen, Otubain Cysteine Peptidase of Leishmania donovani, for the Serodiagnosis of Visceral Leishmaniasis and for Monitoring Treatment Response.

Tests for visceral leishmaniasis (VL) are not uniformly effective for all endemic regions. In a serological assay, a novel antigen, otubain cysteine peptidase, compared with rK39, showed comparable sensitivity with Indian VL serum samples and prominently increased sensitivity with Brazilian samples, as well as improved monitoring of the treatment response.

Evaluation of Cysteine Protease C of Leishmania donovani in Comparison with Glycoprotein 63 and Elongation Factor 1α for Diagnosis of Human Visceral Leishmaniasis and for Posttreatment Follow-Up Response.

Visceral leishmaniasis (VL) is a threat in many developing countries. Much effort has been put to eliminating this disease, for which serodiagnosis remains the mainstay for VL control programs. New and improved antigens as diagnostic candidates are required, though, as the available antigens fail to demonstrate equal optimum performance in all areas of endemicity. Moreover, these diagnoses are dependent on invasive serum sampling. In the current study, we cloned and expressed Leishmania donovani cysteine protease C (CPC) and evaluated its diagnostic and test-of-cure possibilities by detecting the antibody levels in human serum and urine through ELISA and immunoblot assays. Two immunodominant antigens, recombinant glycoprotein 63 (GP63) and elongation factor 1α (EF1α), identified earlier by our group, were also assessed by employing human serum and urine samples. Of these three antigens in ELISAs, CPC demonstrated the highest sensitivities of 98.15% and 96% positive testing in serum and urine of VL patients, respectively. Moreover, CPC yielded 100% specificity with serum and urine of nonendemic healthy controls compared to GP63 and EF1α. Urine samples were found to be more specific than serum for distinguishing endemic healthy controls and other diseases by means of all three antigens. In all cases, CPC gave the most promising results. Unlike serum, urine tests demonstrated a significant decrease in antibody levels for CPC, GP63, and EF1α after 6 months of treatment. The diagnostic and test-of-cure performances of CPC in the immunoblot assay were found to be better than those of GP63 and EF1α. In conclusion, CPC, followed by GP63 and EF1α, may be utilized as candidates for diagnosis of VL and to assess treatment response.

Leishmania donovani mediated higher expression of CCL4 induces differential accumulation of CD4+CD56+NKT and CD8+CD56+NKT cells at infection site.

Sterile cure from visceralized Leishmania donovani (L. donovani) needs Th1 cell support along with the assistance from innate immune cells, NK cells and NKT cells. NKT cells play as a connecting link between innate and adaptive immune cell and support T helper cell function. Earlier, a categorical function of CD56 positive CD4+ or CD8+ NKT cells was reported in visceral leishmaniasis (VL). It was observed in in vitro that CD4+CD56+NKT cells, but not CD8+CD56+NKT cells, were accumulated at the L. donovani infection site. Therefore, in vitro experiments have been carried out to decipher the mechanism behind preferential accumulation of CD4+CD56+NKT cells at infection site. In this study, 1.89 fold higher expression of CCL4/MIP-1ß was noticed in infected macrophages. The higher expression of CCL4 was correlated with preferential accumulation of CCR5+CD4+CD56+NKT cells and apoptosis of CD8+CD56+NKT cells at in vitro infection site. The CD4+CD56+NKT cells were also observed expressing TGF-ß dominantly. Interaction of CCL4 chemotaxis was interrupted by blocking, which led to drift back the TGF-ß producing CD4+CD56+NKT cells and promoted CD8+CD56+NKT cells recruitment in in vitro infection site. CCR5 blockade also reduced CD25 and FoxP3 positive CD4+CD56+NKT cells in in vitro infection site. Therefore, it was concluded that Leishmania promotes strategic expression of CCL4, which alternately attracts CCR5+ cells, mostly expressing regulatory cytokines, at infection site. This reduces the CD8+CD56+NKT cells at infection site through Smad4 mediated TGF-ß expression and activation of caspases. Data indicates that L. donovani induces higher expression of CCL4 in host cell to attract CCR5+ cells under its strategic plan to downregulate host immune response.

Leishmania donovani resistant to Ambisome or Miltefosine exacerbates CD58 expression on NK cells and promotes trans-membrane migration in association with CD2.

Visceral leishmaniasis (VL) is a disease that is associated with compromised immunity and drug un-responsiveness as well as with the emergence of drug resistance in Leishmania donovani (Ld). Ld down-modulates cellular immunity by manipulating signaling agents, including a higher expression of the adhesion molecule CD58. The expression of CD58 and CD2 on natural killer (NK) cells facilitates intercellular adhesion and signaling. The influence of drug-resistant Ld on the expression of CD58 and CD2 was addressed in this study. The mean florescence intensity (MFI) of CD58 but not of CD2 was twofold higher on CD56+ cells during VL, but was down-regulated after treatment. In addition, MFI of CD58 on CD56+ cells was further exacerbated in VL subjects who had relapsed after Ambisome or Miltefosine treatment. The same pattern of CD58 expression was also obtained upon stimulation of healthy peripheral blood mononuclear cells with Miltefosine- or Ambisome-resistant Ld. The ratio of CD56+CD58+IFN-γ+/CD56+CD58+IL-10+ cells was reduced by 6.98-fold after stimulation with Ld. Further, an antagonist to CD58 or its counter-receptor CD2 down-regulated CD56+ NK cell recruitment across a polycarbonate trans-membrane at Ld infection sites. This study reports that factors associated with drug resistance in Ld probably promote higher expression of CD58 on CD56+ cells and their migration to the infection site in association with CD2.

Revealing a Novel Antigen Repressor of Differentiation Kinase 2 for Diagnosis of Human Visceral Leishmaniasis in India.

Visceral leishmaniasis (VL) is one of the major global health concerns due to its association with morbidity and mortality. All available diagnostic tools have been, until now, unable to provide a very specific and cost-effective mode of detection for VL globally. Therefore, the design of robust, specific, and commercially translatable diagnostic tests is urgently required. Currently, we are attempting to identify and explore the diagnostic potential of a novel parasite antigen. Repressor of differentiation kinase 2 (RDK2), a serine/threonine kinase, has a versatile role in parasite life cycle progression. However, its role as a diagnostic candidate for VL has not been investigated. Herein, we cloned and over-expressed LdRDK2 and studied the recombinant RDK2 for the diagnosis of human VL using serum and urine samples. In silico analysis predicted that RDK2 is conserved among Leishmania species with the least conservation in humans. RDK2 developed immune-reactive bands with antibodies present in VL patients' sera, and it demonstrated no cross-reactivity with sera from healthy controls and other diseases. Additionally, RDK2 antigen demonstrated a significant reactivity with IgG antibodies of VL patients' sera, with 78% sensitivity and 86.67% specificity as compared to healthy controls and other diseases. Furthermore, we evaluated its utility for non-invasive diagnosis of VL using patients' urine samples and found 93.8% sensitivity and 85.7% specificity. RDK2 was found to have better sensitivity and treatment response in patients' urine compared to serum samples, indicating its role as a promising point of care (POC) antigen. In a nutshell, we explored the role of RDK2 as a potential diagnostic marker for VL in both invasive and non-invasive modes as well as its utility as a promising POC antigen for treatment response cases.

Development and Clinical Evaluation of Serum and Urine-Based Lateral Flow Tests for Diagnosis of Human Visceral Leishmaniasis.

Visceral leishmaniasis (VL), a fatal parasitic infection, is categorized as being neglected among tropical diseases. The use of conventional tissue aspiration for diagnosis is not possible in every setting. The immunochromatography-based lateral flow assay (LFA) has attracted attention for a long time due to its ability to give results within a few minutes, mainly in resource-poor settings. In the present study, we optimized and developed the LFA to detect anti-Leishmania antibodies for VL diagnosis. The performance of the developed test was evaluated with serum and urine samples of Indian VL patients and Brazilian sera. The new test exploits well-studied and highly-sensitive purified antigens, LAg isolated from Leishmania donovani promastigotes and protein G conjugated colloidal-gold as a signal reporter. The intensity of the bands depicting the antigen-antibody complex was optimized under different experimental conditions and quantitatively analyzed by the ImageJ software. For the diagnosis of human VL in India, LFA was found to be 96.49% sensitive and 95% specific with serum, and 95.12% sensitive and 96.36% specific with urine samples, respectively. The sensitivity and specificity of LFA were 88.57% and 94.73%, respectively, for the diagnosis of Brazilian VL using patients' sera infected with Leishmania infantum. LFA is rapid and simple to apply, suitable for field usage where results can be interpreted visually and particularly sensitive and specific in the diagnosis of human VL. Serum and urine LFA may improve diagnostic outcomes and could be an alternative for VL diagnosis in settings where tissue aspiration is difficult to perform.

CD8dim but not CD8bright cells positive to CD56 dominantly express KIR and are cytotoxic during visceral leishmaniasis.

This study reports a structural and functional heterogeneity of CD8+CD56+NKT cells, which usually decrease quantitatively during visceral leishmaniasis. Based on fluorescence intensity of CD8 receptors on CD56+NKT cells, two populations of CD8+CD56+NKT cells have been identified. These cells were recognized as CD8dimCD56+NKT and CD8brightCD56+NKT cells. We further analyzed the functional nature of CD8dim and CD8bright positive CD56+NKT cells. In comparison to CD8brightCD56+NKT cells, a significantly higher percentage of CD8dimCD56+NKT cells expressed KIR during VL. The percentage of CD8dimCD56+NKT cells expressing KIR was found 4 fold higher in VL as compared to healthy subjects. But, the difference was insignificant in case of CD8brightCD56+NKT cells. CD8+CD56+NKT cells release granzyme B to kill the infected cells. A categorical difference was also observed in the function of CD8dimCD56+NKT and CD8brightCD56+NKT cells during visceral leishmaniasis. The percentage of granzyme B expressing CD8dimCD56+NKT cells was 2.83 fold higher in VL compared to healthy subjects. But, there was no significant difference in granzyme B expressing CD8brightCD56+NKT cells in samples from healthy and VL subjects. However, within VL subject, the percentage of granzyme B expressing CD8dimCD56+NKT cells was 5.7 fold higher in comparison to CD8brightCD56+NKT cells. This study concludes that CD8dimCD56+NKT cells are more cytotoxic than CD8brightCD56+NKT cells during VL.

Immunoproteomic Identification and Characterization of Leishmania Membrane Proteins as Non-Invasive Diagnostic Candidates for Clinical Visceral Leishmaniasis.

Visceral leishmaniasis (VL), a potentially fatal disease is an outcome of infection caused by the parasite Leishmania donovani. The clinical diagnostic tests for this disease are still related to invasive tissue aspiration or serological immunochromatography. Advancements in immunoproteomics such as two-dimensional gel electrophoresis, mass spectrometry, B cell epitope prediction, and peptide synthesis have enabled researchers to discover newer biomarkers for disease diagnosis. In this study, we have screened several urine-reactive leishmanial membrane proteins as potential biomarker candidates. In the immunoblot assay, three proteins 51, 55 and 63 kDa showed 100% reactivity to the urine of 47 VL patients and nonreactive to 18 healthy and other diseases. Mass spectrometry revealed the identity of 51, 55 and 63 kDa proteins as elongation factor 1α (EF1-α), α-tubulin, and glycoprotein 63, respectively. B cell reactive epitopes of these proteins were mapped through bioinformatic tools and one epitope from each protein that had the highest score were synthesized. All the three native electroeluted proteins and their corresponding synthetic peptides were tested through ELISA for reactivity with VL and control urine samples. While all three demonstrated good reactivity, the diagnostic performance of EF1-α was the best. Our findings illustrate the use of urine-based proteomic approach for biomarker discovery in non-invasive clinical diagnosis of VL.

Plasma Gelsolin Level in HIV-1-Infected Patients: An Indicator of Disease Severity.

Plasma gelsolin (pGSN) is a multifunctional protein involved mainly in severing and clearing of actin filaments. Its level correlates with inflammation and several diseases making it a potential biomarker of diagnostic and prognostic values. The pGSN level in groups of treated and untreated HIV-1-infected Indian patients is investigated in this study. This study aims at investigating the levels of pGSN in HIV-1-infected patients across different age, sex, severity of disease, and treatment status. Blood samples of 213 patients were analyzed for CD4 counts by flow cytometry and pGSN was quantified by enzyme-linked immunosorbent assay (ELISA). The level of pGSN is significantly increased in HIV-1 infected patients (227.2 ± 54.3 µg/ml) compared to healthy volunteers (167.9 ± 61.8 µg/ml). The level correlates with CD4 cell counts as patients with lower CD4 counts showed higher pGSN levels and vice versa. Gender does not affect pGSN level; however, antiretroviral (ARV) treatment reduces pGSN toward normal. Within low CD4 cell count group, the untreated patients have 52% higher pGSN than healthy volunteers, whereas with treatment, the difference reduces to 24%. Similarly, high CD4 cell count (>350 cells/mm3) group of patients showed 44% increase in pGSN in untreated patients compared to 21% increase in treated patients. There is an upregulation of pGSN in HIV-1 infection and it is inversely correlated with CD4 cell counts. Treatment with ARV drugs decreases pGSN levels toward normal. The monitoring of pGSN level in HIV-1-infected patients could be an important indicator of severity of disease and recovery during treatment.

Repeated training of accredited social health activists (ASHAs) for improved detection of visceral leishmaniasis cases in Bihar, India.

Accredited Social Health Activists (ASHAs) are incentive-based, female health workers responsible for a village of 1000 population and living in the same community and render valuable services towards maternal and child health care, polio elimination program and other health care-related activities including visceral leishmaniasis (VL). One of the major health concerns is that cases remain in the endemic villages for weeks without treatment causing increased likelihood to treatment failure and disease transmission in the community. To address this problem, we have begun a training program for ASHAs to enhance early detection of potential VL cases and referring them to their local Primary Health Centers (PHCs) for diagnosis and treatment. The result of this training showed increased referral rate to PHCs for diagnosis and treatment. Encouraged with the results from a single training session, we determined in the present study whether repeated training of ASHAs resulted in an a further increase in VL case referral to the local PHCs. After two training sessions, VL referrals by ASHAs increased to 46% as compared to 28% after a single training session in this cohort and a baseline of 7% before training. ASHA training is an effective way to conduct active case detection of VL cases and should be repeated once a year.

Longitudinal Study of Transmission in Households with Visceral Leishmaniasis, Asymptomatic Infections and PKDL in Highly Endemic Villages in Bihar, India.

BACKGROUND: Visceral Leishmaniasis (VL) is a neglected tropical disease that afflicts some of the poorest populations in the world including people living in the Bihar state of India. Due to efforts from local governments, NGOs and international organizations, the number of VL cases has declined in recent years. Despite this progress, the reservoir for transmission remains to be clearly defined since it is unknown what role post kala-azar dermal leishmaniasis (PKDL) and asymptomatic infections play in transmission. This information is vital to establish effective surveillance and monitoring to sustainably eliminate VL. METHODOLOGY/PRINCIPAL FINDINGS: We performed a longitudinal study over a 24-month period to examine VL transmission and seroconversion in households with VL, PKDL and asymptomatic infections in the Saran and Muzaffarpur districts of Bihar. During the initial screening of 5,144 people in 16 highly endemic villages, 195 cases of recently treated VL, 116 healthy rK39 positive cases and 31 PKDL cases were identified. Approximately half of the rK39-positive healthy cases identified during the initial 6-month screening period were from households (HHs) where a VL case had been identified. During the 18-month follow-up period, seroconversion of family members in the HHs with VL cases, PKDL cases, and rK39-positive individuals was similar to control HHs. Therefore, seroconversion was highest in HHs closest to the time of VL disease of a household member and there was no evidence of higher transmission in households with PKDL or healthy rK39-positive HHs. Moreover, within the PKDL HHs, (the initial 31 PKDL cases plus an additional 66 PKDL cases), there were no cases of VL identified during the initial screen or the 18-month follow-up. Notably, 23% of the PKDL cases had no prior history of VL suggesting that infection resulting directly in PKDL is more common than previously estimated. CONCLUSIONS/SIGNIFICANCE: These observations argue that acute VL cases represent the major reservoir for transmission in these villages and early identification and treatment of VL cases should remain a priority for VL elimination. We were unable to obtain evidence that transmission occurs in HHs with a PKDL case.

Noninvasive Diagnosis of Visceral Leishmaniasis: Development and Evaluation of Two Urine-Based Immunoassays for Detection of Leishmania donovani Infection in India.

BACKGROUND: Visceral Leishmaniasis (VL), a severe parasitic disease, could be fatal if diagnosis and treatment is delayed. Post kala-azar dermal leishmaniasis (PKDL), a skin related outcome, is a potential reservoir for the spread of VL. Diagnostic tests available for VL such as tissue aspiration are invasive and painful although they are capable of evaluating the treatment response. Serological tests although less invasive than tissue aspiration are incompetent to assess cure. Parasitological examination of slit-skin smear along with the clinical symptoms is routinely used for diagnosis of PKDL. Therefore, a noninvasive test with acceptable sensitivity and competency, additionally, to decide cure would be an asset in disease management and control. METHODOLOGY/PRINCIPAL FINDINGS: We describe here, the development of antibody-capture ELISA and field adaptable dipstick test as noninvasive diagnostic tools for VL and PKDL and as a test of cure in VL treatment. Sensitivity and specificity of urine-ELISA were 97.94% (95/97) and 100% (75/75) respectively, for VL. Importantly, dipstick test demonstrated 100% sensitivity (97/97) and specificity (75/75) in VL diagnosis. Degree of agreement of the two methods with tissue aspiration was 98.83% (κ = 0.97) and 100% (κ = 1), for ELISA and dipstick test, respectively. Both the tests had 100% positivity for PKDL (14/14) cases. ELISA and dipstick test illustrated treatment efficacy in about 90% (16/18) VL cases when eventually turned negative after six months of treatment. CONCLUSIONS/SIGNIFICANCE: ELISA and dipstick test found immensely effective for diagnosis of VL and PKDL through urine samples thus, may substitute the existing invasive diagnostics. Utility of these tests as indirect methods of monitoring parasite clearance can define infected versus cured. Urine-based dipstick test is simple, sensitive and above all noninvasive method that may help not only in active VL case detection but also to ascertain treatment response. It can therefore, be deployed widely for interventions in disease management of VL particularly in poor resource outskirts.

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

BACKGROUND: One 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 FINDING: Through 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. CONCLUSIONS: Training 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.

Efficacy and safety of amphotericin B emulsion versus liposomal formulation in Indian patients with visceral leishmaniasis: a randomized, open-label study.

BACKGROUND: India is home to 60% of the total global visceral leishmaniasis (VL) population. Use of long-term oral (e.g. miltefosine) and parenteral drugs, considered the mainstay for treatment of VL, is now faced with increased resistance, decreased efficacy, low compliance and safety issues. The authors evaluated the efficacy and safety of an alternate treatment option, i.e. single infusion of preformed amphotericin B (AmB) lipid emulsion (ABLE) in comparison with that of liposomal formulation (LAmB). METHODS: In this multicentric, open-label study, 500 patients with VL were randomly assigned in a 3:1 ratio to receive 15 mg/kg single infusion of either ABLE (N = 376) or LAmB (N = 124). Initial cure (Day 30/45), clinical improvement (Day 30) and long term definitive cure (Day 180) were assessed. FINDINGS: A total of 326 (86.7%) patients in the ABLE group and 122 (98.4%) patients in the LAmB group completed the study. Initial cure was achieved by 95.9% of patients in the ABLE group compared to 100% in the LAmB group (p = 0.028; 95% CI: -0.0663, -0.0150). Clinical improvement was comparable between treatments (ABLE: 98.9% vs. LAmB: 98.4%). Definitive cure was achieved in 85.9% with ABLE compared to 98.4% with LAmB. Infusion-related pyrexia (37.2% vs. 32.3%) and chills (18.4% vs. 18.5%) were comparable between ABLE and LAmB, respectively. Treatment-related serious adverse events were fewer in ABLE (0.3%) compared to LAmB (1.6%). Two deaths occurred in the ABLE group, of which one was probably related to the study drug. Nephrotoxicity and hepatotoxicity was not observed in either group. CONCLUSIONS: ABLE 15 mg/kg single infusion had favorable efficacy and was well tolerated. Considering the demographic profile of the population in this region, a single dose treatment offers advantages in terms of compliance, cost and applicability. TRIAL REGISTRATION: www.clinicaltrials.gov NCT00876824.

Diagnosis of visceral leishmaniasis in Bihar India: comparison of the rK39 rapid diagnostic test on whole blood versus serum.

BACKGROUND: Antibody-detecting rapid diagnostic tests (RDTs) against rK39 are available to aid in the diagnosis of visceral leishmaniasis (VL). Although these rK39 RDTs have been developed, validated and approved for use with serum, they are universally performed using whole blood. It was therefore necessary to determine whether this RDT is as sensitive on whole blood as on serum. METHOD AND PRINCIPAL FINDINGS: In this study we compared the rK39 RDT on serum and blood samples from 624 individuals with symptoms of VL attending the outpatient clinic at the Rajendra Memorial Research Institute of Medical Sciences, Patna, India. A total of 251 cases (40%) were both serum and blood-positive and 26 cases (4%) were identified as blood-negative and serum-positive. These 26 individuals in general had low titer antibodies against rK39 as determined by ELISA and follow-up on most of these individuals revealed none had persistent VL symptoms. The Cohen kappa index comparing blood and serum was 0.88 indicating excellent concordance. CONCLUSION: Although the concordance was excellent, it is possible to miss rK39 positive individuals when using blood and the titer of anti-rK39 antibodies is low. We recommend that when an individual from an endemic area has obvious clinical symptoms of VL and the whole blood rK39 RDT is negative, that the test should be redone 2-3 weeks later if the symptoms persist.