Diagnosis Of Post-kala-azar Dermal Leishmaniasis Research Articles

Evaluation of blood based quantitative PCR as a molecular diagnostic tool for post kala-azar dermal leishmaniasis (PKDL).

Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis. 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR. The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease.

Post-kala-azar dermal leishmaniasis (PKDL) drug efficacy study landscape: A systematic scoping review of clinical trials and observational studies to assess the feasibility of establishing an individual participant-level data (IPD) platform.

Post-kala-azar dermal leishmaniasis (PKDL) is a dermatosis which can occur after successful treatment of visceral leishmaniasis (VL) and is a public health problem in VL endemic areas. We conducted a systematic scoping review to assess the characteristics of published PKDL clinical studies, understand the scope of research and explore the feasibility and value of developing a PKDL individual patient data (IPD) platform. A systematic review of published literature was conducted to identify PKDL clinical studies by searching the following databases: PubMed, Scopus, Ovid Embase, Web of Science Core Collection, WHO Global Index Medicus, PASCAL, Clinicaltrials.gov, Ovid Global Health, Cochrane Database and CENTRAL, and the WHO International Clinical Trials Registry Platform. Only prospective studies in humans with PKDL diagnosis, treatment, and follow-up measurements between January 1973 and March 2023 were included. Extracted data includes variables on patient characteristics, treatment regimens, diagnostic methods, geographical locations, efficacy endpoints, adverse events and statistical methodology. A total of 3,418 records were screened, of which 56 unique studies (n = 2,486 patients) were included in this review. Out of the 56 studies, 36 (64.3%) were from India (1983-2022), 12 (21.4%) from Sudan (1992-2021), 6 (10.7%) were from Bangladesh (1991-2019), and 2 (3.6%) from Nepal (2001-2007). Five (8.9%) studies were published between 1981-1990 (n = 193 patients), 10 (17.9%) between 1991-2000 (n = 230 patients), 10 (17.9%) between 2001-2010 (n = 198 patients), and 31 (55.4%) from 2011 onwards (n = 1,865 patients). Eight (14.3%) were randomised clinical trials, and 48 (85.7%) were non-randomised studies. The median post-treatment follow-up duration was 365 days (range: 90-540 days) in 8 RCTs and 360 days (range: 28-2,373 days) in 48 non-randomised studies. Disease diagnosis was based on clinical criterion in 3 (5.4%) studies, a mixture of clinical and parasitological methods in 47 (83.9%) and was unclear in 6 (10.7%) studies. Major drugs used for treatment were miltefosine (n = 636 patients), liposomal amphotericin B (L-AmB) (n = 508 patients), and antinomy regimens (n = 454 patients). Ten other drug regimens were tested in 270 patients with less than 60 patients per regimen. Our review identified studies with very limited sample size for the three major drugs (miltefosine, L-AmB, and pentavalent antimony), while the number of patients combined across studies suggest that the IPD platform would be valuable. With the support of relevant stakeholders, the global PKDL community and sufficient financing, a PKDL IPD platform can be realised. This will allow for exploration of different aspects of treatment safety and efficacy, which can potentially guide future healthcare decisions and clinical practices.

Generalized old world leishmaniasis: first Moroccan case in an immunocompetent adult?

RésuméIntroductionLa leishmaniose dermique post kala-azar (LDPK) est un syndrome cutané observé après un traitement de leishmaniose viscérale (LV). Nous décrivons un cas probable chez un adulte immunocompétent.ObservationIl s'agit d'un homme âgé de 36 ans, originaire du sud du Maroc, avec antécédent de LV deux ans auparavant, traitée par antimoniate de méglumine et amphotéricine B avec une bonne évolution, hospitalisé en dermatologie pour un placard papulo-nodulaire érythémateux de la face. Le tableau dermatologique remontait à 6 mois avec une atteinte inaugurale de la muqueuse buccale. L'examen de la muqueuse buccale retrouvait une ulcération du tiers postérieur de la langue et un aspect papillomateux du voile du palais. La biopsie et le frottis cutanés retrouvaient des amastigotes de Leishmania. La PCR ITS1 était positive (genre Leishmania). Le diagnostic de LDPK était évoqué. Le patient a bénéficié d'injections intra-musculaires d'antimoniate de méglumine avec une bonne évolution.ConclusionÀ notre connaissance, il s'agit du premier cas de leishmaniose généralisée évoquant une LDPK décrit au Maroc chez un adulte immunocompétent.

Real-Time Fluorimetry Loop-Mediated Isothermal Amplification for Diagnosis of Leishmaniasis and as a Tool for Assessment of Cure for Post-Kala-Azar Dermal Leishmaniasis.

Despite the dwindling number of visceral leishmaniasis (VL) cases in India, there is an urgent need for early and unequivocal diagnostics for controlling and preventing the reemergence of VL. Post-kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, serves as a reservoir of the parasite. Diagnosis of PKDL, especially the macular variant, is challenging and poses impediment toward attainment of VL elimination. In this study, a real-time fluorimetry loop-mediated isothermal amplification (RealAmp) assay has been established for the detection of different clinical manifestations of leishmaniasis. The study included 150 leishmaniasis patients (25 VL, 25 cutaneous leishmaniasis [CL], and 100-PKDL) along with 120 controls. The assay demonstrated sensitivity of 100% (95% CI: 86.68-100) for diagnosis of VL and PKDL (95% CI: 79.61-100) and 96% (95% CI: 86.68-100) for CL with 100% specificity. Moreover, considering the cardinal role of PKDL, diagnosis using minimally invasive slit aspirate was explored, which demonstrated remarkable sensitivity of 96% (95% CI: 87.64-98.47). As a test of cure for PKDL, RealAmp successfully detected parasite in two of posttreatment cases who later reported relapse on follow-up. Also, direct sample lysis using slit aspirate was attempted in a small group that yielded sensitivity of 89% (95% CI: 67.20-96.90). RealAmp depicted excellent diagnostic accuracy in the diagnosis of leishmaniasis in concordance with the established SYBR Green I-based (Molecular Probes, Eugene, OR) visual loop-mediated isothermal amplification (LAMP) and the reference comparator real-time PCR. The study endorsed the employment of LAMP either as visual-LAMP or RealAmp for an accurate and expeditious diagnosis of PKDL and as a tool for assessment of cure.

Advancement in Molecular Diagnosis of Post Kala-Azar Dermal Leishmaniasis.

Post kala-azar dermal leishmaniasis (PKDL), a clinical sequela of visceral leishmaniasis (VL), plays a critical role in the anthroponotic transmission of VL, particularly in the Indian subcontinent (ISC). The early, accurate, and feasible diagnosis of PKDL is essential for the attainment and sustenance of VL elimination goal in ISC. PKDL poses a stumbling block for this goal, considering the heterogeneity presented with regard to time after cure of VL and onset of PKDL, chronicity, and clinical variations. In most of the endemic regions the diagnosis is based on clinical examination, previous history of VL, by ruling out other disorders, and by the response to treatment. The conventional microscopic examination involving the demonstration of Leishman–Donovan bodies (LDB) in macrophages is pathognomonic, however, the method faces constraints in terms of being invasive, less sensitive, technically demanding, and difficult to be applied in field conditions. Serological evidences are of limited use because antileishmanial antibodies remain positive for years after VL treatment. Molecular tools such as PCR, nested-PCR, Q-PCR overcome these constraints and have become increasingly popular due to their high sensitivity and specificity along with their applicability in diverse clinical samples. Molecular methods not only play a key role in early detection but also provide quantification and monitoring of treatment effectiveness.NCBI PubMed search tool was used for locating, selecting, and extracting research articles pertinent for this review article by using various related terminologies on the molecular diagnosis of leishmaniasis.

Histopathological study of post kala-azar dermal leishmaniasis and its importance for diagnosis in a peripheral tertiary institution

Histopathological study of post kala-azar dermal leishmaniasis and its importance for diagnosis in a peripheral tertiary institution - IJCED- Print ISSN No: - 2581-4710 Online ISSN No:- 2581-4729 Article DOI No:- 10.18231/2581-4729.2019.0003, IP Indian Journal of Clinical and Experimental Dermatology-IP Indian J Clin E

Evaluation of anti-gal enzyme-linked immunosorbent assay for the diagnosis of Indian post-kala-azar dermal leishmaniasis.

Elimination of kala azar from India is challenging as there are potential reservoirs of Leishmania donovani in patients with post-kala-azar dermal leishmaniasis (PKDL). The vast repertoire of carbohydrate moieties on L. donovani is known to elicit specific and strong humoral responses in patients with kala azar. The present study was undertaken to evaluate the diagnostic performances of anti-gal antibodies using enzyme-linked immunosorbent assay for successful serological diagnosis of PKDL in Indian patients and to differentiate cases of past cured visceral leishmaniasis infections. We developed Gal enzyme-linked immunosorbent assay to measure specific anti-gal IgG isotype in the sera of 71 Indian patients with PKDL. The diagnostic efficacy of the newly developed assay was evaluated for precision, sensitivity and accuracy. Gal2 enzyme-linked immunosorbent assay revealed three-fold increased anti-gal titers in 71 patients with active PKDL compared to controls. Subclass enzyme-linked immunosorbent assay analysis further revealed enhanced IgG2 and IgG3 anti-gal titers in patients with PKDL compared to control subjects. The rank order for specificity and sensitivity for IgG subclasses was IgG3>IgG2>IgG4>IgG1. The area under the curve values of 0.98 and 0.99 were obtained for IgG and IgG3 Gal2 enzyme-linked immunosorbent assays respectively. Overall sensitivity and specificity were 95.7% (95% CI: 88.1-99.1) and 98.1% (95% confidence interval: 90.1-99.9), and 98.5% (95% CI: 92.4-99.9) and 98.1% (95% CI: 90.1-99.9), respectively. Intra-assay coefficient of variation was 1.5% and inter-assay coefficient of variation was 11.7%. The Gal2 enzyme-linked immunosorbent assay needs to be further investigated in mass surveys. Taken together, anti-gal titers detected through Gal2 enzyme-linked immunosorbent assay can serve as an effective diagnostic tool in disease elimination setting and help in better case management in endemic districts.

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

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

Glycoproteins in circulating immune complexes are biomarkers of patients with Indian PKDL: A study from endemic districts of West Bengal, India.

BackgroundPost Kala Azar Dermal Leishmaniasis (PKDL) occurs as dermal consequence of previous Visceral Leishmaniasis (VL) infection and serves as an important reservoir for transmission of VL. Diagnosis of PKDL is often challenging for its symptomatic resemblance to other co-endemic diseases like Leprosy or Vitiligo. Parasitological examination by slit-skin smear and culture are the standard methods but lack high sensitivity. Thus, for efficient control of VL, reliable diagnostic and prognostic assay of PKDL are required.ObjectivePreviously, glycoproteins (9-OAcSA) have been reported as promising biomarkers of Indian VL patients. However, till date, the status of glycans in Indian PKDL patients remains unexplored. Accordingly, in this study, the glyco-profile of PKDL Circulating Immune Complexes (CICs) as compared to other cross diseases like Vitiligo and Leprosyhas been investigated. Further, a novel Glyco CIC assay has been developed for efficient Indian PKDL patient diagnosis.Methods/principal findingIn the present study, 90 PKDL patients were enrolled from 3 VL endemic districts of West Bengal during 2015–16. Glycosylation profile of isolated CICs from sera of PKDL patients were initially analyzed through gradient SDS gel electrophoresis followed by PAS silver double staining, which revealed the presence of several glycan rich PKDL specific proteins of varying molecular weights. To further characterize the glyco-profile of acid dissociated affinity purified immuno-reactive antigens present in the CICs, glycosylation was demonstrated in these purified CIC antigens by DIG glycan differentiation kit with or without glycosidase as well as neuraminidase treatment. Diagnostic evaluation of the newly developed colorimetric Glyco CIC assay through Receiver Operating Characteristic (ROC) curve analysis revealed excellent (0.99) AUC value as compared to other conventional serodiagnostic assays like PEG CIC, Parasite ELISA (IgG and IgM). Additionally, longitudinal monitoring of 18 PKDL patients further revealed its good prognostic utility.ConclusionThese results highlight the glycosylation status of CICs among Indian PKDL patients present in all the studied endemic districts of West Bengal. These PKDL biomarkers were completely absent in cross diseases like Vitiligo and Leprosy. Further, the newly developed Glyco CIC assay had an improved sensitivity of 95.6%, specificity of 99.3%, NPV of 97.1% and PPV of 98.9%.

Revisiting the role of the slit-skin smear in the diagnosis of Indian post-kala-azar dermal leishmaniasis.

Post kala azar dermal leishmaniasis (PKDL) is a neglected dermatosis that develops as a sequel to kala azar after apparent complete treatment. Being a non life threatening condition, patients often delay treatment thereby maintaining a reservoir of infection. The diagnosis of PKDL rests on the demonstration of the parasite in tissue smears, immune diagnosis by detection of parasite antigen or antibody in blood, or detection and quantitation of parasite DNA in tissue specimens. Sophisticated molecular tests are not only expensive but also need skilled hands and expensive equipment. To be useful, diagnostic methods must be accurate, simple and affordable for the population for which they are intended. This study was designed to assess functionality and operational feasibility of slit-skin smear examination. Sensitivity and specificity was evaluated by performing slit-skin smear and histo-pathological examination in 46 PKDL patients and the results were compared with the parasite load in both the slit aspirate and tissue biopsy specimens by performing quantitative Real-time PCR (Q-PCR). The slit-skin smear examination was more sensitive than tissue biopsy microscopy. The parasite loads significantly differed among various types of clinical lesions (P < 0.05). The threshold of parasite load for detection by SSS microscopy was 4 parasites/μl in slit aspirate and 60 parasites/μg tissue DNA in tissue biopsy while that for tissue microscopy was 63 parasites/μl and 502 parasites/μg tissue DNA respectively. As detection of Leishmania donovani bodies may be challenging in inexperienced hands, the microscopic structure of these has been detailed along with a comprehensive discussion of pre analytical, analytical and post analytical variables affecting its identification. To facilitate the diagnosis of PKDL, some scenarios have been suggested taking into consideration the clinical, epidemiological, immunological and microscopic aspects. Such evidence based medicine helps minimize intuition, systematize clinical experience and provides a diagnostic rationale as sufficient grounds for a clinical decision.

Post-Kala-Azar Dermal Leishmaniasis Associated With Oral Lesions: A Rare Case Report

Post-kala-azar dermal leishmaniasis (PKDL) is a dermatosis that usually occurs after visceral leishmaniasis (VL) caused by Leishmania donovani and characterized by macular, maculopapular, and nodular skin lesions on the whole body surface. It is a late cutaneous manifestation of VL in untreated or inadequately treated patients, first described by Brahmachari in 1922. In India, PKDL occurs in 6–20% of the cases of VL, 6 months to 5 years after the apparently successful treatment of VL. The diagnosis of PKDL presents a challenge due to low parasite burden in the lesions. We report a case of PKDL that presented with polymorphic skin lesions along with the involvement of the oral cavity including an insight into the recent advances in diagnosis, pathogenesis, and case management.

PKDL--A Silent Parasite Pool for Transmission of Leishmaniasis in Kala-azar Endemic Areas of Malda District, West Bengal, India.

Post Kala-azar Dermal Leishmaniasis (PKDL) is a chronic but not life-threatening disease; patients generally do not demand treatment, deserve much more attention because PKDL is highly relevant in the context of Visceral Leishmaniasis (VL) elimination. There is no standard guideline for diagnosis and treatment for PKDL. A species-specific PCR on slit skin smear demonstrated a sensitivity of 93.8%, but it has not been applied for routine diagnostic purpose. The study was conducted to determine the actual disease burden in an endemic area of Malda district, West Bengal, comparison of the three diagnostic tools for PKDL case detection and pattern of lesion regression after treatment. The prevalence of PKDL was determined by active surveillance and confirmed by PCR based diagnosis. Patients were treated with either sodium stibogluconate (SSG) or oral miltefosine and followed up for two years to observe lesion regression period. Twenty six PKDL cases were detected with a prevalence rate of 27.5% among the antileishmanial antibody positive cases. Among three diagnostic methods used, PCR is highly sensitive (88.46%) for case confirmation. In majority of the cases skin lesions persisted after treatment completion which gradually disappeared during 6–12 months post treatment period. Reappearance of lesions noted in two cases after 1.5 years of miltefosine treatment. A significant number of PKDL patients would remain undiagnosed without active mass surveys. Such surveys are required in other endemic areas to attain the ultimate goal of eliminating Kala-azar. PCR-based method is helpful in confirming diagnosis of PKDL, referral laboratory at district or state level can achieve it. So a well-designed study with higher number of samples is essential to establish when/whether PKDL patients are free from parasite after treatment and to determine which PKDL patients need treatment for longer period.

A Study on Health Seeking Behaviors of Patients of Post-Kala-Azar Dermal Leishmaniasis.

Post-Kala-Azar Dermal Leishmaniasis (PKDL) remains a major public health threat in Bangladesh. A cross-sectional study was carried out in Surya Kanta Kala azar Research Centre (SKKRC), Mymensingh, from January 2012 to July 2013 to evaluate the health seeking behaviour and the length of delay of PKDL management. The consecutive 200 diagnosed PKDL cases that got treatment in SKKRC hospital were subjected to evaluation. Most (98%) of the patients were not aware and had no knowledge about PKDL, though 87.5% had a history of history of Kala-azar treatment. Many patients reported first to village doctor (15.5%), the pharmacy shop (10%), or traditional health provider (7.5%) upon recognition of symptom. The time between the initial symptom recognition and first medical consultation (patient delay) ranged from 10 days to 4745 days (13 years) with a median of 373 days (mean: 696; IQR: 138 to 900 days). The time between first medical consultations to definite treatment (system delay) ranged from 0 days to 1971 days (5.4 years), with a median delay of 14 days (mean: 46.48; IQR: 7 to 44 days) that was reported in this study. Age, education, occupation, and residential status had significant association with patient delay (P < 0.05). Educational status, occupation, number of treatment providers, and first health care provider had a significant association with system delay (P < 0.05). Success in PKDL diagnosis and treatment requires specific behavior from patients and health care providers which facilitate those practices.

POST KALA-AZAR DERMAL LEISHMANIASIS WITH ULCERATION ON FOOT: AN ATYPICAL CASE PRESENTATION SUCCESSFULLY TREATED WITH MILTEFOSINE

Visceral Leishmaniasis (VL) or Kala-Azar (KA) is highly endemic in the Indian subcontinent particularly in Bihar. Post Kala-azar Dermal Leishmaniasis (PKDL) is a known reservoir for leishmania parasites for transmission of VL. The kala-azar elimination program was launched by the government of three countries of the Indian subcontinent in the year 2005, which aims to eliminate kala-azar by 2015 and PKDL by 2018. PKDL diagnosis and management has not been addressed properly. In this case report we have described a case of PKDL with ulcer and atypical presentation who was subsequently diagnosed by demonstration of Leishmania donovani bodies in the skin snips through microscopy, kinetoplast-DNA nested PCR and clinically. The patient was treated with miltefosine capsules in the dose of 50 mg twice-a-day for twelve-weeks and responded completely. This case report assumes great importance as prompt diagnosis and treatment of PKDL is essential in the kala-azar elimination program.

Post-kala-Azar dermal leishmaniasis: A diagnostic dilemma in a nonendemic area.

Post-kala-Azar dermal leishmaniasis: A diagnostic dilemma in a nonendemic area.

Comparative Evaluation of PCR and Imprint Smear Microscopy Analyses of Skin Biopsy Specimens in Diagnosis of Macular, Papular, and Mixed Papulo-Nodular Lesions of Post-Kala-Azar Dermal Leishmaniasis

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), particularly the macular form, is difficult when based on microscopy. This study compared the results of nested PCR (91.9% positive samples) with imprint smear microscopy (70.9% positive samples) for 62 PKDL samples. We found that nested PCR, which indicated 87.5% positivity for the macular lesions, compared to 41.6% positivity by imprint smear microscopy, is an efficient method for early diagnosis of PKDL.

Fine‐needle sampling provides appreciable diagnostic yield in lesions of post kala azar dermal leishmaniasis: Analysis of four cases from North Eastern India

Post-kala-azar dermal leishmaniasis (PKDL) is a sequel of visceral leishmaniasis (VL), usually occurring 6 months to 3 years after VL. Spectrum of cutaneous lesions in PKDL can be hypopigmented macules, nodules, plaques, or erythema. It is usually diagnosed clinically, supplemented by ancillary techniques like skin smear examination, histopathology, polymerase chain reaction, and monoclonal antibody test. Literature on the role of cytology in the diagnosis of PKDL is extremely limited. Here we highlight the appreciable yield of fine-needle sampling in four cases of PKDL, which may be considered as a useful diagnostic aid.

Reliable diagnosis of post‐kala‐azar dermal leishmaniasis (PKDL) using slit aspirate specimen to avoid invasive sampling procedures

Confirmatory diagnosis of post-kala-azar dermal leishmaniasis (PKDL) is primarily based on invasive skin biopsy procedure. We evaluated the utility of minimally invasive slit aspirate specimen for serological and molecular diagnosis of PKDL. We compared the PKDL diagnosis using slit aspirate and skin biopsy specimens from the same patients. Serological diagnosis using rK39 strip test was performed with serum and slit aspirate sample; molecular diagnosis for parasite detection and quantification was carried out by quantitative real-time PCR (Q-PCR) with skin biopsy and slit aspirate sample. The rK39 serological strip test was positive in all PKDL cases with both slit aspirate and serum samples (n=50) and negative in all control cases (n=24), giving a sensitivity of 100% (95% CI: 92.9-100%) and a specificity of 100% (95% CI: 86.2-100%). Quantitative-PCR detected parasite in all PKDL slit aspirates (n=50, sensitivity=100%, 95% CI: 92.9-100%) and tissue biopsies (n=46, sensitivity=100%, 95% CI: 92.3-100; it was negative in all controls including dermal tissues (n=24) and slit aspirates (n=24), giving specificity of 100% (95% CI: 86.2-100%). The parasite load in tissue and slit aspirate samples was significantly (P<0.0001) correlated (r=0.82). Slit aspirates are a simpler and minimally invasive sampling technique for initial screening by serology followed by confirmatory diagnosis of PKDL with microscopy and/or Q-PCR. The simplified procedure has the potential for epidemiological studies and assessment of cure in PKDL.

PCR-Based Detection of Leishmania DNA in Skin Samples of Post Kala-Azar Dermal Leishmaniasis Patients from an Endemic Area of Bangladesh

Post kala-azar dermal leishmaniasis (PKDL) is a sequel of visceral leishmaniasis (VL) and PKDL patients are an important reservoir for anthroponotic transmission of VL. Therefore, diagnosis and treatment of PKDL is important for the kala-azar elimination program in South Asia, including Bangladesh. While definitive diagnosis of PKDL is still based on microscopy, despite the low sensitivity of this method of diagnosis, PCR for identification of kinetoplast DNA (kDNA) from Leishmania parasites is expected to be a rapid and sensitive diagnostic method. We attempted PCR-based diagnosis from skin biopsy specimens and compared PCR to other available detection methods in order to determine the acceptability and feasibility of the PCR diagnostic method in an endemic area of VL in Bangladesh. Both skin biopsy specimens and blood samples were collected from 110 patients suspected to have PKDL from 6 subdistrict health complexes in Mymensingh, Bangladesh. Using microscopy, we identified 32 samples (29.1%) that were positive for Leishmania. Immunochromatography tests indicated that 85 samples (77.3%) were positive for Leishmania. In contrast, a total of 104 (94.5%) samples tested positive using nested PCR, while unaffected portions of skin from PKDL patients tested negative. Sequencing analysis of the PCR products indicated that the amplified portion had more than 98% nucleotide sequence identity to the Leishmania donovani reference strain, D10. These findings indicate that the PCR method using a skin biopsy is highly sensitive and useful for confirmatory diagnosis of PKDL.

Enhanced case detection and improved diagnosis of PKDL in a Kala-azar-endemic area of Bangladesh.

ObjectivesTo support the Bangladesh National Kala-azar Elimination Programme (NKEP), we investigated the feasibility of using trained village volunteers for detecting post-kala-azar dermal leishmaniasis (PKDL) cases, using polymerase chain reaction (PCR) for confirmation of diagnosis and treatment compliance by PKDL patients in Kanthal union of Trishal sub-district, Mymensingh, Bangladesh.MethodsIn this cross-sectional study, Field Research Assistants (FRAs) conducted census in the study area, and the research team trained village volunteers on how to look for PKDL suspects. The trained village volunteers (TVVs) visited each household in the study area for PKDL suspects and referred the suspected PKDL cases to the study clinic. The suspected cases underwent physical examinations by a qualified doctor and rK39 strip testing by the FRAs and, if positive, slit skin examination (SSE), culture, and PCR of skin specimens and peripheral buffy coat were done. Those with evidence of Leishmania donovani (LD) were referred for treatment. All the cases were followed for one year.ResultsThe total population of the study area was 29,226 from 6,566 households. The TVVs referred 52 PKDL suspects. Probable PKDL was diagnosed in 18 of the 52 PKDL suspect cases, and PKDL was confirmed in 9 of the 18 probable PKDL cases. The prevalence of probable PKDL was 6.2 per 10,000 people in the study area. Thirteen PKDL suspects self-reported from outside the study area, and probable and confirmed PKDL was diagnosed in 10 of the 13 suspects and in 5 of 10 probable PKDL cases respectively. All probable PKDL cases had hypopigmented macules. The median time for PKDL development was 36 months (IQR, 24–48). Evidence of the LD parasite was documented by SSE and PCR in 3.6% and 64.3% of the cases, respectively. PCR positivity was associated with gender and severity of disease. Those who were untreated had an increased risk (odds ratio = 3.33, 95%CI 1.29–8.59) of having persistent skin lesions compared to those who were treated. Patients' treatment-seeking behavior and treatment compliance were poor.ConclusionImproved detection of PKDL cases by TVVs is feasible and useful. The NKEP should promote PCR for the diagnosis of PKDL and should find ways for improving treatment compliance by patients.