Post-kala-azar Dermal Leishmaniasis Research Articles

Quantifying the Infectiousness of Post-Kala-Azar Dermal Leishmaniasis Toward Sand Flies.

On the Indian subcontinent, visceral leishmaniasis (VL) incidence is on track to reach elimination goals by 2020 in nearly all endemic districts. Although not included in official targets, previous data suggest post-kala-azar dermal leishmaniasis (PKDL) patients can act as an infection reservoir. We conducted xenodiagnosis on 47 PKDL patients and 15 VL patients using laboratory-reared Phlebotomus argentipes. In direct xenodiagnosis, flies were allowed to feed on the patient's skin for 15 minutes. For indirect xenodiagnosis, flies were fed through a membrane on the patient's blood. Five days later, blood-fed flies were dissected and examined by microscopy and/or polymerase chain reaction (PCR). A 3-mm skin snip biopsy (PKDL) or venous blood (VL) was processed by quantitative PCR. Twenty-seven PKDL patients (57.4%) had positive results by direct and/or indirect xenodiagnosis. Direct was significantly more sensitive than indirect xenodiagnosis (55.3% vs 6.4%, P < .0001). Those with positive xenodiagnosis had median skin parasite loads >1 log10 unit higher than those with negative results (2.88 vs 1.66, P < .0001). In a multivariable model, parasite load, nodular lesions, and positive skin microscopy were significantly associated with positive xenodiagnosis. Blood parasite load was the strongest predictor for VL. Compared to VL, nodular PKDL was more likely and macular PKDL less likely to result in positive xenodiagnosis, but neither difference reached statistical significance. Nodular and macular PKDL, and VL, can be infectious to sand flies. Active PKDL case detection and prompt treatment should be instituted and maintained as an integral part of VL control and elimination programs.

Development of a sandwich ELISA to detect Leishmania 40S ribosomal protein S12 antigen from blood samples of visceral leishmaniasis patients

BackgroundVisceral leishmaniasis (VL), caused by Leishmania donovani complex parasites, is a neglected parasitic disease that is generally fatal if untreated. Despite decades of research to develop a sensitive VL diagnostic test, definitive diagnosis of VL still mainly relies on the visualization of the parasite in aspirates from the spleen, liver or bone marrow, an invasive and dangerous process with variable sensitivity. A sensitive assay that can detect Leishmania antigen from blood samples will help confirm cause, cure or recurrence of VL.MethodsIn this study, rabbit polyclonal antibodies were raised against eight recombinant Leishmania proteins that are highly abundant in Leishmania. The antibodies were purified and labeled with biotin for developing a prototype sandwich enzyme-linked immunosorbent assay (ELISA).ResultsThe ELISA for the Leishmania 40S ribosomal protein S12 detected target antigen with the highest sensitivity and specificity and could detect 1 pg of purified protein or as few as 60 L. donovani parasites. The 40S ribosomal protein S12 sandwich ELISA could detect the target antigen from Peripheral Blood Mononuclear Cell (PBMC) samples in 68% of VL patients and post-kala-azar dermal leishmaniasis (PKDL) patients, providing an estimation of parasitemia ranging from 15 to 80 amastigotes per ml of blood.ConclusionThese results indicate that the 40S ribosomal protein S12 sandwich ELISA warrants further tests with more clinical samples of VL patients and other parasitic diseases. It is hopeful that this ELISA could become a useful tool for confirming VL diagnosis, monitoring treatment progress, disease recurrence and possibly detecting asymptomatic Leishmania infections with a high parasite load.

Phagocytes and the Leishmania Parasite: A Marriage of Convenience

ABSTRACTThe current drive by the Government of India to eliminate Leishmaniasis has pinpointed post-kala azar dermal leishmaniasis (PKDL) as the strongest contender for the disease reservoir. This emphasizes the necessity to consider the eradication of PKDL as top priority, and hinges on its early diagnosis and management. We undertook this challenge and have provided insights into Leishmania biology, by focusing our efforts in (i) delineating the immunopathogenesis of PKDL, a disease unique to South Asia (ii) developing diagnostic/prognostic tools for monitoring antileishmanial treatment in patients with visceral leishmaniasis and PKDL. In order to delineate the immunopathogenesis of PKDL, it was established that the parasite adopts multiple approaches to deviously manipulate host monocytes/macrophages, and thus facilitate parasite survival and disease progression. The parasite adopts a multipronged approach that includes attenuation of the oxidative burst within phagocytes, polarization of monocytes/ macrophages towards alternate activation, enhancement of CD8 T-cell exhaustion and a decreased presence of Langerhans cells. Identification of these immunological changes have allowed for development of biomarkers that have been exploited to develop diagnostic and prognostic markers for monitoring the disease progression, either in terms of antibody based markers, or quantification of the parasite load, the latter being the most definitive approach. Measurement of parasite load has proved to be an effective tool for monitoring the effectiveness of chemotherapy. Taken together, the identification of biomarkers and new chemotherapeutic modalities has helped in improved management and potential elimination of leishmaniasis.

Atypical leishmaniasis: A global perspective with emphasis on the Indian subcontinent.

BackgroundAmong the neglected tropical diseases, leishmaniasis continues to be prevalent in many tropical and subtropical countries despite international, national, and local efforts towards its control and elimination over the last decade. This warrants a critical evaluation of such factors as under-reporting, asymptomatic infections, post kala azar dermal leishmaniasis (PKDL) cases, and drug resistance. In this review, we highlight lesser-understood atypical presentations of the disease involving atypical parasite strains against a background of classical leishmaniasis with a focus on the Indian subcontinent.Methods and findingsA literature review based on endemic areas, the nature of disease manifestation, and underlying causative parasite was performed with data collected from WHO reports for each country. Searches on PubMed included the term ‘‘leishmaniasis” and “leishmaniasis epidemiology” alone and in combination with each of the endemic countries, Leishmania species, cutaneous, visceral, endemic, non-endemic, typical, classical, atypical, and unusual with no date limit and published in English up to September 2017. Our findings portray a scenario with a wider distribution of the disease in new endemic foci, with new discoveries of parasite-driven atypical disease manifestations in different regions of the world. Unlike the classical picture, some Leishmania species are associated with more than one disease presentation, e.g., the L. donovani complex, generally associated with the visceral form, is now also associated with a cutaneous disease presentation, while L. tropica species complex, known to cause cutaneous disease, can cause viscerotropic disease. This phenomenon points towards the discovery of novel parasite variants as etiologic agents of atypical disease manifestations and represents an excellent opportunity to identify and study genes that control disease virulence and tropism.ConclusionsThe increased recognition of atypical leishmaniasis as an outcome of parasite variants has major implications for leishmaniasis control and elimination. Identifying molecular correlates of parasite isolates from distinct regions associated with different disease phenotypes is required to understand the current epidemiology of leishmaniasis in regions with atypical disease.

Knowledge, stigma, health seeking behaviour and its determinants among patients with post kalaazar dermal leishmaniasis, Bihar, India.

BackgroundLesishmaniasis is a neglected tropical disease endemic in Bihar, India. Inappropriate health seeking behaviour of post kala-azar dermal leishmaniasis (PKDL) patients may increase the disease duration, severity and transmissibility. Simultaneously, lack of knowledge and perceived stigma may also increase the length of delay in receiving treatment. This ultimately effects the kala-azar elimination program.MethodsA cross sectional study was conducted in 120 confirmed PKDL patients, aged 18 years and older. Data related to knowledge and health seeking behaviour was collected by a pre-tested questionnaire. EMIC stigma scale was used for assessing the perceived stigma. Patients were personally interviewed after taking informed consent. Data analysis was done by using SPSS 16 software.ResultsThe time between appearance of symptoms and first medical consultation (patient delay) ranged from 15 days to 5475 days (15 years) with a median of 285 days. The time between first medical consultations to onset of specific treatment (system delay) ranged from 2 to 5475 days with a median of 365 days. Many patients approached first to quacks (8.4%), homeopathic and ayurvedic practitioners (25.8%) upon recognition of symptoms. Majority of the patients (68.3%) had poor knowledge about PKDL and its vector. Type of skin lesions and gender had significant association with patient delay and system delay respectively (p<0.05). Distance to primary health centre (PHC) had significant association with patients delay as well as system delay (p<0.05). Patients with younger age, unmarried and polymorphic lesions had higher stigma (p<0.05). Patients with PKDL feel stigmatized in different areas.ConclusionPKDL treatment delays were unacceptably high and patients had poor knowledge compounded with feelings of stigmatization. To reduce the delay, a system may be evolved to establish some sort of public-private collaboration, besides awareness programs should be tailored, and implemented for improving the patient education regarding the disease and its linkage with VL.

Using focused pharmacovigilance for ensuring patient safety against antileishmanial drugs in Bangladesh\u2019s National Kala-azar Elimination Programme

BackgroundAdverse effects of antileishmanial drugs can affect patients’ quality of life and adherence to therapy for visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). In Bangladesh, there are 26 treatment centers that manage leishmaniasis cases coming from 100 endemic upazilas (subdistricts) of 26 districts (these include VL, PKDL, treatment failure, and relapse VL and cutaneous leishmaniasis cases). This study aimed to investigate the feasibility of using focused pharmacovigilance for VL (VLPV) in Bangladesh’s National Kala-azar Elimination Programme for the early detection and prevention of expected and unexpected adverse drug reactions (ADRs).MethodsThis activity has been going on since December 2014. Activity area includes secondary public hospital or Upazila health complex (UHC) in hundred sub districts and Surya Kanta Kala-azar Research Center (SKKRC) in Mymensingh District, a specialized center for management of complicated VL and PKDL cases. Communicable Disease Control (CDC) of the Directorate General of Health Services (DGHS) assigned twenty five of hundred UHCs and SKKRC (total 26) as treatment centers depending on their suitable geographical location. This was implemented for better management of VL cases with Liposomal Amphotericin B (AmBisome®) to ensure patient convenience and proper utilization of this expensive donated drug. A VLPV expert committee and a UHC VLPV team were established, an operational manual and pharmacovigilance report forms were developed, training and refresher training of health personnel took place at UHCs and at the central level, collected information such as patient data including demographics, treatment history and response, adverse events were analyzed. This report includes information for the period from December 2014 to December 2016.ResultsFrom December 2014 to December 2016, 1327 leishmaniasis patients were treated and 1066 (80%) were available for VLPV. Out of these, 57, 33, 9, and 1% were new VL, PKDL, VL relapse, and other cases, respectively. Liposomal amphotericin B was mostly used (82%) for case management, followed by miltefosine (20%) and paromomycin (3%). Out of the 1066 patients, 26% experienced ADRs. The most frequent ADR was fever (17%, 176/1066), followed by vomiting (5%, 51/1066). Thirteen serious adverse events (SAEs) (eight deaths and five unexpected SAEs) were observed. The expert committee assessed that three of the deaths and all unexpected SAEs were possibly related to treatment. Out of the five unexpected SAEs, four were miltefosine-induced ophthalmic complications and the other was an AmBisome®-induced avascular necrosis of the nasal alae. The Directorate General of the Drug Administration entered the ADRs into the World Health Organization Uppsala Monitoring Centre (WHO-UMC) VigiFlow database.ConclusionsThis study found that VLPV through NKEP is feasible and should be continued as a routine activity into the public health system of Bangladesh to ensure patient safety against anti-leishmanial drugs.

Treatment of widespread nodular post kala-azar dermal leishmaniasis with extended- dose liposomal amphotericin B in Bangladesh: A series of four cases

Background: Post Kala-azar Dermal Leishmaniasis (PKDL) is a sequela of Visceral Leishmaniasis (VL) and suggested as reservoirs of parasite. Currently, Miltefosine (MF), Amphotericin B Deoxycholate (ABD) and Liposomal Amphotericin B (LAmB) are recommended by World health organization (WHO) as treatment options for PKDL. But single MF or LAmB often causes incomplete cure specially cases with extensive lesion. To overcome this problem here we describe four cases of PKDL who had widespread nodular and macular lesions and were treated with two cycles of LAmB doses with 20 mg/kg body weight divided into 4 equal doses and administered every alternate day. Methods & Materials: Patients admitted to the Surya kanta kala-azar research centre (SKKRC) with extensive nodular or mixed lesion were considered to treat with LAmB. Hospital physician collected patients’ history and performed physical examination. Suspected cases were confirmed by microscopic examination of slit skin smear. At first, LAmB treatment was initiated at the dose of 20 mg/kg into four divided doses every alternate day. Before starting treatment all haematological and biochemical markers were checked. Second cycle was initiated for those who displayed marked improvement but not complete recovery. Finally, a follow up physical and microscopic examination was also done to evaluate the treatment outcome. Results: Of them, two had previous VL history and two had no previous VL history at all. The lesions started from chin with macular type, later distributed to other parts of the body with both macular and nodular type. The lesions were non-itching, touch sensitive and rk 39 strip test was positive. All of them were confirmed through microscopic examination of slit skin smear. Their haematological and biochemical markers were within normal limit. After first cycle, all of them showed initial progress and after second cycle all of them attained complete cure. Fever with shivering was the only documented adverse event during treatment and was managed by paracetamol. Conclusion: The cases highlighted in this report explore the success of utilising extended LAmB dose schedule for treating a patient with widespread lesions.The triumph of this alternate treatment schedule for PKDL needs to be supported with clinical trials that evaluate its efficacy and safety.

Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing

Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is caused by Leishmania donovani. In addition to fatal VL, these parasites also cause skin diseases in immune-competent and -suppressed people, post-kala azar dermal leishmaniasis (PKDL) and HIV/VL co-infections, respectively. Genetic polymorphism in 36 Ethiopian and Sudanese L. donovani strains from VL, PKDL and HIV/VL patients was examined using Amplified Fragment Length Polymorphism (AFLP), kDNA minicircle sequencing and Southern blotting. Strains were isolated from different patient tissues: in VL from lymph node, spleen or bone marrow; and in HIV/VL from skin, spleen or bone marrow. When VL and PKDL strains from the same region in Sudan were examined by Southern blotting using a DNA probe to the L. donovani 28S rRNA gene only minor differences were observed. kDNA sequence analysis distributed the strains in no particular order among four clusters (A – D), while AFLP analysis grouped the strains according to geographical origin into two major clades, Southern Ethiopia (SE) and Sudan/Northern Ethiopia (SD/NE). Strains in the latter clade were further divided into subpopulations by zymodeme, geography and year of isolation, but not by clinical symptoms. However, skin isolates showed significantly (p < 0.0001) fewer polymorphic AFLP fragments (average 10 strains = 348.6 ± 8.1) than VL strains (average 26 strains = 383.5 ± 3.8).

Cytokines and chemokines differentially regulate innate immune cell trafficking during post kala-azar dermal leishmaniasis.

Post kala-azar dermal leishmaniasis (PKDL) is often considered to be the anthroponotic reservoir of visceral leishmaniasis (VL) in India. A better understanding of the host immune-response in dermal lesions of PKDL patients is therefore of utmost significance to minimize such patients and to restrict VL transmission. Although the innate immune response is known to play an important role in parasite clearance from dermal lesions, the actual contribution of innate cells to the pathogenicity of PKDL is poorly understood. The present study explored the immune-pathogenesis of PKDL patients to understand the expression of CD62L, CD11b, CXCL8/IL-8, and MIP1-α and their contribution in signaling during innate cell trafficking. Twenty-five individuals were enrolled, who comprised eight active and untreated macular cases, seven active and untreated cases with papulo-nodular PKDL manifestations, five successfully treated post PKDL cases and five healthy individuals from a non-endemic region of Bihar, India. The immunological investigation was performed on biopsy specimens prepared with a disaggregation technique and blood samples. We observed that the PMNs in nodular patients displayed decreased L-selectin (CD62L) levels and increased integrin (CD11b) expression compared with those in macular patients. Further analysis showed that lower PMN extravasation in macular patients occurred because of inadequate CXCL8/ IL-8 release. In summary, Leishmania donovani (L. donovani) infection in macular PKDL patients decreased leucocyte rolling (L-selectin shedding) and induced up-regulation of the cellular signaling factors involved in pathogenesis (ERK1/2) as well as down regulated the signaling elements (p38 MAPK) involved in the Th1 response, especially in PMNs.

Safety and Effectiveness of Short-Course AmBisome in the Treatment of Post-Kala-Azar Dermal Leishmaniasis: A Prospective Cohort Study in Bangladesh.

A safe and effective short-course treatment regimen for post-kala-azar dermal leishmaniasis (PKDL) is considered essential for achieving and sustaining elimination of visceral leishmaniasis (VL) in the Indian subcontinent [1, 2]. Here, single-dose liposomal amphotericin B (AmBisome) has been adopted as a first-line regimen for VL; however the effectiveness and safety of AmBisome for PKDL has not been formally evaluated. The safety and effectiveness of AmBisome 15 mg/kg, given over 15 days in 5 biweekly infusions of 3 mg/kg on an outpatient basis, was evaluated between April and November 2014 in patients with clinically diagnosed PKDL, aged ≥12 years and residing in a highly VL-endemic area in Bangladesh. This was a prospective cohort observational study, with the objective to assess final cure 12 months after treatment. Clinical response was monitored at 1, 3, 6, and 12 months, and safety during treatment and up to 1 month after treatment. Of the 280 patients meeting the inclusion criteria, 273 were assessed at 12 months. A complete or major improvement of lesions was seen in 245 patients (89.7%); 213 (78.0%) were considered completely cured. Lesions did not improve in 28 (10.3%) and new lesions appeared in 13 (4.8%). All patients completed treatment without severe or serious adverse events. A short-course 15-mg/kg AmBisome regimen proved safe and effective in the treatment of clinically diagnosed PKDL in Bangladesh, and should be considered a treatment option for routine programmatic use in the VL elimination effort in the Indian subcontinent.

Deciphering the Role of Post-Kala-Azar Dermal Leishmaniasis Patients in the Transmission of Leishmania Donovani: Implications for the Kala-Azar Elimination Program

Background: In the Indian subcontinent, visceral leishmaniasis (VL) incidence is on track to reach elimination goals by 2020 in nearly all endemic districts. Although not included in official targets, previous data suggest post-kala-azar dermal leishmaniasis (PKDL) patients can act as an infection reservoir. Methods: We conducted xenodiagnosis of 47 PKDL and 15 kala-azar patients using laboratory-reared Phlebotomus argentipes. In direct xenodiagnosis, flies were given access to the the patient's skin to feed for 15 minutes. For indirect xenodiagnosis, flies were fed through a membrane on the patient's blood. Five days later, blood-fed flies were dissected and examined by microscopy and/or PCR. A 3-mm skin snip biopsy (PKDL) or venous blood (kala-azar) was processed by quantitative PCR. Findings: Twenty-seven PKDL patients (57.4%) had positive results by direct and/or indirect xenodiagnosis. Direct was significantly more sensitive than indirect xenodiagnosis (55.3% vs 6.4%, p 1 log10 unit higher than those with negative results (2.88 vs 1.66, p<0.0001). In a multivariable model, parasite load, nodular lesions and positive skin microscopy were significantly associated with positive xenodiagnosis. Blood parasite load was the strongest predictor for kala-azar. Compared to kala-azar, nodular PKDL was more likely and macular PKDL less likely to result in positive xenodiagnosis, but neither difference reached statistical significance. Interpretation: Both nodular and macular PKDL can be infectious to sand flies. Active PKDL case detection and prompt treatment should be instituted and maintained as an integral part of VL control and elimination programs. Funding: This work was supported by the World Health Organization Special Programme for Research and Training in Tropical Diseases, Switzerland; Spanish Foundation for lnternational Cooperation, Health and Social Affairs; and icddr,b core donors. The funding sources had no role in the study design, collection, analysis and interpretation of the data, preparation of the manuscript, or the decision to submit for publication. Conflict of Interest: Dr. Bern reports personal fees from DNDi, outside the submitted work. All other authors: No reported conflicts of interest. Ethical Approval Statement: The protocol was approved by the Ethical Review Committee of ICDDR,B (#PR-14010). All patients provided written informed consent.

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%.

Visceral leishmaniasis: An immunological viewpoint on asymptomatic infections and post kala azar dermal leishmaniasis

Elimination of visceral leishmaniasis is a priority programme in Indian subcontinent. The World Health Organization has set a new target to eliminate kala-azar by the year 2020 as previous target elimination year (2015) has passed. The elimination programme has successfully curbed the rate of infection in endemic regions; however, there are still few challenges in its route. The current drug control regime is extremely limited and comprises only two (amphotericin B and miltefosine) drugs, which are also susceptible for parasites resistance. Moreover, these drugs do not produce sterile cure, and cured patients may develop post kala-azar dermal leishmaniasis even after a decade of cure leaving behind a potent source of parasitic reservoirs for further disease transmission. A significant proportion of endemic population remain seropositive but aymptomatic for many years without any clinical symptom that serve as latent parasitic reservoirs. The lack of tools to identify live parasites in asymptomatic infections and there association in disease transmission, parameters of sterile cure along with post kala-azar dermal leishmaniasis progression remain a major threat in its elimination. In this review, we discuss the potential of host immune inhibitory mechanisms to identify immune correlates of protective immunity to understand the mystery of asymptomatic infections, sterile cure and post kala azar dermal leishmaniasis.

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.

Visual Detection of Leishmania donovani and Leptomonas seymouri in Co–Infected Samples by Their Specific DNA Biomarker and Gold Nanoparticle

AbstractVisceral leishmaniasis (VL) is a fatal tropical disease that often relapses in apparently recovered patients as post kalaazar dermal leishmaniasis (PKDL) in Indian subcontinent. Recently, the co‐infection of Leishmania donovani and another tripanosmatid, Leptomonas seymouri has been reported in VL and PKDL in this region. The coexistence of Leishmania imposter, Leptomonas in the clinical isolates and its follow up cultures has been challenging in immunological and vaccine development research against leishmaniasis, where maintenance of pure Leishmania culture is a prerequisite. The impact of this newly observed parasitic co‐habitation in diagnosis and management of VL and PKDL is yet to be ascertained. However, an early detection of this emerging co‐infection has become intriguing for research and medical management of the disease. Internal transcribed spacer‐1 (ITS‐1), in which Leishmania and Leptomonas differ in sequences, were used as the specific markers in the current assay. The color of gold nanoparticle visually got changed as target DNA isolated from either of the species interacted with designed, species‐specific oligonucleotide probes added externally. The current colorimetric assay to detect the co‐infection of Leishmania and Leptomonas is a robust, sensitive, high throughput, low cost and rapid method requiring no sophisticated instruments.

Post-kala-azar dermal leishmaniasis in the Indian subcontinent: A threat to the South-East Asia Region Kala-azar Elimination Programme.

BackgroundThe South-East Asia Region Kala-azar Elimination Programme (KAEP) is expected to enter the consolidation phase in 2017, which focuses on case detection, vector control, and identifying potential sources of infection. Post-kala-azar dermal leishmaniasis (PKDL) is thought to play a role in the recurrence of visceral leishmaniasis (VL)/kala-azar outbreaks, and control of PKDL is among the priorities of the KAEP.Methodology and principal findingWe reviewed the literature with regard to PKDL in Asia and interpreted the findings in relation to current intervention methods in the KAEP in order to make recommendations. There is a considerable knowledge gap regarding the pathophysiology of VL and PKDL, especially the underlying immune responses. Risk factors (of which previous VL treatments may be most important) are poorly understood and need to be better defined. The role of PKDL patients in transmission is largely unknown, and there is insufficient information about the importance of duration, distribution and severity of the rash, time of onset, and self-healing. Current intervention methods focus on active case detection and treatment of all PKDL cases with miltefosine while there is increasing drug resistance. The prevention of PKDL by improved VL treatment currently receives insufficient attention.Conclusion and significancePKDL is a heterogeneous and dynamic condition, and patients differ with regard to time of onset after VL, chronicity, and distribution and appearance of the rash, as well as immune responses (including tendency to self-heal), all of which may vary over time. It is essential to fully describe the pathophysiology in order to make informed decisions on the most cost-effective approach. Emphasis should be on early detection of those who contribute to transmission and those who are in need of treatment, for whom short-course, effective, and safe drug regimens should be available. The prevention of PKDL should be emphasised by innovative and improved treatment for VL, which may include immunomodulation.

Prevalence of visceral leishmaniasis, risk factors and associated disorders: Knowledge of inhabitants and professionals in Fulbaria, Mymensingh

A total of 215 cases were recorded from the rural areas of Fulbaria upazila during June 2015 to May 2016. The prevalence of kala-azar was higher in male (51.22%) than female (36.96%). In the area, 10.1 - 20 years age group was the most prevalent group and the prevalence was found to decrease with the increase of age. Post Kala-azar Dermal Leishmaniasis (PKDL) was developed in 38.14% of kala-azar patients of Fulbaria upazila. The prevalence of kala-azar was found 63.89% in mud house dwellers and when living in tin shed house but with earthen floor, the prevalence was 58.79%. On the other hand, 10.31% had single infection with kala-azar, 21.65% had double infection and 68.04% had multiple infection. Living in proximity to prior case, climatic change, racked mud house, humid area, cattle sheds made of cracked mud walls, lack of awareness and knowledge regarding kala azar were the risk factors. Total 45 professionals were interviewed in the Upazila Health Complex and Upazila Sub-Centers of the study area through a preset questionnaire. Of them, 71.11% had the appropriate acquaintance about the agent (Leishmania donovani) of kala-azar and 35.29% professionals had the correct comprehension regarding the reservoir of kala-azar (man). Most of them (91.11%) recommended rK 39 dipstick test as the single most important diagnostic blood test and Miltefosine and Amphotericin B are the most recommended drugs for treatment of kala-azar.Bangladesh J. Zool. 45(1): 73-83, 2017

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment.

Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL.

Monitoring of Parasite Kinetics in Indian Post-Kala-azar Dermal Leishmaniasis.

The potential reservoirs of leishmaniasis in South Asia include relapsed cases of visceral leishmaniasis (VL), patients with post-kala-azar dermal leishmaniasis (PKDL), and an asymptomatically infected population. Therefore, assessment of cure in terms of parasite clearance, early detection of PKDL, and asymptomatic VL are pivotal for ensuring elimination. This study aimed to monitor the efficacy of miltefosine and liposomal amphotericin B (LAmB) in PKDL based on parasite load. Patients with PKDL were recruited from the dermatology outpatient departments or during active field surveys. Skin biopsies were collected at disease presentation, immediately at the end of treatment, and 6 months later. The presence of parasite DNA was assessed by internal transcribed spacer-1 polymerase chain reaction, and quantified by amplification of parasite kinetoplastid DNA. At disease presentation (n = 184), the median parasite load was 5229 (interquartile range [IQR], 896-50898)/μg genomic DNA (gDNA). Miltefosine cleared the parasites to <10 in the macular (n = 17) and polymorphic (n = 21) variants, and remained so up to 6 months later (<10 parasites). LAmB reduced the parasite burden substantially in macular (n = 34; 2128 [IQR, 544-5763]/µg gDNA) and polymorphic PKDL (n = 36; 2541 [IQR, 650-9073]/µg gDNA). Importantly, in patients who returned 6 months later (n = 38), a resurgence of parasites was evident, as the parasites increased to 5665 (IQR, 1840-17067)/µg gDNA. This study established that quantifying parasite load is an effective approach for monitoring patients with PKDL, wherein miltefosine demonstrated near-total parasite clearance and resolution of symptoms. However, in cases treated with LAmB, the persistence of parasites suggested treatment inadequacy. This needs immediate redressal in view of the leishmaniasis elimination program targeted for 2020.

Successful Treatment of Post Kala-azar Dermal Leishmaniasis and Disseminated Tuberculosis Co-infection With Liposomal Amphotericin B and Anti-tubercular Drugs in Bangladesh

Abstract We report a case of a 40-year-old male patient residing in a hyperendemic area of Bangladesh. The patient was diagnosed with disseminated tuberculosis and post kala-azar dermal leishmaniasis at Surya Kanta Kala-azar Research Centre. He had thickening, furrowing, and hyperpigmentation of the skin over both lower limbs and had nodular lesions on the face and earlobes and macular lesion on the back of the chest and limbs with intact sensation. Right-sided moderate pleural effusion, mild ascites, and mild hepatosplenomegaly were also found. He had positive result of Xpert MTB/RIF test from pleural fluid and tuberculin skin test. The results of rapid diagnostic test rK39, slit skin smear microscopy for Leishman-Donovan bodies, and polymerase chain reaction for skin sample of post kala-azar dermal leishmaniasis were found positive. Liposomal amphotericin B and anti-tubercular (Category 1) drugs were used simultaneously without any serious adverse effect, and improvement was found at a follow-up visit after 6 months of the treatment.