Diagnosis Of Post-kala-azar Dermal Leishmaniasis Research Articles

Increasing Incidence of Post–Kala‐Azar Dermal Leishmaniasis in a Population‐Based Study in Bangladesh

Post-kala-azar dermal leishmaniasis (PKDL) occurs after kala-azar treatment and acts as a durable infection reservoir. On the basis of active case finding among 22,699 respondents, 813 (3.6%) had had kala-azar since 2002, of whom 79 (9.7%) developed PKDL. Eight additional patients with PKDL had no history of kala-azar. Annual kala-azar incidence peaked at 85 cases per 10,000 person-years in 2004 and fell to 46 cases per 10,000 person-years in 2007, but PKDL incidence rose from 1 case per 10,000 person-years in 2002-2004 to 21 cases per 10,000 person-years in 2007. The rising PKDL incidence threatens the regional visceral leishmaniasis elimination initiative and underscores the urgent need for more effective PKDL diagnosis and treatment.

LeishmaniaPromastigote Membrane Antigen-Based Enzyme-Linked Immunosorbent Assay and Immunoblotting for Differential Diagnosis of Indian Post-Kala-Azar Dermal Leishmaniasis

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani, is difficult, as the dermal lesions are of several types and resemble those caused by other skin diseases, especially leprosy. Since the disease generally appears very late after the clinical cure of kala-azar in India, it is also difficult to correlate PKDL with a previous exposure to L. donovani. Very few attempts have been made so far to diagnose PKDL serologically, and the diagnostic methods vary in their sensitivities and specificities. Diagnosis of PKDL through sophisticated PCR methods, although highly sensitive, has limited practical use. We have developed a serodiagnostic method using an enzyme-linked immunosorbent assay to detect specific immunoglobulin (Ig) isotypes and IgG subclass antibodies in the sera of Indian PKDL patients. Our assay, which uses L. donovani promastigote membrane antigens, was 100% sensitive for the detection of IgG and 96.7% specific for the detection of IgG and IgG1. Optical density values for individual patients, however, demonstrated wide variations. Western blot analysis based on IgG reactivity could differentiate patients with PKDL from control subjects, which included patients with leprosy, patients from areas where kala-azar is endemic, and healthy subjects, by the detection of polypeptides of 67, 72, and 120 kDa. The recognition patterns of the majority of serum samples from patients with PKDL were also distinct from those of the serum samples from patients with visceral leishmaniasis (VL), at least for a 31-kDa polypeptide. To further differentiate patients with PKDL from those with active and cured VL, we analyzed the specific titers of the Ig isotypes and IgG subclasses. High levels of IgG, IgG1, IgG2, and IgG3 antibodies significantly differentiated patients with PKDL from patients cured of VL. The absence of antileishmanial IgE and IgG4 in patients with PKDL differentiated these patients from those with active VL. These results imply intrinsic differences in the antibodies generated in the sera from patients with PKDL and VL.

Identification of parasite antigen, correlation of parasite density and inflammation in skin lesions of post kala-azar dermal leishmaniasis.

Post kala-azar dermal leishmaniasis (PKDL) is an unusual dermatosis following kala-azar (KA). Demonstration of the amastigotes in lesions plays an important role in the diagnosis of PKDL. It was aimed to evaluate the utility of an antibody G2D10 in detecting leishmania parasite antigen, to correlate the parasite number/percentage of parasites with the inflammation, and to assess the epidemiological significance associated with the location of the parasites. The study was conducted on 50 cases. Hematoxylin and eosin (H & E) stains and immunohistochemical (IHC) stains, using G2D10 antibody, was performed on the skin biopsies. The number of parasites and density of inflammation were semiquantitatively assessed. Leishmania donovan bodies (LDBs) were identified in 50% of cases with the H & E compared to 80% positivity with the IHC. All 50 cases showed inflammation in the superficial dermis (SD). About 44% showed dense inflammation compared to 16% sparse and 40% moderate inflammation. Parasite percentage was maximum in the SD (100%) compared to 75 and 42% in the mid and deep dermis, respectively. The IHC showed a higher percentage of LDB localization (80 vs. 50%). Density of inflammation was maximum in the SD. The parasite percentage was correlated with the inflammation. Location of parasites could have an epidemiological significance.

Evaluation of enzyme-linked immunosorbent assay for diagnosis of post-kala-azar dermal leishmaniasis with crude or recombinant k39 antigen.

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL.

The development of post-kala-azar dermal leishmaniasis (PKDL) is associated with acquisition of Leishmania reactivity by peripheral blood mononuclear cells (PBMC).

PKDL develops in about 50% of Sudanese patients treated for visceral leishmaniasis (kala-azar). Patients with kala-azar were entered into this study and followed for a period of up to 2 years. During follow up 12 patients developed PKDL and eight did not. Proliferative responses and cytokine production to Leishmania donovani and control antigens were measured in vitro using PBMC isolated at the time of diagnosis of kala-azar, after treatment of visceral leishmaniasis, during follow up, and at the time of diagnosis of PKDL. Proliferative responses and interferon-gamma (IFN-gamma) production were low at diagnosis and increased after treatment of kala-azar in both patients who developed (group 1) and those who did not develop PKDL later (group 2). In group 1, development of PKDL was always associated by an increased PBMC response to Leishmania antigen in proliferation and IFN-gamma production assays. There were no differences in Leishmania antigen-induced production of IL-4, IL-5 and IL-10 between or within the two groups. We have previously shown that Leishmania parasites spread to the skin during visceral leishmaniasis and proposed that PKDL was the result of an immunological attack on parasites, which have survived in the skin despite the drug treatment. The finding that PKDL develops after treatment of kala-azar as Leishmania-reactive T cells start to circulate in peripheral blood in sufficient numbers to be detected in in vitro assays supports this hypothesis.

Use of PCR for diagnosis of post-kala-azar dermal leishmaniasis.

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (n = 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with Mycobacterium leprae could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.

Nodular post-kala-azar dermal leishmaniasis: a distinct histopathological entity.

Post-kala-azar dermal leishmaniasis (PKDL) is an infrequently occurring sequel to treated visceral leishmaniasis. Diagnosis, particularly in non-endemic areas, is difficult because the clinical appearances may be subtle and simulate lepromatous leprosy. The histopathology of the condition has been a neglected subject. Nodular lesions constitute one of the large variety of lesions that can be seen in PKDL. This paper describes the histopathology of such lesions in 26 patients seen over a period of approximately 8 years in a non-endemic setting. All the biopsies had strikingly similar light microscopic features with characteristic findings: a dense lymphohistiocytic infiltrate beneath an atrophic epidermis, pronounced follicular plugging, vascular hyalinization and collagen changes and negative Fite stain. These allow a definite diagnosis of PKDL even in the absence of demonstrable Leishman-Donovan (L-D) bodies.

Reactivity of various leishmanial antigens in a direct agglutination test and their value in differentiating post-kala azar dermal leishmaniasis from leprosy and other skin conditions.

A direct agglutination test (DAT) for the detection of post-kala azar dermal leishmaniasis (PKDL) was evaluated in conditions that simulate the disease clinically or immunologically. A reference strain of Leishmania donovani (LEM 1399), and antigen preparations from Leishmania isolates from Bangladeshi patients with post-kala azar dermal leishmaniasis or visceral leishmaniasis were used. A titre of at least 51,200 was obtained in tests of patients with PKDL with all three antigens, whereas a maximum titre of 1600 was recorded in patients with cutaneous leishmaniasis, mucocutaneous leishmaniasis or leprosy. Antigens from dermal isolates of L. tropica (LV 140) and L. braziliensis (LV 65) yielded titres of 1600-6400 in patients with PKDL. The lowest titre recorded in 70 patients tested with the homologous PKDL antigen was 409,600. In patients with leprosy, cutaneous leishmaniasis, syphilis, onchocerciasis, tuberculosis, blastomycosis or vitiligo, titres ranged from 100 to 1600. Tha DAT is better than current parasitological and histopathological methods for the diagnosis of PKDL in areas in which leprosy is co-endemic.