Parasite detection in patients with post kala-azar dermal leishmaniasis in India: a comparison between molecular and immunological methods.

AIMS: To evaluate the sensitivity and specificity of serological, immunohistochemical, and molecular methods in the diagnosis of post kala-azar dermal leishmaniasis (PKDL). METHODS: Twenty five patients with confirmed PKDL and 25 controls were included in the study. G2D10, a monoclonal antibody against Leishmania, was used for the immunohistochemical (IHC) staining of lesion sections to visualise anti-Leishmania donovani antibodies. The diagnostic usefulness of IHC was compared with enzyme linked immunosorbent assay (ELISA) with a recombinant (rk39) antigen, and a species specific polymerase chain reaction (PCR) assay, amplifying a kinetoplast minicircle DNA sequence. RESULTS: IHC detected 22 of 25 PKDL cases, giving a sensitivity of 88%. The diagnostic sensitivity of both the ELISA and PCR tests was higher (96%). All of the 25 controls examined were negative in PCR, indicating 100% specificity of the test, whereas ELISA showed 96% specificity. CONCLUSIONS: IHC with G2D10 significantly enhances the sensitivity of detection of PKDL over routine haematoxylin and eosin staining. ELISA with a recombinant antigen is an economical and practical assay. PCR is the most sensitive and specific diagnostic method for PKDL. The tests described would facilitate the recognition of patients with PKDL, enabling timely treatment, which would contribute greatly to the control of kala-azar.

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

BACKGROUND: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Emperipolesis in a common breast malignancy. A case report.

BACKGROUND: Emperipolesis is a phenomenon characterized by the presence of leukocytes/lymphocytes within the cytoplasm of other cells. The present report describes this unusual observation within epithelial cancer cells of the breast. CASE: A 52-year-old female presented with a hard, adherent lump over the right breast for one year. Fine needle aspiration and histopathologic examination of the tumor showed features of infiltrating duct carcinoma with emperipolesis as a striking feature of the tumor cells. The tumor showed a near-total response to neoadjuvant chemotherapy. CONCLUSION: The mechanism and biologic significance of emperipolesis in producing a near-total response to neoadjuvant chemotherapy in the present case suggest its role in inducing a tumoricidal effect, possibly involving a cascade of chemokines.

Lichen scrofulosorum - a series of eight cases.

Lichen scrofulosorum is a rare entity of children and young adults. The cutaneous lesions are typically symptomless papular eruptions, associated with a strong Mantoux reaction, tuberculosis of lymph nodes and/or other organs. Eight patients between the ages of 5 and 36 years were included in this series. Lesions were commonly seen on the trunk, though a case of involvement of the palms and soles was also observed. Pulmonary tuberculosis was seen in 5 adults; 1 patient each had pleural effusion, tuberculous adenitis and tuberculoma, respectively. Histopathological examination typically showed epithelioid cell granulomas around hair follicles. The histogenesis is unclear, a type IV hypersensitivity reaction provoked by chronic tubercular infection is probable. The patients responded well to antituberculous therapy.

Sporotrichoid presentations in leprosy.

Two adult patients of leprosy, one woman and one man, presented with a clinical picture simulating sporotrichosis. The skin and regional nerve trunk was affected in one, and in the other the disease was confined to the nerve. Both had features of an upgrading reaction following anti-leprosy therapy; this was seen as erosion and scarring of the plaque, and acute onset of abscesses along the easily palpable and thickened nerve that ruptured through the skin. The diagnosis was supported by histopathology. In the light of other infections that give rise to a sporotrichoid pattern of infection it is concluded that leprosy should also be included in this category so that early diagnosis and use of corticosteroids can be implemented quickly to prevent nerve destruction.

A study of cutaneous tuberculosis in children.

Sixty-three children out of a total of 199 patients seen with cutaneous tuberculosis during a 7-year period were included in this study. Culture was positive in only four, and the diagnosis was based on clinical examination, tuberculin reaction, histopathology, and response to antitubercular therapy. Forty had lupus vulgaris (LV) and 23 scrofuloderma (SD). The lower half of the body was predominantly affected in those with LV, and keratotic and hypertrophic forms were frequently encountered. LV planus mainly affected the face. Ulcerative and atrophic types of LV were infrequent. Extensive lesions in three children led to disfiguring scars and contractures. Scrofuloderma often involved the cervical group of lymph nodes followed by the inguinal, submandibular, and axillary groups. As compared to skin tuberculosis in adults, regional lymph node involvement in LV was more common, and a combination of both LV and SD was less frequent in children. No difference in clinical presentation could be detected between the BCG vaccinated and unvaccinated children. Tuberculous infection either in the lungs or the bones was present in eight children. An HIV test done in five patients with widespread lesions was negative. Irregular therapy or late diagnosis leading to serious complications, inadequate parental or community support, and lack of awareness among practitioners are the problems to be remedied.

Disabilities in multibacillary leprosy patients: before, during and after multidrug therapy.

One hundred fifty-one patients (125 males and 26 females) of multibacillary leprosy (LL 88, BL 40, BB 23), registered during 1986-1992 for multidrug therapy (MDT), were analysed with reference to their disabilities before, during and after MDT. At induction 48 (31.7%) had no disability (Gr 0), 59 (39.0%) had only peripheral anaesthesia (Gr 1) and 44 (29.1%) had Gr 2 and 3 deformities with or without anaesthesia. The parallel analysis of the three groups, with nearly equal duration of symptoms, revealed that new deformities developed in only a few cases during and after MDT, least in the Gr 0 group. The crude fresh deformity incidence was 59.2 per 1,000 person years of observation. The rate of recovery from anaesthesia was higher (64%) in Gr 1 group than that (44%) in group with Gr 2, 3 deformities. No significant difference was observed between the incidence of Gr 2 deformities developed before, during and after MDT (incidence of claw-hands 9.2% before and 7.9% during and after MDT, trophic ulcers 13.9% before and 17.8% during and after MDT). Out of 19 cases which developed motor weakness during MDT and follow-up, 10 (52.6%) were instances of quite nerve paralysis. Occupational factors influenced the development of deformities but not the sex and bacterial load. Generally, the lower the Grade of disability at induction of patient for MDT, the lower the chances of new disability development and higher the chances of recovery from sensory impairments.

Addition of immunotherapy with Mycobacterium w vaccine to multi-drug therapy benefits multibacillary leprosy patients.

Immunotherapy with a vaccine consisting of autoclaved Mycobacterium w, was given in addition to standard chemotherapy (multidrug therapy (MDT)) to 93 multibacillary (MB) leprosy patients. One hundred and seven patients with similar types of disease served as controls and received MDT + placebo injections. The study was a double-blind randomised trial. On opening the codes, results obtained were in concordance with those in a single-blind trial which has been extensively reported. Bacteriological clearances were significantly more rapid in vaccinated patients (p < 0.03). Thirty-five LL or BL patients with a high bacterial index (BI) of 6 were completely cleared of acid-fast bacilli (AFB) after eight doses of vaccine. Only 8 patients in the control group became bacteriologically negative in the same time period. They all had BIs < 4. Associated with decreasing BI was accelerated clinical regression of lesions after vaccination and lepromin conversion rates of 100% for BB, 71% for BL and 70% for LL. A significant number of immunised patients showed histological improvement (p < 0.004). Thirty-six showed a complete disappearance of dermal granulomas and a picture of non-specific infiltration. The vaccine did not precipitate neuritis or deformities; episodes were noted in vaccinated patients as were incidences of Type 2 reaction. The overall improvement was reflected by a shorter duration of treatment and faster release of vaccinated patients.

Localized lepromatous leprosy and its response to chemo-immunotherapy.

BACKGROUND: This is an unusual presentation of lepromatous leprosy (LL) in a young boy, 12 years of age. The study forms part of a large scale immunotherapeutic trial with Mycobacterium w (M.w) antileprosy vaccine. The trial is being conducted in two major hospitals in New Delhi, India. MATERIALS AND METHODS: This patient presented with three lesions: one on each forearm and the third on the left leg. He was classified initially as borderline tuberculoid leprosy. Slit-skin smears and histopathology from the lesions proved the diagnosis to be lepromatous leprosy with a bacterial index (BI) 6+. The initial lepromin test was negative. The patient was treated with chemo-immunotherapy (standard multidrug therapy and immunotherapy with Mycobacterium w vaccine). RESULTS: Investigations after 1 year (15 months) of multi-drug therapy and three doses of vaccine, showed a remarkable fall in the BI from 6 to 0 in the lesions, a lepromin positivity of 5 mm, and a histological upgrading from lepromatous leprosy to borderline tuberculoid. Immunologic studies at 15 months revealed a good LTT response and high levels of cytokines, specifically IL-2 and IFN-gamma. CONCLUSIONS: This report presents an LL patient with disease limited to a few sites. It stresses the importance of slit-smear and biopsy in all patients of leprosy, and it highlights the upgrading observed on administration of chemo-immunotherapy.

Study of HLA class II alleles by PCR oligotyping in leprosy patients from north India.

Host factors seem to play an important role in determining the immune response and the differential manifestations of lepromatous (LL) and tuberculoid (TT) leprosy. In order to investigate the role of immunogenetic factors in determining the form of leprosy, the HLA class II alleles of DRB1, DRB3, DRB5, DQA1, DQB1 and DPB1 were studied by a PCR oligotyping technique in 93 patients and 47 healthy controls. DRB1*1501 and DRB1*1502 (two of five tested subsets of the serologically defined DR2) accounted for 81.5% of the multibacillary patients (relative risk 16.3) and 60.7% of the TT patients (relative risk 5.7) compared to 21.3% in normal, ethnically- and geographically-matched controls. The much stronger association of DRB1*1501 with the multibacillary form than with the TT type of leprosy suggests a possible role in the differential immune response to M. leprae antigens. DQB1*0601 was found significantly more often than in controls throughout the leprosy spectrum, while DQA1*0103 was most frequent in the LL group and DQA1*0102 was selectively increased in the borderline lepromatous (BL) patients. On the other hand, DRB1*0701, DQB1*0201 and DQA1*0201 were decreased in the multibacillary leprosy patients (MLP) compared to TT patients and controls, and DQB1*0503 was selectively decreased in TT patients, suggesting that these HLA alleles might play a role in modulating the immune response that determines the form of leprosy that develops in each patient.

Reversal reaction in multibacillary leprosy patients following MDT with and without immunotherapy with a candidate for an antileprosy vaccine, Mycobacterium w.

Immunotherapy with a candidate for an antileprosy vaccine, Mycobacterium w, was given in addition to standard multidrug therapy (MDT) to 53 multibacillary lepromin negative patients belonging to BB, BL and LL types of leprosy (vaccine group). An equal control group received MDT and injections of micronized starch as placebo. Both the vaccine and placebo were administered intradermally every 3 months. The patients were evaluated at determined intervals by clinical, bacteriological and histopathological parameters and lepromin testing. Reactional episodes were analysed with reference to incidence, onset, frequency and severity during and after release from treatment (RFT). Incidence of reversal reaction (RR) was marginally higher in the vaccine group (22.6% vaccine group vs 15% control group). All cases with a history of downgrading type 1 reaction developed RR during therapy. Most episodes occurred within the 1st year of the commencement of therapy--50% developing within 3 months. Late reversal reaction (after RFT) were observed in 3.8% of cases in both groups, and 50% of the reactors in the control group and 33% in the vaccine group had repeated reactional episodes. Incidence of neuritis associated with RR as well as isolated neuritis was similar in both groups.

Immunotherapy with Mycobacterium w vaccine decreases the incidence and severity of type 2 (ENL) reactions.

Immunotherapy with Mycobacterium w (M.w) vaccine was given to 45 patients with multibacillary (MB) leprosy; 41 similarly classified patients served as controls. All patients received standard multidrug therapy (MDT). Incidence, severity and frequency of type 2 (ENL) reactional episodes were monitored in both groups in a follow-up extending up to 4 years. Reactions were seen in fewer vaccinated (10/37) BL and LL patients than in the control group (12/34). A total of 20 episodes were recorded in the vaccine group as against 29 in the controls, 75% of reactions were mild in vaccinated and 51.72% were mild in the control group patients, and 3 patients in the control group had more than 3 reactional episodes. None of the vaccinated patients showed this. No additional incidence of neuritis were seen among vaccinated individuals during reactional episodes.