Monitoring the Long-Term Effectiveness of Miltefosine in Indian Post-Kala-Azar Dermal Leishmaniasis.

Post-kala-azar dermal leishmaniasis (PKDL), the dermal sequel to visceral leishmaniasis (VL), is characterized by hypopigmented macules (macular) and/or papules and nodules (polymorphic). Post-kala-azar dermal leishmaniasis plays a significant role in disease transmission, emphasizing the need for monitoring chemotherapeutic effectiveness. Accordingly, this study aimed to quantify the parasite burden in PKDL patients after treatment with miltefosine by a quantitative polymerase chain reaction (qPCR). A Leishmania kinetoplastid gene-targeted qPCR was undertaken using DNA from skin biopsy specimens of patients with PKDL at three time points, i.e., at disease presentation (week 0, n = 157, group 1), upon completion of treatment (week 12, n = 39, group 2), and at any time point 6 months after completion of treatment (week ≥36, n = 54, group 3). A cycle threshold (Ct) <30 was considered the cutoff for positivity, and load was quantified as the number of parasites/µg genomic DNA (gDNA); cure was considered when samples had a Ct >30. The parasite load at disease presentation (group 1) was 10,769 (1,339-80,441)/µg gDNA (median [interquartile range]). In groups 2 and 3, qPCR results were negative in 35/39 cases (89.7%) and 48/54 cases (88.8%), respectively. In the 10/93 (10.8%) qPCR-positive cases, the parasite burdens in groups 2 and 3 were 2,420 (1,205-5,661)/µg gDNA and 22,195 (5,524-100,106)/µg gDNA, respectively. Serial monitoring was undertaken in 45 randomly selected cases that had completed treatment; all cases in groups 2 or 3 had a Ct >30, indicating cure. Overall, qPCR confirmed an 89.2% cure (as 83/93 cases showed parasite clearance), and the persistent qPCR positivity was attributed to nonadherence to treatment or unresponsiveness to miltefosine and remains to be investigated.

Status of B-Lymphocyte Subsets and Their Homing Markers in Patients With Post-Kala-Azar Dermal Leishmaniasis.

In visceral leishmaniasis, the Type II helper T cell predominance results in B cell modulation and enhancement of anti-leishmanial IgG. However, information regarding its dermal sequel, post-kala-azar dermal leishmaniasis (PKDL), remains limited. Accordingly, this study aimed to elucidate the B cell-mediated antibody-dependent/independent immune profiles of PKDL patients. In the peripheral blood of PKDL patients, immunophenotyping of B cell subsets was performed by flow cytometry and by immunohistochemistry at lesional sites. The functionality of B cells was assessed in terms of skin IgG by immunofluorescence, while the circulating levels of B cell chemoattractants (CCL20, CXCL13, CCL17, CCL22, CCL19, CCL27, CXCL9, CXCL10 and CXCL11) were evaluated by a multiplex assay. In patients with PKDL as compared with healthy controls, there was a significant decrease in pan CD19+ B cells. However, within the CD19+ B cell population, there was a significantly raised proportion of switched memory B cells (CD19+IgD-CD27+) and plasma cells (CD19+IgD-CD38+CD27+). This was corroborated at lesional sites where a higher expression of CD20+ B cells and CD138+ plasma cells was evident; they were Ki67 negative and demonstrated a raised IgG. The circulating levels of B cell chemoattractants were raised and correlated positively with lesional CD20+ B cells. The increased levels of B cell homing markers possibly accounted for their enhanced presence at the lesional sites. There was a high proportion of plasma cells, which accounted for the increased presence of IgG that possibly facilitated parasite persistence and disease progression.

Molecular monitoring of treatment efficacy in human visceral leishmaniasis.

BACKGROUND: Focused efforts of the visceral leishmaniasis elimination program have led to a drastic decline in cases, and the present challenge is disease monitoring, which this study aimed to assess. METHODS: A Leishmania kinetoplastid-targeted qPCR quantified parasite load at disease presentation, and following treatment completion (n=49); an additional 80 cases were monitored after completion of treatment. RESULTS: The parasite load at disease presentation was 13 461.00 (2560.00-37764.00)/µg gDNA, which upon completion of treatment reduced in 47 of 49 cases to 1(1-1)/µg gDNA, p<0.0001. In 80 cases that presented >2 months post-treatment, their parasite burden similarly decreased to 1(1-1)/µg gDNA except in 6 of 80 cases, which were qPCR positive. CONCLUSION: In 129 cases of visceral leishmaniasis, qPCR by quantification of parasite burden proved effective for monitoring treatment.

Comprehensive investigation of fever cases enrolled during 2019 dengue outbreaks from three hyperendemic regions of North 24 Parganas district of West Bengal, India.

For the past several decades, dengue fever has been emerging in epidemic proportions in several regions of the world. During August-September 2019, an increasing number of fever cases were being reported from some areas of North 24 Parganas district of West Bengal, India. Accordingly, outbreak investigation of fever cases from these affected areas of Bongoan, Barasat, and Habra was carried out. To characterize clinical and biochemical features of fever cases as well as to investigate the utility of CRP as a Dengue severity marker in resource-limited settings. We systematically enrolled 108 patients from the affected region of North 24 Parganas. Standard diagnostic assays along with routine serological and biochemical parameters were performed. Of the 108 patients, 77 (71%) were confirmed with Dengue infection followed by 22 (20%) DENV seronegative and 9 (8%) coinfected DENV cases. Among the 77 confirmed Dengue patients, 53 (69%) had primary infection while 24 (31%) had secondary infection. Among the DENV clinical symptoms, fever (r = 0.50; p = 0.004), headache (r = 0.40; p = 0.03) and abdominal pain (r = -0.40; p = 0.02) were found to bear significant correlation with DENV viral load. The predominant circulating serotype was found to be DENV2. CRP Dengue severity cut-off level of 10.15 mg/L (AUC: 0.85; 86% sensitivity, 77% specificity) was obtained. CRP had correlation with viral load (r = 0.4, p = 0.05) within febrile phase of infection. The performance of biomarkers can be influenced by local epidemiology, geography, and several patient factors, therefore, CRP Dengue severity cut-off value may be region-specific. This study for the first time attempts to estimate CRP Dengue severity cut-off value based on routine immunoturbidometric evaluation from Dengue Hyperendemic zones of North 24 Parganas, West Bengal, Eastern India.

Liposomal amphotericin B is more effective in polymorphic lesions of post kala-azar dermal leishmaniasis.

BACKGROUND: Post kala-azar dermal leishmaniasis (PKDL) is thought to be the reservoir of infection for visceral leishmaniasis in South Asia. The development of strategies for the diagnosis and treatment of PKDL are important for the implementation of the visceral leishmaniasis elimination program. AIMS: Liposomal amphotericin B (L-AMB) has been an overwhelming success in the treatment of visceral leishmaniasis. However, the empirical three-week regimen of L-AMB proposed for PKDL was shown to be inadequate, especially in the macular variant. This study aimed to delineate response of the different variants of PKDL to L-AMB. METHODS: Skin biopsies were collected from PKDL cases at disease presentation and upon completion of treatment with L-AMB. Parasite DNA was detected by Internal Transcribed Spacer-1 PCR (ITS-1 PCR) and quantified by amplification of parasite kDNA. CD68 + macrophages were estimated in tissue sections by immunohistochemistry. RESULTS: Treatment with L-AMB decreased the parasite load by 97% in polymorphic cases but only by 45% in macular cases. The median parasite load (89965 vs 5445 parasites/µg of genomic DNA) as well as infiltration by CD68+ cells before treatment was much greater in the polymorphic cases. LIMITATIONS: Although monitoring of the parasite load for 12 months post-treatment would have been ideal, this was not possible owing to logistical issues as well as the invasive nature of biopsy collection procedure. CONCLUSION: A dramatic decrease in the parasite burden was noted in patients with polymorphic lesions. Although patients with macular disease also had a decrease in parasite burden, this was not as marked as in the polymorphic cases. There was also a significantly greater infiltration of CD68 + macrophages in polymorphic PKDL before therapy.

Clinical Proteomics Profiling for Biomarker Identification Among Patients Suffering With Indian Post Kala Azar Dermal Leishmaniasis.

Background: Post Kala Azar Dermal Leishmaniasis (PKDL) is a non-fatal dermal sequel of Visceral Leishmaniasis (VL), affecting individuals worldwide. Available diagnostic tools lack sensitivity and specificity toward identifying macular (MAC) PKDL patients, due to low parasite load in patients' sample. Confirmatory test like punch biopsy are invasive and painful. Considering the rural nature of this disease and the prevailing situation of diagnostic scenario, PKDL patients mostly remains unattended from receiving proper medical care. They in turn act as "mobile parasite reservoir," responsible for VL transmission among healthy individuals (HI). This study aims to identify PKDL disease specific glycated protein biomarkers, utilizing the powerful LC-MS/MS technology, which is the tool of choice to efficiently identify and quantify disease specific protein biomarkers. These identified PKDL disease specific novel glycoproteins could be developed in future as immunochromatographic based assay for efficient case detection. Methodology: Previously our lab had identified importance of glycated (Circulating Immune Complexes) CICs, among PKDL patients. This study aims to further characterize disease specific glycated protein biomarkers, among MAC PKDL patients for both diagnostic and prognostic evaluation of the disease. LC-MS/MS based comparative spectral count analysis of MAC PKDL to polymorphic (POLY) PKDL, HI, and Cured (CR) individuals were performed. Proteins level alterations among all study groups were confirmed by Western blot and enzyme-linked immunosorbant Assay (ELISA). Results: Among MAC PKDL patients 43, 60, 90 proteins were altered compared to POLY PKDL, HI, and CR groups, respectively. Filtering for the most significant proteins, Plasminogen (PLG) and Vitronectin (VTN) were identified which promisingly identified MAC PKDL cases. Active surveillance results from endemic districts of West Bengal revealed drastic rise of MAC PKDL cases, alarming the urgency for field adaptive efficient biomarker. Conclusion: This current study aims to establish PLG and VTN as novel diagnostic and prognostic protein biomarker for MAC and POLY PKDL cases management.

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

BACKGROUND: 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. AIM: 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. METHODS: 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. RESULTS: 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%. LIMITATIONS: The Gal2 enzyme-linked immunosorbent assay needs to be further investigated in mass surveys. CONCLUSION: 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.

Dermatological Implications of Galectin-3 in Circulation: An Evaluation From the Perspective of Patients With Differential Manifestations of Post-Kala-Azar Dermal Leishmaniasis.

Galectin-3, a ß-galactoside-binding lectin, has been implicated in vast repertoire of inflammatory and immunomodulatory processes including skin diseases. However, galectin-3 has not been comprehensively studied in infectious diseases. This study emphasizes on fascinating aspects of galectin-3 expression in dermal infection by studying post-kala-azar dermal leishmaniasis (PKDL), an intracellular infection caused by Leishmania donovani. Indian PKDL is a well-recognized parasitic dermatosis, with a high risk of anthroponotic transmission of L. donovani in causing leishmaniasis. This study aims to investigate the levels of galectin-3 and galectin-3-binding site expression in circulation of different forms of Indian patients with PKDL. Thirty-seven confirmed untreated PKDL patients, comprising 20 polymorphic and 17 macular PKDL manifestations, were evaluated for the levels of sera galectin-3 with respect to 28 age- and sex-matched healthy controls from endemic areas. Result shows a significant increment (P < 0.001) in circulatory galectin-3 levels in PKDL variants as compared to healthy controls. In addition, there were heightened levels of galectin-3 and galectin-3-binding sites on cellular infiltrates on lesional sites. Furthermore, there was a positive correlation between frequencies of mononuclear cells and galectin-3 during microcirculation in lesions. Data were well corroborated with positive correlation of IL-10 and IFN-γ with sera galectin-3 levels. Moreover, flow cytometry demonstrated the enhanced expression levels of the galectin-3-binding site in circulation in patients with PKDL as compared to healthy controls. Taken together, elevated levels of galectin-3 reflect its involvement in PKDL pathogenesis.

Active surveillance identified a neglected burden of macular cases of Post Kala-azar Dermal Leishmaniasis in West Bengal.

BACKGROUND: Post Kala-azar Dermal Leishmaniasis (PKDL) develops in patients apparently cured of Visceral Leishmaniasis (VL), and is the strongest contender for being the disease reservoir. Therefore, existence of a few cases is sufficient to trigger an epidemic of VL in a given community, emphasizing the need for its active detection and in turn ensuring success of the current elimination program. This study explored the impact of active surveillance on the demographic profile of PKDL patients in West Bengal. METHODOLOGY/PRINCIPAL FINDINGS: Patients with PKDL were recruited through passive (2003-date, n = 100) and active surveillance (2015-date, n = 202), the former from outpatient departments of dermatology in medical colleges in West Bengal and the latter through an active door-to-door survey in four VL hyper-endemic districts of West Bengal. Passive surveillance indicated a male preponderance and a predominance of polymorphic lesions, whereas active surveillance indicated absence of any gender bias and more importantly, macular PKDL constituted almost 50% of the population burden. In terms of polymorphic vs. macular PKDL, the former appeared at a later age, their disease duration was longer and had a higher parasite burden. In the polymorphic variant, the lesional distribution was asymmetrical, comprised of papules/nodules/macules that were present mainly in sun-exposed areas whereas in macular cases, the hypopigmented patches were diffusely present all over the body. CONCLUSIONS/SIGNIFICANCE: Active surveillance unraveled a disease component whose demographic profile showed important differences with PKDL cases who sought treatment in government hospitals. Detection of a higher proportion of macular cases indicates that this variant is not an uncommon presentation as conventionally stated in text books, and should be studied in greater detail to ensure success of the ongoing Leishmaniasis elimination programme.

Socio-demographic and Clinico-Epidemiological Study of Scrub Typhus in Two Tertiary Care Hospitals of Kolkata.

Background and Aims: Scrub typhus is the commonest of the rickettsial diseases in India and is difficult to diagnose. Untreated cases have fatality rates of 30-45%. Eschar is present in 7-97% cases. Pneumonia and acute respiratory distress syndrome (ARDS) are frequent complications. Serum immunoglobulin M capture ELISA is the most sensitive test. Doxycycline is the drug of choice. Our objectives were to study the socio-demographic and clinic-epidemiological profiles of scrub typhus cases in two tertiary care hospitals in Kolkata, India. This was the first study of scrub typhus in Southern West Bengal and its neighboring areas. . Methods: Study was conducted over 16 months and all fever cases of Tropical Medicine / Medicine outpatients' clinics were evaluated. Results: Fourteen cases were diagnosed. 78.6% were from rural areas and 35.7% were farmers. Headache and fever were the commonest presenting complaints while eschar was found in only 21.4%. Serum IgM scrub typhus antibody was positive in all cases . Conclusion: Scrub typhus should be a differential diagnosis in acute febrile illness cases, as early diagnosis and therapy prevents complications.

A cross-sectional study on renal involvement among HIV-infected patients attending a tertiary care hospital in Kolkata.

Background and objectives: The HIV-associated renal diseases represent a spectrum. Indian data on this is sparse. This study was undertaken to find out the prevalence and clinicopathological spectrum of renal involvement in HIV among antiretroviral therapy (ART) naïve patients (Group 1) and among those on ART (Group 2). Methods: Systematic random sampling was undertaken to select 109 patients each from virology outpatient department (VOPD) and ART centre of a tertiary care hospital. They were screened and further investigated if renal involvement was found. Results: Renal involvement was present in 25/109 (22.94%) and 15/109 (13.76%) patients of Groups 1 and 2, respectively. Among patients of Groups 1 and 2, 9/24 (37.5%) and 2/13 (15.4%), respectively, had clinically significant proteinuria, but none in the nephrotic range. Statistically significant relationships of renal involvement were observed with CD4 count <100/µl and with low BMI. Of the patients of Group 2, 20% of those on a tenofovir-based regimen had renal involvement with tubular changes, while only 4.6% of those on other regimens had renal involvement. This difference was statistically significant (p<0.05; OR=5.25). Conclusion: Renal involvement was less common among those on ART. Low CD4 count and body mass index (BMI) were associated with renal dysfunction. Patients on a tenofovir-based regimen had more renal involvement compared with not on a tenofovir-based regimen.

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

BACKGROUND: Post 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. OBJECTIVE: Previously, 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 FINDING: In 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. CONCLUSION: These 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%.

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

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Status of circulating immune complexes, IL8 titers and cryoglobulins in patients with dengue infection.

Dengue, a serious viral infection caused by the mosquito vector, Aedes aegyptii, affects about 390 million people annually from more than 125 countries across the globe. However, until now, there is no reliable clinical or laboratory indicator to accurately predict the development of dengue severity. Here, we explored critical pathophysiological determinants like IL8, circulating immune complex (CIC) and cryoglobulin in dengue-infected patients for identification of novel dengue severity biomarker(s). Totally, 100 clinically suspected dengue cases were tested by NS1 ELISA and MAC ELISA for dengue virus aetiology. For control, 49 healthy volunteers were included. Blood profiling (complete hemogram and liver function test) of patient population were done using automated cell counter and standard auto analyzer based biochemical analysis. Serum CIC was quantified by PEG precipitation. Serum cryoglobulins were estimated by Folin assay. Levels of serum IL-8 were assessed by standard sandwich ELISA kits. Patient CIC were further characterized by SDS Gel electrophoresis. Forty per cent of the cases tested positive, of which 11 patients had severe clinical manifestation. The mean ±SEM of cryoglobulin concentration for DHF, DF, and HC were 1.30 ± 0.31, 0.59 ± 0.08 and 0.143 ± 0.009 µg/µl, respectively. Thus, DHF and DF patients have shown 9- and 2.2-fold increase in cryoglobulin levels; and 18- and 5-fold increased CIC, respectively compared to HC patients. The mean ±SEM of CIC-PEG index for DHF, DF and HC were 491 ± 41.22, 146 ± 14.19 and 27.98 ± 2.56, respectively. Raised levels of IL8 titers were also found in all 11 DHF patients. Peak levels of CIC, cryoglobulin and IL8 titers were associated with thrombocytopenia. SDS PAGE analysis of CIC from DHF revealed the presence of at least six protein bands that were not observed in samples from DF and HC. Prediction efficacy of IL8, CIC and cryoglobulin for DHF was determined using the receiver operator characteristic curve (ROC). The area under the curve was 1.00 for IL8, 0.99 for CIC and 0.74 for cryoglobulins. Overall, the results suggest that CIC, IL-8 and cryoglobulins may serve as important laboratory parameters to monitor dengue infection progression.

Tuberculoid leprosy and cytomegalovirus retinitis as immune restoration disease in a patient with AIDS.

Here we report a unique case of tuberculoid leprosy and cytomegalovirus retinitis in a 27-year-old female patient with AIDS, suggestive of highly active antiretroviral therapy (HAART)-induced immune restoration disease. After initiation of HAART, the patient presented with decreased visual acuity, hypoesthetic patch with local nerve thickening, and an increase in her CD4+ T cell count. On further investigations cytomegalovirus retinitis and tuberculoid leprosy were confirmed. To our knowledge no case with such a co-existence has previously been reported.

Management of snakebite cases by national treatment protocol at Jalpaiguri District Hospital in West Bengal in the year 2010--a retrospective study.

Snakebite remains a public health problem in India, occurring most frequently in the summer and rainy seasons. Bites are maximal in lower limbs. Victims are typically male and between 17 and 27 years of age. Children and the elderly have higher mortality. The worst affected states are Kerala, Maharashtra, Tamil Nadu, Orissa, Assam and West Bengal. There was no uniform guideline for treatment of snakebite cases. The five common venomous Indian snakes biting humans are common cobra, krait, Russell's viper, saw scaled viper and the hump nose pit viper. Seventy per cent of all snakebites are non-venomous. Even in bites by venomous snakes, envenomation occurs in only 50% of cases. Immobilisation is much more important than tight ligature, which may cause gangrene. Only a minority need antivenom, which is expensive, short in supply and may cause severe reaction. Antivenom treatment is recommended on the basis of local and systemic signs and symptoms and 20 minutes whole blood clotting test (20WBCT). Delay in starting AVS treatment is the main cause of mortality and morbidity. Skin test is of no value. But antivenom should not be used unless specifically indicated. The "Do it RIGHT" approach of national treatment protocol indicates the initial steps to be taken before reaching a hospital or primary healthcare facility. And it resulted in a 66% decline in the amount of ASV administration and an absolute reduction of mortality by 24%. However first aid treatment of the bitten limb/area with broad-spectrum antibiotics, injection tetanus antitoxin and Supportive treatment with blood transfusion, ventilatory support, anticholinesterase and peritoneal dialysis may also be required.