Mehebubar Rahman

Genomic Characterization of Nipah Virus, West Bengal, India

An intrafamilial outbreak in West Bengal, India, involving 5 deaths and person-to-person transmission was attributed to Nipah virus. Full-genome sequence of Nipah virus (18,252 nt) amplified from lung tissue showed 99.2% nt and 99.8% aa identity with the Bangladesh-2004 isolate, suggesting a common source of the virus.

Enhanced Lesional Foxp3 Expression and Peripheral Anergic Lymphocytes Indicate a Role for Regulatory T Cells in Indian Post-Kala-Azar Dermal Leishmaniasis

Indian post-kala-azar dermal leishmaniasis (PKDL) is a low-frequency (5-10%) dermal sequela of visceral leishmaniasis (VL) caused by Leishmania donovani; importantly, affected individuals are speculated to be parasite reservoirs. Insight into its immunopathogenesis could translate into rational immunomodulatory therapeutic approaches against leishmaniases. In patients with PKDL (n=21), peripheral lymphocytes were analyzed for surface markers, intracellular cytokines, and lymphoproliferative responses using flow cytometry. In lesional tissue biopsies (n=12), expression of counter-regulatory cytokines (IFN-gamma and IL-10) and the T-regulatory transcription factor forkhead box protein 3 (Foxp3) was analyzed using reverse transcriptase-PCR, along with immunohistochemical detection (n=8) of CD3 and Foxp3 positivity. In patients with PKDL, circulating CD8(+)CD28(-) and antigen-induced IL-10(+)CD3(+) lymphocytes were increased and receded with treatment. CD8(+) lymphocytes showed impaired proliferative responses to L. donovani antigen (LDA) and phytohemagglutinin, which were reinstated after treatment. At presentation, the upregulated lesional IFN-gamma and IL-10 messenger RNA (mRNA), Foxp3 mRNA, and protein were curtailed after treatment. In Indian patients with PKDL, increased frequency of the CD8(+)CD28(-) phenotype, enhanced antigen-specific IL-10 production, and accompanying anergy of circulating lymphocytes suggest their regulatory nature. Furthermore, the concomitantly elevated lesional expression of Foxp3 suggests their possible recruitment into the lesional site, which would sustain disease pathology.

Short-Course Treatment Regimen of Indian Visceral Leishmaniasis with an Indian Liposomal Amphotericin B Preparation (Fungisome™)

India bears the burden of about half of global visceral leishmaniasis (VL) cases with emerging problems of stibanate resistance. Liposomal preparations have improved treatment outcome through shorter duration of therapy and lower toxicity compared with conventional amphotericin B. We report the efficacy of two short-course regimens of an Indian preparation of liposomal amphotericin B (Fungisome™) for VL caused by Leishmania donovani in India. An open-label, randomized, single-center comparative study was undertaken from 2008 to 2011, involving 120 treatment naive non-human immunodeficiency virus VL patients randomly allocated to two groups. Fungisome™ was given, in groups A (N = 60), 5 mg/kg daily for 2 days and B (N = 60), 7.5 mg/kg daily for 2 days, as intravenous infusion. Initial cure rate was 100% in both the groups after 1 month posttreatment. At 6 months after completion of treatment, definitive cure rate was group A 90% (54/60, 95% confidence interval (CI): 80.55-95.72%); group B: 100% (95% CI: 95.92-100%); (P = 0.027). No serious adverse events occurred in either group. The short-course, 2-day regimen of 15 mg/kg Fungisome™ infusion is easy to administer, effective, and safe for treatment of VL caused by L. donovani in India.

Easy Test for Visceral Leishmaniasis and Post–Kala-azar Dermal Leishmaniasis

To the Editor: Diagnosis of visceral leishmaniasis (VL), fatal if untreated, is complex because the symptoms are the same for many fever-associated ailments. Despite limitations, diagnosis remains based on finding Leishmania amastigotes in spleen and/or bone marrow aspirates (1). Sophisticated laboratory methods, although sensitive, are costly. The immunochromatographic strip test that uses recombinant K39 antigen (rK39), although satisfactory in India, is less sensitive in Africa, Latin America, and Mediterranean regions (2). Post–kala-azar dermal leishmaniasis (PKDL), a sequel to VL in India and Africa, is often confused with other skin diseases (3,4). Diagnosis of VL in dogs in Latin America and Mediterranean countries remains confusing because of rampant asymptomatic infections and elevated antibodies against Leishmania spp (5). Earlier we reported the diagnostic potential of L. donovani (MHOM/IN/83/AG83) promastigote membrane antigens (LAg) (3,6). Here we report applicability of LAg-based ELISA and dipstick systems even at primary health centers. Using randomized sampling, we tested samples from 122 kala-azar patients from India, 20 PKDL patients from India, and 40 VL patients from Brazil. VL was confirmed by finding parasites in aspirates. Serum samples were collected before chemotherapy was given. PKDL was diagnosed as described (3). Control samples were collected from 24 healthy persons from non–disease-endemic areas in India; 15 healthy persons from disease-endemic areas in India; 20 healthy persons from disease-endemic areas in Brazil; and 21 persons with Hansen disease, 7 with filariasis, 4 with tuberculosis, 1 with lymphoma, 1 with leukemia, 2 with virus-induced fever, and 5 with malaria. Consent was obtained from all human donors. This study was approved by Ethical Committee on Human Subjects at Indian Institute of Chemical Biology and the Ethical Board for Human Subjects and Animal Experimentation of the Federal University of Piaui. We developed a diagnostic ELISA with modifications of our previous method (6). Microtiter plates were coated with 2.5 μg LAg at pH 7.5 (100 μL/well) and kept at 4°C overnight, after which they were blocked with 1% bovine serum albumin, dried, and stored at 4°C as precoated plates. The assay performed at room temperature took ≈2.5 h. Test and control serum samples (1:1,000 dilution, 100 μL/well) were applied to the plates for 45 min and shaken occasionally. Horseradish peroxidase–conjugated goat anti-human immunoglobulin (Ig) G (Genei, Bangalore, India) was applied at 1:5,000 (100 μL/well) for 45 min. Color development with ortho-phenylenediamine (Sigma-Aldrich, St. Louis, MO, USA) was allowed for 5–10 min. Positive results were determined by comparing colors with those on a card previously prepared for positive and negative wells. ELISA, performed for the VL and PKDL patients from India, was 100% sensitive (percentage of patients with confirmed disease and positive test results) and 96.3% specific (percentage of negative controls with negative test results) (Figure, panel A); sensitivity and specificity were higher than that reported earlier (6) and by other studies that used crude leishmanial antigens (2). One sample from each of filariasis, lymphoma, and disease-endemic area controls was marginally false positive. Seropositivity was diagnosed for 1 patient who had a negative spleen aspirate but clinical signs of VL and for 1 patient who refused spleen or bone marrow aspiration. Figure Representative results of ELISA and dipstick testing. A) Samples underwent ELISA in duplicate. Upper panel, positive samples in duplicate (1–2 and 3–4) in wells A–H, except A1–A2 (blank), and G3–G4 and H3–H4 ... To avoid any visible cross-reaction in the dipstick assay, we optimized LAg concentration, test serum dilution, and control serum dilution. Optimum concentration for human studies is 500 μg/mL LAg, 1:2,000 serum dilutions, 1:2,000 horseradish peroxidase–conjugated goat anti-human IgG, and 15 mg 3,3′-diaminobenzidine (Sigma-Aldrich) as substrate in 30 mL Tris-buffered saline. LAg was bound to a long nitrocellulose piece at the test line (line on which LAg is coated). Goat anti-human IgG (Genei) at 1:25 was coated as an internal control line. Free sites were blocked with 2% bovine serum albumin containing 0.01% NaN3 and were air dried. LAg-coated membranes were affixed to the end of a plastic support (with a free end as handle) with double-adhesive tape, cut into 4-mm–wide sticks, and stored at room temperature. During the testing process at room temperature, dipsticks were incubated in diluted serum for 30 min, washed 2×, incubated for 30 min with secondary antibody, washed 3×, and incubated in substrate solution for 1 min. Finally, dipsticks were washed in water, dried on tissue paper, and examined for specific reaction. When stored at room temperature without desiccation, dipsticks performed consistently for 12 months. Dipsticks appeared equally sensitive and specific (100%) for VL from India and Brazil and for PKDL. Because internal control lines remained positive, analyses were considered valid (Figure, panel B). LAg dipsticks are more sensitive for diagnosing VL in Brazil than rK39 (7) and cost ≈70× less (8). Although further validation with a larger sample size and healthy controls from disease-endemic areas and controls for other diseases is warranted, these easy, simple, and low-cost methods could emerge as efficient tools for diagnosis of VL and PKDL.

Alternative treatment approach to cerebral toxoplasmosis in HIV/AIDS: experience from a resource-poor setting

The current standard treatment for cerebral toxoplasmosis (pyrimethamine/sulfadiazine) often encounters problems of poor tolerability, adverse effects, frequent dropouts and non-availability of pyrimethamine/sulfadiazine in some parts of India. We have had to use the combination of two effective alternative agents for toxoplasmosis, cotrimoxazole and clindamycin, on compassionate grounds. This retrospective observational study reports superior efficacy and better tolerability of cotrimoxazole/clindamycin compared to the recommended regimen. Primary end-point (complete response) was defined as more than 50% improvement of clinical status or more than 50% decrease in the size of brain lesions after two weeks of treatment initiation. Complete response occurred more commonly with cotrimoxazole/clindamycin than with pyrimethamine/sulfadiazine group (80% vs. 31.25%, respectively, relative risk 2.56, 95% confidence interval: 1.21–5.43). There was a trend towards higher on-treatment mortality in the pyrimethamine/sulfadiazine group in comparison to the cotrimoxazole/clindamycin (mortality rate 37.5% in pyrimethamine/sulfadiazine vs 12% in cotrimoxazole/clindamycin, p = 0.07, relative risk = 3.125, 95% confidence interval: 0.91–10.75). Overall, 62.5% (10/16) of patients on pyrimethamine/sulfadiazine suffered drug-related adverse reactions compared to 24% (6/25) on cotrimoxazole/clindamycin (p = 0.02, relative risk = 2.60, 95% confidence interval: 1.17–5.76). The commonest complication of pyrimethamine/sulfadiazine was severe thrombocytopenia with major bleeding (4/16, 25%). We propose that the new combination chemotherapy, which is widely available, effective and safe, can be used in developing countries.

Epidemiological Investigation of an Outbreak of Acute Encephalitis Syndrome (AES) in Malda District of West Bengal, India

Background: An unusual outbreak of acute encephalitis syndrome (AES) with high case fatality was reported from Kaliachak- I, II and III Blocks of Malda District of West Bengal in the month of June 2014 affecting 72 children with 34 deaths. The purpose of this study was to investigate the outbreak in the light of epidemiological as well as etiological determinants. Methods: The investigating team collected clinical and epidemiological data from the cases admitted at Malda Medical College and at the Kaliachak BPHC. Different clinical samples, (serum, CSF etc) collected from cases as well as control population were screened for different pathological, biochemical and microbiological parameters. Additionally, the CSF specimens were also processed for the isolation of viruses by inoculating in the chorio-allantoic membrane (CAM) of embryonated chick eggs and intracerebral inoculation of suckling mice. Statistical methods included calculation of proportions (percentages), different test of significance (t-test, chi square etc). Results: All children were from age group of 9 months to 10 years (median=3, mean=3.73, SD=1.98) and belonged to low socioeconomic background of litchi growing belt of Malda. Most of the cases were male (65% approx). Case Fatality Rate was 47.2%. The main presenting features were sudden onset of convulsions (100%) in the early hours of dawn followed by rapid progression to unconsciousness (100%) and decerebrate rigidity (47%). Fever was present in around one third of cases. Hypoglycaemia and leucocytosis were two predominant features. Clinical samples subjected to molecular and serological testing, were all found negative for known viruses causing acute encephalitis. 3 out of 4 CSF samples produced demonstrable pocks in Chorio allantoic membrane of the embryonated eggs although the pock count varied from 4- 22 per CAM. Significantly low blood glucose level was found in the controls from litchi belt areas as compared to the controls of non-litchi belt areas of Malda. Conclusion: The evidence gathered so far pointed towards a viral etiology although the causative virus remained unidentified. Hypoglycaemia probably induced by litchi fruit might have aggravated the encephalitis rather than actually causing it.

Protective Efficacy of Secondary Prophylaxis Against Visceral Leishmaniasis in Human Immunodeficiency Virus Coinfected Patients Over the Past 10 Years in Eastern India.

Coinfection with visceral leishmaniasis (VL) and human immunodeficiency virus (HIV) leads to frequent treatment failure, relapse, and death. In this retrospective analysis from eastern India (2005-2015), our primary objective was to ascertain the protective efficacy of secondary prophylaxis with monthly amphotericin B (AmB) given in patients with HIV-VL coinfection toward reducing relapse and mortality rates. The secondary objective was to compare clinical features, laboratory findings, and treatment outcomes in HIV-VL patients in contrast to VL monoinfection. Overall, 53 cases of HIV-VL and 460 cases of VL monoinfection were identified after excluding incomplete records. Initial cure rate was 96.23% in HIV-VL (27 received liposomal AmB and 26 AmB deoxycholate). All patients with initial cure (N = 51) were given antiretroviral therapy. Secondary prophylaxis (N = 27) was provided with monthly 1 mg/kg AmB (15 liposomal, 12 deoxycholate). No relapse or death was noted within 6 months in the secondary prophylaxis group (relapse: none versus 18/24 [75%]; mortality: none versus 11/24 [45.8%]; P < 0.001 for both). Secondary prophylaxis remained the sole significant predictor against death in multivariate Cox regression model (hazard ratio = 0.09 [95% confidence interval = 0.03-0.31]; P < 0.001). HIV-VL patients had higher 6-month relapse rate, less relapse-free 12-month survival, and higher mortality (P < 0.001 each) than VL monoinfection. In conclusion, it appears from this study that secondary prophylaxis with monthly AmB might be effective in preventing relapse and mortality in HIV-VL.

RNA-Seq analysis of peripheral blood mononuclear cells reveals unique transcriptional signatures associated with disease progression in dengue patients

&NA; Patients infected with Dengue virus usually present a mild, self‐limiting febrile dengue infection (DI) that occasionally leads to a potentially lethal complication, called the severe dengue (DS). The ability to identify the prognostic markers of DS could allow an improved disease intervention and management. To identify the transcriptional signatures associated with the dengue disease progression, we carried out the high‐throughput sequencing of the RNA isolated from the peripheral blood mononuclear cells (PBMCs) of the dengue patients of varying severity and compared with that in the patients with other febrile illnesses (OFIs) or the healthy controls. The transcriptional signatures that discriminated the DS patients from OFI and DI patients were broadly related to the pathways involving glycine, serine, and threonine metabolisms, extracellular matrix organization, ubiquitination, and cytokines and inflammatory response. Several upregulated genes in the inflammatory process (MPO, DEFA4, ELANE, AUZ1, CTSG, OLFM4, SLC16A14, and CRISP3) that were associated with the dengue disease progression are known to facilitate leukocyte‐mediated migration, and neutrophil activation and degranulation process. High activity of MPO and ELANE in the plasma samples of the follow‐up and recovered dengue patients, as well as and the presence of a larger amount of cell‐free dsDNA in the DS patients, suggested an association of neutrophil‐mediated immunity with dengue disease progression. Careful monitoring of some of these gene transcripts, and control of the activity of proteins encoded by them, may have a great translational significance for the prognosis and management of the dengue patients.

Nephritic syndrome and anasarca in a case of lymphatic filariasis: A rare association

Abstract Lymphatic filariasis is caused by a nematode, namely, Wuchereria bancrofti, Brugia malayi and Brugia timori . Lymphatic filariasis is a spectrum of illness and can manifest as, asymptomatic microfilaria, acute lymphatic filariasis, chronic lymphatic filariasis, tropical pulmonary eosinophilia and some systemic manifestations which involves joint, heart, kidney, nerve etc . Here, we present a case of anasarca with nephritic syndrome caused by lymphatic filariasis which is a rare presentation and only few cases have been reported in India so far.

Absolute CD4 counts in monoinfected chronic hepatitis B patients in the advanced immune-active stage.

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