Niti Singh

Predicting kala-azar disease manifestations in asymptomatic patients with latent Leishmania donovani infection by detection of antibody against recombinant K39 antigen.

Clinically visceral leishmaniasis is suspected in only a fraction of infected persons, as the majority of these may not have clinical manifestations and remain asymptomatic. There is scanty information on diagnosing latent infections and predicting disease in asymptomatic persons. We therefore carried out a study on asymptomatic contacts of patients with visceral leishmaniasis and post-kala-azar dermal leishmaniasis by using methods for detection of antibody to recombinant K39 (rK39) antigen. A total of 240 patients with leishmaniasis and 150 asymptomatic contacts were tested for anti-rK39 immunoglobulin G (IgG) and IgA antibodies. Fifty-five asymptomatic persons were found to be seropositive. These individuals were monitored every 3 months for 1 year. On follow-up, 43.9% of the asymptomatic seropositive contacts developed kala-azar within the first 3 months, and a cumulative total of 69% developed kala-azar within 1 year. The rest remained asymptomatic and self-healed the infection. The sensitivity and specificity of rK39 enzyme-linked immunosorbent assay (ELISA) and dipstick tests were 100%, while an in-house-developed latex agglutination test had 80% sensitivity. The antibody profile showed that the IgG anti-rK39 antibodies reached a titer of up to 10(-6) within 6 months of infection, started declining thereafter, and completely disappeared in 2 to 3 years in successfully treated cases. Significant titers of IgA antibodies were detectable a little earlier than those of IgG antibodies and were undetectable after 6 months. The study showed that mass screening of family members and contacts by using anti-rK39 ELISA could be a highly reliable tool for early diagnosis and to plan prophylactic treatment of latently infected asymptomatic carriers to eradicate kala-azar.

Animal Reservoirs of Visceral Leishmaniasis in India

Abstract:  Visceral leishmaniasis (VL) is a disease that has both zoonotic and anthroponotic etiologies. In India, VL is endemic, considered to be anthroponotic, and caused by Leishmania donovani. Anthroponotic diseases are maintained by transmission from human to human and to a lesser extent from human to animals. Serum samples from 1,220 animals from 7 human VL endemic districts of Bihar, India, were tested for antibodies to a recombinant kinetoplast antigen (rK39 antigen) present in amastigotes of visceralizing Leishmania species, i.e., L. donovani complex. Additionally, PCR was used to examine samples positive by rK39 antigen serology. Antibodies to rK39 indicative of VL were detected in 33 of 1,220 animals. Thirty-one of 867 goats (Capra hircus), 1 of 161 cattle (Bos indicus), and 1 of 54 wild rats (Rattus sp.) were positive by rK39 serology. None of 106 chickens (Gallus domesticus), 26 sheep (Ovis aries), 3 water buffaloes (Bubalus bubalus), or 3 dogs (Canis familiaris) was positive by rK39 serology. Leishmania donovani DNA was detected by PCR in 20 rK39 positive blood samples from goats and 1 sample from a cow. The present study indicates that goats are potential animal reservoirs of human VL in India.

Fever of unknown origin : a prospective study

Fever of unknown origin (FUO) is a problem frequently faced by clinicians all over the world. One hundred and twenty-one cases of FUO presenting to a large teaching hospital in northern India were prospectively studied over a period of 2 years. Infections were the commonest cause accounting for 43.8% cases of FUO, with tuberculosis (TB) being the commonest infection encountered. Collagen vascular diseases and tumours accounted for 15.7 and 8.3% cases, respectively. No cause could be found out in a substantial number of cases (19%) even after invasive investigations. Knowledge of the current patterns of FUO is important since many patients present with potentially treatable diseases.

Comparative proteomic analysis of sequential isolates of Mycobacterium tuberculosis from a patient with pulmonary tuberculosis turning from drug sensitive to multidrug resistant

Background & objectives: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. The intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.

Trichuris vulpis infection in an indian tribal population

Stools from 28 of the 82 inhabitants on remote Little Andaman Island in India were examined for parasite eggs and cysts. Trichuris trichiura eggs were found in 27, Trichuris vulpis eggs in 5, Strongyloides stercoralis larvae in 3, hookworm eggs in 15, Entamoeba histolytica and Entamoeba coli cysts each in 9, Giardia lamblia in 6, Retortamonas sp. in 3, Iodoamoeba sp. in 2, and Chilomastix sp. in 2 stools. Ascaris lumbricoides eggs were not seen. The occurrence of T. vulpis eggs in 5 stools and the absence of A. lumbricoides eggs were considered unusual findings.

A successful management of dengue fever in pregnancy: report of two cases.

In the most recent outbreak of dengue fever in India, we encountered at least seven cases of fever with thrombocytopenia in pregnancy but only two were seropositive for dengue. In one of the cases there was postpartum hemorrhage while in the other case there was perinatal transmission to the neonate requiring platelet transfusions. The diagnostic difficulties in pregnancy and the management are discussed.

Genetic diversity and drug susceptibility profile of Mycobacterium tuberculosis isolated from different regions of India.

OBJECTIVES Molecular genotyping profiles of Mycobacterium tuberculosis (MTB) provide a valuable insight into the evolution and transmission of the bacilli. Due to the lack of comprehensive national level data from India on this subject, we performed this study to determine the recent trends and distribution of various MTB lineages circulating in India. METHODS A total of 628 MTB isolates were obtained from North, West, South, Central and Eastern India. Spoligotyping and drug susceptibility testing was performed by using manufacturers instructions. RESULTS Spoligotyping detected 102 distinct spoligo-patterns. A total of 536 (85.3%) isolates were distributed into 85 SITs which matched the pre-existing database, whereas 17 SITs were newly created for 34 (5.4%) isolates. Overall, CAS family genotype was predominant, comprising 222 (35.4%) isolates, followed by EAI in 152 (24.2%), Beijing in 108 (17.2%), Manu in 41 (6.5%), T in 30 (4.8%), H in 6 (0.9%), X in 3 (0.5%) and one (0.2%) each in Ural and AFRI. Drug susceptibility testing identified 134 (21.3%) isolates as multi drug resistant (MDR). CONCLUSIONS The CAS lineage had a pan India presence but EAI lineage was confined to southern parts of India. Beijing genotype of MTB was significantly associated (p-value <0.0001) with MDR.

Poor Performance of Serological Tests in the Diagnosis of Pulmonary Tuberculosis: Evidence from a Contact Tracing Field Study

Background Delayed or missed diagnosis of TB continues to fuel the global TB epidemic, especially in resource limited settings. Use of serology for the diagnosis of tuberculosis, commonly used in India, is another factor. In the present study a commercially available serodiagnostic assay was assessed for its diagnostic value in combination with smear, culture and clinical manifestations. Methodology/Principal Findings A total of 2300 subjects were recruited for the study, but 1041 subjects were excluded for various reasons. Thus 1259 subjects were included in the study of which 470 were pulmonary tuberculosis cases (440 of 470 were culture-positive) and 789 were their asymptomatic contacts. A house-to-house survey method was used. Blood samples were tested for IgM, IgA, and IgG antibodies using the Pathozyme Myco M (IgM), Myco A (IgA) and Myco G (IgG) enzyme immunoassay (EIA). Out of 470 PTB cases, BCG scar was positive in 82.34%. The Mantoux test and smear positivity rates in PTB cases were 94.3% (430/456), and 65.32% (307/470), respectively. Among the asymptomatic contacts, BCG scar was positive in 95.3% and Mantoux test was positive in 80.66% (442/548) contacts. No contact was found falsely smear positive. The sensitivity of IgM, IgA, and IgG EIA tests was 48.7%, 25.7% and 24.4%, respectively, while the specificity was 71.5%, 80.5%, 76.6%, respectively. Performance of EIAs was not affected by the previous BCG vaccination. However, prior BCG vaccination was statistically significantly (p = 0.005) associated with Mantoux test positivity in PTB cases but not in contacts (p = 0.127). The agreement between serology and Mantoux test was not significant. Conclusion The commercial serological test evaluated showed poor sensitivity and specificity and suggests no utility for detection of pulmonary tuberculosis.

Loop-mediated isothermal amplification assay for rapid and sensitive diagnosis of tuberculosis

OBJECTIVES Loop-mediated isothermal amplification (LAMP) is a newly developed molecular method that can be performed isothermally. We developed and evaluated a LAMP assay using novel primers to diagnose tuberculosis directly from clinical samples. MATERIALS Primers were designed to amplify the specific novel esat-6 gene target of Mycobacterium tuberculosis (MTB). Quantitated DNA was used to determine analytical sensitivity and specificity was evaluated by testing 29 NTM and 37 other bacterial species. After standardization, its sensitivity and specificity were evaluated on samples from 118 TB suspected and 31 non-TB patients and compared it with smear, culture and mPCR methods. RESULTS LAMP was able to detect 5 fg DNA (one MTB) within 21 min and found to be 10 times more sensitive than mPCR and showed 100% specificity against NTM and other bacterial species. In clinical samples, LAMP showed highest MTB detection rate (52.5%) as compared to mPCR (44%) and culture (30.5%). On culture positive and mPCR positive samples, the sensitivity of LAMP was found to be 100% (95% CI 90.2-100) and 96.1% (95% CI 86.7-99.5) respectively with 93.5% (95% CI 78.5-99.2) of overall specificity. CONCLUSION LAMP was found to be more sensitive than culture and mPCR for the detection of MTB. It showed specificity comparable to mPCR but was rapid and cost effective.

Incidence and Prevalence of Tuberculosis among Household Contacts of Pulmonary Tuberculosis Patients in a Peri-Urban Population of South Delhi, India

Background Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of mortality and morbidity across all age groups throughout the world, especially in developing countries. Methodology/Principal Findings In this study, we have included 432 open index cases with their 1608 household contacts in a prospective cohort study conducted from May 2007 to March 2009. The follow-up period was 2 years. All Index cases were diagnosed on the basis of suggestive signs and symptoms and sputum being AFB positive. Among the 432 index patients, 250 (57.9%) were males and 182 (42.1%) females; with mean age of 34±14.4 yr and 26±11.1 yr, respectively. Out of 1608 household contacts, 866 (53.9%) were males and 742 (46.1%) females; with mean age of 26.5±15.8 and 26.5±16.0 yr, respectively. Of the total 432 households, 304 (70.4%) had ≤4 members and 128 (29.6%) had ≥5 members. The median size of the family was four. Of the 1608 contacts, 1206 were able to provide sputum samples, of whom 83 (6.9%) were found MTB culture positive. Household contacts belonging to adult age group were predominantly (74, 89.2%) infected as compared to the children (9, 10.8%). On screening the contact relationship status with index patients, 52 (62.7%) were first-degree relatives, 18 (34.6%) second-degree relatives and 12 (14.5%) spouses who got infected from their respective index patients. Co-prevalent and incident tuberculosis was found in 52 (4.3%) and 31 (2.6%) contacts, respectively. In incident cases, the diagnosis could be made between 4 to 24 months of follow-up, after their baseline evaluation. Conclusion Active household contact investigation is a powerful tool to detect and treat tuberculosis at early stages and the only method to control TB in high-TB-burden countries.