Sarman Singh

Genome-wide analysis of the host intracellular network that regulates survival of Mycobacterium tuberculosis

We performed a genome-wide siRNA screen to identify host factors that regulated pathogen load in human macrophages infected with a virulent strain of Mycobacterium tuberculosis. Iterative rounds of confirmation, followed by validation, identified 275 such molecules that were all found to functionally associate with each other through a dense network of interactions. This network then yielded to a molecular description of the host cell functional modules that were both engaged and perturbed by the pathogen. Importantly, a subscreen against a panel of field isolates revealed that the molecular composition of the host interface varied with both genotype and the phenotypic properties of the pathogen. An analysis of these differences, however, permitted identification of those host factors that were invariantly involved, regardless of the diversification in adaptive mechanisms employed by the pathogen. Interestingly, these factors were found to predominantly function through the regulation of autophagy.

Chemotherapy of Leishmaniasis: Past, Present and Future

Leishmaniasis is a parasitic disease caused by hemoflagellate, Leishmania spp. The parasite is transmitted by the bite of an infected female phlebotomine sandfly. The disease is prevalent throughout the world and in at least 88 countries. Nearly 25 compounds are reported to have anti-leishmanial effects but not all are in use. The pentavalent antimony compounds have remained mainstay for nearly 75 years. Pentavalent antimony is a prodrug that is reduced by glutathione to active trivalent species catalyzed by thiol-dependent-reductase. However, emergence of resistance led to the use of other compounds--amphotericin B, pentamidine, paromomycin, allopurinol etc. Amphotericin B, an antifungal macrolide polyene is characterized by the hydrophilic polyhydroxyl and hydrophobic polyene faces on it long axis. Presently, it is the only drug with highest cure rate. It acts on membrane sterols resulting in parasite cell lysis. Its lipid formulations have been developed to minimize side effects. Other anti-fungals like ketoconazole, fluconazole and terbinafine are found less effective. Recently, anticancer alkylphosphocholines have been found most effective oral compounds. These act as membrane synthetic ether-lipid analogues, and consist of alkyl chains in the lipid portions. Most promising of these are miltefosine (hexadecylphosphocholine), edelfosine (ET-18-OCH(3)) and ilmofosine (BM 41.440). However, the recent focus has been on identifying newer therapeutic targets in the parasite such as DNA topoisomerases. The present review describes the current understanding of different drugs against leishmaniasis, their chemistry, mode of action and the mechanism of resistance in the parasite. Future perspectives in the area of new anti-leishmanial drug targets are also enumerated. However, due to the vastness of the topic main emphasis is given on visceral leishmaniasis.

Recent advances in the diagnosis of leishmaniasis.

Leishmaniasis is a parasitic disease caused by a haemoflagellate Leishmania. There are more than 21 species causing human infection. The infection is transmitted to humans through the bites of female sandflies belonging to 30 species. The disease manifests mainly in 3 forms: the visceral, the cutaneous and the mucocutaneous leishmaniasis. The diagnosis of visceral form is conventionally made by the demonstration of amastigotes of the parasite in the aspirated fluid from the bone marrow, the spleen, and rarely from the lymph nodes, or the liver. The parasite demonstration and isolation rates are rather poor from cutaneous and mucocutaneous lesions due to low parasite load and high rate of culture contamination. Recently several recombinant proteins have been developed to accomplish accurate diagnosis. Recombinant kinesin protein of 39 kDa called rK 39 is the most promising of these molecules. The antigen used in various test formats has been proved highly sensitive and specific for visceral leishmaniasis. It is useful in the diagnosis of HIV-Leishmania co-infection and as a prognostic marker. Molecular techniques targeting various genes of the parasite have also been reported, the PCR being the most common molecular technique successfully used for diagnosis and for differentiation of species.

DIAGNOSTIC AND PROGNOSTIC VALUE OF K39 RECOMBINANT ANTIGEN IN INDIAN LEISHMANIASIS

The recombinant product (rK39) of the 39 amino acid repeats encoded by a kinesin-like gene of visceral Leishmania spp. was further evaluated by enzyme-linked immunosorbent assay (ELISA) for its diagnostic potential in Indian kala-azar (VL) and post kala-azar dermal leishmaniasis (PKDL). Anti-rK39 antibodies were highly positive in 20 symptomatic cases, including 6 resistant to single or double chemotherapy, but became negligible or absent in 9 recently cured patients. Endpoint titration of samples from the 20 active cases showed that the anti-rK39 IgG titers fell within a wide range of 10(-2) to > 10(-6), and that their mean was > 1 order of magnitude higher than in VL reported previously. The anti-rK39 IgG titers were correlated with parasite burden found in the patients and remained undiminished in those refractory to chemotherapy. These results indicate that: (1) the K39 epitope is conserved in Indian strains of Leishmania donovani, (2) the extremely high levels of K39 antibodies in both VL and PKDL suggest the application of rK39 for sensitive and specific serodiagnosis, and (3) rK39 ELISA is also valuable for prognostic evaluation of both diseases.

Mother-to-child transmission of hepatitis E virus infection

Objectives : Water borne or enterically transmitted non-A-non-B hepatitis is a major public health problem in India. Many of these cases carry fatal outcome. The hepatitis E virus (HEV) has been considered to be the most important causative agent of this entity. The severity and fatality rates of HEV infection are reported to be rather more in pregnant women. However, there is meager information from India, on mother to child transmission of this agent.Methods : During 1997-98, we studied 60 pregnant women suspected to have acute viral hepatitis to understand the frequency of various viral etiologies, disease course and outcome of the pregnancy. Six cord blood samples were tested for IgG, and IgM antibodies against hepatropic viral agents and also for hepatitis E virus RNA by RT-nested PCR using ORF-1 as target.Results : Of the 60 pregnant patients hospitalised at All India Institute of Medical Sciences, New Delhi for acute hepatitis, 22 (37%) were positive for IgM anti-HEV antibodies and 10% were infected with hepatitis B virus. Co-infection of HEV with Hepatitis B and C was seen in 1 and 2 patients, respectively. Most (72%) of the HEV infected patients were in third trimester of pregnancy (P<0.05). Of the 6 cord blood samples tested 3 (50%) were positive for HEV RNA. Though, all mothers were RNA positive, half of the babies did not get infectedin utero with HEV. Fourteen of the 22 (63.6%) HEV infected mothers developed fulminant hepatic failure and all died.Conclusion : The mortality rate in HIV infected mothers was 100%. Mother to child transmission of hepatitis E virus infection was established in 50%

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.

Chronic diarrhoea in HIV patients: Prevalence of coccidian parasites

The purpose of this study was to determine the prevalence of intestinal parasites in HIV patients with or without diarrhoea and to see an association between diarrhoea and the coccidian parasites in our setting. Stool samples from 113 HIV patients, 34 chronic diarrhoea and 79 without any history of diarrhoea were collected and examined for enteric parasites by microscopy. One hundred and thirteen control samples from HIV negative patients complaining of prolonged diarrhoea were also collected and analysed. Prevalence of coccidian parasites in HIV and non-HIV patients; with and without diarrhoea was compared using chi-square tests. Enteric parasites were detected in 55.8% HIV patients with diarrhoea compared to 16.4% in patients without diarrhoea (P<0.001). Isospora belli was found in 41.1% (14/34) of chronic diarrhoea and 6.3% (5/79) in non-diarrhoeal cases (P<0.001). Cryptosporidium was detected in 20.6% (7/34) of chronic diarrhoea and 2.5% (2/79) in non-diarrhoeal cases (P<0.001). Cyclospora cayetanensis associated diarrhoea was detected in only one case of chronic diarrhoea (2.9%). CD4+ T-cell count was lower (180 cells/microL) in diarrhoeal HIV patients as compared to non-diarrhoeal patients. Coccidian parasites were seen at a mean CD4+ T-cell count of 186.3 cells/microL. This study concluded that Isospora belli was the predominant parasite followed by Cryptosporidium spp. and both were strongly associated with diarrhoea among HIV patients.

Non-tuberculous mycobacteria in TB-endemic countries: are we neglecting the danger?

There are more than 120 members of the genus Mycobacterium, which are diverse in pathogenicity, in vivo adaptation, virulence, response to drugs, and growth characteristics. Mycobacteria other than M. tuberculosis complex and M. leprosy are known as Non-Tuberculous Mycobacteria (NTM) and are known by various acronyms. They attracted abrupt attention only after the AIDS epidemic, but most of the reports were published from TB non-endemic countries [1] and only rarely from TB-endemic countries. This is probably because the chances of missing NTM species are higher in TB-endemic countries, which are poorly equipped and overburdened with other diseases (Box 1). The information regarding their true incidence and prevalence in these countries is scarce [2]. In the absence of such authentic information, the current dogma has been that the NTM are of the least consequence. However, we do not agree with this myth and wish to present our viewpoint on this important aspect and emphasize the need for a fresh look at this neglected aspect. Box 1. Possible Factors for Under-reporting of NTM from TB-Endemic Countries NTM infections are not reportable in any country. Awareness is lacking among treating physicians and microbiologists. Laboratory infrastructure is lacking for culture and identification of non-tuberculous mycobacteria. High burden of TB and HIV attracts the bulk of the attention of the health care system; governmental fiscal inputs toward the costs of these neglected infections continue to be neglected. Standardized or accepted criteria to define NTM respiratory disease are lacking. Prevalence of NTM Infections before and after the AIDS Epidemic We searched methodological search terms and phrases such as “non-tuberculous mycobacteria and AIDS” in Medline records and found that 3,020 articles were published between 1981 and 2009. Using the same phrase, only 59 articles were published between 1900 and 1981, indicating a clear upsurge of NTM disease in the post-AIDS era. However, most of these publications were from TB non-endemic countries [1]–[5], but not much significance could be adhered to these isolations [2]. The disseminated NTM infection is typically seen when the CD4+ T lymphocyte number falls below 50 µl. For this reason, it is argued that in TB-HIV co-endemic countries, AIDS patients usually die of tuberculosis or other infections before their CD4+ count falls low enough for NTM to cause a disease (Box 2). Nevertheless, we feel that besides this argument, in a majority of the patients, the diagnosis of NTM disease gets missed in these countries. Box 2. Facts about Non-tuberculous Mycobacterial Disease AIDS patients are significantly more vulnerable to NTM infections due to severe T cell immunodeficiency. Solid organ transplant patients, even though immunocompromised, are not at as high a risk as their HIV-positive counterparts. Although some genetic and anatomical factors predispose to NTM, no proven associations have been proven among geographical, occupational, or ethnic factors and NTM infections. Anatomical abnormalities and other co-morbidities such as chronic obstructive pulmonary disease (COPD), bronchiectasis, cystic fibrosis (CF), pneumoconiosis, past history of TB, pulmonary alveolar proteinosis, and esophageal motility disorders are well-established predisposing conditions. Disseminated NTM infections have been associated with specific genetic syndromes such as mutations in interferon (IFN)-γ, interleukin (IL)-12 synthesis, and in response pathways and the nuclear factor-κB essential modulator (NEMO). Conventional methods are not sufficiently sensitive to estimate prevalence and incidence of NTM infections. Monoplex TB-specific PCR needs to be replaced by multiplex PCR systems on relevant clinical samples along with blood and urine samples in tertiary-care settings. Multiplex PCR primers have been designed to amplify genus-specific regions, M. tuberculosis complex specific, M. avium complex specific, M fortuitum complex specific, and species-specific gene targets, that can be performed in a single tube. Geographical Distribution of NTM Some workers also consider that the low detection rate of NTM is due to diversity in the environmental and climatic conditions in the HIV-TB-endemic countries, but this argument is not supported by the literature [3]–[5]. In most of the surveys, the rate of human NTM infections is estimated by non-specific antibody assays or skin tests [6]. Hence, these findings may not be a reliable source of information. The International Union against Tuberculosis and Lung Diseases (IUATLD) reviewed data from 14 countries and found that the M. avium complex (MAC) was the most frequently isolated species in all these countries, which included China, India, and Korea. While M. fortuitum was the most frequently encountered species in Belgium (2.1%), the Czech Republic (17.5%), Denmark (5.3%), Finland (6.7%), France (6.5%), Germany (12.2%), Italy (2.5%), Portugal (16.5%), Spain (10.8%), Switzerland (17.5%), Turkey (33.9%), and the United Kingdom (6.0%), undoubtedly, environment is the main reservoir of NTM. There is no evidence of human-to-human or animal-to-human transmission [6]. Most infections are acquired either from the water (treated or untreated) or soil. MAC and M. fortuitum are frequently isolated from the drinking water distribution systems and swimming pools in both developing and developed countries.

Safety of 3 Different Reintroduction Regimens of Antituberculosis Drugs after Development of Antituberculosis Treatment–Induced Hepatotoxicity

BACKGROUND Drug-induced hepatotoxicity (DIH) is the most common adverse drug reaction leading to interruption of antituberculosis treatment. Worldwide, different reintroduction regimens have been advocated, but no consensus guidelines are available. Reintroduction of antituberculosis drugs in patients with DIH has never been studied systematically. We aimed to compare the safety of 3 different reintroduction regimens of antituberculosis drugs in patients with antituberculosis DIH. METHODS A total of 175 patients with a diagnosis of antituberculosis DIH were randomized to receive 1 of 3 different predefined reintroduction regimens of antituberculosis drugs and were evaluated prospectively. Patients in arm I were given isoniazid, rifampicin, and pyrazinamide simultaneously at full dosage from day 1. In arm II, drugs were administered in a manner similar to that recommended in the American Thoracic Society guidelines for reintroduction. In arm III, drugs were administered in accordance with British Thoracic Society guidelines. RESULTS Nineteen patients (10.9%) had recurrence of DIH during follow-up. Eight, 6, and 5 patients had recurrence of hepatitis in arms I, II, and III, respectively (P = .69). Of all the clinical and laboratory parameters, pretreatment serum albumin level was the only statistically significant predictor of future recurrence of DIH on reintroduction of antituberculosis drugs (P < .01). CONCLUSIONS The recurrence rate of hepatotoxicity was not significantly different between the 3 groups. According to the findings of the present study, all 3 of the potentially hepatotoxic drugs (isoniazid, rifampicin, and pyrazinamide) can be reintroduced simultaneously at full dosage safely from day 1, especially for patients with bilateral extensive pulmonary tuberculosis, to halt disease transmission or to treat patients with life-threatening tuberculosis. TRIAL REGISTRATION ClinicalTrials.gov identifier number: NCT00405301.

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims:  Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management.