Rakesh Yadav

Prevalence of multidrug resistance in Mycobacterium tuberculosis isolates from HIV seropositive and seronegative patients with pulmonary tuberculosis in north India

BackgroundMultidrug resistant (MDR) and extensively-drug resistant (XDR) tuberculosis (TB) are a serious threat to the national TB control programs of developing countries, and the situation is further worsened by the human immunodeficiency virus (HIV) pandemic. The literature regarding MDR/XDR-TB is, however, scanty from most parts of India. We carried out this study to assess the prevalence of MDR/XDR-TB in new and previously treated cases of pulmonary TB and in HIV seropositive and seronegative patients.MethodsSputum and blood specimens were obtained from 2100 patients suspected of pulmonary tuberculosis and subjected to sputum microscopy and culture for TB, and HIV serology at our tertiary care centre in north India. The culture positive Mycobacterium tuberculosis isolates were subjected to drug susceptibility testing (DST) for first line anti-tuberculosis drugs, and the MDR isolates were further subjected to second line DST. Various parameters of the patients’ were analyzed viz. clinical presentation, radiology, previous treatment history, demographic and socioeconomic data and microbiology results.ResultsOf the 2100 patients, sputum specimens of 256 were smear positive for acid-fast bacilli (AFB), 271 (12.9%) grew Mycobacterium spp., and M. tuberculosis was isolated in 219 (10.42%). Of the 219 patients infected with M. tuberculosis, 20.1% (44/219) were found to be seropositive for HIV. Overall, MDR-TB was observed in 17.4% (39/219) isolates. There were 121 newly diagnosed and 98 previously treated patients, of which MDR-TB was found to be associated with 9.9% (12/121) and 27.6% (27/98) cases respectively. There was significantly higher association of MDR-TB (12/44, 27.3%) with HIV seropositive patients as compared to HIV seronegative patients (27/175, 15.4%) after controlling previous treatment status, age, and sex (odd’s ratio, 2.3 [95% CI, 1.000-5.350]; p-value, 0.05). No XDR-TB was found among the MDR-TB isolates.ConclusionThe present study demonstrated a high prevalence of drug resistance amongst pulmonary TB isolates of M. tuberculosis from north India as compared to the WHO estimates for India in 2010, though this could possibly be attributed to the clustering of more serious or referred cases at our tertiary care centre. The prevalence of MDR-TB in HIV seropositive patients was significantly higher than seronegative individuals. The study emphasizes the need to monitor the trends of drug resistance in TB in various populations in order to timely implement appropriate interventions to curb the menace of MDR-TB.

Development and evaluation of multiplex PCR in rapid diagnosis of abdominal tuberculosis

The clinical features of abdominal tuberculosis (TB) are non-specific and establishing a diagnosis remains a challenge. A delay in diagnosis is likely to increase the morbidity in these patients. We developed a multiplex polymerase chain reaction (PCR) using 16SrRNA, IS6110, and devR, and evaluated it in comparison with other conventional tests in clinical suspects of abdominal TB. A total of 183 patients with clinical suspicion of abdominal TB (96 patients with intestinal TB and 87 with peritoneal TB) were enrolled for the study. Endoscopic or intraoperative biopsies were collected from patients suspected of intestinal TB and ascitic fluid was collected from patients with a suspicion of peritoneal TB. Of the intestinal tuberculosis group, there were 40 confirmed cases and 56 controls, while of the peritoneal tuberculosis group there were 37 confirmed cases and 50 controls. Multiplex PCR showed a high sensitivity and specificity in both the intestinal TB and peritoneal TB groups. When combined with histopathology, multiplex PCR could detect 97.5% of all the cases in the intestinal tuberculosis group, while in combination adenosine deaminase levels (ADA) in cases of peritoneal tuberculosis it increased the specificity of diagnosis of peritoneal tuberculosis to 95%. In combination with histopathology in suspected intestinal TB cases, and ADA testing in suspected peritoneal TB cases, it can be used as a highly sensitive, specific, and rapid diagnostic tool with the ability to supplement the limitations of other diagnostic modalities.

Development of a robust QSAR model to predict the affinity of pyrrolidine analogs for dipeptidyl peptidase IV (DPP- IV)

QSAR analysis using multiple linear regression and partial least squares methods were conducted on a data set of 47 pyrrolidine analogs acting as DPP IV inhibitors. The QSAR models generated (both MLR and PLS) were robust with statistically significant s, F, r, r2 and r2 (CV) values. The analysis helped to ascertain the role of shape flexibility index, Ipso atom E-state index and electrostatic parameters like dipole moment, in determining the activity of DPP IV inhibitors. In addition to QSAR modeling, Lipinski’s rule of five was also employed to check the pharmacokinetic profile of DPP IV inhibitors. Since none of the compounds violated the Lipinski’s rule of five indicating that the DPP IV inhibitors reported herein have sound pharmacokinetic profile and can be considered as potential drug candidates for diabetes mellitus Type II.

Transcriptome analysis of mycobacteria in sputum samples of pulmonary tuberculosis patients

Pulmonary tuberculosis, the disease caused by Mycobacterium tuberculosis, still retains a top rank among the deadliest communicable diseases. Sputum expectorated during the disease continues to be a primary diagnostic specimen and also serves as a reservoir of bacteria. The expression pattern of mycobacteria in sputum will lead to an insight into bacterial adaptation at the most highly transmissible stage of infection and can also help in identifying newer diagnostic as well as drug targets. Thus, in the present study, a whole genome microarray of Mycobacterium tuberculosis was used to elucidate the transcriptional profile of mycobacteria in the sputum samples of smear positive pulmonary tuberculosis patients. Overall, the mycobacteria in sputum appeared to be in a low energy and low replicative state as compared to in vitro grown log phase M. tb with downregulation of genes involved in ATP synthesis, aerobic respiration and translational machinery. Simultaneously, downregulation was also seen in the genes involved in secretion machinery of mycobacteria along with the downregulation of genes involved in the synthesis of phthiocerol dimycocerosate and phenol glycolipids. In contrast, the majority of the genes which showed an upregulation in sputum mycobacteria were of unknown function. Further identification of these genes may provide new insights into the mycobacterial behavior during this phase of infection and may help in deciphering candidates for development of better diagnostic and drug candidates.

Evaluation of in-house loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of M. tuberculosis in pulmonary specimens.

Loop‐mediated isothermal amplification (LAMP) assay has come forward as a rapid, cost‐effective molecular technique for diagnosis of tuberculosis (TB) in developing countries. This study evaluated Mycobacterium tuberculosis–specific in‐house LAMP assay targeting 16s rRNA and compared it with other conventional tests and nucleic acid amplification assay (IS6110 PCR).

Identification of Novel HIV 1- Protease Inhibitors: Application of Ligand and Structure Based Pharmacophore Mapping and Virtual Screening

A combined ligand and structure-based drug design approach provides a synergistic advantage over either methods performed individually. Present work bestows a good assembly of ligand and structure-based pharmacophore generation concept. Ligand-oriented study was accomplished by employing the HypoGen module of Catalyst in which we have translated the experimental findings into 3-D pharmacophore models by identifying key features (four point pharmacophore) necessary for interaction of the inhibitors with the active site of HIV-1 protease enzyme using a training set of 33 compounds belonging to the cyclic cyanoguanidines and cyclic urea derivatives. The most predictive pharmacophore model (hypothesis 1), consisting of four features, namely, two hydrogen bond acceptors and two hydrophobic, showed a correlation (r) of 0.90 and a root mean square of 0.71 and cost difference of 56.59 bits between null cost and fixed cost. The model was validated using CatScramble technique, internal and external test set prediction. In the second phase of our study, a structure-based five feature pharmacophore hypothesis was generated which signifies the importance of hydrogen bond donor, hydrogen bond acceptors and hydrophobic interaction between the HIV-1 protease enzyme and its inhibitors. This work has taken a significant step towards the full integration of ligand and structure-based drug design methodologies as pharmacophoric features retrieved from structure-based strategy complemented the features from ligand-based study hence proving the accuracy of the developed models. The ligand-based pharmacophore model was used in virtual screening of Maybridge and NCI compound database resulting in the identification of four structurally diverse druggable compounds with nM activities.

Ligand-based drug design studies using predictive pharmacophore model generation on 4H-1,2,4-triazoles as AT1 receptor antagonists

Angiotensin II subtype I receptor constitutes a successful target for the treatment of hypertension and cardiovascular diseases. In this study, the pharmacophore search was used to identify structural features that are common in the set of 4H-1,2,4-triazoles. The HypoGen algorithms implemented in the Catalyst software package was employed to create quantitative models. The common feature model for AT1 included two ring aromatic features and two hydrophobic features with statistical values of 0.88 as correlation coefficient for training set. The best model Hypo 1 was validated using a test set of 38 compounds and cat-scramble validation method.

In silico structure-based drug design approach to develop novel pharmacophore model of human peroxisome proliferator-activated receptor γ agonists

A structure-based pharmacophore generation approach was employed to identify novel structural characteristics and scaffolds for peroxisome proliferator-activated receptor γ (PPARγ). The structure-based six feature pharmacophore hypothesis generated in the present study signifies the importance of hydrogen bond donors, hydrogen bond acceptors and hydrophobic interaction between the PPARγ structure and its agonists. The interaction shown by the compounds provides an important insight into the mechanism involved in the ligand receptor interaction. The model will aid in the development of oral hypoglycemic agents with improved efficacy and reduced side effects and can be used to search comparatively larger compound libraries/databases to retrieve more potent and structurally diverse group of compounds.

Common binding requirements of PPAR-α/δ/γ pan agonists: quantitative structure–activity relationship analysis of indanylacetic acid derivatives carrying 4-thiazolyl-phenoxy tail group

A QSAR study on a series of indanylacetic acid derivatives with activity against PPAR-α, δ, and γ was made using combination of various physiochemical descriptors. Several statistical regression expressions were obtained using stepwise multiple linear regression (MLR) analysis and partial least square (PLS) method. The highly predictive and validated models generated through classical 2D molecular descriptors provided deeper insights about the binding of these small molecules to the human nuclear receptor PPAR-α, δ, and γ. The results reveal that dipole moment and number of hydrogen bond donors are important descriptors in determining effective binding of PPAR agonists to all three subtypes.

Rapid identification of medically important mosquitoes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

BackgroundAccurate and rapid identification of dipteran vectors is integral for entomological surveys and is a vital component of control programs for mosquito-borne diseases. Conventionally, morphological features are used for mosquito identification, which suffer from biological and geographical variations and lack of standardization. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for protein profiling of mosquito species from North India with the aim of creating a MALDI-TOF MS database and evaluating it.MethodsMosquito larvae were collected from different rural and urban areas and reared to adult stages. The adult mosquitoes of four medically important genera, Anopheles, Aedes, Culex and Armigerus, were morphologically identified to the species level and confirmed by ITS2-specific PCR sequencing. The cephalothoraces of the adult specimens were subjected to MALDI-TOF analysis and the signature peak spectra were selected for creation of database, which was then evaluated to identify 60 blinded mosquito specimens.ResultsReproducible MALDI-TOF MS spectra spanning over 2–14 kDa m/z range were produced for nine mosquito species: Anopheles (An. stephensi, An. culicifacies and An. annularis); Aedes (Ae. aegypti and Ae. albopictus); Culex (Cx. quinquefasciatus, Cx. vishnui and Cx. tritaenorhynchus); and Armigerus (Ar. subalbatus). Genus- and species-specific peaks were identified to create the database and a score of > 1.8 was used to denote reliable identification. The average numbers of peaks obtained were 55–60 for Anopheles, 80–100 for Aedes, 30–60 for Culex and 45–50 peaks for Armigeres species. Of the 60 coded samples, 58 (96.67%) were correctly identified by MALDI-TOF MS with a score > 1.8, while there were two unreliable identifications (both Cx. quinquefasciatus with scores < 1.8).ConclusionsMALDI-TOF MS appears to be a pragmatic technique for accurate and rapid identification of mosquito species. The database needs to be expanded to include species from different geographical regions and also different life-cycle stages to fully harness the technique for entomological surveillance programs.