Mohd Azam

Prevalence of leptospira in acute hepatitis syndrome and assessment of IL-8 and TNF-alpha level in leptospiral hepatitis.

Abstract To study the prevalence of leptospira in acute hepatitis syndrome and to assess interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha levels in the pathogenesis of hepatitis due to leptospiral infection. Two hundred and forty-seven consecutive cases with symptoms of acute hepatitis and 30 healthy controls were enrolled in the study and detailed clinical history was elicited from them. Enzyme-linked immunosorbent assays (ELISAs) for HAV, HBV, HCV and HEV were performed to rule out common viral aetiology of hepatitis. IgM antibodies to leptospira were detected by ELISA. IL-8 and TNF-alpha levels were estimated in leptospira-positive cases and healthy controls by ELISA. Out of 247 cases of acute hepatitis, 46 (18·62%) were observed to be positive for IgM antibodies for leptospira. The mean age of these patients was 31·99±0·28 years (25 males and 21 females; M/F ratio: 1·19∶1). The mean ALT, AST and ASP were raised in the majority of patients. IL-8 was found to be elevated (130·81 pg/ml) in a large majority of cases 41/46, 89·1% (P<0·001). Patients with more severe symptoms were associated with higher levels of IL-8. One mortality was observed due to leptospira. Unpredictably, TNF-alpha level was largely suppressed (45·63 pg/ml) in most of the leptospira-positive patients in comparison with healthy controls. Leptospira-induced hepatitis should be actively looked for in patients negative for A–E viral hepatitis. IL-8 appears to play an important role in the pathogenesis of leptospiral hepatitis. High TNF-alpha should alert clinicians for aggressive in hospital management of patients.

Zero prevalence of primary drug resistance-associated mutations to protease inhibitors in HIV-1 drug-naive patients in and around Aligarh, India.

INTRODUCTION This study aimed to evaluate the prevalence of resistance mutations in the protease gene of HIV-1 strains isolated from north Indian antiretroviral (ARV) treatment-naive patients and to assess the phylogenetic relatedness of these strains with known HIV-1 strains. METHODOLOGY Fifty-four HIV-1 strains isolated from treatment-naive patients (n = 54) were included in this study. Resistance genotyping for the protease gene was performed using semi-nested PCR and DNA sequencing. The sequences were aligned (ClustalW) and a phylogenetic tree was built (MEGA 4 software). Drug resistance (DR) pattern was analyzed using the Stanford HIV-DR database and the IAS-USA mutation list. For subtyping purposes, all the nucleotide sequences were submitted to the REGA HIV-1 subtyping tool version 2.0l. RESULTS All the strains (100%) were found to belong to the C subtype and to harbor at least two secondary mutations in the protease gene. The most frequent mutations were H69K and I93L (52 of 52 strains), followed by I15V (80.7%), L19I (69.2%), M36I (67.3%), R41K (94.2%), L63P (61.5%), and L89M (82.7%). CONCLUSION This study confirms that HIV-1 subtype C predominates in northern India. Protease secondary mutations associated with drug resistance to protease inhibitors (PIs) were present with high frequency in the HIV-1 C subtype strains isolated from north Indian ARV treatment-naive patients, but no primary resistance mutations were found in this region. We suggest that resistance testing in HIV-1 infected patients should ideally be performed before the initiation of therapy to tailor the treatment for the individual to achieve the optimal therapeutic outcome.

Emergence of Hepatitis B Virus Genotype F in Aligarh Region of North India

Introduction. HBV genotypes and subtypes are useful clinical and epidemiological markers. In this study prevalent HBV genotypes were assessed in relation to serological profile and clinical status. Material & Methods. 107 cases of HBV were genotyped. Detailed clinical history was elicited from them. HBsAg, HBeAg, anti-HBs, anti-HBe, and anti-HBc-IgM were assessed. HBV genotyping was performed using Kirschbergs type specific primers (TSP-PCR), heminested PCR, and Naitos monoplex PCR. Nucleotide sequencing was performed. Results. A total of 97 (91%) were genotyped following the methods of Kirschberg et al./Naito et al. Genotype D was by far the most prevalent genotype 91 (85.04%) in this region. A surprising finding was the detection of genotype F in 5 (4.67%) of our patients. Genotype A strangely was observed only in one case. In 85.7% genotype D was associated with moderate to severe liver disease, 43.9% HBeAg, and 18.7% anti-HBc-IgM positivity. Majority of genotype F (80%) was seen in mild to moderate liver disease. It was strongly associated with HBeAg 60% and 20% anti-HBc-IgM positivity. Conclusion. Emergence of genotype F in India merits further study regarding its clinical implications and treatment modalities. Knowledge about HBV genotypes can direct a clinician towards more informed management of HBV patients.

Prevalence of drug resistance mutations in HIV-1 protease gene from North India

Results Among 35 patients, there were 17 drug naive and 18 first line drugs experienced HIV-1 infected patients (22 males & 13 females; mean age: 35.95 years; mean CD4 cells: 216.4cells/mm). Majority of our patients showed mutations at T12S/T (82.85%), K14R (40%), I15V (71.42%), L19I/T/V/M (97.14%), M36I (71.42%), R41K (88.57%), L63P (65.71%), H63K (100%) and L89M (74.28%) positions in both group of patients while other mutations were at positions 35, 37, 45, 60, 62, 77, and 82 in few cases. Interestingly, one first line drug experienced patient showed major DR mutations at D30N and M46I positions. Majority (94.28%) was belonging to subtype C and 2 patients were belonging to subtypes A (A1).

Understanding the mode of binding mechanism of doripenem to human serum albumin: Spectroscopic and molecular docking approaches

The infections caused by multidrug resistant bacteria are widely treated with carabapenem antibiotics as a drug of choice, and human serum albumin (HSA) plays a vital role in binding with drugs and affecting its rate of delivery and efficacy. So, we have initiated this study to characterize the mechanism of doripenem binding and to locate its site of binding on HSA by using spectroscopic and docking approaches. The binding of doripenem leads to alteration of the environment surrounding Trp‐214 residue of HSA as observed by UV spectroscopic study. Fluorescence spectroscopic study revealed considerable interaction and complex formation of doripenem and HSA as indicated by Ksv and Kq values of the order of 104 M−1 and 1012 M−1 s−1, respectively. Furthermore, doripenem quenches the fluorescence of HSA spontaneously on a single binding site with binding constant of the order of 103 M−1, through an exothermic process. Van der Waals forces and hydrogen bonding are the major forces operating to stabilize HSA‐doripenem complex. Circular dichroism spectroscopic study showed changes in the structure of HSA upon doripenem binding. Drug displacement and molecular docking studies revealed that the binding site of doripenem on HSA is located on subdomain IB and III A. This study concludes that, due to significant interaction of doripenem on either subdomain IB or IIIA of HSA, the availability of doripenem on the target site may be compromised. Hence, there is a possibility of unavailability of threshold amount of drug to be reached to the target; consequently, resistance may develop in the bacterial population.

Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity

New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1Q123A were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution.

Updates on the pathogenicity status of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a pathogenic bacterial species that causes infections and diseases in both plants and animals, including several human diseases, especially in immune-compromised patients, and many hospital-acquired infections. Given that P. aeruginosa is an opportunistic pathogen, the occurrence of antimicrobial resistance makes it difficult to treat and eradicate. Antimicrobial resistance in P. aeruginosa is categorized as intrinsic, acquired, or adaptive. Here, we different aspects of resistance and pathogenicity in P. aeruginosa, such as the role of outer membrane proteins, transcriptional regulators, efflux pumps, enzymes, and biofilms in antimicrobial resistance. We also highlight quorum-sensing (QS) genes, their protein secretion, and role in pathogenicity; different QS inhibitors; and the influence of QS on the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system and virulence factor production.