Devendra Desai

Application of Multiplex ARMS and SSCP/HD Analysis in Molecular Diagnosis of Cystic Fibrosis in Indian Patients

AbstractBackground: Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The most severe, ΔF508, mutation accounts for nearly 70% of CF cases worldwide. Besides ΔF508, there are other point mutations, namely G542X, G551D, R553X, N1303K, and 621+1(G→T), which are common among Caucasians. Additionally, a polyT polymorphism in intron 8 is also involved in the pathogenesis of CF. However, neither the prevalence nor the types of mutations causing CF in India are known. In this study, we aimed at estimating the frequency of the above common mutations and polymorphism in clinically suspected CF cases. We also carried out partial analysis of the CFTR gene, limited to exons 10 and 11, to identify other variations in these exons. Methods: The multiplex amplification refractory mutation system (ARMS) test was applied for rapid simultaneous analysis of six most common CF mutations, in 100 normal and 39 elevated sweat chloride cases. The scanning of exons 10 and 11 was carried out by single-stranded conformation polymorphism/heteroduplex (SSCP/HD) analysis, followed by DNA sequencing in 50 normal and 37 elevated sweat chloride cases. A single ARMS-polymerase chain reaction assay was used to distinguish the 5T, 7T, and 9T alleles in 100 normal and 33 elevated sweat chloride cases. Results: The multiplex ARMS analysis identified the ΔF508 mutation at an allele frequency of 24% in Indian CF cases. However, the other predominant CF mutations were found to be absent. The 7T polyT variant was observed to be the most common allele, followed by the 9T, and 5T, which was the lowest. The ΔF508 mutation was observed in all instances with the 9T variant. The SSCP/HD and DNA sequencing additionally revealed a known polymorphism (M470V, exon 10) and a known mutation [1525-1(G→A), intron 9]. The 1525-1(G→A) mutation, observed in a single 4-year-old male, is predicted to code for a class II defective CFTR protein. Conclusion: The findings of this study suggest a difference in relative frequencies and spectrum of CFTR mutations in Indian CF cases. A larger screening study of the entire CFTR gene in clinically well defined CF cases is required to delineate common mutations in the CFTR gene and enable molecular diagnosis of CF in India.

Acute renal tubular injury with acute hepatitis A infection: Is it just a coincidence?

SUMMARY:  Acute renal failure has rarely been reported in association with acute hepatitis A infection. The commonest form of renal injury in such patients has been found to be acute tubular necrosis. We report two cases of hepatitis A infection in which acute renal failure occurred very early in the course of the illness and had a clinical presentation and recovery pattern suggestive of acute tubular necrosis. In both patients, the clinical course of renal dysfunction was almost parallel to the course of hepatic dysfunction. Patient 1 needed dialysis, whereas patient 2 did not need dialysis and had a very rapid recovery from renal function in spite of having more severe azotaemia. Patient 2 was administered acetylcysteine in high doses for suspected fulminant hepatic failure. A potential benefit of a high dose of acetylcysteine in recovery of renal function from acute tubular necrosis is postulated.

Comparison of Mycobacterium tuberculosis culture using liquid culture medium and Lowenstein Jensen medium in abdominal tuberculosis

BackgroundTraditionally, the Lowenstein Jensen (LJ) medium has been used for culturing Mycobacterium tuberculosis. In abdominal tuberculosis (TB), the reported yield from tissue culture is between 20% and 60%. Liquid cultures are reported to give a higher yield but there is little data available in abdominal TB.AimTo compare the yield of TB culture with BACTEC 460TB liquid medium and LJ medium for patients with suspected abdominal TB and determine cost effectiveness.MethodsThis prospective study was done in consecutive cases with clinical, radiological, endoscopic/surgical, and histological suspicion of abdominal TB. Tissue biopsies obtained at colonoscopy or surgery were processed and plated on LJ medium as well as the BACTEC 460TB system. NAP (ρ-nitro-α-acetylamino-β-hydroxy-propiophenone) differentiation was carried out to determine species. The cost of each method and cost per yield were calculated.ResultsOf the 29 cases, 22 cases (76%) were positive on BACTEC 460TB culture while 14 (48%) were positive on LJ medium giving a 64% increment in yield. However, the culture of one patient grew on LJ medium, where the BACTEC 460TB was negative. The additional cost of BACTEC 460TB is Rs. 460 and LJ is Rs. 40.ConclusionsSamples from patients with abdominal TB should be processed on both liquid and LJ medium. For high yield, the use of a liquid culture medium system is essential.

Rate of recurrence in Indian patients presenting with acute pancreatitis and identification of chronicity on follow up: Possible risk factors for progression

BackgroundTo study the profile and long-term outcome of Indian patients presenting with acute pancreatitis and the possible risk factors for progression.MethodsConsecutive patients with acute or recurrent acute pancreatitis seen in our department during July 2013 to December 2014 were included. Details of past episodes were collected and patients were followed up till March 2015.ResultsIn the 97 patients included (mean age 47.2 [SD 16.9] years; 74 men), gallstones (37 [38.1%]) and alcohol (19 [19.6%]) were the major identified etiologies; the idiopathic (31 [32%]) group constituted a third of patients. Recurrences were more common with idiopathic etiology (14 patients out of 30 had recurrences [46.7%]) as compared to alcoholic (5 out of 19 [26.3%]) and biliary (4 out of 37 [10.8%]) pancreatitis and with mild index episode. Following the episode of acute pancreatitis, identification of chronic pancreatitis was more common with alcoholic (6 out of 18 [33%]) and idiopathic (9 out of 30 [30%]) etiology as compared to other etiologies. Longer duration of follow up, but not number of recurrent episodes, was associated with identification of chronicity in patients presenting as acute pancreatitis.ConclusionsOut of 97 patients with acute pancreatitis, 27 (27.8%) developed recurrences with risk factors being idiopathic etiology and mild index episode. Eighteen of 97 (18.6%) patients had evidence of chronic pancreatitis on follow up, risk factors being the alcoholic and idiopathic varieties, and longer duration of follow up.

Drug resistance in intestinal tuberculosis: A reason to worry?: Drug resistance in intestinal tuberculosis

Emergence of drug resistance in intestinal tuberculosis (ITB) makes the treatment of this condition challenging. While there is growing evidence of multiple and extensive drug resistance in pulmonary and glandular tuberculosis (TB), literature regarding susceptibility and resistance patterns in ITB is scarce. The aim of the current paper was to study the prevalence of drug resistance in patients with ITB.

Thiopurine metabolite level and toxicity in Indians with inflammatory bowel disease

A lower dose requirement and higher toxicity of thiopurine is reported in Asian patients with inflammatory bowel disease (IBD) as compared with Caucasian patients. These reports are based on thiopurine methyltransferase measurement studies rather than metabolite estimation.

Imaging of the small intestine in Crohn’s disease: Joint position statement of the Indian Society of Gastroenterology and Indian Radiological and Imaging Association

The Indian Society of Gastroenterology (ISG) Task Force on Inflammatory Bowel Disease and the Indian Radiological and Imaging Association (IRIA) developed combined ISG-IRIA evidence-based best-practice guidelines for imaging of the small intestine in patients with suspected or known Crohn’s disease. These 29 position statements, developed through a modified Delphi process, are intended to serve as reference for teaching, clinical practice, and research.

Author’s response to: Acid-fast bacilli culture positivity and drug resistance in abdominal tuberculosis

We read with interest the correspondence regarding “Acid-fast bacilli: culture positivity and drug resistance in abdominal tuberculosis”. It is well known that breakthrough contamination occurs in TB Mycobacteria Growth Indicator Tube (MGIT) cultures up to 8.1 %; this is even higher for all combined solid media at 21 %. At our CAPand NABLaccredited mycobacteriology lab, stringent quality control/ assurance practices are in place for every batch of cultures put up to avoid any cross contamination with appropriate negative controls being performed at least four times a day to prevent any breakthrough contamination. We also appreciate the concerns regarding high rates of resistance. However, we are a tertiary care center with a referral bias towards nonresponders. Prevalence of resistant strains at our institution is hence on the higher side and may not necessarily represent the prevalence in the community. Split-sample analysis of phenotypic drug susceptibility testing (DST) with molecular tests including pyrosequencing is regularly done with good concordance. MGIT DST for SIRE (streptomycin, isoniazid, rifampicin, and ethambutol) currently is a reliable option as in vitro DST results based on the proportion method (which combines the critical concentration of primary antituberculous drugs with a standardized inoculummade from a pure culture of the recovered strain) has been shown to correlate well with clinical response to anti-TB chemotherapy. As regards pyrazinamide, phenotypic susceptibility testing overall agreement of the BACTEC MGIT 960 system with the BACTEC 460TB system for PZA testing was found to be 96.6 % [1].