Characterization of RPO B gene for detection of rifampicin drug resistance by SSCP and sequence analysis.

Purpose: Because of the emergence of multidrug-resistant tuberculosis in recent times, the rapid detection of resistance to the first-line anti-tuberculosis drug rifampicin was felt worldwide. Accordingly, this study was conducted to evaluate the diagnostic potential of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for checking its utility as a rapid screening test for determination of rifampicin drug resistance. Materials and Methods: A total of 34 isolates of Mycobacterium tuberculosis ( M. tuberculosis ) (22 rifampicin resistant, 11 rifampicin sensitive and one control H37Rv) strains were analysed by PCR-SSCP and DNA sequencing within the 157-bp region of the rpo B gene (Ala 500 -Val 550 ). Results: Rifampicin resistance was detected successfully by PCR-SSCP in 20/22(90.90%) of rifampicin-resistant strains showing a total of nine different mutations in seven codon positions: codon 513 (CAA→CCA), 516 (GAC→GTC), 507 (GGC→GAC), 526 (CAC→GAC, TAC), 531 (TCG→TTG, TGG), 522 (TCG→TGG) and 533 (GTG→CCG). Two rifampicin-resistant strains showed an identical PCR-SSCP pattern with the wild type H37Rv; 77.27% rifampicin-resistant strains showed a single point mutation and 9.09% had no mutation. Three rifampicin-resistant strains showed characteristic double mutations at codon positions 526 and 531. Sensitivity and specificity were calculated as 90.90% and 100%. Conclusions: Rifampicin-resistant genotypes were mainly found in codon positions 516, 526 and 531. PCR-SSCP seems to be an efficacious method of predicting rifampicin resistance and substantially reduces the time required for susceptibility testing from 4 to 6 weeks to a few weeks.

Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples.

PURPOSE: The correlation between the presence of specific gene sequence of M. tuberculosis and specific diagnosis of clinical tuberculosis is not known. This study compared the results of polymerase chain reaction (PCR) amplification of M. tuberculosis specific DNA sequences (IS6110, 65kDa, 38kDa and mRNA coding for 85B protein) from different clinical samples of pulmonary and extrapulmonary tuberculosis. METHODS: One hundred and seventy-two clinical samples from suspected tuberculosis patients were tested for smear examination, culture (LJ and rapid BACTEC 460 TB system) and PCR. PCR was performed with specific primers for the targets: IS6110, 65 kDa, 38 kDa and 85 B. RESULTS: Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture (P < 0.05). Smear positive samples (56) and the samples (36) showing positive results by conventional methods (smear and LJ medium culture) and BACTEC were found to be positive by all PCR protocols. No significant difference was found between the four PCR protocols (P> 0.05). The primer specific for amplifying the 123bp IS6110 fragment gave the highest positivity (83%), followed by 65kDa, 38kDa and 85B RT-PCR in descending order. CONCLUSIONS: These data suggest that the presence of IS6110 correlates more closely with the diagnosis of clinical tuberculosis than that of 65kDa, 38kDa and 85B proteins.

Protein antigen b (Pab) based PCR test in diagnosis of pulmonary and extra-pulmonary tuberculosis.

BACKGROUND AND OBJECTIVES: Diagnosis of tuberculosis (TB) is largely based on microscopy and culture examination which are either less sensitive, or time consuming. In the present study a PCR (polymerase chain reaction) test based on DNA sequence coding for a 38-kilodalton protein antigen b (Pab) ,specific for Mycobacterium tuberculosis was compared with Ziehl-Neelsen (ZN) stained AFB (acid fast bacilli) smear examination, culture based on conventional Lowenstein-Jensen (LJ) medium and radiometric BACTEC 460 system for the diagnosis of TB using clinical samples obtained from pulmonary and extra-pulmonary cases of TB. METHODS: Clinical samples obtained from 168 patients of suspected TB (pulmonary and extrapulmonary) were subjected to ZN smear examination, LJ culture, radiometric BACTEC culture and a PCR test by amplifying 419 bp sequence coding for Pab, a glycoprotein of molecular weight 38 kDa. RESULTS: A significant difference was seen in the sensitivity of different tests, the figures being 74.2 per cent for PCR test, 53.4 per cent for BACTEC culture, 47.1 per cent for LJ medium based culture and 35.2 per cent for ZN smear examination (P<0.05). However, there was no significant difference between different tests as far as specificity was concerned. PCR test sensitivity in pulmonary and extra-pulmonary clinical samples were 74.3 and 71.5 per cent respectively, being significantly higher (P<0.05) when compared with sensitivity of other tests. The mean detection time for M. tuberculosis was 24.0 days by LJ media culture, 12.8 days by BACTEC culture and less than 1 day by smear examination and PCR test. INTERPRETATION AND CONCLUSION: PCR test is more sensitive than ZN smear examination, LJ medium culture and BACTEC culture for diagnosing TB in pulmonary and extra-pulmonary clinical samples.

Comparison of various microbiological tests including polymerase chain reaction for the diagnosis of osteoarticular tuberculosis.

PURPOSE: To evaluate the utility of the polymerase chain reaction (PCR) test for diagnosing osteoarticular tuberculosis (TB). METHODS: Clinical samples (synovial tissue and synovial fluid) obtained from 23 cases of suspected osteoarticular tuberculosis were subjected to Ziehl Neelsen (ZN) smear examination, radiometric BACTEC culture and PCR test for tuberculosis by amplifying 65 kDa antigen coding region of Mycobacterium tuberculosis (M.tb) genome. RESULTS: PCR test was found to be much sensitive than the ZN smear examination and BACTEC culture (p<0.05) in the diagnosis of osteoarticular TB. In synovial fluid samples, PCR was positive in 73.9%, ZN smear examination in 17.39% and BACTEC culture in 39.13% of cases. The positivities were relatively lower with synovial tissue samples, the corresponding figures being 60.8, 8.6 and 26.08% respectively. Moreover, on combining the results of synovial fluid and tissues, the corresponding figures further increased to 78.2, 21.7 and 43.3% respectively. Further, sensitivity and specificity for PCR employing BACTEC culture as the "gold standard" was 100% respectively. Using BACTEC culture, the earliest positivity was seen in three days using synovial tissue specimen and 13 days with synovial fluid, the average detection times being 23.2 days and 32.6 days respectively. On the other hand, PCR test gave a positive result within 24 hours. CONCLUSIONS: PCR test was shown to be much more sensitive than ZN smear examination and BACTEC culture test for diagnosing osteoarticular tuberculosis.

Comparison of the conventional diagnostic modalities, bactec culture and polymerase chain reaction test for diagnosis of tuberculosis.

PURPOSE: To evaluate the performance of 65 kDa antigen based PCR assay in clinical samples obtained from pulmonary and extrapulmonary cases of tuberculosis. METHODS: One hundred and fifty six samples were processed for detection of Mycobacterium tuberculosis by ZN smear examination, LJ medium culture, BACTEC radiometric culture and PCR tests. RESULTS: A significant difference was seen in the sensitivities of different tests, the figures being 74.4% for PCR test, 33.79% for ZN smear examination, 48.9% for LJ culture and 55.8% for BACTEC culture (P< 0.05). However, there was no significant difference (P>0.05) as far as specificity of different tests was concerned. PCR test sensitivity in pulmonary and extrapulmonary clinical samples were 72.7% and 75.9% respectively and found to be significantly higher (P< 0.05) when compared with those of other tests. The mean detection time for M.tuberculosis was 24.03 days by LJ medium culture, 12.89 days by BACTEC culture and less than one day by PCR test. CONCLUSIONS: PCR is a rapid and sensitive method for the early diagnosis of pulmonary and extrapulmonary tuberculosis.

Experience of surgical management of pseudo-aneurysms of branches of the coeliac axis in a North Indian Hospital.

BACKGROUND: Bleeding splanchnic artery pseudo-aneurysm is a rare but frequently fatal complication that can be successfully managed by angiographic embolization. However, certain patients because of hemodynamic instability, non-availability of technique or angiographic failure may require primary surgical intervention. METHOD: Retrospective review of 13 patients presenting with exsanguinating hemorrhage from ruptured pseudo-aneurysm arising from branches of coeliac axis, managed surgically in absence of angiographic embolization. RESULTS: Splenic artery was most commonly involved (n = 7) followed by hepatic (n = 3), gastroduodenal (n = 2) and left gastric artery (n = 1). The most common underlying aetiology was pancreatitis (n = 8, acute = 2; chronic = 6) followed by iatrogenic (n = 3), liver abscess (n = 1) and gastric ulcer (n = 1). Seven patients presented with upper gastrointestinal (GI) bleed, while 2 each with lower GI bleed, haemobilia and bleeding through tube drains. CT-scan accurately demonstrated the pseudo-aneurysm in 11 (84.6%) patients and additionally demonstrated the underlying pathology. The surgical management chiefly consisted of ligation of offending vessel and additional procedures directed at primary pathology. Overall, 77% patients had a favourable outcome while 23% died consequent to ongoing hemorrhage. CONCLUSION: Pseudo-aneurysm involving the branches of coeliac axis most commonly arises as a result of pancreatitis and affects splenic artery. CT-scan accurately demonstrates pseudo-aneurysm and associated pathology in majority of cases. Primary surgical management in the presence of hemodynamic instability and non-availability of angiographic embolization is a viable alternative.

Paraganglionoma of extrahepatic biliary tract causing obstructive jaundice.

We report a young woman with paraganglionoma arising from the extrahepatic bile duct presenting with acute obstructive jaundice. The patient underwent excision of the gall bladder and extrahepatic bile duct with the tumor, and Roux-en-Y hepaticojejunostomy. She is asymptomatic 9 months later, with normal biochemical investigations and imaging.

Immobilization and properties of β-D-galactosidase from Lactobacillus bulgaricus.

β-D-galactosidase (EC 3.2.1.23) from Lactobacillus bulgaricus (1373) was immobilized by entrapment in a Polyacrylamide gel lattice. The enzymatic properties of the immobilized β-galactosidase were compared with those of the native enzyme. The temperature and pH optima were not affected by the immobilization. After entrapment of the enzyme no significant change was observed in its thermostability. The pH stability of the immobilized enzyme was higher than that of the native enzyme on the acidic side. The Km values for the immobilized and native β-galactosidase with both lactose and o-nitrophenyl-β-D-galactoside as substrates were comparable. The immobilized enzyme could be repeatedly used 12 times without any loss of activity. No loss in the activity of the immobilized β-galactosidase was found after its storage for 30 days at 4°C and for 20 days at 25°C.