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The present study was carried out to evaluate the performance of two novel gladiolus hybrid selections IIHRG-7 and IIHRG-11 along with commercial checks, for flower quality traits and Fusarium wilt resistance in completely randomized block design, during 2012-13 to 2014-15. Three years data were pooled and analyzed statistically. The hybrid selections IIHRG-7 and IIHRG-11 had been developed through hybridization by crossing Arka Meera x Picardy and Gold Medal 412 x Arka Poonam, respectively followed by selection. IIHRG-7 has novel flower colour (as per RHS Colour Chart) i.e., Red-Purple (65.B) having Red-Purple (62.A) streaks with Red-Purple (67.B) splash and spike with variegated florets, while, IIHRG-11 has novel floret colour as Red (41.C) having Red (41.A) margin. Blotch Red (46.B) with yellow (13.C) border and resistance to Fusarium wilt disease. These hybrid selections are suitable for cut-flower and flower arrangement purposes. Further, these hybrid selections will be useful for developing new gladiolus hybrid selections with novel traits and resistance to Fusarium wilt disease.

Background: Identification of 14 high-risk human papillomavirus (HR-HPV) is immensely important in elucidating molecular epidemiology, patient monitoring and evidence-based treatment. There is paucity of such data from Chhattisgarh state of Central India. The present study has evaluated tagging oligonucleotide cleavage and extension-mediated Anyplex HR-HPV genotyping assay in identification of 14 HR-HPV genotypes attributable to premalignant and malignant cervical lesion in comparison to GP5+/6+ assay, cytology and colposcopy. Materials and Methods: A total of 185 clinically suspected cases of premalignant and malignant cervical lesion were investigated by HR-HPV genotyping, GP5+/6+, cytology and colposcopy. Results: Genotyping assay showed clinical sensitivity and specificity of 86.5% (confidence interval [CI]: 80.7–91.0) and 100% (CI: 86.3–100) respectively and found noninferior to GP5+/6+ assay (P > 0.05). HR-HPV prevalence was 76.3%, 88.4%, 94.8%, 100% and 100% among cervical intraepithelial neoplasia (CIN) Grade I–III, squamous cell carcinoma and adenocarcinoma cases, respectively. The four most common genotypes detected in CIN I–III were HPV 16 (63.9%), HPV 39 (15.0%), HPV 18 (6.0%) and HPV 33 (5.3%). In cervical cancer (CC) cases, HPV 16 (44.4%), HPV 39 (11.1%), dual infection of HPV 16, 18 (11.1%) and triple infection of HPV 16, 18, 33 (11.1%) were the four most identified genotypic aetiologies. A novel coinfection of HR-HPV 35, 39 were found in two and one cases of CIN I and II. Finding of HPV 39 as the second most prevalent genotype was unusual and underscores the importance of genotyping screening. Conclusion: Anyplex HR-HPV assay is arguably the useful assay for better patient management and can be useful for HR-HPV screening by its unique individual genotype identification of all HR-HPV. Finding of HPV 16, 39, 18, 33 and coinfection of 16,18 and 16, 18, 33 in CIN and CC would help vaccine manufacturer to design specific future HPV polyvalent vaccine preparation to curb down the CC-associated mortality.

Cervical cancer is one of the leading causes of cancer deaths among women. It results due to human papillomavirus (HPV) infection. Cervical intraepithelial neoplasia (CIN) is the preinvasive condition of cervical cancer. Various objective immunohistochemical (IHC) markers have been studied for cervical cancer. This study is aimed at studying the expression of B-cell lymphoma-2 (Bcl-2) IHC marker among preinvasive and invasive lesions of cervical cancer and its association with HPV infection.This prospective study was conducted over a period of 1 year in a tertiary care hospital in central India, included 73 women suffering from CIN and cancer cervix. The expression of Bcl-2 and the presence of HPV genotypes were studied.Out of 73 patients, 34 had cancer cervix, out of which 15 (44%) had Bcl-2 positivity, 24 had CIN 1, out of which 13 (54%) had Bcl-2 positivity, 10 had CIN 2, out of which 4 (40%) had Bcl-2 positivity and 5 had CIN 3, out of which 3 (60%) had Bcl-2 positivity. No significant difference was found in Bcl-2 positivity among CIN-1, CIN-2, CIN-3, and cancer cervix cases with a Chi-square value of 1.116 and P = 0.77. HPV positivity was found in 41 (56%) out of 73 patients where HPV 16 subtype was the most common (31.5%), followed by HPV 18 (13.7%). No significant association between HPV positivity and Bcl-2 positivity was found with P = 0.34.Bcl-2 IHC seems to have variable expression among CIN cases. Although its expression is low among invasive cancer cases when compared with preinvasive lesions, the difference is not significant. Similarly, no significant association was found between Bcl-2 expression and HPV infections.

BACKGROUND: Enteric fever is the most common cause of community acquired blood stream infections in under developed and developing countries. The enteric fever is exclusive to humans and transmitted through the faeco-oral route. Though India is an endemic zone for enteric fever, the data is very scarce from Central India. The present study was undertaken to determine the prevalence of enteric fever in this region and to know the antimicrobial susceptibility pattern of the isolated typhoidal Salmonellae. MATERIAL AND METHOD: We conducted a retrospective analysis of blood culture positive cases of enteric fever over a period of two years (December 2015 to December 2017). All blood cultures submitted for suspected enteric fever and associated symptoms were included in the study. Relevant demographic, clinical and laboratory data were analyzed. RESULT: A total of 51 cases (3.56%) were of typhoidal Salmonella from a total of 1430 blood culture submission. Salmonella Typhi were 70.5% while Salmonella Paratyphi A were 29.5% of the total isolated Salmonellae. The most vulnerable age group was 10-19years (41.2%). The mean minimum inhibitory concentration of ciprofloxacin for Salmonella Typhi and Salmonella Paratyphi A are 1.20 and 1.97 μg/ml respectively. All the isolates were susceptible to ceftriaxone. Highest isolation was in the July – September quarter (35.3%). CONCLUSION: There is a high prevalence of the disease which needs urgent focus on safe water, sanitation services and also to establish guidelines for empiric therapy for enteric fever.

Introduction: Effective management of Extrapulmonary Tuberculosis (EPTB) requires simultaneous identification of Mycobacterium tuberculosis (M.tuberculosis) complex, NonTuberculous Mycobacteria (NTM) and Rifampicin (RIF) sensitivity pattern for early and effective evidence based Antitubercular Treatment (ATT). Aim: To evaluate the diagnostic potential of multiplex Real-Time Polymerase Chain Reaction (mRT-PCR) versus GeneXpert and conventional microscopy and culture. Materials and Methods: A total of 110 multivaried extrapulmonary specimens from an equal number of patients with strong clinical/radiological/histopatholgical evidence of EPTB were subjected to conventional microscopy, liquid culture MGIT 960 system, GeneXpert and mRT-PCR. Results: Highest positivity of 88.8% (97/110) was shown by mRT-PCR followed by GeneXpert (52.72%, 58/110), liquid culture MGIT 960 system (44.54%, 49/110) and microscopy (4.54%, 5/110), (p

Pasteurella species are zoonotic bacterial pathogens implicated very infrequently in various human infections following animal bites or licks usually of dogs and cats. This case report described a rare clinical presentation of dacryocystitis caused by P.canis in a Human Immunodeficiency Virus (HIV) positive young male patient involved in caring of cattle. It advocates the utmost need of recognizing the wide clinical manifestation spectrum of P.canis even without prior penetrating injury. P.canis associated clinical infection is more extensive than had been thought previously especially in immunocompromised patient. Early accurate identification and evidence based anti-microbial therapy may prove crucial in preventing further potential complications.

Burkholderia cepacia (B.cepacia) and Acinetobacter baumannii (A.baumannii), the highly intrinsically resistant nonfermenters are known to cause frequent infections in immunocomproimsed or hospitalized patients with significant mortality rate. In this rare clinical presentation, both were simultaneously isolated from a case of neonatal sepsis with respiratory failure. The prompt early diagnosis and antibiogram of these nonfermenters were proved to be of tremendous help in the present case with successful treatment outcome of dual infection of B.cepacia and A.baumannii. The present case report strongly emphasises the clinical importance of early accurate diagnosis of these emerging potential fatal non-fermenters which could otherwise prove fatal in case of any slight delay or misidentification. Implementation of strict surveillance policy to monitor the growth of these non-fermenters and draconian infection control measure holds a key to success in significant reduction of the associated mortality and morbidity.

Background: Ventilator-associated pneumonia is the second most common nosocomial infection in neonatal intensive care unit (NICU) patients. The recent trend signifies multidrug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa as the most frequent causative agents in these cases. Objectives: The aim of this research was to study the trends in the isolation of MDR pathogens from a newly constructed NICU. Methods: A retrospective analysis of six months data on isolation of MDR pathogens from newborns on mechanical ventilation in a newly established NICU was done. Results: Out of seven, MDR Acinetobacter baumannii was isolated from four (57.14%) newborns on mechanical ventilation. The strain was sensitive only to polymyxin B and colistin and was isolated repeatedly from the initial three samples collected at an interval of two to three days. In one case, Pseudomonas aeruginosa was also isolated in association with Acinetobacter with same sensitivity pattern. While expecting recovery from illness in two babies, sudden change in the type of microbial flora was noticed in the samples collected on the 4th instance. This time, the initial polymyxin-sensitive organisms were replaced by intrinsically polymyxin resistant organisms like Serratia species and Burkholderia cepacia. Conclusions: Repeated isolation of MDR Acinetobacter baumannii along with other resistant phenotypes of gram-negative bacteria is a cause of concern for any newly established NICU setup. Long-term use of reserved antibiotics leads to selection pressure, resulting in establishment of the relatively less common environmental opportunists as new, more troublesome pathogens. It may further complicate the treatment decisions and lengthen the hospital stay and associated morbidity.

Background: Gallbladder is rarely injured as it is embedded in the liver and well protected by ribcage. Gallbladder perforation is a rare complication of acute calculus cholecystitis. Spontaneous gallbladder perforation is even rarer.Case Series: We present our experience of 4 cases of spontaneous gallbladder perforation. The patients were mostly males (3:1) with age ranging from 42-64 years. All the patients presented with abdominal pain, distension, ileus and fever ranging from 2-5 days duration. There was no prior history of biliary colic. Examination revealed abdominal tenderness and ileus. Abdominal radiographs showed multiple air-fluid levels and no pneumoperitoneum. Sonography and CT scan of abdomen revealed pericholecystic fluid, intra-abdominal collection with no evidence of gallstones. Provisional diagnosis was peritonitis in 3 and acute pancreatitis in one case. Image guided abdominal paracentesis yielded bilious fluid. In view of lack of clinical improvement and aspiration of bilious content all the patients were subjected to laparotomy. Gallbladder perforation in fundus region without stones and intra-abdominal bile collection was noted in all the patients. Cholecystectomy with peritoneal lavage and abdominal drainage was performed in all cases. Postoperative course was uneventful except wound infection in 2 cases. Histopathology report revealed features of acute cholecystitis in 2 and acute on chronic cholecystitis in the remaining 2 patients.Conclusion: Diagnosis of spontaneous gallbladder perforation should be suspected in middle aged patients who present with acute abdomen but does not have classical signs of peritonitis and in whom paracentesis has yielded bilious fluid.

SUMMARYWe investigated an unprecedented outbreak of fulminant hepatitis B virus (HBV) that occurred in Modasa, Gujarat (India) in 2009. Genomic analysis of all fulminant hepatic failure cases confirmed exclusive predominance of subgenotype D1. A1762T, G1764A basal core promoter (BCP) mutations, insertion of isoleucine after nt 1843, stop codon mutation G1896A, G1862T transversion plus seven other mutations in the core gene caused inhibition of HBeAg expression implicating them as circulatingprecore/BCP mutant virus. Two rare mutations at amino acids 89 (Ile→Ala) and 119 (Leu→Ser) in addition to other mutations in thepolymerase (pol)gene may have caused some alteration in either of fourpolgene domains to affect encapsidation of pregenomic RNA to enhance pathogenicity. Sequence similarity among patients' sequences suggested an involvement of a single hepatitis B mutant strain/source to corroborate the finding of gross and continued usage of HBV mutant-contaminated syringes/needles by a physician which resulted in this unprecedented outbreak of fulminant hepatitis B. The fulminant exacerbation of the disease might be attributed to mutations in the BCP/precore/coreandpolgenes that may have occurred due to selection pressure during rapid spread/mutation of the virus.

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 and 83.3% 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 32.6days 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.

Background: Effective management of multidrug-resistant tuberculosis (MDR-TB) requires cost-effective and rapid screening of rifampicin (RIF) and isoniazid (INH) resistance. Accordingly, a highly promising high-resolution melting (HRM) analysis was evaluated in the detection of mutation in rpoB, katG gene and inhA promoter region in Mycobacterium tuberculosis isolates. Methods: A total of 143 M. tuberculosis isolates comprising phenotypically confirmed 94 MDR and 49 sensitive isolates were analyzed by HRM following real-time-polymerase chain reaction in comparison to gold standard of targeted DNA sequencing of rpoB, katG gene and inhA promoter region. Results: HRM correctly identified MDR-TB by rapid and accurate detection of predominantly and infrequently occurring specific single nucleotide polymorphism in rpoB, katG gene and inhA promoter region. rpoB HRM showed sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 98% each respectively. Predominantly, S531 L/W (TCG → TTG/TGG) mutation accounted for 68.47% of RIF resistance followed by H526Y/R (13.04%, CAC → TAC/CGC), D516Y/V/G (10.86%, GAC → TAC/GTC/GGC), Q513P (4.34%, CAA → CCA), and one rare mutation at codon position L533A (CTG → CGG). Combined KatG and inhA HRM sensitivity, specificity, PPV, and NPV were 90%, 100%, 100%, and 84.48% respectively and detected frequent mutation at codon position S315T/I/N (70%, AGC → ACC, AGC → ACT, AGC → AAC) and rare mutation at codon position T314P (3.3%, ACC → CCC) and 329 (2.2%, GAC → GCC) of katG gene. In inhA, mutations were recorded at mostly promoter position − 15 (10%, C → T) and infrequently at − 8 (3.3%, T → G, T → C). HRM assay limitation noticed in recognizing silent mutation in rpoB as a mutant, nondetection of infrequent mutation S310A in katG, and the inability of detecting mutation outside the targeted region of investigated genes. Conclusion: HRM may prove to be a vital molecular assay in rapid screening of TB cases for early detection of MDR TB, leading to early evidenced-based initiation of antitubercular treatment that will significantly reduce MDR transmission.

Study Background: An unprecedented outbreak of enterically transmitted acute hepatitis erupted in Raipur between April-June, 2014 claiming lives of 23 persons including 8 pregnant women. It was investigated by serological, biochemical and molecular methods to reveal its etiological agent, prevalence and genotype responsible for the outbreak. Methods: A total of one hundred six (106) blood samples from equal number of suspected cases of acute hepatitis cases presented in AIIMS Raipur for treatment were investigated for Anti-HEV IgM, Anti-HAV IgM, LFT, RT-PCR and sequencing. Results: Serological analysis incriminated HEV as the chief causative agent of this outbreak with attack rate of 68.8% followed by HAV (9.4%) while dual infection was noted in 2 cases (1.8%). Mortality rate of 2.87 per 10,000 population and incidence rate of 1.7% were recorded. In comparison to children and elderly cases, adults age group were more affected with men had higher attack rate than women and children (p

Background and Objectives Rapid clinical manifestation/progression of the meningococcal meningitis and lacunae in conventional bacteriological test often encourages indiscriminate use of antibiotics much before the etiology is established. Accordingly this study was planned to evaluate ctrA PCR for rapid molecular detection. In addition, multiplex PCR and sequencing was done for serogroup prediction to provide essential epidemiological and laboratory evidence for decision makers of health department of the country for choosing appropriate vaccine and phylogenetic analysis to establish its lineage. Materials and Methods 73 CSF samples, collected from equal number of suspected cases, were investigated by both bacteriological (microscopy, culture, LA and drug sensitivity testing) as well as molecular tests i.e. PCR targeting conserved ctrA gene, multiplex PCR for serogroup characterization and DNA sequencing. Results ctrA PCR revealed sensitivity, specificity, positive predictive value and negative predictive values of 93.15%, 100%,100%, and 88.23% respectively. Multiplex PCR based genogrouping followed by DNA sequencing, BLAST and phylogenetic analysis revealed complete homology with earlier submitted Neisseria meningitidis serogroup A strain Z2491 to suggest the sole involvement of only serogroup A in the outbreak. Two strains showed resistance to cefuroxime, ciprofloxacin, nalidixic acid. Only one strain showed resistance to ciprofloxacin, emphasizing the need for a constant surveillance system. Conclusion These diagnostic molecular tools are of paramount importance in establishing etiology, serogrouping, and epidemiological surveillance especially in developing countries like India.

The level of circulating tetanus toxin, antitoxin and their individual influence on the outcome of tetanus cases were determined in unimmunized 125 neonatal and 39 infant cases of tetanus. PHA (passive haemagglutination) test showed 40% positive cases for toxin while its absence in the remaining cases indicated of either toxin fixation to the central nervous system (CNS) or it got neutralized by antitoxin. TN (toxin neutralization) and PHA test carried out in 46 sera samples revealed a strong positive correlation (r = 0.9) showing that 35/46 (76%) and 38/46 (82.6%) samples were positive for antitoxin, respectively. 25.4% of the neonate and infant cases and 34% of the control group had a protective serum tetanus antitoxin level. 42.5% of the paired sera from unimmunized mothers and their neonates showing nonprotective antitoxin levels suggested that a high level of antitoxin is needed for transplacental transfer, although transfer may not play a decisive role in the resistance against the disease. The presence of toxin or antitoxin in the clinical cases did not affect the outcome of the disease, although in neonates, presence of toxin was found to be a bad prognostic sign. This study explicitly advocates for the need to improve the vaccination coverage strategy.

Ever since the beginning of the epidemic of HIV, one of the poignant aspects of HIV infection is transmission of the virus from mother to child. It is not known whether pregnancy accelerates the progression of HIV infection from a clinically asymptomatic stage to a progressive clinical phase. Present study was carried out to understand disease progression in pregnant women from India. We studied co-receptor utilization (the major determinant of HIV disease progression), N-glycosylation sites, and sequence variability. Blood samples were collected from 25 HIV sero-positive patients, eleven from the antenatal risk group (experimental group), nine from heterosexual male, and five from heterosexual female risk group (control group). Partial env gene was amplified by PCR and sequenced. BLAST search and phylogenetic analysis were used to determine the subtype. The deduced amino acid sequence of the V3 region was used to predict co-receptor, determine sequence variability and N-glycosylation site. The experimental group comprising the antenatal risk group did not exhibit any difference in terms of co-receptor, N-glycosylation, and sequence variability when compared with the control, non-pregnant group. Pregnancy does not seem to accelerate the clinical course of HIV infection. The female body during the gestation phase possibly acquires certain strategies to impede or at least alleviate the disease progression during the crucial immune-compromised pregnancy phase, which would otherwise adversely affect the mother as well as the fetus during the infection.

Objective To evaluate the clinical utility of PCR compared with other available diagnostic modalities in prompt diagnosis of female genital tuberculosis causing infertility. Study design Prospective case-controlled trial. Premenstrual endometrial biopsy specimens were collected from 150 infertile women of reproductive age group suspected of having genital tuberculosis. All patients underwent diagnostic endoscopy (laparoscopy and hysteroscopy) and the samples obtained were subjected to microscopy, culture by the BACTEC 460 TB System, histopathology and polymerase chain reaction (PCR) for detection of 165bp region of 65kDa gene of Mycobacterium tuberculosis . The results were correlated with the laparoscopic findings. Results While the laparoscopy/hysteroscopy findings were indicative of tuberculosis in 12.6% of cases, 14.6% of the specimens showed evidence of 65kDa gene of M. tuberculosis and only 3.33%, 1.33% and 0.66% were positive by culture, smear and histopathology, respectively. Conclusion Since laparoscopy, hysteroscopy other endoscopic procedures are associated with operative risks and may cause flaring of infection, and other conventional laboratory tests including histopathology have poor sensitivity, PCR-based detection of 65kDa gene of M. tuberculosis in endometrial biopsy specimens could be a promising molecular diagnostic technique compared to conventional methods of diagnosis.

Background and Objectives: Rapid clinical manifestation/progression of the meningococcal meningitis and lacunae in conventional bacteriological test often encourages indiscriminate use of antibiotics much before the etiology is established. This study was planned to evaluate ctrA PCR for rapid molecular detection. In addition, multiplex PCR and sequencing was done for serogroup prediction to provide essential epidemiological and laboratory evidence for decision makers of health department of the country for choosing appropriate vaccine and phylogenetic analysis to establish lineage. Materials and Methods: 73 CSF samples, collected from equal number of suspected cases, were investigated by both bacteriological (microscopy, culture, LA and drug sensitivity testing) as well as molecular tests i.e. PCR targeting conserved ctrA gene, multiplex PCR for serogroup characterization and DNA sequencing. Results: ctrA PCR revealed sensitivity, specificity, positive predictive value and negative predictive values of 93.15%, 100%,100%, and 88.23% respectively. Multiplex PCR based genogrouping followed by DNA sequencing, BLAST and phylogenetic analysis revealed complete homology with earlier submitted Neisseria meningitidis serogroup A strain Z2491 to suggest the sole involvement of only serogroup A in the outbreak. Two strains showed resistance to cefuroxime, ciprofloxacin, nalidixic acid. Only one strain showed resistance to ciprofloxacin, emphasizing the need for a constant surveillance system. Conclusion: These diagnostic molecular tools are of paramount importance in establishing etiology, serogrouping, and epidemiological surveillance especially in developing countries like India.

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.

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.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.Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture (P0.05). 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 (P0.05). 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.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.

To evaluate the performance of 65 kDa antigen based PCR assay in clinical samples obtained from pulmonary and extrapulmonary cases of tuberculosis.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.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 (P0.05). However, there was no significant difference (P0.05) 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 (P0.05) 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.PCR is a rapid and sensitive method for the early diagnosis of pulmonary and extrapulmonary tuberculosis.