Sunil Gupta

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.

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.

Serological diagnosis of childhood tuberculosis by estimation of mycobacterial antigen 60-specific immunoglobulins in the serum

SETTING An ELISA assay based on mycobacterial antigen 60 (A60) for the estimation of specific immunoglobulins in the serum has been used successfully for the rapid diagnosis of tuberculosis in studies done predominantly in Western countries. In a recent Indian study, encouraging results were reported in adult tuberculosis. OBJECTIVE To evaluate the utility of this ELISA test for rapid diagnosis of different clinical forms of tuberculosis in Indian children. DESIGN ELISA test based on mycobacterial A60 was used to estimate specific IgM, IgA and IgG antibodies in the sera obtained from 452 cases of tuberculosis and 161 controls in the paediatric population of Delhi, India. RESULTS Of the 161 controls, only 7.4% were positive for IgM, 4.3% for IgG, 3.7% for IgA and 8% when a combination of IgM and IgA was considered. Of 58 cases of definite pulmonary tuberculosis, 55.2% were positive for IgM, 32.7% for IgG, 36.2% for IgA and a high positivity of 72.4% was seen when IgA and IgM estimations were combined. The corresponding figures in 150 cases of definite extrapulmonary tuberculosis were 57.3%, 36.6%, 38% and 76.6%. A relatively weak serology was observed in 244 cases of probable tuberculosis. A very high positivity (95%) was seen in acid-fast bacilli-positive cases of tuberculosis. CONCLUSIONS Our findings point to a very good specificity (92%) and a reasonably good sensitivity (75.5%) of the test when combined IgM and IgA antibody titres are considered in the diagnosis of childhood tuberculosis.

Diagnosis of tuberculosis in an era of HIV pandemic: A review of current status and future prospects

HIV and tuberculosis co-infection interact in fundamentally important ways. This interaction is evident patho-physiologically, clinically and epidemiologically. There are several differences between HIV-infected and HIV-uninfected patients with tuberculosis (TB) that have practical diagnostic implications. TB is more likely to be disseminated in nature and more difficult to diagnose by conventional diagnostic procedures as immunosuppression progresses. As TB rates continue to increase in HIV-endemic regions, improved diagnostic techniques merit consideration as TB-control strategies. There is a need to develop more user friendly techniques, which can be adapted for use in the high-burden and low-income countries. This review focuses on the diagnostic challenges in HIV-TB co-infection with an update on the current techniques and future prospects in an era of HIV pandemic.

A severe and explosive outbreak of hepatitis B in a rural population in Sirsa district, Haryana, India: unnecessary therapeutic injections were a major risk factor

Most outbreaks of viral hepatitis in India are caused by hepatitis E. This report describes an outbreak of hepatitis B in a rural population in Haryana state in 1997. At least 54 cases of jaundice occurred in Dhottar village (population 3096) during a period of 8 months; 18 (33.3%) of them died. Virtually all fatal cases were adults and tested positive for HBsAg (other markers not done). About 88% (21/24) of surviving cases had acute or persistent HBV/HCV infections; 54% (13/24) had acute hepatitis B. Many other villages reported sporadic cases and deaths. Data were pooled from these villages for analysis of risk factors. Acute hepatitis B cases had received injections before illness more frequently (11/19) than those found negative for acute or persistent HBV/HCV infections (3/17) (P = 0.01). Although a few cases had other risk factors, these were equally prevalent in two groups. The results linked the outbreak to the use of unnecessary therapeutic injections.

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.