PCR colorimetric dot-blot assay and clinical pretest probability for diagnosis of Pulmonary Tuberculosis in Smear-Negative patients

Background  

Smear-negative pulmonary tuberculosis (SNPTB) accounts for 30% of Pulmonary Tuberculosis (PTB) cases reported annually in developing nations. Polymerase chain reaction (PCR) may provide an alternative for the rapid detection of Mycobacterium tuberculosis (MTB); however little data are available regarding the clinical utility of PCR in SNPTB, in a setting with a high burden of TB/HIV co-infection.     

Detection of Mycobacterium tuberculosis by a polymerase chain reaction colorimetric dot-blot assay.

SETTING: A public health laboratory in a tuberculosis-endemic region in Brazil. OBJECTIVE: To evaluate the accuracy of a combined polymerase chain reaction (PCR) colorimetric dot-blot protocol for Mycobacterium tuberculosis detection in clinical samples in a public health laboratory. DESIGN: Eighty clinical samples (13 cerebrospinal fluid, 31 induced sputum, 17 expectorated sputum, eight bronchoalveolar lavage and 11 pleural fluid) were assayed with the developed protocol. The accuracy of polymerase chain reaction (PCR) dot-blot methodology was compared to PCR agarose gel electrophoresis (PCR-AG) using as a gold standard the bacteriological result (culture and biochemical identification) combined with clinical follow-up. One internal region of the IS6110 repetitive element of the M. tuberculosis complex was selected for amplification and the amplified product transferred to nylon membranes to be detected by biotinylated DNA probe. RESULTS: Overall sensitivity and specificity obtained were respectively 90% and 97% for PCR-AG and 95% and 97% for the PCR dot-blot. Among the 56 respiratory specimens, the sensitivity and specificity results for PCR-AG were respectively 88% and 95%, and for PCR dot-blot they were 94% and 95%. Among the 24 non-respiratory specimens the sensitivity and specificity results were respectively 83% and 100% for PCR-AG, and 100% and 100% for the PCR dot-blot protocol. CONCLUSION: The results demonstrated that the PCR dot-blot assay may be helpful in the diagnosis of tuberculosis, and feasible even in resource-poor countries.     

Truncation in the tcdC region of the Clostridium difficile PathLoc of clinical isolates does not predict increased biological activity of Toxin B or Toxin A

Background

The increased severity of disease associated with the NAP1 strain of Clostridium difficile has been attributed to mutations to the tcdC gene which codes for a negative regulator of toxin production. To assess the role of hyper-production of Toxins A and B in clinical isolates of Clostridium difficile, two NAP1-related and five NAP1 non-related strains were compared.

Methods

Sequencing was performed on tcdC, tcdR, and tcdE to determine if there were differences that might account for hyper-production of Toxin A and Toxin B in NAP1-related strains. Biological activity of Toxin B was evaluated using the HFF cell CPE assay and Toxin A biological activity was assessed using the Caco-2 Trans-membrane resistance assay.

Results

Our results confirm that Toxin A and Toxin B production in NAP1-related strains and ATCC 43255 occurs earlier in the exponential growth phase compared to most NAP1-nonrelated clinical isolates. Despite the hyper-production observed in ATCC 43255 it had no mutations in tcdC, tcdR or tcdE. Analysis of the other clinical isolates indicated that the kinetics and ultimate final concentration of Toxin A and B did not correlate with the presence or lack of alterations in tcdC, tcdR or tcdE.

Conclusion

Our data do not support a direct role for alterations in the tcdC gene as a predictor of hyperproduction of Toxin A and B in NAP1-related strains.     

Sunburn,sunscreens, and phenotypes: some risk factors for cutaneous melanoma in southern Brazil

BACKGROUND: The risk factors for cutaneous malignant melanoma have been studied in populations from numerous countries around the world. There are no published studies on the risk factors for this malignancy in Brazil, the largest country in South America. METHODS: A case-control study of all melanoma patients attending a university hospital in Porto Alegre, Brazil, was conducted over a 3-year period from 1995 to 1998. Phototype, hair and eye color, solar habits, history of sunburn, use of sunscreens, and the number of nevi were evaluated through a questionnaire and full body skin examination. Bivariate analysis and a logistic regression model were used to evaluate the data. RESULTS: One hundred and three malignant melanoma patients and 206 matched controls were enrolled in the study. The female to male ratio was 2 : 1. Light phototypes were more prone to the development of cutaneous melanoma. Although stronger in the bivariate analysis, in the logistic regression model, phototypes I or II and ephelides emerged only as moderate risk factors; light eye color and light hair color were not independently significant, with adjusted odds ratios (OR) close to zero. Commonly acquired nevi (CAN) showed a significant and strong effect in the bivariate analysis only when the "30 or more" category was compared to baseline. In the logistic regression model, the presence of a large number of CAN showed an association with increased levels of risk, although these findings did not reach classical significance. Dysplastic or atypical nevi seemed to contribute more strongly, although still with a moderate excess of relative risk. When the use of sunscreens was compared to no use at all, it appeared to show progressive protection as the solar protection factor (SPF) increased. Only SPF15 or greater (SPF15+) showed strong and significant protection when compared to baseline. Physical measures offered a weaker level of protection. Nevertheless, there was a significant increase in the risk of melanoma for those with a large number of sunburn episodes. It was found that 30 or more alleged episodes of sunburn showed a very strong OR of 11.4 (95% confidence interval, 2.6-50.5), the most significant in the study. CONCLUSIONS: Phototypes I and II, freckles, a large number of acquired nevi, dysplastic nevi, and inadequate photoprotection appeared as risk factors with moderate strength for cutaneous malignant melanoma in the studied population. The color of the eyes and hair showed a very weak statistical significance as a risk factor. Sunscreens showed progressive significance corresponding to an increase in SPF, the best scores in statistical protection being achieved in users of sunscreens with SPF15 or greater. Frequent sunburn episodes appeared as the most important risk factor associated with malignant melanoma in this sample of the white population in southern Brazil.     

Pleural fluid ADA,IgA‐ELISA and PCR sensitivities for the diagnosis of pleural tuberculosis

The diagnosis of pleural tuberculosis (pTB) is difficult, and more sensitive and specific techniques are needed. In the period August 1998 to November 2002, we evaluated 132 patients with a pleural effusion submitted to a thoracentesis and pleural biopsy in a tertiary care hospital in Rio de Janeiro, Brazil. Three tests were performed and compared in the pleural fluid: ADA activity measurement, IgA‐ELISA for two combined specific Mycobacterium tuberculosis antigens, and polymerase chain reaction (PCR) for detection of M. tuberculosis DNA. Ninety‐five patients (72%) were given a final diagnosis of pTB. Overall histopathologic sensitivity was 77%. The sensitivities of pleural fluid culture and AFB smear were 42% and 1%, respectively. Twenty‐one (22%) additional patients had a clinical diagnosis of pTB. Median follow‐up time of all TB patients after the completion of antituberculous treatment was 13 months. Sensitivities of ADA, IgA‐ELISA and PCR were 91%, 78% and 82%, while specificities were 93%, 96% and 85%, respectively. Only ADA sensitivity was significantly higher than the histopathologic examination (McNemar χ² test; p = 0.002) and also significantly higher than ELISA (p = 0.049), but not higher than PCR (p = 0.143). We conclude that the routine use of ADA activity measurement in pleural fluid can obviate the need for a pleural biopsy in the initial diagnostic approach to pleural effusions, while IgA‐ELISA and PCR techniques, potentially more specific tests, need further refinement to improve their accuracy.     

Pleural fluid ADA, IgA-ELISA and PCR sensitivities for the diagnosis of pleural tuberculosis

The diagnosis of pleural tuberculosis (pTB) is difficult, and more sensitive and specific techniques are needed. In the period August 1998 to November 2002, we evaluated 132 patients with a pleural effusion submitted to a thoracentesis and pleural biopsy in a tertiary care hospital in Rio de Janeiro, Brazil. Three tests were performed and compared in the pleural fluid: ADA activity measurement, IgA-ELISA for two combined specific Mycobacterium tuberculosis antigens, and polymerase chain reaction (PCR) for detection of M. tuberculosis DNA. Ninety-five patients (72%) were given a final diagnosis of pTB. Overall histopathologic sensitivity was 77%. The sensitivities of pleural fluid culture and AFB smear were 42% and 1%, respectively. Twenty-one (22%) additional patients had a clinical diagnosis of pTB. Median follow-up time of all TB patients after the completion of antituberculous treatment was 13 months. Sensitivities of ADA, IgA-ELISA and PCR were 91%, 78% and 82%, while specificities were 93%, 96% and 85%, respectively. Only ADA sensitivity was significantly higher than the histopathologic examination (McNemar chi2 test; p = 0.002) and also significantly higher than ELISA (p = 0.049), but not higher than PCR (p = 0.143). We conclude that the routine use of ADA activity measurement in pleural fluid can obviate the need for a pleural biopsy in the initial diagnostic approach to pleural effusions, while IgA-ELISA and PCR techniques, potentially more specific tests, need further refinement to improve their accuracy.