Predictive value of the acid fast smear for detection of Mycobacterium tuberculosis in respiratory specimens in a Reference Center of HIV/Aids in Rio de Janeiro, Brazil

In order to evaluate the predictive value of acid fast bacilii (AFB) smear for the diagnosis of Mycobacterium tuberculosis in respiratory specimens in a setting with a high prevalence of Aids and an unknown prevalence of nontuberculous mycobacteria (NTM), we retrospectively examined specimens cultured for mycobacteria between 1 September 1993 and 30 September 1994 and medical records of patients with positive culture in a General Hospital, Aids reference in Rio de Janeiro, Brazil. Seventy three per cent (1517/2077) of samples were respiratory specimens and mycobacteria were recovered from 20.6% (313/1517) of these. M. tuberculosis was identified in 94.2% (295/313) and NTM in 5.8% (18/313). The yield of positive AFB smear and of positive culture was 6.1% (93/1517) and 20.6% (313/1517), respectively. The positive predictive value (PPV) of AFB for M. tuberculosis was 98.4% in expectorated sputum and 96.4% in bronchoalveolar lavage. Forty four percent (130/295) of specimens with positive culture for M. tuberculosis and 66.7% (12/18) for NTM were from patients HIV positive. The conclusion was that in our study population, the PPV of AFB for M. tuberculosis in respiratory specimens was high and the prevalence of NTM was low despite the high prevalence of HIV positive.

Comparison of quantiferon-TB gold with tuberculin skin test for detecting latent tuberculosis infection prior to liver transplantation.

Screening for latent tuberculosis infection (LTBI) is recommended prior to organ transplantation. The Quantiferon-TB Gold assay (QFT-G) may be more accurate than the tuberculin skin test (TST) in the detection of LTBI. We prospectively compared the results of QFT-G to TST in patients with chronic liver disease awaiting transplantation. Patients were screened for LTBI with both the QFT-G test and a TST. Concordance between test results and predictors of a discordant result were determined. Of the 153 evaluable patients, 37 (24.2%) had a positive TST and 34 (22.2%) had a positive QFT-G. Overall agreement between tests was 85.1% (kappa= 0.60, p < 0.0001). Discordant test results were seen in 12 TST positive/QFT-G negative patients and in 9 TST negative/QFT-G positive patients. Prior BCG vaccination was not associated with discordant test results. Twelve patients (7.8%), all with a negative TST, had an indeterminate result of the QFT-G and this was more likely in patients with a low lymphocyte count (p = 0.01) and a high MELD score (p = 0.001). In patients awaiting liver transplantation, both the TST and QFT-G were comparable for the diagnosis of LTBI with reasonable concordance between tests. Indeterminate QFT-G result was more likely in those with more advanced liver disease.

Standartization of broth microdilution method for Mycobacterium tuberculosis

Indirect drug susceptibility tests of Mycobacterium tuberculosis was done to investigate the accuracy and feasibility of a broth microdilution method (BMM) for determining minimal inhibitory concentrations of conventional drugs against M. tuberculosis. Test drugs included isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S) and pyrazinamide (Z). Fifty isolates of M. tuberculosis from patients who had never received drug therapy, and H37Rv strain for control, were evaluated in the system. When comparing this method with the gold standard proportional method in Lowenstein-Jensen medium, sensitivity of 100% for all drugs and specifities of 91, 100, 96, 98 and 85% were observed respectively for H, R, E, S and Z. The BMM was read faster (14-20 days) than the proportional method (20-28 days). The microdilution method evaluated allows the testing of multiple drugs in multiple concentrations. It is easy to perform and does not require special equipment or expensive supplies. In contrast to radiometric method it does not use radioactive material.

Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1

Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification.

Risk Factors for tuberculosis among human immunodeficiency virus-infected persons. A case-control study in Belo Horizonte, Minas Gerais, Brazil (1985-1996)

The objective of this study was to identify tuberculosis risk factors and possible surrogate markers among human immunodeficiency virus (HIV)-infected persons. A retrospective case-control study was carried out at the HIV outpatient clinic of the Universidade Federal de Minas Gerais in Belo Horizonte. We reviewed the demographic, social-economical and medical data of 477 HIV-infected individuals evaluated from 1985 to 1996. The variables were submitted to an univariate and stratified analysis. Aids related complex (ARC), past history of pneumonia, past history of hospitalization, CD4 count and no antiretroviral use were identified as possible effect modifiers and confounding variables, and were submitted to logistic regression analysis by the stepwise method. ARC had an odds ratio (OR) of 3.5 (CI 95% - 1.2-10.8) for tuberculosis development. Past history of pneumonia (OR 1.7 - CI 95% 0.6-5.2) and the CD4 count (OR 0.4 - CI 0.2-1.2) had no statistical significance. These results show that ARC is an important clinical surrogate for tuberculosis in HIV-infected patients. Despite the need of confirmation in future studies, these results suggest that the ideal moment for tuberculosis chemoprophylaxis could be previous to the introduction of antiretroviral treatment or even just after the diagnosis of HIV infection.

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuber-culosis infection.

Active and passive screening for tuberculosis in Vaud Canton, Switzerland

Résumé Même si l'incidence de la tuberculose est basse en Suisse, cette maladie reste un problème de santé publique en raison des migrations de populations provenant de pays où l'incidence de la tuberculose est élevée. Les immigrants, à leur arrivée en terre helvétique, doivent s'annoncer auprès d'un des cinq centres d'enregistrement pour les réfugiés (Vallorbe, Bâle, Kreuzlingen, Altstätten et Chiasso) et subir un contrôle médical qui comprend un test tuberculinique et une radiographie du thorax afin de détecter des anomalies compatibles avec une tuberculose. Les requérants avec des signes de maladie sont immédiatement pris en charge dans le but d'éviter une dissémination du bacille de Koch. Cette* étude rétrospective compare la présentation bactériologique et clinique de la tuberculose ainsi que le résultat du traitement de cette maladie chez les immigrants diagnostiqués par dépistage actif (= immigrants venant d'être enregistrés comme requérants d'asile en Suisse) avec d'autres patients diagnostiqués par dépistage passif (= patients suisses, travailleurs étrangers résidents en Suisse ainsi que d'autres étrangers incluant les touristes, les étudiants, les immigrants illégaux ainsi que 11 requérants d'asile détectés tardivement et passivement après leur entrée en Suisse). Parmi les 179 patients, 78% sont des étrangers. La médiane d'âge de la population étrangère atteinte de tuberculose est de 29 ans contre 64 ans pour les Suisses. Le dépistage actif a été effectué auprès de 71 requérants d'asile chez lesquels 49.3% [CI : 37.4 - 61.2] n'avaient pas de symptômes contre 17.6% [Cl : 10.3 - 24.9] chez les 108 passifs. La durée des symptômes était de 2 mois dans le groupe des actifs versus 2.5 mois chez les passifs (ns). L'analyse bactériologique est positive à l'examen direct ou à la culture chez 63.4% des actifs contre 70.4% des passifs (ns). La confirmation bactériologique de la tuberculose chez des patients asymptomatiques s'élevait à 42.2% [Cl : 27.2-57.2] chez les actifs contre 13% [Cl : 5.31-20.7] chez les passifs. Le plus grand danger de dissémination est couru par les patients avec un examen direct positif dont la proportion des asymptomatiques était de 22.2% ([Cl : 9.6-34.8] dans le groupe des actifs contre 11.7% [CI : 4.4-19.0] dans le groupe des passifs. Le résultat du traitement, comprenant les patients guéris (avec confirmation bactériologique de guérison) ainsi que les patients ayant accompli le traitement jusqu'à la fin (mais sans confirmation bactériologique) est similaire dans les groupes des actifs et passifs. Le résultat différent selon le statut légal avec 88% pour les travailleurs étrangers, 85% pour les Suisses, 78% pour les autres étrangers et 83% pour les réfugiés. Ces chiffres sont proches des cibles de l'OMS (85%). Le dépistage actif de la tuberculose permet la détection plus précoce des cas de tuberculose que le dépistage passif. Etant donné que les immigrants proviennent de régions où la prévalence de la tuberculose est supérieure à celle de la Suisse, ce dépistage à la frontière permet non seulement de diminuer la dissémination de cette maladie par la prise en charge immédiate des malades et de réduire la durée des symptômes mais encore de détecter des patients ne présentant aucun symptôme malgré une activité bactériologique positive. Les résultats du traitement ne satisfont pas entièrement aux exigences de l'OMS, ce qui peut être expliqué par le fait que la population de patients tuberculeux suisses étant plus âgés que celles des étrangers, le nombre de décès est plus nombreux (soit par la tuberculose, soit par les complications de maladies sous-jacentes) et que le suivi de patients étrangers est plus difficile car certains disparaissent durant le traitement et d'autres sont transférés ailleurs en Suisse ou retournent dans leur pays. Summary Aim: This retrospective study compared the bacteriological and clinical presentation of tuberculosis and the outcome of treatment in immigrant notified for TB after active screening by chest X-ray at the border with other patients detected by passive screening. Design: Retrospective study of all patients notified for TB in Vaud Canton in 2001 and 2002. Result: In Vaud Canton 78% of the 179 patients notified for TB were foreign-born. Among 71 asylum seekers actively screened at the border, 49.3% [CI 37.4 - 61.2] were symptom-free vs 17.6% [CI 10.3 - 24.9] among 108 passively screened patients. In the passively screened group, the proportion of asymptomatic patients was 15.4% for Swiss patients. 8.6% for foreign workers, and 29.4% for other foreigners. The average duration of symptoms before diagnosis among patients with complaints was 2 months in actively screened foreign-born, compared to 2.5 months in passively screened patients (no significant difference by Wilcoxon-Mann-Whitney test). The proportion of pulmonary TB cases with positive smear or culture was 63.4% in actively screened patients vs 70.4% in passively detected cases. Among actively screened patients with bacteriological confirmation, 42.2% [CI 27.2-57,2] were asymptomatic compared to 13% [CI 5.31-20.7] for passively screened patients. Considering only smear positive patients, the proportion of symptom-free patients was 22.2% [CI 9.6-34.8] in 45 actively screened cases vs 11-7% [4.4 - 19.0] for 77 passive screening. Cure and treatment completion rate for new cases reached 88% for foreign workers, 83% for asylum seekers, 85% for Swiss patients, 78% for other foreigners. Conclusions: Actively screened patients were more frequently asymptomatic than passively detected cases, even when considering only patients with bacteriological confirmation. The active screening by chest X-ray of an immigrant population with a high prevalence of tuberculosis allows the early detection and treatment of tuberculosis. This may contribute to the protection of the resident population for infection. The outcome of treatment for tuberculosis was satisfactory in all population groups.

Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France

We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP)-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100% (16/16) from France and 89% (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%), 526, 513 and 533 (18.7% each). In Brazilian strains the most common mutations were in codons 531 (72.2%), 526 (11.1%) and 513 (5.5%). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.

Involvement of Parachlamydia in bovine abortions in Scotland.

Bovine abortion represents a major animal welfare issue and a cause of substantial economic loss yet the rate of successful diagnosis remains low. Chlamydia-related organisms including Parachlamydia have recently emerged as putative cattle abortifacients. Placental tissue samples and fetal lung from bovine abortion submissions across Scotland in Spring 2011 were investigated by histopathology for the presence of suspect Chlamydia-related organisms. Evidence of Chlamydia-related organisms was observed in 21/113 (18.6%) placenta samples. Thirteen of the suspect cases and 18 histopathology negative cases were analysed by molecular and immunohistochemical methods. All samples were PCR positive for Parachlamydia but sequencing revealed high homology between identified environmental 16S sequences in all but three cases. Parachlamydial antigen was detected in 10/31 placental samples (32.2%) with pathology consistent with chlamydial infection. This work supports the need for further surveillance investigations and experimental studies to determine the role of Parachlamydia in bovine abortion.

Infection latente a M. tuberculosis, mise a jour 2011 [Latent M. tuberculosis infection, update 2011].

It is estimated that one third of the world population is latently infected by Mycobacterium tuberculosis and thus at risk of reactivation. Latent tuberculosis (TB) impact in Switzerland is often overlooked. Diagnosis and prophylaxis are insufficiently undertaken, especially for people at higher risk of reactivation due to immunosuppression. Interferon-gamma release assays replace tuberculosis skin tests for diagnosis of latent infection in adults. It is still recommended to treat prophylactically a case of latent TB infection with 9 months of isoniazid; however therapy with rifampicin for 4 months, currently an alternative option, is linked to improved adherence and favorable cost-benefit ratio.

Transmission of tuberculosis in Havana, Cuba: a molecular epidemiological study by IS6110 restriction fragment length polymorphism typing

The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55%) had unique IS6110 restriction fragment length polymorphism (RFLP) patterns, while isolates from 23 others (45%) had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.

Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation.

PURPOSE: To determine whether bovine corneal endothelial (BCE) cells and keratocytes express the inducible form of nitric oxide synthase (NOS) after exposure to cytokines and lipopolysaccharide (LPS), and to study the regulation of NOS by growth factors. METHODS: Cultures of bovine corneal endothelial cells and keratocytes were exposed to increasing concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). At selected intervals after exposure, nitrite levels in the supernatants were evaluated by the Griess reaction. Total RNA was extracted from the cell cultures, and messenger RNA levels for inducible NOS (NOS-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Exposure of BCE cells and keratocytes to LPS and IFN-gamma resulted in an increase of nitrite levels that was potentiate by the addition of TNF-alpha. Analysis by RT-PCR demonstrated that nitrite release was correlated to the expression of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced nitrite release in both cells, whereas transforming growth factor-beta (TGF-beta) slightly potentiated it. Fibroblast growth factor-2 (FGF-2) inhibited LPS-IFN-gamma-induced nitrite release and NOS-2 messenger RNA accumulation in keratocytes but not in BCE cells. CONCLUSIONS: The results demonstrate that in vitro activation of keratocytes and BCE cells by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammatory corneal diseases in vivo.

The candidate tuberculosis vaccine Mtb72F/AS02 in PPD positive adults: A randomized controlled phase I/II study.

Prevention of tuberculosis (TB) through vaccination would substantially reduce the global TB burden. Mtb72F/AS02 is a candidate TB vaccine shown to be immunogenic and well tolerated in PPD-negative adults. We evaluated the safety and immunogenicity of Mtb72F/AS02 in Mycobacterium-primed adults (BCG-vaccinated, or infected adults who had received post-exposure chemoprophylaxis or treatment for pulmonary TB disease). In this observer-blind controlled trial, 20 BCG-vaccinated adults and 18 adults previously infected with Mycobacterium tuberculosis (Mtb), were randomized 3:1 to receive three doses of Mtb72F/AS02 or AS02 at one-month intervals, and followed for 6 months post third vaccination. Mtb72F/AS02 was well tolerated in BCG-vaccinated adults, and tended to be more reactogenic in Mtb-infected adults. Adverse events were mainly self-limiting, resolving without sequelae. No serious adverse events were reported. The adverse events in Mtb72F/AS02 vaccinees were not clearly associated with vaccine-induced responses (as assessed by proinflammatory cytokines, total IgE and C-reactive protein levels). No Th2 T-cell responses, or vaccine-induced T-cell responses to Mtb antigens (CFP-10/PPD/ESAT-6) were detected by ICS. In both cohorts, Mtb72F/AS02 induced persistent polyfunctional Mtb72F-specific CD4(+) T-cell responses and anti-Mtb72F humoral responses. IFN-γ was detectable in serum one day post each vaccination. Further evaluation of the candidate vaccine, Mtb72F/AS02, is warranted. Trial registration: ClinicalTrials.gov identifier: NCT00146744.

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.