Manual liquid culture on simple Middlebrook 7H9 or MGIT for the diagnosis of smear-negative pulmonary tuberculosis.

OBJECTIVES: To compare the performance of liquid culture on simple Middlebrook 7H9 to the one of manual mycobacterial growth indicator tube (MGIT) and solid culture on Ogawa for the diagnosis of smear-negative tuberculosis (SN-TB) in a high-burden, resource-constrained setting. METHODS: Sputum samples from patients with clinical suspicion of SN-PTB admitted to two-third-level hospitals in Lima between September 2005 and May 2008 were cultured in parallel on simple Middlebrook 7H9, manual MGIT and Ogawa. A case of SN-TB was defined as one with a positive culture in any medium. RESULTS: Among samples from 542 patients, 151 (28%) cases of SN-TB were identified. The sensitivity of Middlebrook 7H9 (0.76, 95% CI 0.69-0.83) was not substantially different from that of MGIT (0.85, 95% CI 0.79-0.91). Ogawa had the lowest sensitivity (0.63, 95% CI 0.55-0.71). The median turnaround time was similar for both liquid media (18 days), and it was shorter than that of Ogawa (30 days). CONCLUSIONS: Culture on simple Middlebrook 7H9 performs almost as well as MGIT, at a probably more affordable cost. Further studies on the cost-effectiveness of this overlooked technique should be performed.

Functional Analysis Using Whole-Genome Sequencing of a Drug-Sensitive Mycobacterium tuberculosis Strain from Peru.

We report the whole-genome sequence of a Latin American-Mediterranean (LAM) lineage drug-sensitive Mycobacterium tuberculosis strain from Peru, INS-SEN. The functional analysis revealed more mutations in secondary metabolite biosynthesis, transport, and catabolism (clusters of orthologous groups [COG] category Q) than for other LAM-sensitive strains. This study contributes to the understanding of the genomic diversity of drug-sensitive M. tuberculosis.

Evidence of Clonal Expansion in the Genome of a Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolate from Peru.

We report the genome sequence of Mycobacterium tuberculosis INS-MDR from Peru, a multidrug-resistant tuberculosis (MDR-TB) and Latin American-Mediterranean (LAM) lineage strain. Our analysis showed mutations related to drug resistance in the rpoB (D516V), katG (S315T), kasA (G269S), and pncA (Q10R) genes. Our evidence suggests that INS-MDR may be a clonal expansion related to the African strain KZN 1435.

Genetic diversity, population structure and drug resistance of Mycobacterium tuberculosis in Peru.

This paper presents the first evaluation of the molecular epidemiology of Mycobacterium tuberculosis in Peru. We characterised 323 isolates using spoligotyping and mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTR) typing. We aimed to determine the levels of genetic diversity and genetic differentiation among and within Peruvian isolates and the epidemiological factors which may be driving patterns of population structure and evolution of M. tuberculosis in Peru. Our results compared to the fourth international spoligotyping database (SpolDB4) and MIRU-VNTRplus, show that the main M. tuberculosis families present are Latin American-Mediterranean, Haarlem, T, and Beijing. Bayesian clustering recovered 15 groups in the Peruvian M. tuberculosis isolates, among which two were composed mainly of orphans, implying the presence of native "Peruvian" strains not previously reported. Variable levels of association with drug resistance were observed, with Beijing genotypes not showing any association with multidrug resistance, while in other groups MIRU-VNTR loci 2, 23, 31, and 40 were found to be associated with the multidrug-resistant tuberculosis (MDR-TB) phenotype, suggesting that a linkage disequibrium between these MIRU and drug resistance loci may be present. Genetic differentiation was present among drug resistant and sensitive strains. Ethambutol appeared to be the main driver of differentiation, suggesting that strong selection pressure could have been exerted by drug treatment in Peru over recent years.

Quality assessment of smear microscopy by stratified lot sampling of treatment follow-up slides.

SETTING: Ten peripheral laboratories performing routine acid-fast bacilli (AFB) smear microscopy in Lima, Peru. OBJECTIVES: To test whether external quality assessment (EQA) rechecking of AFB smears becomes more efficient with stratified lot sampling of treatment follow-up smears. DESIGN: In 2 consecutive years, a stratified lot sample of 36 treatment follow-up slides and 24 diagnostic slides were randomly selected and blindly rechecked. A second controller determined the final result for discordant slides. Feedback was provided to laboratory technicians during supervisory visits. RESULTS: More false-negative errors were found in the follow-up slides than in the tuberculosis suspect slides: 25 vs. 3. This represented a yield of 3.5% in 720 follow-up slides and only 0.6% in 480 diagnostic slides. Positive predictive values were high in both years. Respectively three and eight laboratories did not reach a relative sensitivity of >65% during the first and second year, and a clear improvement was seen in only one laboratory. Excessive workload seemed to preclude raising the level of routine performance. CONCLUSIONS: EQA with stratified lot sampling of treatment follow-up slides proved very efficient and effective for identifying laboratories with substandard performance in a setting with low positivity rates in routine diagnostic smears.

A clinical prediction rule for pulmonary tuberculosis in emergency departments.

SETTING: University-affiliated hospital located in an area with a high incidence of pulmonary tuberculosis (PTB). OBJECTIVE: To develop a clinical prediction rule (CPR) based on information obtainable on admission, to permit rapid identification of patients with PTB. DESIGN: Information from patients with respiratory symptoms who attended the emergency department of Cayetano Heredia Hospital, Lima, Peru, was collected prospectively. Clinical symptoms, past medical history, demographic data and results of chest X-rays (CXRs), sputum smear and culture in Löwenstein-Jensen media were obtained. Based on logistic regression, we constructed a scoring system to predict PTB. RESULTS: A total of 345 patients were enrolled in the study, including 109 (31%) culture-proven PTB cases. In logistic regression analysis, we found age, previous history of PTB, weight loss, presence of cavities, upper lobe infiltrate and miliary pattern on CXR as independent predictors of PTB. We designed a scoring system with these variables, taking into account their statistical weight. The score attained 93% sensitivity and 42% specificity. CONCLUSION: The CPR that was developed performed well in our population. It merits further validation in other settings. It should not, however, replace, but should complement sputum microscopy when deciding on isolation, and it does not preclude microbiology in making a definitive diagnosis.