Simultaneous identification of mycobacterial isolates to the species level and determination of tuberculosis drug resistance by PCR followed by electrospray ionization mass spectrometry.

Mycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used to determine the appropriate treatment regimen for patients often take months following the presumptive diagnosis of tuberculosis. Furthermore, the misidentification of nontuberculosis mycobacteria (NTM) in patients presumably suffering from tuberculosis results in additional human and health care costs. The mechanisms of resistance for several drugs used to treat Mycobacterium tuberculosis are well understood and therefore should be amenable to determination by rapid molecular methods. We describe here the use of PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) in an assay that simultaneously determines INH and RIF resistance in Mycobacterium tuberculosis and identifies and determines the species of NTMs. The assay panel included 16 primer pairs in eight multiplexed reactions and was validated using a collection of 1,340 DNA samples from cultured specimens collected in the New York City area, the Republic of Georgia, and South Africa. Compared with phenotypic data, the PCR/ESI-MS assay had 89.3% sensitivity and 95.8% specificity in the determination of INH resistance and 96.3% sensitivity and 98.6% specificity in the determination of RIF resistance. Based on a set of 264 previously characterized liquid culture specimens, the PCR/ESI-MS method had 97.0% sensitivity and 99.9% specificity for determination of NTM identity. The assay also provides information on ethambutol, fluoroquinolone, and diarylquinoline resistance and lineage-specific polymorphisms, to yield highly discriminative digital signatures potentially suitable for epidemiology tracking.

Mycobacterium tuberculosis Beijing lineage favors the spread of multidrug-resistant tuberculosis in the Republic of Georgia.

High rates and transmission of multidrug-resistant (MDR) tuberculosis (TB) have been associated with the Mycobacterium tuberculosis complex (MTBC) Beijing lineage, pointing to the importance of pathogen genetic factors for the modulation of infection outcome and epidemiology. We present here an in-depth analysis of the population structure of MTBC strains from the Republic of Georgia, a high-incidence setting at the Black Sea Coast. Phylogenetic lineages were identified based on 24-locus MIRU-VNTR (for mycobacterial interspersed repetitive unit-variable number tandem repeat) and spoligotyping analysis. Clusters of strains with identical genotyping profiles were determined as an indicator for the rate of recent transmission. Among the 183 M. tuberculosis isolates investigated, the most prominent lineage found was Beijing (26%), followed by the LAM (18%), Ural (12%), and Haarlem (5%) strains. A closely related previously undefined phylogenetic group (62 strains) showed a genotyping pattern similar to laboratory strain H37RV and was denominated as "Georgia-H37RV-like." Although isoniazid resistance was found among strains of different lineages, MDR TB was nearly completely restricted to Beijing strains (P < 0.0001). Approximately 50% of the isolates were grouped in clusters, indicating a high rate of recent transmission. Our data indicate that, in addition to the confirmation of the importance of Beijing genotype strains for the TB epidemiology in former Soviet Union countries, a high-population diversity with strains of the LAM, Ural, Haarlem, and a previously undefined lineage represents nearly two-thirds of the strains found in Georgia. Higher rates among previously treated and MDR TB patients point to a higher potential of lineage Beijing to escape therapy and develop MDR TB.

Monitoring therapeutic efficacy by real-time detection of Mycobacterium tuberculosis mRNA in sputum.

BACKGROUND: Current laboratory methods for monitoring the response to therapy for tuberculosis (TB) rely on mycobacterial culture. Their clinical usefulness is therefore limited by the slow growth rate of Mycobacterium tuberculosis. Rapid methods to reliably quantify the response to anti-TB drugs are desirable. METHODS: We developed 2 real-time PCR assays that use hydrolysis probes to target DNA of the IS6110 insertion element and mRNA for antigen 85B. The nucleic acids are extracted directly from concentrated sputum samples decontaminated with sodium hydroxide and N-acetyl-L-cysteine. We prospectively compared these assays with results obtained by sputum mycobacterial culture for patients receiving anti-TB therapy. RESULTS: Sixty-five patients with newly diagnosed TB and receiving a standardized first-line anti-TB drug regimen were evaluated at week 2 and at months 1, 2, and 4 after therapy initiation. Both the DNA PCR assay (98.5% positive) and the mRNA reverse-transcription PCR (RT-PCR) assay (95.4% positive) were better than standard Ziehl-Neelsen staining techniques (83.1%) for detecting M. tuberculosis in culture-positive sputum samples. The overall agreement between culture and mRNA RT-PCR results for all 286 sputum samples was 87.1%, and compared with culture, the mRNA RT-PCR assay's diagnostic sensitivity and specificity were 85.2% and 88.6%, respectively. For monitoring efficacy of therapy, mRNA RT-PCR results paralleled those of culture at the follow-up time points. CONCLUSIONS: The continued presence of viable M. tuberculosis according to culture and results obtained by RT-PCR analysis of antigen 85B mRNA correlated clinically with resistance to anti-TB drugs, whereas the DNA PCR assay showed a high false-positive rate. This mRNA RT-PCR assay may allow rapid monitoring of the response to anti-TB therapy.

Prevalence of and molecular basis for tuberculosis drug resistance in the Republic of Georgia: validation of a QIAplex system for detection of drug resistance-related mutations.

We developed a QIAplex system for the simultaneous detection of 24 Mycobacterium tuberculosis gene mutations responsible for resistance to isoniazid (INH), rifampin (RIF), streptomycin (STM), and ethambutol (EMB) in 196 M. tuberculosis isolates recovered in the Republic of Georgia. In comparison to phenotypic susceptibility tests, the QIAplex showed sensitivity and specificity of 85.4% and 96.1% for INH, 94.4% and 99.4% for RIF, 69.6% and 99.2% for STM, 50.0% and 98.8% for EBM, and 86.7% and 100.0% for multidrug resistance, respectively. The dominant resistance mutations revealed were a mutation in katG resulting in S315T (katG S315T), rpsL K43R, and rpoB S531L. Mutations katG S315G and S315T and rpoB S531L were detected with higher frequencies in pretreated patients than in naive patients (P < 0.05). Simultaneous detection of 24 common drug resistance-related mutations provides a molecular tool for studying and monitoring M. tuberculosis resistance mechanism and epidemiology.

High prevalence of multidrug-resistant tuberculosis in Georgia.

BACKGROUND: Tuberculosis (TB) has emerged as a serious public health problem in the country of Georgia. However, little or no data exist on rates and risk factors for drug-resistant TB, including multidrug-resistant (MDR)-TB, in Georgia. OBJECTIVE: To assess the prevalence and risk factors for drug-resistant TB. METHODS: A cross-sectional prospective survey of patients with suspected pulmonary TB was carried out at four sentinel sites (Tbilisi, Zugdidi, Kutaisi, and Batumi) in Georgia between January 1, 2001 and December 31, 2004. RESULTS: Among 1422 patients with suspected pulmonary TB, 996 (70.0%) were culture positive; 931/996 (93.5%) had drug susceptibility testing performed. Overall, 64.0% of patients (48.3% of new and 85.3% of retreatment cases) had positive cultures for Mycobacterium tuberculosis resistant to >or=1 first-line antituberculosis drugs. The overall prevalence of MDR-TB was 28.1% (10.5% of newly diagnosed patients and 53.1% of retreatment cases). In multivariate analysis, risk factors for MDR-TB included: being a retreatment case (prevalence ratio (PR)=5.28, 95% CI 3.95-7.07), history of injection drug use (PR=1.59, 95% CI 1.21-2.09), and female gender (PR=1.36, 95% CI 1.12-1.65). CONCLUSIONS: MDR-TB has emerged as a serious public health problem in Georgia and will greatly impact TB control strategies.

Electrocardiographic changes in patients with acute stroke: a systematic review.

Repolarization and ischemic-like electrocardiographic (ECG) changes observed during acute phase of stroke may cause diagnostic and management dilemmas for the clinician. In this systematic review, we have compiled all information available in the literature on the prevalence of these ECG changes and QT prolongation during the acute phase of stroke and their coexistence with other abnormal cardiac findings. Abnormalities, such as ischemic-like ECG changes and/or QT prolongation, were found in 76% (95% CI 73-90) of patients with subarachnoid hemorrhage, irrespective of whether they had preexisting heart disease or not. Such ECG changes were present in more than 90% of unselected patients with ischemic stroke and intracerebral hemorrhage, but the prevalence was much lower after exclusion of patients with preexisting heart disease. Compared with other abnormal cardiac findings (cardiac wall motion abnormality detected by echocardiography, elevated levels of biochemical markers of myocardial injury, autopsy findings, thallium scintigraphy), these ECG changes were characterized by a high sensitivity but a very low specificity. Thus, in patients with subarachnoid hemorrhage, repolarization and ischemic-like ECG changes are mainly direct consequences of the cerebral condition and their absence essentially rules out cardiac abnormalities. In patients with ischemic stroke and intracerebral hemorrhage, these ECG abnormalities (and QT prolongation) most often represent preexisting coronary artery disease. The specificity of ECG changes to diagnose acute myocardial infarction is low in the acute phase of stroke.