Tuberculosis (TB) is an important public health problem and remains one of the most threatening curable infectious diseases, despite improvements in diagnostic and drug susceptibility tests. The effective control of TB is based on the immediate detection of Mycobacterium tuberculosis, followed by the prompt implementation of adequate antituberculous therapy (29).The emergence of strains resistant to the major anti-TB drugs speeds up the need for rapid methods for the identification of resistant M. tuberculosis strains in order to treat the disease effectively and, at the same time, prevent the spread of resistant strains (6,8,9,16). Multidrug-resistant (MDR) M. tuberculosis strains, which are resistant at least to rifampin (RIF) and isoniazid (INH), have emerged worldwide and seriously threaten TB control and prevention programs (30).The main mutations that confer RIF resistance are located in the rpoB gene, specifically, in the well-defined 81-bp core region (22,24). About 95% of RIF-resistant strains have a mutation in this region, which facilitates the rapid development of approaches for the detection of resistance to this drug (23,24,26). However, the molecular basis of resistance to

The purpose of this study was to evaluate the GenoType MTBDRsl assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to fluoroquinolones (FLQ), injectable second-line antibiotics [kanamycin (KM) and capreomycin (CM)], and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 34 clinical strains were characterized with the Bactec 460 TB system. Fifty-four clinical samples from 16 patients (5 were smear negative and 49 were smear positive) were also tested directly. The corresponding isolates of the clinical specimens were also analyzed with the Bactec 460TB. When there was a discrepancy between assays, pyrosequencing was performed. The overall rates of concordance of the MTBDRsl and the Bactec 460TB for the detection of FLQ, KM/CM, and EMB susceptibility in clinical strains were 72.4% (21/29), 88.8% (24/27), and 67.6% (23/34), whereas for clinical samples, rates were 86.5% (45/52), 92.3% (48/52), and 56% (28/50), respectively. In conclusion, the GenoType MTBDRsl assay may be a useful tool for making early decisions regarding KM/CM susceptibility and to a lesser extent regarding FLQ and EMB susceptibility. The test is able to detect mutations in both clinical strains and samples with a short turnaround time. However, for correct management of patients with extensively drugresistant tuberculosis, results must be confirmed by a phenotypical method. E fficient tuberculosis (TB) control is based on an early diagnosis followed by the rapid identification of drug resistance, in order to treat patients adequately, break the chain of transmission, and avoid the spread of resistant strains (38). Multidrug-resistant (MDR) Mycobacterium tuberculosis strains resistant at least to isoniazid (INH) and rifampin (RIF), which are two of the main firstline anti-TB drugs, have emerged worldwide and seriously threaten TB control and prevention programs. At the same time, the emergence of extensively drug-resistant tuberculosis (XDR TB), defined as MDR TB with additional resistance to fluoroquinolones (FLQ) [moxifloxacin (MOX), ofloxacin (OFL), and levofloxacin] and at least one of the three injectable second-line drugs [amikacin (AM), kanamycin (KM), and capreomycin (CM)], has also become an important global health problem.Conventional methods for detecting XDR strains are sequential, because they are applied once a strain has grown in solid or liquid medium and has been shown to be resistant to first-line drugs, mainly RIF and INH. As a consequence, the pattern of resistance to second-line drugs becomes available later. In addition, methods of detecting resistance to second-line drugs are not fully standardized (19,39), so the comparison of resistance incidences between different geographical settings is difficult.Regarding first-line drugs, mutations related to INH and RIF resistance have been extensively investigated and involve mainly the rpoB, katG, and inhA genes (30). Ethambutol (EMB) is another first-line drug, and its resistance has been rel...

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The aim of this study was to evaluate a pyrosequencing method for the detection of Mycobacterium tuberculosis isolates resistant to rifampin and isoniazid using both clinical strains and clinical samples, comparing the results with those of the Bactec 460TB and GenoType MTBDRplus assays. In comparison to Bactec 460TB as the gold standard, the sensitivity of pyrosequencing for detecting isoniazid and rifampin resistance was 76.9% and 97.2%, respectively, for clinical strains, and the specificity was 97.2 and 97.9%, respectively. For clinical specimens, the sensitivity and specificity for both drugs were 85.7% and 100%, respectively. The overall concordance between pyrosequencing and the GenoType MTBDRplus assay for clinical strains was 99.1%, and for clinical samples, it was 98.2%. Pyrosequencing is a valuable tool for rifampin and isoniazid resistance detection.Tuberculosis control and prevention programs are based on an early diagnosis followed by rapid identification of drug resistance (26). The detection of drug-resistant Mycobacterium tuberculosis strains is usually performed by time-consuming phenotypic methods. In this sense, new molecular methods for the detection of mutations conferring drug resistance to the two main first-line antituberculous drugs, rifampin (RIF) (rpoB) (18,20,23) and isoniazid (INH) (katG and the inhA promoter region), have been developed (8,18,19,21,22).Pyrosequencing is a semiautomated sequencing assay based on real-time monitoring of DNA synthesis, optimized to analyze single-nucleotide polymorphisms (SNPs) and short DNA sequences (9). The detection of single polymorphisms by pyrosequencing has allowed the identification, resistance detection, and typing of a wide range of microorganisms, including M. tuberculosis (2,3,5,11,12,14,17,27).The objective of this study was to evaluate the ability of pyrosequencing to detect the most frequent mutations in rpoB, katG, and inhA in clinical strains of M. tuberculosis and directly from clinical specimens, comparing the results with phenotypic and genotypic standard methods (genotyping and GenoType MTBDRplus; Hain Lifescience GmbH, Nehren, Germany). To our knowledge, this is the first study comparing pyrosequencing results with those obtained using GenoType MTBDRplus.Clinical strains. Ninety well-characterized M. tuberculosis strains were phenotypically studied using the radiometric method of the Bactec 460TB system (Becton Dickinson, Towson, MD). Among the 90 characterized strains, 11 were fully susceptible and 79 were resistant (43 RIF s /INH r and 36 RIF r / INH r ). All clinical strains were genotypically characterized with the GenoType assay. In addition, for 67 of 79 INH-and/or RIF-resistant strains, genotypic characterization by sequencing was also available. Commercial methods were performed following the manufacturer's instructions.Clinical specimens. Forty-eight clinical specimens were retrospectively selected from 26 patients during clinical routine (Fig. 1). The Ziehl-Neelsen smears were graded on a scale from 0 to 4ϩ (15). The s...

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Cervical cancer is the second most prevalent type of cancer in women worldwide. A total of 500,000 new cases are diagnosed each year and cause more than 270,000 deaths (15). Since the 1940s, screening programs for cervical cancer prevention, mainly based on the Papanicolau smear test, have been implemented in resource-rich countries, resulting in a remarkable decrease in its incidence and related mortality (16). However, this test has a limited sensitivity, especially for detecting precancerous lesions (1, 6).Genital human papillomavirus (HPV) is a highly common sexually transmitted infection. Although most HPV infections are transient and asymptomatic, epidemiological studies worldwide have demonstrated that persistent infection with certain genotypes is the necessary cause for the development of cervical cancer and its precursor lesions (3,19,24). More than 100 HPV types have been described and classified into high-risk types (HR-HPV) and low-risk types (LR-HPV) according to the probability of developing cervical cancer (14). Therefore, in addition to the Papanicolau smear test, HPV detection assays have been implemented in many countries to improve cervical cancer screening. These assays have a higher sensitivity than the Papanicolau smear test for the detection of women at risk of developing precancerous lesions (12).Since HPV cannot be grown in conventional cell cultures and serological assays are unreliable, molecular techniques constitute the best choice to diagnose HPV infection. Currently, the only assay that has been approved by the U.S. Food and Drug Administration for the detection of HPV-DNA is the Hybrid Capture II system (HC-II; Digene Corp., Gaithersburg, MD). This signal amplification assay was designed to detect LR-HPV and HR-HPV genotypes in two different kits but does not provide genotype information.The interest of HPV genotyping has increased in light of the recently licensed HPV bivalent and tetravalent vaccines (9, 23). Genotyping also allows clinicians to monitor patients according to the oncogenic risk of the HPV types identified. Several genotyping assays have been developed over the last years with a variety of amplification and detection strategies (reviewed in

We developed a novel method, PyroTyping, for discrimination of Mycobacterium tuberculosis isolates combining pyrosequencing and IS6110 polymorphism. A total of 100 isolates were analysed with IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, mycobacterial interspersed repetitive units – variable number tandem repeats (MIRU-VNTR), and PyroTyping. PyroTyping results regarding clustering or discrimination of the isolates were highly concordant with the other typing methods performed. PyroTyping is more rapid than RFLP and presents the same discriminatory power, thus, it may be useful for taking timely decisions for tuberculosis control.