High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Isolates

We evaluated high-resolution melting (HRM) curve analysis as a tool for detecting rifampin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis in an accurate, affordable, and rapid manner. Two hundred seventeen M. tuberculosis clinical isolates of known resistance phenotype were used. Twenty-nine known rpoB mutant DNAs, including rare mutations, were also included. Four pairs of primers were designed: rpoB-F/R (for codons 516 to 539 of rpoB), rpoB-516F/R (for codons 508 to 536 of rpoB), katG-F/R (for the codon 315 region of katG), and inhA-F/R (for the nucleotide substitution of C to T at position −15 of inhA). An HRM curve was generated for each isolate after real-time PCR differentiated the mutant from the wild-type strains. DNA sequencing of the target regions was performed to confirm the results of the HRM curve analysis. All but one of the 73 RIF-resistant (RIF-R) strains and all 124 RIF-susceptible (RIF-S) isolates were correctly identified by HRM curve analysis of rpoB. Twenty-seven of 29 known rpoB mutants were detected. In HRM curve analysis of katG and inhA, 90 INH-R strains that harbored katG or inhA mutations, or both, and all INH-S strains were correctly identified. Ten phenotypically INH-R strains not harboring katG or inhA mutations were not detected. The HRM curve analysis will be a useful method for detection of RIF and INH resistance in M. tuberculosis in a rapid, accurate, simple, and cost-effective manner.The rates of mortality and morbidity from tuberculosis (TB) remain high, despite intense worldwide efforts. One of the major factors sustaining the current TB epidemic is the increasing drug resistance of Mycobacterium tuberculosis strains (2). In the early 1990s, multidrug-resistant (MDR) TB cases that were resistant to at least rifampin (RIF) plus isoniazid (INH) arose (6). When the frequency and distribution of extensively drug-resistant (XDR) TB cases were assessed in 2004 by the U.S. Centers for Disease Control and Prevention and the World Health Organization, several cases of drug-resistant tuberculosis consistent with an XDR phenotype were found (7). This study revealed that 20% of the isolates met the MDR criteria; 2% of those were classifiable as XDR; and 4%, 15%, and 19% of the XDR TB cases were from the United States, South Korea, and Latvia, respectively (7). Thus, it is crucial that rapid drug susceptibility tests be developed to prevent the spread of MDR and XDR TB.Although drug susceptibility testing (DST) is a prerequisite for accurate results, such testing requires much time and labor (3). Therefore, several molecular techniques have been applied to detect mutations related to drug resistance (5, 10). Resistance to RIF and INH, the mainstays of antituberculosis treatment, is mainly attributable to mutations in genes encoding the drug target or drug-converting enzymes (8). Early studies demonstrated that 95% of the resistance to RIF is associated with mutation of the RIF resistance-determining region of rpoB, whereas mutations in katG and the regulatory zone of inhA are most frequently associated with INH resistance (11).The oligonucleotide chip method and real-time PCR have been used for detection of drug-resistant M. tuberculosis (17, 21, 27, 29). A novel method of high-resolution melting (HRM) curve analysis is an accurate and simple technique for analyzing the genotype without the need for specific probes. The dye LC Green, SYTO9, or Eva Green saturates amplified DNA, unlike SYBR green dye, during homogeneous melting curve analysis. Also, HRM curve analysis generates a difference plot curve, which analyzes nucleic acid sequences with high accuracy. Application of genotyping by HRM curve analysis has followed (13, 19). The aim of the study described here was to develop a useful molecular tool for the identification of drug resistance in M. tuberculosis in an accurate, rapid, and cost-effective manner.     

Performance Assessment of the GenoType MTBDRplus Test and DNA Sequencing in Detection of Multidrug-Resistant Mycobacterium tuberculosis

To facilitate the management of multidrug-resistant (MDR) tuberculosis, two nucleic acid sequence-based methods, the GenoType MTBDRplus test and DNA sequencing, were assessed for the rapid detection of drug-resistant Mycobacterium tuberculosis for the first time in the Asia-Pacific region. The performances of these two assays in detecting the presence of rifampin (rifampicin) (RIF) and isoniazid (INH) resistance-associated mutations in the rpoB, katG, inhA regulatory region, inhA, and oxyR-ahpC genes were compared to that of a conventional agar proportion drug susceptibility test. A total of 242 MDR and 30 pansusceptible M. tuberculosis isolates were evaluated in this study. The sensitivities obtained for RIF-resistant detection by the GenoType MTBDRplus test and by resistance gene sequencing were 95.5% and 97.9%, respectively. The sensitivities for INH resistance detection by the GenoType MTBDRplus test and by resistance gene sequencing were 81.8% and 93.4%, respectively. Together, the sensitivity for MDR tuberculosis detection was 78.5% with the GenoType MTBDRplus test and 91.3% by resistance gene sequencing. The specificity for RIF resistance, INH resistance, and MDR detection was 100% by both methods. The GenoType MTBDRplus test has the advantage of a short turnaround time for drug-resistant M. tuberculosis detection. Overall, the two assays performed equally well in detecting RIF resistance (P = 0.13). However, DNA sequencing demonstrated superior performance in detecting INH resistance (P < 0.001) and MDR tuberculosis (P < 0.001). We suggest that new alleles of INH resistance genes should be evaluated to improve the sensitivity of the GenoType MTBDRplus test, especially for different geographic areas with genetically diverse M. tuberculosis strains.The emergence of multidrug-resistant tuberculosis (MDR-TB), defined as infection with a Mycobacterium tuberculosis complex isolate resistant to at least isoniazid (INH) and rifampin (rifampicin) (RIF), is a public health concern and threatens global TB control programs (22). In Taiwan, approximately 15,000 new TB cases are diagnosed annually, of which an estimated 4% are MDR-TB (12). Therefore, the Taiwan Centers for Disease Control (CDC) not only has strengthened directly observed treatment in the management of TB as of 2006, to prevent MDR generation, but also has implemented a DOTS-Plus (directly observed treatment, short-course) strategy for the management of MDR-TB patients as of 2007 (8). However, this program can be hampered by delayed laboratory diagnosis. The completion of diagnosis by conventional methods and drug susceptibility testing (DST) of M. tuberculosis normally take months.The World Health Organization and partners have endorsed the use of the molecular test GenoType MTBDRplus (Hain Lifescience GmbH, Nehren, Germany) for rapid detection of high-risk MDR-TB cases, even directly from certain clinical specimens (1, 4, 6, 10, 15, 21). The GenoType MTBDRplus test is a PCR-based amplification and reverse blotting assay that employs specific probes hybridized to nitrocellulose strips to detect RIF and INH resistance. The assay detects mutations in the rpoB gene for RIF resistance, in the katG gene for high-level INH resistance, and in the inhA regulatory region gene for low-level INH resistance. To evaluate the reliability of the assay, DNA sequencing analyses of rpoB for RIF and katG, the inhA regulatory region gene, inhA, or oxyR-ahpC for INH were conducted in parallel.Our previous study demonstrated the genetic diversity of MDR M. tuberculosis isolates with novel alleles in the rpoB gene in Taiwan (11). Likewise, the distribution of M. tuberculosis isolates differs in different geographic regions (5, 11). The GenoType MTBDRplus test has been assessed in Europe (6, 10, 15, 21), South Africa (4), and the Caribbean (1), but not in the Asia-Pacific region, where there is a high prevalence of Beijing family M. tuberculosis isolates. Here we report the performance of the revised GenoType MTBDRplus test compared to that of DNA sequencing using a culture-based phenotypic DST, which is considered the gold standard for routine clinical practice.     

Evaluation of the Analytical Performance of the Xpert MTB/RIF Assay

We performed the first studies of analytic sensitivity, analytic specificity, and dynamic range for the new Xpert MTB/RIF assay, a nucleic acid amplification-based diagnostic system that detects Mycobacterium tuberculosis and rifampin (RIF) resistance in under 2 h. The sensitivity of the assay was tested with 79 phylogenetically and geographically diverse M. tuberculosis isolates, including 42 drug-susceptible isolates and 37 RIF-resistant isolates containing 13 different rpoB mutations or mutation combinations. The specificity of the assay was tested with 89 nontuberculosis bacteria, fungi, and viruses. The Xpert MTB/RIF assay correctly identified all 79 M. tuberculosis isolates and correctly excluded all 89 nontuberculosis isolates. RIF resistance was correctly identified in all 37 resistant isolates and in none of the 42 susceptible isolates. Dynamic range was assessed by adding 10² to 10⁷ CFU of M. tuberculosis into M. tuberculosis-negative sputum samples. The assay showed a log-linear relationship between cycle threshold and input CFU over the entire concentration range. Resistance detection in the presence of different mixtures of RIF-resistant and RIF-susceptible DNA was assessed. Resistance detection was dependent on the particular mutation and required between 65% and 100% mutant DNA to be present in the sample for 95% certainty of resistance detection. Finally, we studied whether assay specificity could be affected by cross-contaminating amplicons generated by the GenoType MTBDRplus assay. M. tuberculosis was not detected until at least 10⁸ copies of an MTBDRplus amplicon were spiked into 1 ml of sputum, suggesting that false-positive results would be unlikely to occur.Conventional diagnostic methods for Mycobacterium tuberculosis are slow and/or lack sensitivity. A number of new diagnostic approaches have brought incremental improvements to detection and drug susceptibility testing; however, the technical complexity of these assays and their dependence on dedicated laboratory infrastructure have limited their adoption, especially in low-resource, high-burden settings (1, 11, 12, 21). The recently introduced Xpert MTB/RIF (manufactured and marketed by Cepheid, Sunnyvale, CA) assay simultaneously detects the presence of M. tuberculosis and its susceptibility to the important first-line drug rifampin (RIF) (7). A sample processing system and an automated heminested real-time PCR assay are integrated into a single disposable cartridge. The assay can be performed directly from a clinical sputum sample or from a decontaminated sputum pellet and can generally be completed in less than 2 h (7).The Xpert MTB/RIF assay detects M. tuberculosis and RIF resistance by PCR amplification of the rifampin resistance-determining region (RRDR) of the M. tuberculosis rpoB gene and subsequent probing of this region for mutations that are associated with RIF resistance. Approximately 95% of RIF-resistant tuberculosis cases contain mutations in this 81-bp region (16). Our previous work has established that the Xpert MTB/RIF assay has a limit of detection (LOD), defined as the minimum number of bacilli that can be detected with 95% confidence) of 131 CFU per ml of clinical sputum (7). The assay was also able to identify RIF resistance in samples containing 23 common clinically occurring rpoB mutations. None of the 20 nontuberculosis mycobacteria (NTM) species tested, including the NTM species commonly described as causing human disease were falsely identified as M. tuberculosis (7), suggesting high specificity. Several small studies using clinical samples demonstrated 98% to 100% sensitivity overall, 72% sensitivity in smear-negative patients, and a specificity of 100% (7).In the present study, we expand upon our previous work and report on several critical analytical assay performance characteristics, including dynamic range, sensitivity, specificity, RIF resistance detection in heterogeneous samples, and resiliency against cross-contamination by other nucleic acid amplification techniques (NAATs).     

Combined Real-Time PCR and rpoB Gene Pyrosequencing for Rapid Identification of Mycobacterium tuberculosis and Determination of Rifampin Resistance Directly in Clinical Specimens

Our laboratory has developed a rapid, sensitive, and specific molecular approach for detection in clinical specimens, within 48 h of receipt, of both Mycobacterium tuberculosis complex (MTBC) DNA and mutations within the 81-bp core region of the rpoB gene that are associated with rifampin (RIF) resistance. This approach, which combines an initial real-time PCR with internal inhibition assessment and a pyrosequencing assay, was validated for direct use with clinical specimens. To assess the suitability of real-time PCR for use with respiratory, nonrespiratory, acid-fast bacillus (AFB)-positive and AFB-negative specimens, we evaluated specimens received in our laboratory between 11 October 2007 and 30 June 2009. With culture used as the “gold standard,” the sensitivity, specificity, and positive and negative predictive values were determined for 1,316 specimens to be as follows: for respiratory specimens, 94.7%, 99.9%, 99.6%, and 98.6%, respectively; for nonrespiratory specimens, 88.5%, 100.0%, 100.0%, and 96.9%, respectively; for AFB-positive specimens, 99.6%, 100.0%, 100.0%, and 97.7%, respectively; and for AFB-negative specimens, 75.4%, 99.9%, 98.0%, and 98.4%, respectively. PCR inhibition was determined to be minimal in this assay, occurring in 0.2% of tests. The rpoB gene pyrosequencing assay was evaluated in a similar prospective study, in which 148 clinical specimens positive for MTBC DNA by real-time PCR were tested. The final results revealed that the results of direct testing of clinical specimens by the pyrosequencing assay were 98.6% concordant with the results of conventional testing for susceptibility to RIF in liquid culture and that our assay displayed adequate sensitivity for 96.6% of the clinical specimens tested. Used together, these assays provide reliable results that aid with the initial management of patients with suspected tuberculosis prior to the availability of the results for cultured material, and they also provide the ability to predict RIF resistance in Mycobacterium tuberculosis-positive specimens in as little as 48 h from the time of clinical specimen receipt.One-third of the world''s population is infected with Mycobacterium tuberculosis. In 2007 alone, 9.27 million new cases were identified and 1.6 million deaths occurred. Only 44% (4.1 million cases) of these were acid-fast bacillus (AFB) smear positive (24). The resurgence of tuberculosis (TB) in developed as well as developing countries since 1980 has been associated with the HIV epidemic, the emergence of drug-resistant strains, and increases in emigration from regions with high rates of disease endemicity (6, 9, 11). The rapid detection of M. tuberculosis is essential for disease management, because of the high risk of transmission from person to person. The CDC recommends that clinical specimens received be analyzed simultaneously by culture, AFB staining, and nucleic acid amplification (NAA) protocols (2). Culture is the “gold standard” for final determination, but it is slow and may take up to 2 to 8 weeks. Staining for AFB is rapid but has a low sensitivity and a low specificity, since it does not distinguish nontuberculous mycobacteria (NTM) from members of the M. tuberculosis complex (MTBC). Thus, rapid identification, which is essential for effective control, relies on NAA.A number of studies involving the detection of MTBC by PCR have been reported; these have targeted cfp10 (12), the senX3-regX3 intergenic region (4), the16S rRNA gene (10), and the internal transcribed spacer (ITS) (21). However, the most commonly used target for the identification of MTBC is the multiple-copy-number insertion sequence IS6110 (also known as IS986) (4, 5, 8, 9, 14, 16, 20, 22), which is thought to provide the highest sensitivity.The assessment of rifampin (RIF) resistance in M. tuberculosis-infected patients is critically important to patient management and can affect both the treatment of individual patients and the spread of disease. The standard methods for drug susceptibility testing (DST) of M. tuberculosis can take weeks to months to provide results. Due to the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), there is a critical need for new, rapid, and accurate DST methods. NAA assays for determination of mutations in an 81-bp region of the rpoB gene have been published, and an excellent correlation between the presence of these mutations and RIF resistance has been demonstrated. Mutations in this region are believed to correlate with RIF resistance in 96% of all RIF-resistant M. tuberculosis strains (18). Several tests have been published that perform nucleic acid sequence analysis of mycobacterial isolates by methods that include single-nucleotide polymorphism analysis (3) and pyrosequencing (13, 17, 25) of isolates, but no methodology yet reported has utilized rpoB gene pyrosequencing analysis of the 81-bp core region directly with clinical specimens positive for MTBC by real-time PCR. Here we describe a two-step molecular approach that will detect MTBC DNA and yield rpoB gene mutation analysis results within 48 h from the time of receipt of the clinical specimen.     

Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand,Near-Patient Technology

Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System''s MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay''s buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.An alarming increase in the global incidence of drug-resistant Mycobacterium tuberculosis infection has created a critical need for methods that can rapidly detect M. tuberculosis and identify drug-resistant cases (53). Failure to quickly and effectively recognize and treat patients with drug-resistant tuberculosis (TB), particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, leads to increased mortality, nosocomial outbreaks, and resistance to additional antituberculosis drugs (14, 37). However, MDR and XDR tuberculosis can be effectively treated if properly identified (35). A number of new diagnostic approaches have brought incremental improvements in detection and drug susceptibility testing (2, 9, 19, 24, 37, 41, 46); however, none can realistically provide actionable information within the time frame of a single office or clinic visit. Thus, despite technical advances, rapid diagnostics have not yet been able to have an impact on critical initial decisions regarding hospitalization, isolation, and the choice of treatment regimens for suspected tuberculosis patients.Previously, we showed that direct molecular detection of M. tuberculosis and rifampin resistance could be accomplished simultaneously (27); more recently, our group developed a single-tube, molecular beacon-based real-time PCR assay for the detection of rifampin-resistant M. tuberculosis (42, 43). Mutations in the 81-bp rifampin resistance-determining region (RRDR) of the rpoB gene, which occur in 95 to 98% of all rifampin-resistant strains (and which are almost invariably absent in rifampin-susceptible strains), were detected by five overlapping molecular beacons (34). The assay proved to be simple, rapid, specific, and highly sensitive in tests on isolates of M. tuberculosis from New York City, Madrid (42), India, and Mexico (51). As most rifampin-resistant isolates are also resistant to isoniazid, rifampin resistance can be used as a marker for MDR M. tuberculosis (36, 44, 49). However, like all nucleic acid amplification-based assays for M. tuberculosis detection (15), this assay was too complex and too prone to operator errors, sample cross-contamination, and biohazards for rapid near-patient use.The Cepheid GeneXpert System (Sunnyvale, CA), a single-use sample-processing cartridge system with integrated multicolor real-time PCR capacity (45), has the potential to greatly simplify nucleic acid amplification tests. Here, we utilized this new technology to develop an on-demand, near-patient PCR assay that employs a novel six-color dye set to detect M. tuberculosis and identify rifampin resistance as a surrogate for MDR directly from a patient''s sputum in less than 2 h. The many features of this system, including sample decontamination, hands-free operation, on-board sample processing, and ultrasensitive hemi-nested PCR, enabled us to create a low-complexity assay with a sensitivity that approached certain culture methods. This type of assay may prove to be useful in the initial management of suspected tuberculosis cases in both the United States and the world at large.     

Rapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis by High-Resolution Melting Analysis

We have developed a high-resolution melting (HRM) assay to scan for mutations in the rpoB, inhA, ahpC, and katG genes and/or promoter regions for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis. For assay development, 23 drug-resistant isolates of M. tuberculosis having 29 different mutations, together with 40 drug-susceptible isolates, were utilized. All 29 mutations were accurately detected by our assay. We further validated the assay with a series of 59 samples tested in a blind manner. All sequence alterations that were within the regions targeted by the HRM assay were correctly identified. Compared against results of DNA sequencing, the sensitivity and specificity of our HRM assay were 100%. For the blinded samples, the specificities and sensitivities were 89.3% and 100%, respectively, for detecting rifampin resistance and 98.1% and 83.3%, respectively, for detecting isoniazid resistance, as isolates with mutations in regions not encompassed by our assay were not detected. A C-to-T sequence alteration at position −15 of the ahpC regulatory region, which was previously reported to be associated with isoniazid resistance, may possibly be a polymorphism, as it was detected in an isoniazid-susceptible M. tuberculosis isolate. HRM is a rapid, accurate, simple, closed-tube, and low-cost method. It is thus an ideal assay to be used in countries with a high prevalence of drug-resistant M. tuberculosis and where cost-effectiveness is essential. As a mutation-scanning assay for detecting drug-resistant M. tuberculosis, it can potentially lead to better treatment outcomes resulting from earlier treatment with the appropriate antibiotics.The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant TB (XDR-TB) has hampered the control and treatment of TB (45). MDR-TB is defined as TB that is resistant to at least isoniazid (INH) and rifampin (RIF), two main first-line antitubercular drugs, while XDR-TB is MDR-TB that is additionally resistant to three or more second-line drugs. MDR-TB accounts for an estimated 5% of all TB cases (44); however, patients are often not expeditiously diagnosed, resulting in the delay of appropriate treatment as well as poorer treatment outcomes for patients and the propagation and spread of MDR-TB. Conventional methods for drug susceptibility testing of MDR-TB require an additional culture period, typically between 2 and 5 weeks. An easy-to-implement, cost-effective, and rapid method for drug susceptibility testing is thus of paramount importance to limit the spread of drug-resistant tuberculosis.Drug resistance in Mycobacterium tuberculosis is due to mutations in genes or promoters of genes activating the drug or encoding the drug targets, which are detectable in the majority of drug-resistant isolates (41). Mutations associated with RIF resistance occur mainly in an 81-bp RIF resistance-determining region (RRDR) of the rpoB gene (codons 507 to 533; numbering according to the Escherichia coli rpoB sequence), with >95% of RIF-resistant isolates containing at least one mutation in this region (12, 13, 22, 28, 31, 36, 43). Mutations associated with INH resistance occur mainly in the katG gene (codon 315), the inhA gene and regulatory region, and the ahpC regulatory region (11, 20, 27, 29, 34, 40, 42).While several molecular methods have been previously described for drug susceptibility testing of M. tuberculosis (2, 6, 7, 9, 26, 38), the cost and technical demands of the assays restrict their usage, especially in countries where funds are scarce. Another problem with the majority of PCR-based molecular methods is the requirement of downstream processing of PCR products, which exposes the PCR products to the environment, increasing the risk of cross-contamination of subsequent assays.The high-resolution melting (HRM) analysis is a simple, cost-effective, closed-tube method with sensitivity and specificity reported to be higher than those of denaturing high-performance liquid chromatography (dHPLC) (3). HRM does not require the use of costly fluorescent probes and requires no post-PCR handling, making it an attractive alternative method for genotypic drug susceptibility testing of M. tuberculosis. The method involves performing a PCR with a saturating double-stranded DNA-binding dye such as Syto9, followed by a high-resolution melt analysis, whereby the amplicons are slowly heated to denaturation with real-time monitoring of the decrease in fluorescence during denaturation. By comparing the melting profile of the sample with a reference, any sequence variance can be detected. Homoduplexes are usually detected by a change in melting temperature (T_m), while heteroduplexes are usually detected by a change in the melt curve shape (24). As it is easier to identify a change in melt curve shape (10), the sample and reference DNA can be mixed together and amplified together by PCR to produce heteroduplexes, as in the method we have developed.However, as HRM analysis detects all mutations within the PCR amplicon, known polymorphisms that lie within the amplicon can be excluded by the use of an unlabeled oligonucleotide probe as described by Zhou et al. (48). Briefly, a 3′-blocked unlabeled probe, designed to encompass the polymorphism, is included in the PCR.In this study, we evaluated the efficacy of our assay in comparison with that of standard drug susceptibility testing for the detection of RIF- and/or INH-resistant M. tuberculosis strains from clinical specimens.     

Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin,Isoniazid, and Fluoroquinolones

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.Rifampin (rifampicin), isoniazid, and the fluoroquinolones are the most important initial drug markers for extensively drug-resistant Mycobacterium tuberculosis strains, defined as multidrug-resistant (MDR) isolates (resistant to both isoniazid and rifampin) with additional resistance to a fluoroquinolone and to one of the injectable drugs (2). The fluoroquinolones have become an essential part of treatment regimens for MDR tuberculosis (7, 25). Due to their potency and safety, the new-generation fluoroquinolones are now even being evaluated as first-line medications for tuberculosis (3, 13, 20). Wang et al. further suggested that routine fluoroquinolone resistance testing may have a clinical impact by showing a significant correlation between development of fluoroquinolone and first-line M. tuberculosis drug resistance in an area in which resistant strains are highly endemic (28).The spontaneous acquisition of DNA sequence mutations is the primary genetic basis for the development of M. tuberculosis drug resistance (14). Since sequences are highly conserved, certain mutations correlate well with phenotypic resistance, and a limited number of mutations account for the majority of phenotypic resistance to the important antituberculosis medications, various methods of genotypic testing have successfully been used for the rapid detection of M. tuberculosis resistance (16, 22). The sites that most frequently contain mutations associated with phenotypic resistance, called resistance-determining regions (RDR), differ depending on the drug tested. Among rifampin-resistant isolates worldwide, 95 to 97% harbor mutations in the rifampin RDR, an 81-bp target encompassing codons 507 to 533 of the 3,519-bp rpoB gene (17). Isoniazid resistance has a more complex mechanism, involving several gene targets, the most important of which is codon 315 of the 2,223-bp katG gene, in which mutations are found in up to 50% of resistant isolates (18). Likewise, M. tuberculosis has a quinolone RDR which spans codons 88 to 94 of the 2,517-bp gyrA gene. Mutations in this region, particularly in codon 88, 90, 91, or 94, correlate with high-level resistance and are seen in 42 to 85% of resistant clinical isolates (6).Pyrosequencing, a method of DNA sequencing by synthesis, has been applied to the rapid detection of M. tuberculosis resistance to rifampin, isoniazid, and ethambutol (9, 29). Its main advantage is a much shorter turnaround time than that of conventional drug susceptibility testing, the latter taking 2 to 4 weeks from the time an isolate is obtained in pure culture.After isoniazid and rifampin, the next pivotal drug class in M. tuberculosis treatment is the fluoroquinolone class, as previously discussed (3, 7, 13, 20, 25, 28). Given that most resistance to the latter is determined by mutations that are generally limited to the quinolone RDR of the gyrA gene, it should be feasible and clinically more relevant to develop an assay for rapid resistance testing which includes fluoroquinolone resistance in addition to rifampin and isoniazid resistance.We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones, which we validated against the conventional submerged-disk proportion method. We also improved on the previously reported pyrosequencing assay by reducing the number of primers required to sequence for rifampin resistance (9).     

Feasibility of the GenoType MTBDRsl Assay for Fluoroquinolone,Amikacin-Capreomycin,and Ethambutol Resistance Testing of Mycobacterium tuberculosis Strains and Clinical Specimens

The new GenoType Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay (Hain Lifescience, Nehren, Germany) was tested on 106 clinical isolates and directly on 64 sputum specimens for the ability to detect resistance to fluoroquinolones, injectable drugs (amikacin or capreomycin), and ethambutol in Mycobacterium tuberculosis strains. A total of 63 strains harboring fluoroquinolone, amikacin/capreomycin, or ethambutol resistance and 43 fully susceptible strains were comparatively analyzed with the new MTBDRsl assay, by DNA sequencing, and by conventional drug susceptibility testing in liquid and solid media. No discrepancies were obtained in comparison with the DNA sequencing results. Fluoroquinolone resistance was detected in 29 (90.6%) of 32, amikacin/capreomycin resistance was detected in 39/39 (84.8%/86.7%) of 46/45, and ethambutol resistance was detected in 36 (69.2%) of 52 resistant strains. A total of 64 sputum specimens (42 smear positive, 12 scanty, and 10 smear negative) were tested with the new MTBDRsl assay, and the results were compared with those of conventional drug susceptibility testing. Fluoroquinolone resistance was detected in 8 (88.9%) of 9, amikacin/capreomycin resistance was detected in 6/7 (75.0%/87.5%) of 8, and ethambutol resistance was detected in 10 (38.5%) of 26 resistant strains. No mutation was detected in susceptible strains. The new GenoType MTBDRsl assay represents a reliable tool for the detection of fluoroquinolone and amikacin/capreomycin resistance and to a lesser extent also ethambutol resistance. In combination with a molecular test for detection of rifampin and isoniazid resistance, the potential for the detection of extensively resistant tuberculosis within 1 to 2 days can be postulated.The worldwide emergence of extensively drug-resistant tuberculosis (XDR TB, resistant at least to rifampin and isoniazid, a fluoroquinolone [FLQ], and one of the three injectable second-line drugs amikacin [AM], kanamycin [KM], and capreomycin [CM]) is a serious global health problem (20, 25). In the World Health Organization fourth global report on drug resistance (25), it was documented that more than 45 countries have reported XDR cases. The actual incidence could be underestimated, because second-line drug susceptibility testing (DST) is not available in many countries. To avoid a progressive development similar to that observed in multidrug-resistant TB (resistant at least to rifampin and isoniazid) worldwide, now having the highest rate ever at 5.3%, timely identification of resistant Mycobacterium tuberculosis complex (MTBC) strains is mandatory.Conventional DST for XDR strains is performed sequentially in a two-step procedure beginning with a culture and first-line drug testing, proceeding to further drug testing in the case of multidrug resistance. The time needed for testing, even with the most rapid liquid methods, is still around 1 week per test, constrained by the relatively slow growth of M. tuberculosis (15, 18). The required time can be shortened by fast molecular methods to 1 day per test (3, 8, 21). Since recently broad-based knowledge about mutations that cause resistance to ethambutol (EMB) and some second-line drugs is available. Resistance to FLQs, AM-CM, and EMB in M. tuberculosis is most frequently attributed to mutations in the gyrA, rrs, and embB genes, respectively. First investigations have shown that by targeting mutations in codons 90, 91, and 94 in the gyrA gene, approximately 70 to 90% of all FLQ-resistant strains can be correctly detected (2, 13, 24). Previous reports have linked mutations A1401G, C1402T, and G1484T in the rrs gene to AM, CM, and KAN resistance (1, 11, 12), each of them being responsible for a specific resistance pattern. Mutations G1484T and A1401G were found to cause high-level resistance to all drugs, whereas C1402T causes resistance to only CM and KAN.Furthermore, mutations at embB codon 306 are found in 30 to 68% of EMB-resistant clinical strains (16, 17, 26).PCR-based techniques provide new possibilities for the rapid diagnosis of first- and second-line drug resistance; however, not all mycobacterial laboratories have access to DNA-sequencing facilities. As an alternative, DNA strip assays for the detection of rifampin (INNO-LiPA Rif. TB; Innogenetics, Ghent, Belgium) or rifampin and isoniazid resistance of M. tuberculosis in a single assay (GenoType MTBDR; Hain Lifescience, Nehren, Germany) are now commercially available. These assays have been evaluated for M. tuberculosis cultures and specimens (3, 7, 8, 10, 21). The DNA strip assays are based on PCR or multiplex PCR in combination with reverse hybridization. The existence of a resistant strain is signaled either by the omission of a wild-type band or the appearance of bands representing specific mutations.In order to increase the capacity to detect further drug resistance in M. tuberculosis, the GenoType Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay was developed with a specific focus on the most prevalent gyrA, rrs, and embB gene mutations.The aim of the present study was to determine the sensitivity and specificity of the new MTBDRsl assay for the detection of FLQ, AM, CM, and EMB resistance-associated mutations in culture specimens and directly in smear-positive and -negative clinical specimens.     

High-Level Rifampin Resistance Correlates with Multiple Mutations in the rpoB Gene of Pulmonary Tuberculosis Isolates from the Afghanistan Border of Iran

The aim of this study was to investigate the significance of multiple mutations in the rpoB gene as well as predominant nucleotide changes and their correlation with high levels of resistance to rifampin (rifampicin) in Mycobacterium tuberculosis isolates that were randomly collected from the sputa of 46 patients with primary and secondary cases of active pulmonary tuberculosis from the southern region (Afghanistan border) of Iran where tuberculosis is endemic. Drug susceptibility testing was performed using the CDC standard conventional proportional method. DNA extraction, rpoB gene amplification, and DNA sequencing analysis were performed. Thirty-five (76.09%) isolates were found to have multiple mutations (two to four) in the rpoB (β-subunit) gene. Furthermore, we demonstrate that the combination of mutations with more prevalent nucleotide changes were observed in codons 523, 526, and 531, indicating higher frequencies of mutations among patients with secondary infection. In this study, 76.08% (n = 35) of all isolates found to have mutation combinations involving nucleotide changes in codons 523 (GGG→GCG), 531 (TCG→TTG or TTC), and 526 (CAC→CGC, TTC, AAC, or CAA) demonstrated an association with higher levels of resistance to rifampin (MIC, ≥100 μg/ml).In bacterial populations, the generation of antibiotic resistance depends on the rate of emergence of resistant mutants (1, 19, 23). Correlations between high mutation rates, the geographic distribution of mutations, antibiotic resistance, and virulence in bacteria have been reported in several studies (9, 20, 33, 37). Knowledge of geographic variations is important for monitoring rifampin (rifampicin) resistance within a defined population of patients infected with Mycobacterium tuberculosis, as the prevalence of the mutations studied so far varies for M. tuberculosis strains isolated from different countries (24, 26, 29, 33, 36). In 2004, the prevalence of tuberculosis in Iran was reported to be 17 per 100,000, and at the southern border of Iran (Zabol province) where tuberculosis is endemic, the prevalence was 141 per 100,000 (20). Rifampin resistance is of particular epidemiologic importance, since it represents a valuable surrogate marker for multidrug-resistant (MDR) tuberculosis strains, and the prevalence of MDR strains is a significant obstacle to tuberculosis therapy (4, 21, 26). DNA sequencing studies indicate that more than 95% of rifampin-resistant M. tuberculosis strains have mutations within the 81-bp hot-spot region (codons 507 to 533) of the RNA polymerase β-subunit (rpoB) gene (4, 19, 32). Over the last 15 years, Kapur et al. and Telenti et al. have identified the molecular basis of rifampin resistance in M. tuberculosis (9, 29). Thus, it is important to determine the molecular bases of mutations and their distribution at the level of each country prior to molecular testing introduction for routine diagnostics (9, 11, 13, 15, 16, 23).In this study, we investigated the significance of multiple mutations in the rpoB gene and their correlation with highly prevalent nucleotide changes in codons 523, 531, and 526 and also demonstrated the highly prevalent nucleotide changes observed in the last nine codons of the β-subunit (523 to 531) that are associated with higher levels of resistance to rifampin (MIC, ≥100 μg/ml) in patients bearing secondary M. tuberculosis infection.     

High Diversity of Mycobacterium tuberculosis Genotypes in South Africa and Preponderance of Mixed Infections among ST53 Isolates

The reemergence of tuberculosis (TB) has become a major health problem worldwide, especially in Asia and Africa. Failure to combat this disease due to nonadherence or inappropriate drug regimens has selected for the emergence of multiple-drug-resistant (MDR) TB. The development of new molecular genotyping techniques has revealed the presence of mixed Mycobacterium tuberculosis infections, which may accelerate the emergence of drug-resistant strains. There are some studies describing the local distribution of circulating strains in South Africa, but to date, reports describing the frequency and distribution of M. tuberculosis genotypes, and specifically MDR genotypes, across the different provinces are limited. Thus, 252 isolates (of which 109 were MDR) from eight of the nine provinces of South Africa were analyzed by spoligotyping. Spoligotyping showed 10 different lineages, and ST53 (11.1%) and ST1 (10.3%) were the most frequent genotypes. Of the 75 different spoligopatterns observed, 20 (7.9%) were previously unreported. Analysis of the mycobacterial interspersed repetitive units of variable-number tandem repeats of the ST53 and ST1 isolates revealed that ∼54% of the ST53 isolates were of mixed M. tuberculosis subpopulations. Drug resistance (defined as resistance to at least isoniazid and/or rifampin) could only be linked to a history of previous anti-TB treatment (adjusted odds ratio, 4.0; 95% confidence interval, 2.27 to 7.10; P = <0.0001). This study describes a high diversity of circulating genotypes in South Africa in addition to a high frequency of mixed M. tuberculosis subpopulations among the ST53 isolates. MDR TB in South Africa could not be attributed to the spread of any single lineage.Tuberculosis (TB) is a major cause of illness and death worldwide but especially in Asia and Africa (42). Twenty-two countries designated high TB burden countries account for 80% of all new cases worldwide (42). As of 2008, South Africa was ranked fourth among these, with an incidence rate of 940 cases per 100,000 persons (42) (up from 536 in 2005 [41]). Due to nonadherence to drug regimens or the use of inappropriate drug regimens, the TB epidemic has been largely exacerbated by the emergence of multidrug-resistant (MDR) TB (7).Traditionally, it has been assumed that TB is caused by an infection with a single strain and that recurrences are the result of reactivation of the strain causing the first episode (6, 24, 39). However, it has recently been shown that patients, both human immunodeficiency virus (HIV) positive and HIV negative, in high-incidence settings may have more than one strain in the same sputum sample (24, 39) and that mixed infections may cause complications in the treatment of the disease if a patient is infected with both a sensitive and a resistant Mycobacterium tuberculosis isolate (39). Extensively drug-resistant (XDR) TB strains (defined as resistant to isoniazid [INH] and rifampin [RIF], in addition to any fluoroquinolones and at least one injectable anti-TB drug) (5) were first reported in South Africa in 2006 and later shown to be present in at least 17 countries and on all of the continents (9). Spoligotyping has revealed that the majority of these cases were caused by M. tuberculosis belonging to the KZN family (ST60), which has been known to be prevalent in this area since 1994 (23). Since then, only one other study has been published (19) providing genotypic information on XDR TB strains in South Africa. That study shows that XDR TB strains in South Africa belong to seven different lineages and are present in four of the nine provinces. Such studies highlight the need for standardized and accurate drug susceptibility testing in combination with high-level molecular genotyping in order to carefully monitor new and emerging MDR and XDR TB strains.The “gold standard” for the typing of M. tuberculosis is currently IS6110-based restriction fragment length polymorphism (RFLP). Combined with spoligotyping, this has proven to be very useful for the study of the transmission, evolution, and phylogeny of M. tuberculosis (18). However, RFLP is laborious, requires a large amount of DNA, and has poor interlaboratory reproducibility. Recently, a new genotyping technique based on PCR amplification of mycobacterial interspersed repetitive units of variable-number tandem repeats (MIRU-VNTR) was introduced (31, 32). This method is much faster than IS6110 RFLP and requires less DNA. Fifteen-locus-based MIRU-VNTR analysis has been shown to have slightly better discriminatory power than IS6110 RFLP, especially when combined with spoligotyping (2, 22). In addition to being a rapid and highly discriminatory genotyping method, MIRU-VNTR can also be used for the detection of mixed subpopulations in a single sputum sample (1, 26). Several studies have investigated the differentiation power of different MIRU-VNTR locus combinations for strains of the Beijing lineage (11, 14, 15). The studies suggest that the choice of appropriate MIRU-VNTR loci requires further investigation in diverse M. tuberculosis lineages in countries with low and high TB endemicity.Thus, the objectives of this study were to assess the distribution and diversity of MDR M. tuberculosis genotypes across the South African provinces and to determine if there is an association between MDR TB and a particular M. tuberculosis genotype. We also determined the general population structure of South African M. tuberculosis isolates, irrespective of the drug susceptibility pattern. Furthermore, we assessed the ability of the MIRU-VNTR method to discriminate the most frequent genotypes observed.     

Sputum PCR-Single-Strand Conformational Polymorphism Test for Same-Day Detection of Pyrazinamide Resistance in Tuberculosis Patients

Pyrazinamide is a first-line drug for treating tuberculosis, but pyrazinamide resistance testing is usually too slow to guide initial therapy, so some patients receive inappropriate therapy. We therefore aimed to optimize and evaluate a rapid molecular test for tuberculosis drug resistance to pyrazinamide. Tuberculosis PCR-single-strand conformational polymorphism (PCR-SSCP) was optimized to test for mutations causing pyrazinamide resistance directly from sputum samples and Mycobacterium tuberculosis isolates. The reliability of PCR-SSCP tests for sputum samples (n = 65) and Mycobacterium tuberculosis isolates (n = 185) from 147 patients was compared with four tests for pyrazinamide resistance: Bactec-460 automated culture, the Wayne biochemical test, DNA sequencing for pncA mutations, and traditional microbiological broth culture. PCR-SSCP provided interpretable results for 96% (46/48) of microscopy-positive sputum samples, 76% (13/17) of microscopy-negative sputum samples, and 100% of Mycobacterium tuberculosis isolates. There was 100% agreement between PCR-SSCP results from sputum samples and Mycobacterium tuberculosis isolates and 100% concordance between 50 blinded PCR-SSCP rereadings by three observers. PCR-SSCP agreement with the four other tests for pyrazinamide resistance varied from 89 to 97%. This was similar to how frequently the four other tests for pyrazinamide resistance agreed with each other: 90 to 94% for Bactec-460, 90 to 95% for Wayne, 92 to 95% for sequencing, and 91 to 95% for broth culture. PCR-SSCP took less than 24 hours and cost approximately $3 to $6, in contrast with the other assays, which took 3 to 14 weeks and cost $7 to $47. In conclusion, PCR-SSCP is a relatively reliable, rapid, and inexpensive test for pyrazinamide resistance that indicates which patients should receive pyrazinamide from the start of therapy, potentially preventing months of inappropriate treatment.Tuberculosis kills 1.7 million people annually (2), and antibiotic resistance increasingly complicates control (14). Rapid molecular tests for susceptibility to isoniazid and rifampin (rifampicin) (18) can guide treatment decisions within a day of laboratory diagnosis of tuberculosis disease (3). In contrast, pyrazinamide is another first-line antibiotic included in most tuberculosis treatment regimes, but the results of traditional culture-based pyrazinamide susceptibility testing are infrequently available because they require several weeks and generally involve tuberculosis culture in inhibitory acid media (25, 26). Consequently, little is known about the prevalence of pyrazinamide resistance, although one-third of retreated tuberculosis patients in Peru have pyrazinamide-resistant strains (23).Pyrazinamide resistance is usually caused by a mutation in the pncA gene that disrupts pyrazinamidase activity, preventing conversion of pyrazinamide to its active form (17). The causative mutations may be detected in cultured isolates by sequencing or PCR-single-strand conformational polymorphism (PCR-SSCP) (3, 16). We modified the published protocols for PCR and SSCP (3, 16, 20) to allow pyrazinamide susceptibility testing directly on sputum, as well as enhanced testing of tuberculosis cultures, in order to guide treatment decisions more rapidly and reliably than current techniques.(This research was the focus of a poster presentation at the annual conference of the International Union against Tuberculosis and Lung Disease, Paris, France, October 2008.)     

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.Drug-resistant Mycobacterium tuberculosis complex (MTBC) strains have emerged worldwide as a serious threat for tuberculosis (TB) control. Rates of multidrug-resistant (MDR) strains (i.e., resistance at least to isoniazid [INH] and rifampin [RIF]) have reached levels of up to 14% among patients never treated and up to 40% among previously treated patients in several MDR TB “hot spots” such as such as Karakalpakstan (Uzbekistan) and Kazakhstan in Eastern Europe (10, 37). Every year an estimated 489,000 cases of MDR TB arise globally (36). MDR TB is associated with much poorer treatment outcomes than for drug-susceptible TB, with a much higher risk of developing further resistances (2, 7). Prolonged periods of infectivity result in enhanced transmission of drug-resistant strains, further accelerating the rates of drug resistance (3). Even more worrisome is the emergence of a nearly untreatable form of TB, namely, extensively drug-resistant TB (XDR TB), which is defined as MDR plus additional resistance to any fluoroquinolone and at least one of three injectable drugs (i.e., amikacin, kanamycin, or capreomycin). A recent survey confirmed the worldwide presence of XDR strains, with rates of up 15% of MDR TB cases (30).Considering the difficulties and problems associated with the treatment of resistant TB, high levels of MDR and XDR TB have the clear potential to jeopardize TB control on a local or national level. In addition to various measures for strengthening TB control such as rapid case detection, proper treatment, and rapid detection of drug resistance, the long-term effect of the emergence of drug-resistant strains on the worldwide TB epidemic also depends on the relative fitness of the MDR and XDR strains compared to susceptible strains (3).In clinical MTBC strains, drug resistance results from chromosomal mutations in particular genes that confer resistance, which might also have an effect on bacterial fitness (5, 38). Although initial experiments indicated a lower fitness of e.g., INH-resistant strains, recent results confirm that the fitness of resistant strains depends on the kind of mutations, as well on the strain''s genetic background (13). Furthermore, the initial adverse effects on bacterial fitness might be reversed by compensatory mutations occurring during long-term infection and ongoing transmission. In fact, MDR variants have been described to have even an enhanced fitness compared to susceptible progenitor strains (13). If these figures are used in models for prediction of the MDR TB epidemic, it turns out that even in the case of a well-functioning TB control system, small subpopulations of comparatively fit MDR clones might outcompete susceptible and less-fit resistant strains and become the dominant clones in future with dramatic consequences for TB treatment and control (5).The most striking association between a mycobacterial genetic background and drug resistance documented thus far has been described for strains of the so-called Beijing lineage. These strains have been found to be involved in outbreaks and the transmission of MDR TB in several areas of the world (15). In Eastern Europe, a rising number of studies report a clear association between Beijing genotype infection and drug resistance (8, 11, 15, 28). Furthermore, large clusters of dominant clones have been determined that might indicate the development of “highly transmissible” MDR Beijing strains circulating in the community (28). Similar observations have been recently reported from South Africa, where a rapidly spreading highly resistant clone represents nearly half of all cases in the George subdistrict (35).However, the overall picture of the correlation between bacterial genotype disease characteristics is incomplete. The majority of studies focused on particular strain types such as the Beijing genotype only, basically, because they are easy to recognize by applying genotyping techniques such as IS6110 DNA fingerprint and spoligotyping (34). Based on these markers, a variety of strains were not classifiable into phylogenetic lineages or clonal complexes since the genotyping information was not informative, e.g., due to homoplasy (4, 6). Therefore, the presence of particular genotypes might simply be overlooked and, consequently, the association with clinical characteristics could not be investigated or false associations have been obtained. Since this question is of scientific and public health relevance, further studies addressing the population structure of the MTBC applying more appropriate genetic markers are urgently needed. More recently, a new genotyping techniques based on mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing was developed that allows the simultaneous high-resolution discrimination of clinical isolates for epidemiological studies and a valid phylogenetic strain classification (26, 31).In the present study, we used MIRU-VNTR typing and spoligotyping to investigate the population structure of strains obtained from patients living in the Republic of Georgia, where high rates of MDR TB have been recently reported (14, 20). We specifically analyzed the association between Beijing genotype and drug resistance. Furthermore, the data have been used to classify determine the whole variety of strains circulating in Georgia and to describe new clonal complexes and/or phylogenetic lineages. Phylogenetic strain classifications have been correlated with clinical characteristics.     

Mycobacterium tuberculosis Transmission Is Not Related to Household Genotype in a Setting of High Endemicity

Among the different strains of Mycobacterium tuberculosis, Beijing has been identified as an emerging genotype. Enhanced transmissibility provides a potential mechanism for genotype selection. This study evaluated whether the Beijing genotype is more readily transmitted than other prevalent genotypes to children in contact with an adult tuberculosis (TB) index case in the child''s household. We conducted a prospective, community-based study at two primary health care clinics in Cape Town, South Africa, from January 2003 through December 2004. Bacteriologically confirmed new adult pulmonary TB cases were genotyped by IS6110 DNA fingerprinting; household contacts less than 5 years were traced and screened for M. tuberculosis infection and/or disease. A total of 187 adult index cases were identified from 174 households with children aged less than 5 years. Of 261 child contacts aged 0 to 5 years, 219 (83.9%) were completely evaluated and the isolate from the index case was successfully genotyped. M. tuberculosis infection (induration of ≥10 mm by Mantoux tuberculin skin test) was documented in 118/219 (53.9%) children; 34 (15.5%) had radiographic signs suggestive of active TB. There was no significant difference in the ratio of infected children among those exposed to the Beijing genotype (51/89; 57.3%) and those exposed to non-Beijing genotypes (55/115; 47.8%) (odds ratio, 1.5; 95% confidence interval, 0.8 to 2.7). Genotyping was successful for six children diagnosed with active TB; the isolates from only two children had IS6110 fingerprints that were identical to the IS6110 fingerprint of the isolate from the presumed index case. We found no significant association between the M. tuberculosis genotype and transmissibility within the household. However, undocumented M. tuberculosis exposure may have been a major confounding factor in this setting with a high burden of TB.From an evolutionary perspective, the global tuberculosis (TB) epidemic presents a dynamic picture. Mycobacterium tuberculosis generates significant genetic diversity through deletion, duplication, and recombination events; but unlike most other bacterial pathogens, gene exchange is rare (31, 33). The absence of horizontal gene transfer results in strict clonality with distinct genetic lineages that permit accurate phylogenetic reconstruction. Selection of the most successful genotypes is mediated by genotype-specific differences in host-pathogen interactions (15, 18), some of which have been well characterized in animal models (11, 24, 25). Pathogen-related factors that may contribute to M. tuberculosis genotype selection include variability in transmissibility (the ability to spread from person to person), pathogenicity (the ability to cause clinical disease), the level of protection afforded by Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccination, and the acquisition of drug resistance.The Beijing genotype predominates in parts of East Asia (17, 23, 38, 41), northern Eurasia (12, 31), and southern Africa (8, 39). Beijing has been regarded as an emerging genotype on the basis of its global distribution, its association with young age (4), and its proportional increase in prevalence over time (8, 39). An increased ability to circumvent the protection afforded by BCG vaccination is suggested by the positive association (of the Beijing genotype) with the presence of a BCG scar in human populations (4) and has been observed in mice (24), although more recent findings challenge this observation (20). Multiple mechanisms have been explored to explain the potential link between the emergence of the Beijing genotype and low-level BCG protection (1), which may provide the Beijing genotype with a selective advantage in populations in which universal BCG vaccination is practiced.The association between the Beijing genotype and drug-resistant TB is well documented in multiple settings (2, 9, 31, 32, 37). Although it has been demonstrated that the acquisition of drug resistance is usually associated with a fitness cost, this finding seems variable and strain dependent (14) and may be insufficient to prevent transmission (16). Some Beijing genotypes retain their fitness in vitro, despite the acquisition of drug resistance (36), while compensatory evolution may account for significantly higher levels of fitness in clinical strains than in their progenitors (16). The geographic clustering of drug-resistant cases with evidence of clonal expansion suggests the successful transmission of drug-resistant Beijing genotypes (37). This is supported by the frequency with which isolates of the Beijing genotype are identified among children with drug-resistant TB (30), which indicates successful transmission within the community (34).Variable transmissibility, irrespective of drug resistance, represents a relatively unexplored potential mechanism for the emergence of an M. tuberculosis genotype. Conventional molecular tools are limited by an inability to distinguish factors related to transmissibility from those related to pathogenicity, since only patients with active disease can be evaluated. The value of experimental animal models is equally limited, since artificially induced infection does not allow simulation of the natural airborne transmission of M. tuberculosis. The household provides an appropriate setting in which variables related to recent M. tuberculosis transmission in the human host may be studied and allows the evaluation of young children likely to have been infected through contact with others at the household level.The study described here aimed to determine whether the Beijing genotype is more readily transmitted than other prevalent genotypes to children in household contact with an adult TB index case.     

Spoligotype-Based Comparative Population Structure Analysis of Multidrug-Resistant and Isoniazid-Monoresistant Mycobacterium tuberculosis Complex Clinical Isolates in Poland

The spoligotyping-based population structure of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated in Poland (n = 46), representing all culture-positive MDR tuberculosis (MDR-TB) cases, was compared to that of isoniazid (INH)-monoresistant strains (n = 71) isolated in 2004. The latter data set from a previous study (E. Augustynowicz-Kopeć, T. Jagielski, and Z. Zwolska, J. Clin. Microbiol. 2008, 46:4041-4044) represented 87% of all INH-monoresistant strains. The clustering rates and genotypic-diversity indexes for the 2 subpopulations were not significantly different (P = 0.05). The results were entered in the SITVIT2 database to assign specific shared type designations, corresponding genotypic lineages, and geographical distributions and compared to available data from neighboring countries (Germany, n = 704; Czech Republic, n = 530; Sweden, n = 379; Kaliningrad, Russia, n = 90) and strains from previous studies in Poland (n = 317). MDR strains resulted in 27 patterns (20 unique strains within the study and 7 clusters containing 2 to 6 isolates per cluster with a clustering rate of 56.5%) and belonged to the following genotypic lineages: ill-defined T family (28.3%), Haarlem (17.4%), Latin American and Mediterranean (LAM) (13%), Beijing (8.7%), S family (4.35%), and the X clade (2.17%). Comparison of the genetic structure of the MDR strains with that of INH-monoresistant strains showed that a total of 9 patterns were shared by both groups; these represented 1/3 of the MDR strains and 2/3 of the INH-monoresistant strains. Interestingly, 76.1% of the MDR isolates and 71.8% of the INH-resistant isolates yielded spoligotypes that were previously reported from Poland. The observation that nearly half of the spoligotypes identified among both MDR (48.1%) and INH-monoresistant (43.3%) M. tuberculosis isolates were present in Poland''s neighboring countries suggested that a significant proportion of MDR and INH-resistant TB cases in Poland were caused by strains actively circulating in Poland or its neighbors. Our results corroborate the leading role of the T and Haarlem genotypes in the epidemiology of drug-resistant TB in Poland. Nevertheless, the LAM and Beijing family strains that infected, correspondingly, 13% and 9% of patients with MDR-TB were absent among the strains from patients with INH-monoresistant TB, suggesting that a proportion of MDR-TB cases in Poland are due to ongoing transmission of MDR clones exhibiting specific genotypes. Study of the population genetic relationships between MDR and INH-monoresistant strains by drawing minimum spanning trees showed that ill-defined T1 sublineage strains (1/3 of all INH-monoresistant strains), represented by its prototype, SIT53, constituted the central node of the tree, followed by strains belonging to the well-defined H3, H1, and S subgroups. However, the MDR group, in addition, contained LAM (n = 6) and Beijing (n = 4) lineage isolates. With the exception of the 4 Beijing lineage strains in the latter group and a single orphan isolate in the INH-monoresistant group, none of the remaining 112/117 isolates belonged to principal genetic group 1 (PGG1) in our study. Given the high rate of clustering and the near absence of immigrants in the study, the persistence of MDR-TB in Poland seems to result from active transmission of MDR strains within the autochthonous population, the bulk of it caused by evolutionarily recent tubercle bacilli.Tuberculosis (TB) remains a substantial global health problem. With more than 9 million new cases and almost 2 million deaths every year (50), TB holds one of the leading positions among infectious causes of morbidity and mortality worldwide. One of the reasons for the continuing TB prevalence is the emergence and spread of drug-resistant (DR) and, especially, multidrug-resistant (MDR) (i.e., resistant to at least isoniazid [INH] and rifampin [RMP]) Mycobacterium tuberculosis strains. According to a recent World Health Organization (WHO) report, the global population weighted percentages for any resistance and MDR among all TB cases are 20% and 5.3%, respectively (49). Poland, the largest and most populous country in Central Europe (38,530,000 inhabitants in 2002), has witnessed a dramatic fall in the incidence of TB over the last few decades. While in 1957 the notification rate for all clinical forms was 290.4 per 100,000 population, it had dropped almost 10-fold by the end of 1990s (43). However, the TB notification rate in Poland is still relatively high, nearly twice the mean European Union (EU) rate (24.6/100,000 versus 12.8/100,000 in 2004) (14).Since the early years of chemotherapy for TB, the prevalence of primary drug-resistant TB in Poland has been closely monitored. In 1994, Poland joined the WHO/International Union against Tuberculosis and Lung Disease (IUATLD) global project on anti-TB drug resistance surveillance, and during 1996 and 1997, the first national survey on primary and acquired drug resistance was performed. The subsequent study, completed in 2001, revealed a 2-fold increase in the primary resistance rate in comparison with that in 1997, i.e., 6.1% versus 3.6%, respectively. Likewise, the prevalence of primary multidrug resistance doubled, from 0.6% in 1997 to 1.2% in 2000 (3). In 2004, there was a slight decrease to 5.6% in the percentage of TB cases with primary drug resistance and quite a significant fall to 0.3% in the share of multidrug-resistant tuberculosis (MDR-TB) cases compared to 2000 (49). These figures place Poland among the countries with low to moderate DR-TB rates in the world. However, inadequacies in the registration of TB patients may contribute to an underestimation of drug-resistant cases. The most detrimental impact on the epidemiology of TB in Poland might be the spread of DR-TB from outside the country. This is because Poland shares borders with countries of the former Soviet Union, such as Lithuania, Ukraine, and Russia, where the incidence of DR-TB, including MDR-TB, is exceptionally high (49). Given the geographical epidemiological context, the surveillance capacity for TB in Poland has to be particularly well established.Studying genetic relationships among M. tuberculosis strains has provided important insights into the spread and transmission of TB (8, 15, 26, 30, 31, 34). Spoligotyping targeting the polymorphism of the chromosomal direct-repeat locus is one of the most extensively used techniques for genotypic differentiation between M. tuberculosis strains (27). Primarily used for first-line screening of outbreaks (22, 40), spoligotyping is ideally followed by more discriminatory second-line IS6110 restriction fragment length polymorphism (RFLP) or mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing (9, 24). It has also proven useful for tracking laboratory cross-contaminations (36) and describing the global spread of TB (9, 17). Major advantages of the spoligotyping technique are its simplicity, rapidity, reproducibility, and portability, due to the digital nature of the generated results. Of particular importance is the existence of the international spoligotype database, which provides useful information on the epidemiology and phylogeny of the circulating genotypes of tubercle bacilli worldwide (9).This study aimed to investigate the spoligotyping-based population structure of MDR M. tuberculosis clinical isolates isolated in Poland in 2004 compared to that of INH-monoresistant strains isolated in the same year. Indeed, as reviewed by Dye and Espinal (12), a high proportion of TB patients carrying INH-monoresistant strains acquire RMP resistance after treatment failure, suggesting that the development of INH resistance usually precedes that to RMP.     

Variation in Gamma Interferon Responses to Different Infecting Strains of Mycobacterium tuberculosis in Acid-Fast Bacillus Smear-Positive Patients and Household Contacts in Antananarivo,Madagascar

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop tuberculosis (TB), though many may become latently infected. More precise measurement of the human immune response to M. tuberculosis infection may help us understand this difference and potentially identify those subjects most at risk of developing active disease. Gamma interferon (IFN-γ) production has been widely used as a proxy marker to study infection and to examine the human immune response to specific M. tuberculosis antigens. It has been suggested that genetically distinct M. tuberculosis strains may invoke different immune responses, although how these differences influence the immune responses and clinical outcome in human tuberculosis is still poorly understood. We therefore evaluated the antigen-specific IFN-γ production responses in peripheral blood mononuclear cells from two cohorts of subjects recruited in Antananarivo, Madagascar, from 2004 to 2006 and examined the influence of the infecting M. tuberculosis strains on this response. The cohorts were sputum-positive index cases and their household contacts. Clinical strains isolated from the TB patients were typed by spoligotyping. Comparison of the IFN-γ responses with the spoligotype of the infecting clinical strains showed that “modern” M. tuberculosis strains, like Beijing and Central Asian (CAS) strains, tended to induce lower IFN-γ responses than “ancient” strains, like East African-Indian (EAI) strains, in index cases and their household contacts. These results suggest that new strains may have evolved to induce a host response different from that of ancient strains. These findings could have important implications in the development of therapeutic and diagnostic strategies.Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major cause of global morbidity and mortality throughout the world. It is estimated that there are in excess of new 8 million cases of TB each year, and this represents just the tip of the iceberg. Infection with M. tuberculosis leads to clinically active TB in about 5 to 10% of exposed individuals. A much higher proportion of exposed individuals apparently become latently infected, and these individuals may remain noninfectious and symptom free for years. Approximately one-third of the world population is thought to be latently infected with M. tuberculosis. However, under some circumstances (in about 5% of the latently infected people), the host immune response is perturbed and latent M. tuberculosis infection may develop into clinically active TB (52). This process is most prominent in individuals coinfected with human immunodeficiency virus (HIV), but it can also occur with impairment of the immune system associated with old age, malnutrition, anti-inflammatory drug treatment, etc. Reactivation of latent disease is thought to contribute roughly half of all TB cases, and thus, understanding the factors controlling the development of acute primary TB or latent infection is crucial to TB control (64).Gamma interferon (IFN-γ) production has been widely used to study infection and to examine the human immune response to specific M. tuberculosis antigens. The 6-kDa early secreted antigenic target (ESAT-6) antigen, encoded by genes located within region of difference 1 (RD1) of the M. tuberculosis genome, is much more specific for M. tuberculosis than purified protein derivative (PPD), as these genes were deleted from M. bovis in the development of BCG substrains or are not found in most environmental mycobacteria (29, 53). Some studies showed that the level of IFN-γ release in response to ESAT-6 could identify TB contacts at risk of developing active disease after recent infection (3, 18, 30). CFP7 or TB10.4 is an immunodominant antigen recognized by TB patients and M. bovis BCG-vaccinated subjects, while ESAT-6 is specific to TB patients and induces a strong IFN-γ response (51). Moreover, since CFP7 induces strong protection against infection by M. tuberculosis, it was proposed to be a TB vaccine candidate (1, 19).There is a growing number of observations indicating that TB cases resulting from infection with epidemic strains, such as the W-Beijing strains (22, 35, 39, 44), may display a more severe pathology or more severe symptoms. Beijing strains were also found to induce higher fevers in pulmonary TB patients than other strains (62). In addition, the Beijing genotype, which is responsible for more than 80% of TB cases in China, was associated with virulence and high transmissibility (7, 28). The same has been found more recently with the RD(Rio) strains belonging to the Latin America-Mediterranean (LAM) family (38). Despite the fact that other epidemiological and clinical studies have failed to confirm any association between the mycobacterial genotype and the clinical presentation (8, 41, 43), the immunological aspects of infection with these strains is still of interest and poorly described.Epidemiological studies carried out in Madagascar showed no association between IS6110 patterns and clinical tuberculosis presentation (47), but they did reveal a heterologous population of M. tuberculosis strains, including the existence of a high frequency of unusual genotypes, such as the shared type 109 from the EAI8-MDG family (SpolDB4) (10) or strains with a single copy of IS6110 (24, 46). Since there are limited data on the correlation of the strain genotype with clinical features or the host immune response in patients and their contacts (57, 59), we investigated the IFN-γ response to the ESAT-6, CFP7, and PPD antigens in pulmonary TB patients and their household contacts (as this is commonly used as a biomarker to identify M. tuberculosis infection) and examined the influence of the M. tuberculosis genotype on this response.     

Strains of Mycobacterium tuberculosis from Western Maharashtra,India, Exhibit a High Degree of Diversity and Strain-Specific Associations with Drug Resistance,Cavitary Disease,and Treatment Failure

We performed spoligotyping of Mycobacterium tuberculosis isolates from 833 systematically sampled pulmonary tuberculosis (TB) patients in urban Mumbai, India (723 patients), and adjacent rural areas in western India (110 patients). The urban cohort consisted of two groups of patients, new cases (646 patients) and first-time treatment failures (77 patients), while only new cases were recruited in the rural areas. The isolates from urban new cases showed 71% clustering, with 168 Manu1, 62 CAS, 22 Beijing, and 30 EAI-5 isolates. The isolates from first-time treatment failures were 69% clustered, with 14 Manu1, 8 CAS, 8 Beijing, and 6 EAI-5 isolates. The proportion of Beijing strains was higher in this group than in urban new cases (odds ratio [OR], 3.29; 95% confidence limit [95% CL], 1.29 to 8.14; P = 0.003). The isolates from rural new cases showed 69% clustering, with 38 Manu1, 7 CAS, and 1 EAI-5 isolate. Beijing was absent in the rural cohort. Manu1 was found to be more common in the rural cohort (OR, 0.67; 95% CL, 0.42 to 1.05; P = 0.06). In total, 71% of isolates were clustered into 58 spoligotypes with 4 predominant strains, Manu1 (26%), CAS (9%), EAI-5 (4%), and Beijing (4%), along with 246 unique spoligotypes. In the isolates from urban new cases, we found Beijing to be associated with multidrug resistance (MDR) (OR, 3.40; 95% CL, 1.20 to 9.62; P = 0.02). CAS was found to be associated with pansensitivity (OR, 1.83; 95% CL, 1.03 to 3.24; P = 0.03) and cavities as seen on chest radiographs (OR, 2.72; 95% CL, 1.34 to 5.53; P = 0.006). We recorded 239 new spoligotypes yet unreported in the global databases, suggesting that the local TB strains exhibit a high degree of diversity.The resurgence of tuberculosis (TB) fuelled by multidrug resistance (MDR) and extensive drug resistance has caused significant concern among health care practitioners (36, 37). There have been renewed efforts to understand the biology of the pathogen alongside its epidemiology. Such data have come mostly from regions of sporadic incidence or from populations where the disease is driven by high HIV prevalence (8). Data from some of the highest-disease-burden regions, where tuberculosis has remained endemic, are scarce.India is one such region where Mycobacterium tuberculosis has remained in equilibrium with the population, resulting in an area of tuberculosis endemicity (6, 27). Under such conditions, the strain diversity is expected to be different compared to that for epidemic or sporadic incidents, where a few specific strain types dominate (8, 23); in a setting where tuberculosis is endemic, the pathogen and the host are expected to evolve simultaneously for long durations, resulting in a set of varied strain types (6, 20, 21).Studies from India show differential strain predominance between the southern and northern regions of the country. While the central Asian strain (CAS) is dominant in the north, East African Indian strains (EAI) are observed more frequently in the southern regions (29). Most studies from India as well as specifically from Mumbai, India, showed CAS and Manu1 as the predominant spoligotypes along with EAI as a third large strain lineage (3, 16, 18, 22, 28, 29, 30). Another study from a tertiary care center in Mumbai reported a high proportion of Beijing strains (23%) in a cohort associated with a high proportion of MDR (1). Interestingly, TbD1-positive strains of tuberculosis (such as EAI) predominate in India, whereas TbD1-negative strains of M. tuberculosis are more common in the rest of the world (11).Although previous studies provided preliminary data from various sites in India, they did not reflect strain variability from a single cosmopolitan region. Additionally, studies from Mumbai were biased toward MDR cases (1), had small sample sizes (16), or were derived from a cohort of chronic (re-treated) TB cases accessing tertiary care hospitals (22).Epidemiological studies of M. tuberculosis have been facilitated by a variety of genotyping tools. IS6110 restriction fragment length polymorphism (RFLP) remains the gold standard due to its high level of discrimination (15) but is time-consuming and less suitable in populations with low copy numbers (7). Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing (10, 19, 31) is a high-throughput and discriminatory method, but the best combination of MIRU loci is yet to be achieved, and combinations may differ between populations (33). We used spoligotyping as a primary fingerprint method, due to its relatively high throughput nature. Spoligotyping has a lower discriminatory power than MIRU-VNTR typing, making it less suitable for determining strain transmission. However, spoligotyping served as a useful primary fingerprinting tool allowing comparisons of strain types from strains around the world through updated global databases (5, 17, 35).In this study, we describe the distribution of strain genotypes from a systematic collection of strains from urban Mumbai and two neighboring rural areas. Mumbai is a location where a confluence of people from all parts of the country live in poor, congested neighborhoods with high population densities, up to 64,168 people per square kilometer in one of the city wards as per the 2001 census (26). These conditions, coupled with a high proportion of MDR cases in the region (2, 9), were cause for concern and underlined the need for more information on local circulating strains. We wished to determine the distribution of strains among newly diagnosed TB patients in the region to extend previous observations (16, 22).This study describes the spoligotypes present in the cohort and the extent of their clustering. We further analyzed the association of spoligotypes with other parameters, namely, age, gender, geographical origin of the host, radiology, and multidrug resistance, to obtain deeper insights into strain behavior.     

Prospective Universal Application of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping To Characterize Mycobacterium tuberculosis Isolates for Fast Identification of Clustered and Orphan Cases

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.Molecular tools have been widely used to characterize Mycobacterium tuberculosis isolates, with the aim of better understanding the epidemiology of tuberculosis (TB) (1, 6, 8, 18, 23). This has enabled us to document suspected outbreaks (4, 28, 34), identify risk factors associated with TB transmission (13, 20, 36), and evaluate the efficiency of control programs by observing the dynamics of clustered cases (9, 12, 17, 22, 24).Restriction fragment length polymorphism (RFLP) analysis based on the IS6110 sequence is the reference genotyping method for M. tuberculosis (35). However, its limitations (mainly response times) make its adaptation unsuitable for real-time intervention epidemiological schemes. New genotyping techniques based on PCR have recently been developed and are more suitable for these purposes.One of the most promising PCR-based methods is mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping (21, 30-32). A novel format based on 15 loci has improved upon the initial 12-loci version. Its discriminatory power has been found to be equivalent to that of the standard approach on the basis of reference method, and its response time could be competitive. However, very few long-term analyses apply this technique universally in parallel to the reference method to identify advantages and pitfalls (1, 25, 27, 33).In order to compare both techniques, we selected the province of Almería, in southeast Spain, because of the complexity of its socioepidemiological population profile, which challenges us to develop new and more-efficient methods of surveying TB transmission. In Almería, around 60% of the cases involve immigrants who are dispersed throughout the province and who are highly mobile and difficult to access. Our group had already developed a new advanced system for studying clustered cases by active compilation of data through a newly developed computer application, GenContacTB, and standardized interviews of the patients on the basis of nominal and photographic identification (26). These aspects make Almería a suitable context for exploring novel, rapid M. tuberculosis-genotyping tools. Our study aimed to evaluate MIRU-VNTR genotyping with a set of 15 loci (MIRU-15) over a 39-month period by using a prospective design for most of the period (data for 2005 were retrospectively studied to increase analytical power), and the coverage of the population was universal (all M. tuberculosis isolates were included).     

Evaluation of Colorimetric Methods Using Nicotinamide for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis

The direct detection of pyrazinamide resistance in Mycobacterium tuberculosis is sufficiently difficult that many laboratories do not attempt it. Most pyrazinamide resistance is caused by mutations that inactivate the pyrazinamidase enzyme needed to convert the prodrug pyrazinamide to its active form. We evaluated two newer and simpler methods to assess pyrazinamidase activity, the nitrate reductase and malachite green microtube assays, using nicotinamide in place of pyrazinamide. A total of 102 strains were tested by these methods and the results compared with those obtained by the classic Wayne assay. Mutations in the pncA gene were identified by sequencing the pncA genes from all isolates in which pyrazinamide resistance was detected by any of the three methods. Both the nitrate reductase and malachite green microtube assays showed sensitivities of 93.75% and specificities of 97.67%. Mutations in the pncA gene were found in 14 of 16 strains that were pyrazinamide resistant and in 1 of 4 strains that were sensitive by the Wayne assay. Both of these simple methods, used with nicotinamide, are promising and inexpensive alternatives for the rapid detection of pyrazinamide resistance in limited-resource countries.Due to its activity against semidormant bacilli sequestered within macrophages, pyrazinamide (PZA) is one of the most effect frontline drugs used in the short-course chemotherapy of tuberculosis (TB) and also in retreatment regimens for multidrug-resistant TB patients (6, 36). However, the detection of PZA resistance is difficult and often unreliable because the drug is active only at acid pH (5.5), which also affects the growth of Mycobacterium tuberculosis (5, 37). For this reason, many laboratories do not perform PZA susceptibility testing, and therefore the true extent of global PZA resistance is largely unknown. The radiometric Bactec 460 system (Becton Dickinson, Sparks, MD), using a special acid liquid medium, has been considered the reference method for detection of PZA resistance, but it requires the use of costly and problematic radioisotopes (20, 30). Other commercial tests, such the nonradiometric Bactec MGIT 960 method (Becton Dickinson, Sparks, MD), utilize protocols adapted for PZA susceptibility testing, but they are also expensive and impractical for routine use in resource-poor settings (14, 27).PZA is a prodrug that requires activation by the M. tuberculosis pyrazinamidase (PZase), and most PZA-resistant strains have mutations in pncA, the gene encoding this enzyme, that result in the loss or reduction of PZase activity (7). Thus, an indirect approach to detecting resistance is to assess PZase activity (22). The classic way to detect PZase activity is with the Wayne assay (12, 35), which monitors the hydrolysis of PZA to the active acid form, pyrazinoic acid (POA), through the color change of a ferrous ammonium phosphate solution added to the medium.Nicotinamide (NIC), a structural analogue of PZA with some activity against M. tuberculosis, is also converted to its active acid form, nicotinic acid, by the M. tuberculosis PZase (12, 24). Strains of M. tuberculosis that are resistant to PZA are also resistant to NIC (18, 20, 28), and the conversion of NIC into nicotinic acid by PZase occurs at a physiological pH that does not hinder bacterial growth. In recent studies, PZA resistance was rapidly and accurately detected with the inexpensive resazurin microtiter assay (REMA) and nitrate reductase assay (NRA), using NIC as a surrogate for PZA to avoid the need for acidification of the medium (18, 20). Another alternative colorimetric method, reported by Farnia et al., uses malachite green to test for susceptibility to first- and second-line anti-TB drugs (4). Malachite green is a triphenylmethane dye with a dark green color that becomes colorless during M. tuberculosis metabolism (9).In this study we evaluated the use of NIC in the NRA and malachite green microtube (MGMT) assay for the detection of PZA resistance and compared the results with those obtained by the Wayne assay, which served as a gold standard. We also sequenced the pncA genes from strains determined to be resistant by any of the three methods.     

Integration of Mycobacterium tuberculosis Drug Susceptibility Testing and Genotyping with Epidemiological Data Analysis To Gain Insight into the Epidemiology of Drug-Resistant Tuberculosis in Malatya,Turkey

Drug-resistant tuberculosis (TB) presents a major challenge to global TB control. To gain a better understanding of drug-resistant TB epidemiology in Malatya, Turkey, we conducted the present study using 397 Mycobacterium tuberculosis clinical isolates collected from Malatya, Turkey, in recent years (2000-2007). Resistance to any anti-TB drug was found in 29% (114 of 397) of the study isolates, while the multidrug resistance (MDR) rate was ∼4.5% (18 of 397). Resistances to isoniazid (15.5%) and streptomycin (13.4%) were about twice as high as resistance to rifampin (RMP) (6.3%) and ethambutol (EMB) (6.0%). Importantly, 28% (7 of 25) of the RMP-resistant isolates were non-MDR isolates, as when a significant proportion of RMP-resistant isolates in a population are non-MDR, the predictive value of molecular detection of RMP resistance for MDR can be significantly reduced. Both identical and varied drug resistance patterns were seen in the same genotyping-defined clusters, suggesting that both primary and acquired resistance have contributed to the drug-resistant TB epidemic in Malatya, Turkey. In addition, drug-resistant cases were found to be more likely to be males (odds ratio [95% confidence interval], 1.82 [1.13, 2.94]), suggesting a potential role of gender in the epidemiology of drug-resistant TB in the study population. This study demonstrates that the integration of drug susceptibility testing with genotyping and epidemiological data analysis represents a useful approach to studying the epidemiology of drug-resistant TB.Tuberculosis (TB) remains an important global public health problem, and global TB control is further challenged by the rising epidemics of drug-resistant TB worldwide (29). In 2008, the World Health Organization (WHO) reported that worldwide resistance to any of the anti-TB drugs accounted for 20% of all reported TB cases while an estimated 5.3% of all reported TB cases had multidrug resistance (MDR), defined as resistance to, at least, isoniazid (INH) and rifampin (RMP) (28). Furthermore, globally, only 10% of the roughly 500,000 people who develop MDR TB each year receive treatment, leading to more possible cases of MDR TB (9). Because of the disparities in TB control around the world, a better understanding of the dynamics and driving forces of drug-resistant TB epidemics would contribute to the development of more effective strategies for global TB control.Turkey, with a population of around 70 million, had an annual TB incidence of 27.9 per 100,000 people in 2007 (6). Although the TB incidence in Turkey has decreased by half since 1985, several studies have shown the proportion of drug-resistant TB cases to be higher than the global average (3, 5, 10, 11, 20, 24, 26). In 2005, Surucuoglu and colleagues reported that the rate of resistance to any anti-TB drugs among 355 isolates of Mycobacterium tuberculosis obtained from western Turkey was 21.1%, while another study found that among the 1,513 TB cases diagnosed in Istanbul, Turkey, in 2005, 19% were resistant to at least one drug (20, 25). These rates are similar to the rates of drug resistance found in some of the Eastern European countries that are considered to have the highest drug resistance rates in the world (3). The high drug resistance rate poses a major challenge to the control of TB in Turkey. Previous studies of different populations have found that both host and microbial factors can play a role in drug-resistant TB epidemics. Microbial factors such as specific spoligotype families have been implicated as risk factors. TB genotypes belonging to the Beijing and Latin American and Mediterranean (LAM) families are associated with drug resistance (18, 23).Although previous studies reported high rates of drug-resistant TB infection in different regions of Turkey and several studies reported genotyping results of M. tuberculosis isolates collected from different regions of Turkey (10-12), few assessed associations of microbial and host characteristics with drug-resistant TB cases and attempted to determine the factors driving the epidemic of drug-resistant M. tuberculosis infection in the population. To gain a better understanding of the epidemiology of drug-resistant TB in Malatya, Turkey, we analyzed 397 M. tuberculosis clinical isolates collected from Malatya, Turkey, during the time period between 1 January 2000 and 31 December 2007 and their corresponding epidemiological data. Malatya is the third biggest city in Turkey and has a population of approximately 722,000 and an annual TB incidence of 28.5 per 100,000 (6).     

Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China

A total of 2,346 Mycobacterium tuberculosis isolates from 13 provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 2,153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SpolDB4.0 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 were novel. These 160 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (74.08%), followed by the T family (14.11%). CAS family strains were found only in the Xinjiang and Tibet regions, while EAI family strains were found only in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study.Tuberculosis (TB) continues to be a major public health problem in China. Based on the data from a nationwide random survey of the epidemiology of TB in China in 2000, there were probably 4.51 million active-TB patients in the country, including 1.50 million smear-positive cases, which were the infectious sources (16). From 2006 to 2009, more than 1 million new TB cases emerged each year. Consequently, the task of controlling TB in China remains difficult.The genotyping of Mycobacterium tuberculosis strains is important for TB control because it allows the detection of suspected outbreaks and the tracing of transmission chains. It is also important to monitor species diversity, as well as to identify secondary infections (4, 7, 13, 19). Insertion sequence (IS) 6110 restriction fragment length polymorphism (IS6110 RFLP) is thought of as the gold standard genotyping method for M. tuberculosis strain genotype identification (6, 13, 21). However, the method is time-consuming, labor-intensive, and costly. Furthermore, it is difficult to compare results between laboratories. Spacer oligonucleotide typing (spoligotyping), which is based on the analysis of polymorphisms of direct-repeat (DR) regions comprised of 36-bp DRs interspersed with 35- to 41-bp unique spacer sequences, is a good alternative to traditional IS6110 RFLP fingerprinting because of its simplicity, speed, and reliability (9, 11). Spoligotyping is useful for classifying M. tuberculosis strains into spoligotype families and subfamilies according to the presence or absence of spacer regions (24). Brosch et al. reported that M. tuberculosis can be divided into ancestral or modern strains based on M. tuberculosis-specific deletion 1 (TbD1) region analysis. The TbD1 region is present in ancestral M. tuberculosis strains but is absent from modern ones. These ancestral strains predominantly originated from endemic foci, whereas modern M. tuberculosis strains represent epidemic strains that were introduced into the same geographical regions more recently as a consequence of the worldwide spread of the tuberculosis epidemic (4).Presently, an international spoligotype database, SpolDB4.0, has been established. Although the updated SpolDB4.0 version reflects the global distribution of M. tuberculosis spoligotypes, it contains little information regarding M. tuberculosis strains in China (5). In this study, we typed 2,346 M. tuberculosis clinical isolates from 13 different provinces across China between 2005 and 2007 using spoligotyping to study M. tuberculosis diversity in China.