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.     

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.     

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.     

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.     

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).     

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.)     

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.     

Humoral Immune Responses against the Mycobacterium tuberculosis 38-Kilodalton,MTB48, and CFP-10/ESAT-6 Antigens in Tuberculosis

The diagnosis of smear-negative and culture-negative patients with active tuberculosis (TB) is challenging. The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been an important diagnostic aid. However, detection of antibody responses to a single antigen usually has a low sensitivity for diagnosis of TB. In this study, humoral immune responses against recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 (culture filtrate protein 10/6-kDa early secreted antigen target of M. tuberculosis) antigens in 250 Chinese TB patients and 260 healthy subjects were evaluated by an enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against those antigens in TB patients, even in bacterium-negative ones, were significantly higher than those in healthy subjects (P < 0.001). The serodiagnostic sensitivities to detect antibodies against individual antigens, i.e., recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 antigens, in TB patients were 73.6%, 73.2%, and 60.4%, respectively, with specificities of 85.4%, 77.7%, and 73.8%, respectively. Importantly, the sensitivity to positively detect humoral responses to one of the antigens increased further. Our data suggest that the humoral immune responses to M. tuberculosis antigens in TB patients are heterogeneous. The 38-kDa, MTB48, and CFP-10/ESAT-6 antigens can be used as the cocktail antigens in the serodiagnosis of active TB, especially for smear- or culture-negative TB cases.The control of tuberculosis (TB) remains challenging in China (18). Currently, the diagnosis of active TB mainly relies on clinical symptoms, radiologic findings, and the detection of Mycobacterium tuberculosis in clinical samples using smear staining and mycobacterial culture. However, the diagnosis of TB in smear- and culture-negative TB patients is difficult. The detection of M. tuberculosis-specific antibodies in human sera has been an important aid in diagnosis of TB. Notably, several antigens have been demonstrated to have merit in TB diagnosis, including the 38-kDa protein, which is commonly used in serodiagnostic tests (4, 5, 8, 13, 19, 22, 23). Previous studies suggest that the antibody responses to M. tuberculosis antigens are heterogeneous among individuals (17) so that the detection of antibodies against a single antigen usually has a low sensitivity for diagnosis of TB, especially for bacterium-negative cases. Therefore, it may be valuable to evaluate antibodies against the 38-kDa antigen and other major antigens for the diagnosis of active TB (14, 15).Notably, the MTB48, CFP-10 (culture filtrate protein 10), and ESAT-6 (6-kDa early secreted antigen target of M. tuberculosis) genes are conserved in M. tuberculosis and Mycobacterium bovis isolates but partially deleted or absent in M. bovis BCG as well as in most nontuberculous mycobacteria (NTM) (1-3, 10, 16). Importantly, the proteins encoded by these genes are immunogenic (7, 9, 12, 16). In this study, we cloned the 38-kDa, MTB48, CFP-10, and ESAT-6 genes and generated recombinant 38-kDa, MTB48, and CFP-10/ESAT-6 fusion proteins in Escherichia coli. Subsequently, we developed an enzyme-linked immunosorbent assay (ELISA) for the characterization of serum antibodies against 38-kDa, MTB48, and CFP-10/ESAT-6 antigens in a population of 250 active TB patients and 260 healthy subjects. We found that characterization of antibodies against multiple M. tuberculosis antigens were valuable for the diagnosis of active TB.     

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.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

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.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

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).     

Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-Year Analysis of Susceptibilities of Candida Species to Fluconazole and Voriconazole as Determined by CLSI Standardized Disk Diffusion

Fluconazole in vitro susceptibility test results for 256,882 isolates of Candida spp. were collected from 142 sites in 41 countries from June 1997 to December 2007. Data were collected for 197,619 isolates tested with voriconazole from 2001 to 2007. A total of 31 different species of Candida were isolated. Increased rates of isolation of the common non-albicans species C. glabrata (10.2% to 11.7%), C. tropicalis (5.4% to 8.0%), and C. parapsilosis (4.8% to 5.6%) were noted when the time periods 1997 to 2000 and 2005 to 2007 were compared. Investigators tested clinical isolates of Candida spp. by the CLSI M44-A disk diffusion method. Overall, 90.2% of Candida isolates tested were susceptible (S) to fluconazole; however, 13 of 31 species identified exhibited decreased susceptibility (<75% S), similar to that seen with the resistant (R) species C. glabrata and C. krusei. Among 197,619 isolates of Candida spp. tested against voriconazole, 95.0% were S and 3% were R. About 30% of fluconazole-R isolates of C. albicans, C. glabrata, C. tropicalis, C. rugosa, C. lipolytica, C. pelliculosa, C. apicola, C. haemulonii, C. humicola, C. lambica, and C. ciferrii remained S to voriconazole. An increase in fluconazole resistance over time was seen with C. parapsilosis, C. guilliermondii, C. lusitaniae, C. sake, and C. pelliculosa. Among the emerging fluconazole-R species were C. guilliermondii (11.4% R), C. inconspicua (53.2% R), C. rugosa (41.8% R), and C. norvegensis (40.7% R). The rates of isolation of C. rugosa, C. inconspicua, and C. norvegensis increased by 5- to 10-fold over the 10.5-year study period. C. guilliermondii and C. rugosa were most prominent in Latin America, whereas C. inconspicua and C. norvegensis were most common in Eastern European countries. This survey identifies several less-common species of Candida with decreased susceptibility to azoles. These organisms may pose a future threat to optimal antifungal therapy and underscore the importance of prompt and accurate species identification and antifungal susceptibility testing.Antifungal susceptibility testing is playing an increasing role as a means to track the development of antifungal resistance in epidemiological studies (2, 10, 12, 17, 27, 45-47, 55, 63). One of the important by-products of the standardization of antifungal susceptibility testing has been the ability to conduct surveillance for antifungal resistance using uniform methods (44). Meaningful large-scale surveys of antifungal susceptibility and resistance conducted over time would not be possible without a standardized broth microdilution (BMD) or disk diffusion (DD) method for performing the in vitro studies (12, 38, 60). Global surveillance programs such as the ARTEMIS antifungal surveillance program for DD testing (49, 57, 60) and MIC testing (12, 13), the European Confederation of Medical Mycology (ECMM) survey of candidemia (68), and the SENTRY Antifungal Surveillance Program (36-38) promote the use of standardized DD and BMD methods and provide useful and consistent antifungal susceptibility data from a broad international network of hospitals and laboratories.The ARTEMIS global antifungal surveillance program is among the most comprehensive and long-running fungal surveillance programs (12, 45, 57, 58, 60). The ARTEMIS program was designed to address many of the potential limitations of resistance surveillance studies (26): (i) it is both longitudinal (1997 to present) and global (142 participating sites in 41 countries) in scope, (ii) it employs standardized DD (7) and BMD (9) antifungal susceptibility test methods, (iii) both internal quality control (QC) performed in each participating laboratory and external quality assurance measures are used to validate test results (48, 50, 61), (iv) results are recorded electronically using the Biomic image analysis plate reader (Giles Scientific, Santa Barbara, CA) and are stored in a central database, and (v) both Candida and non-Candida (60) yeast isolates obtained from consecutive clinical samples from all body sites are tested locally, thus avoiding misleading results based on biased selective testing. Thus, the ARTEMIS program generates massive amounts of data that have been externally validated and that can be used to identify temporal and geographic trends in the species distribution of Candida and other opportunistic yeasts, as well as the resistance profiles of these organisms to fluconazole and voriconazole as determined by standardized Clinical and Laboratory Standards Institute (CLSI) DD methods.In the present study, we expand the ARTEMIS database to include the time period from June 1997 through December 2007 and a total of 256,882 isolates of Candida from 142 study sites in 41 countries. We provide comparative susceptibility data for fluconazole and voriconazole for more than 190,000 isolates collected from 2001 to 2007 and include an analysis of resistance rates by year, geographic location, hospital location, and specimen type for selected species.     

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.     

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.     

Polymorphic Exact Tandem Repeat A (PETRA): a Newly Defined Lineage of Mycobacterium tuberculosis in Israel Originating Predominantly in Sub-Saharan Africa

As part of the Israel National Program for Prevention and Control of Tuberculosis, the molecular epidemiology of new tuberculosis cases is monitored. Prospective screening showed that about 20% of all new cases of culture-positive tuberculosis (43 of 222) in Israel in the year 2008 were caused by certain Mycobacterium tuberculosis strains of the central Asian (CAS) spoligotype lineage. The identity and similarity of these strains by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing form a lineage we call PETRA for polymorphic at locus ETR A. The name PETRA was given to 79 strains we have found since the year 2000, because the largest number of strains with MIRU-VNTR profiles identical other than at locus A formed three groups, including 5 of 10 strains that had deleted the ETR A region from their genomes. No PETRA strain was found to be multiple drug resistant (resistant to both isoniazid and rifampin [rifampicin]). Most patients (75% [58 of 77 patients of known origin]) infected with PETRA were of sub-Saharan African origins. The genotypes associated with the 79 PETRA lineage strains presented in this paper suggest that the PETRA lineage is a large, major contributor to new tuberculosis cases in Israel.Mycobacterium tuberculosis is a major cause of world death and morbidity due to bacterial infection (15). It is estimated that almost a third of the world''s population, 2 billion persons, has been exposed to M. tuberculosis and that M. tuberculosis causes about 9 million persons to become ill each year; of these, nearly 2 million die from tuberculosis (15). Israel is a country with a low incidence of tuberculosis, but the recent absorption of almost a fifth of the population from countries with a high incidence of tuberculosis has presented a challenge for the control, prevention, and treatment of tuberculosis. In particular, the introduction of multiple-drug-resistant (MDR) M. tuberculosis primarily from former Soviet Union countries (unpublished data) and of the non-MDR but highly infectious central Asian (CAS) spoligotype lineage MT, primarily from sub-Saharan African (SSA) countries (this study), continues to challenge the public health system in Israel.CAS spoligotype sublineages (5, 6, 61) have been reported to be prevalent in patients from SSA countries (3, 29, 58). Extrapulmonary tuberculosis has been reported from CAS spoligotype M. tuberculosis (41). It was recently reported that the CAS spoligotype lineage contributes to the tuberculosis burden in the Middle East (3).Genotyping on culture-positive Mycobacterium tuberculosis complex (MTC) has been performed at our National Mycobacterium Reference Laboratory since 1997 using restriction fragment length polymorphism (RFLP) typing (75, 76) as the gold standard. Later, 43-spacer spoligotyping (19) was introduced, followed by typing of 16 mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) loci (12 MIRU-VNTR [47, 66] loci, 3 exact tandem-repeat [ETR] loci [25], and the QUB11b [68] locus). For the year 2000, a retrospective analysis of all new culture-positive pulmonary MTC cases in Israel was performed using RFLP typing. From 2001 to 2005 and in 2007, MTC strains were genotyped according to clinical and epidemiological needs. Most new MTC strains of 2006 were MIRU-VNTR genotyped. Since the beginning of 2008, all new culture-positive MTC cases are being MIRU-VNTR genotyped in a prospective screen employing high-throughput capillary electrophoresis with 16 capillaries (4, 65, 67, 68).This paper presents the results obtained from the prospective MIRU-VNTR molecular epidemiological screening of all patients with new cases of culture-positive tuberculosis in Israel in the year 2008 and in addition some results obtained from the retrospective screening of previous years in Israel. These results lead to the definition of a new M. tuberculosis MIRU-VNTR lineage (with CAS spoligotypes) which is named PETRA (for polymorphic ETR A) here. Evidence is presented that the M. tuberculosis PETRA lineage is a major contributor of tuberculosis in Israel and is of predominantly SSA origins. Some PETRA lineage strains have deleted the chromosomal region containing locus ETR A.     

Evaluation of Sensitivity of Multiplex PCR for Detection of Mycobacterium tuberculosis and Pneumocystis jirovecii in Clinical Samples

A multiplex PCR assay for the simultaneous detection of Mycobacterium tuberculosis and Pneumocystis jirovecii was developed using IS6110-based detection for M. tuberculosis and mitochondrial large-subunit (mtLSU) rRNA gene detection for P. jirovecii. Ninety-five pulmonary blinded samples were examined using the developed multiplex PCR assay, and the results were compared with those obtained by the single nested PCRs targeting IS6110 for M. tuberculosis and mtLSU rRNA for P. jirovecii. Of the 95 pulmonary samples tested, the multiplex nested PCR developed here could detect 36 cases of M. tuberculosis infection, 35 cases of P. jirovecii infection, and 17 cases of M. tuberculosis and P. jirovecii coinfections. The sensitivities of the multiplex nested PCR in detecting M. tuberculosis and P. jirovecii were 92.1% and 81.4%, respectively, whereas the specificities in detecting M. tuberculosis and P. jirovecii were 98.2% and 100%, respectively.Pulmonary tuberculosis (TB) and Pneumocystis jirovecii pneumonia are two of the most common opportunistic infections found in association with AIDS worldwide (4), including Thailand (2, 3, 6, 10). About one-third of the world''s population and one-third of people infected with HIV are infected with Mycobacterium tuberculosis. The World Health Organization (WHO) reported that globally 9.2 million new cases of TB and 1.7 million deaths from TB occurred in 2006, and of these, 0.7 million cases and 0.2 million deaths, respectively, were in HIV-positive people (21). At present, Pneumocystis pneumonia, caused by Pneumocystis jirovecii (previously known as P. carinii f. sp. hominis), remains one of the most common AIDS-defining illnesses and is a frequent cause of morbidity and mortality in HIV-infected patients (7). Geographically, TB is the most common respiratory opportunistic infection in people infected with HIV worldwide, especially in the developing world (1, 4, 6, 8, 9, 12), whereas P. jirovecii pneumonia is more prevalent in industrialized countries (4, 5, 13, 16). In Thailand, TB has been the most common opportunistic infection in people with AIDS, whereas P. jirovecii pneumonia has been the second most common (12, 18). The total number of the two infections represents one-half of opportunistic infections in AIDS cases.TB and P. jirovecii pneumonia can clinically and radiologically mimic each other, including having similar presentations in patients, and they cannot always be diagnosed by clinical presentation or sputum examination. In addition, coinfection in individuals may also occur. Therefore, accurate and rapid diagnosis is required. The molecular means of diagnosis is considered to be a reliable technique, and it is essential that it be developed or improved to simultaneously diagnose TB and P. jirovecii pneumonia. Having a technique for differential diagnosis of the two infections would contribute to the ability to provide immediate treatment, controlling the diseases and decreasing the rates of transmission. The aim of the present study was to develop a multiplex PCR technique for the detection of M. tuberculosis and P. jirovecii simultaneously in clinical samples. In the present study, the development of a multiplex PCR involved selection of the appropriate genes, as well as the optimum PCR mixture and PCR thermal profile. The multiplex PCR was applied to test its sensitivity and specificity with clinical specimens.     

Kinetics of a Tuberculosis-Specific Gamma Interferon Release Assay in Military Personnel with a Positive Tuberculin Skin Test

Treatment of latent Mycobacterium tuberculosis infection on the basis of the tuberculin skin test (TST) result is inaccurate due to the false-positive TST results that occur after Mycobacterium bovis BCG vaccination or exposure to nontuberculous mycobacteria (NTM). Gamma interferon release assays (IGRAs) are based on M. tuberculosis-specific antigens. In a previous study among BCG-naïve military employees, a positive TST result after deployment was mostly associated with a negative IGRA result, suggesting exposure to NTM. Data regarding the kinetics of IGRAs are limited and controversial. The present study aimed to reassess the rate of false-positive TST results and to evaluate the kinetics of the Quantiferon TB Gold In-Tube assay (QFT-Git) in military personnel with a positive TST result. QFT-Git was performed at the time of inclusion in the study and was repeated after 2, 6, 12, and 18 or 24 months. Of 192 participants, 17 were recruits and 175 were screened after deployment (n = 169) or because of travel or health care work. Baseline positive QFT-Git results were observed in 7/17 (41.2%) and 12/174 (6.9%) participants, respectively. During follow-up, a negative QFT-Git result remained negative in 163/165 (98.8%) participants. Of 18 subjects with an initial positive QFT-Git result, reversion to a negative result occurred in 1/6 (16%) recruits, whereas it occurred in 8/12 (66%) subjects after deployment or with other risk factors (P = 0.046). The quantitative result was significantly lower in subjects with reversion than in those with consistent positive results (P = 0.017). This study confirmed a low rate of positive QFT-Git results among military personnel with a positive TST result after deployment, supporting the hypothesis of exposure to NTM. Reversion of the majority of initially low-positive QFT-Git results indicates that QFT-Git may be useful for the diagnosis of later reinfections.Each year, about 3,000 Dutch army personnel are deployed to regions where tuberculosis (TB) is highly endemic. Screening of military personnel for latent Mycobacterium tuberculosis infection (LTBI) has thus far been based on the tuberculin skin test (TST). The Netherlands is a country with a low prevalence of TB, with a yearly incidence of 5.9 cases/100,000 population in 2007, only one-third of which occurred among native Dutch persons (Tuberculosis in The Netherlands 2007 [www.kncvtbc.nl]). Personnel are screened by the TST upon initial recruitment into the army, after deployment, or in the presence of other risk factors for TB exposure. Military personnel with TST conversion are prescribed isoniazid for 6 months to prevent TB disease. The risk of progression from untreated LTBI to active TB is generally believed to be about 10%, with half of the cases occurring within 2 years after infection. However, the risks observed in different studies comparing subjects treated with isoniazid or placebo varied widely, depending on the setting and the characteristics of the study population (38). A major disadvantage of the current policy is that a substantial proportion of TST conversions in this setting are thought to be caused by exposure to nontuberculous mycobacteria (NTM), skewing the risk-benefit ratio of preventive treatment (8). In addition, increasing proportions of the Dutch population and Dutch military recruits originate from countries where M. bovis BCG vaccination is routinely used. In BCG-vaccinated Dutch military personnel or those with a previous positive TST result, TST is not performed, as a rule, and chest radiography is used as an alternative, but radiography lacks sensitivity for the detection of LTBI. Finally, a positive TST result often remains positive, thus precluding the detection of reinfection.In order to overcome the disadvantages of the TST, gamma interferon (IFN-γ) release assays (IGRAs) that use M. tuberculosis-specific antigens and that are not affected by BCG and most NTM were developed (2-4, 32, 34). In contact investigations, the results of IGRAs had a better correlation with measures of exposure (5, 19, 21, 43). Since 2005, IGRAs have increasingly been used for the detection of LTBIs either as a replacement of or as adjunct to the TST (18, 26, 28). Of the two presently commercially available IGRAs, the Quantiferon TB Gold In-Tube assay (QFT-Git) is a robust whole-blood-based test suitable for use for large-scale testing (5, 7, 22). In a previous study, QFT-Git was positive for only a minority of military personnel with a positive TST result after deployment (13). Those results were considered to be related to NTM exposure, in accordance with the high proportion of false-positive TST responses assessed by dual skin testing of army recruits by the use of tuberculin and atypical sensitin (8). The destinations of deployment at the time of the earlier study were mainly Iraq and Bosnia (13), but the destination has changed to Afghanistan in the past few years. As the incidence of TB is higher in Afghanistan than in most of the countries where the military personnel were deployed, in order to justify a change in treatment policy, the previously observed low rate of positive IGRA results in association with a positive TST result needed to be studied in the current setting. In the previous study (13), QFT-Git was performed only once, and the subjects were not assessed for eventual later conversion or reversion. Previous studies of the kinetics of IGRAs gave variable and partly conflicting results (9, 12, 14, 15, 17, 20, 29, 30, 35-37, 40), although the main trend was for high-positive results to usually remain positive, and reversion can occur when the results are low or moderately positive (12, 15, 20, 29, 30, 33, 35-37, 42). In a setting of LTBIs, the relevance of follow-up testing by IGRAs may lie in the possibility of detecting later reinfection if reversion to a negative result has been documented.The aims of this study were to study the kinetics of QFT-Git during at least 6 months of follow-up in order to evaluate the possibility of detection of later reinfection and to confirm the previously observed very low rate of positive QFT-Git results in military personnel with a positive TST result after deployment.