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-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.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

Rapid identification of mycobacterial whole cells in solid and liquid culture media by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycobacterial cells. We engineered a strategy based on specific profiles in order to identify the most clinically relevant species of mycobacteria. To validate the mycobacterial database, a total of 311 strains belonging to 31 distinct species and 4 species complexes grown in Löwenstein-Jensen (LJ) and liquid (mycobacterium growth indicator tube [MGIT]) media were analyzed. No extraction step was required. Correct identifications were obtained for 97% of strains from LJ and 77% from MGIT media. No misidentification was noted. Our results, based on a very simple protocol, suggest that this system may represent a serious alternative for clinical laboratories to identify mycobacterial species.The genus Mycobacterium encompasses over 100 species. The most important mycobacterial diseases are predominantly caused by the Mycobacterium tuberculosis complex. The incidence of other mycobacterial diseases due to nontuberculous mycobacteria (NTM) appears to be increasing in recent years due to the increasing number of immunocompromised individuals (1). Because the treatment of these infections differs depending on the isolated species, the correct and rapid identification of causative organisms is essential.Conventional methods for the identification of mycobacteria were classically based on biochemical tests. They required several weeks for adequate growth, and sometimes accurate identification was not possible. Difficulties, such as the lack of adequate reproducibility, the variability of phenotypes, and the fact that phenotype information is limited to common species, may lead to ambiguous or erroneous results (29). New strategies have been developed in the last decades using molecular biology tools (6, 10, 16, 24). The techniques based on DNA hybridization are sensitive, fast, and simple, but the available commercial assays (AccuProbe; Gen-Probe, San Diego, CA) are able to identify only four species and two complexes of mycobacteria (10). Techniques requiring amplification followed by a hybridization step on a solid support are more complete than probes, but commercially available kits are limited to 5 (GenoType MTBC; Hain Lifescience GmbH, Nehren, Germany), 16 (Inno-LiPa Mycobacteria v2; Innogenetics, Gent, Belgium), or 30 (GenoType Mycobacterium; Hain Lifescience GmbH, Germany) species (19, 22, 30). Systems based on sequencing or enzymatic restriction targeting the hsp65, 16S rRNA, sod, and rpoB genes allow good identification of all mycobacteria at the species level but remain limited to specialized laboratories (14, 17, 23, 31, 36, 37). In addition, they are expensive and time-consuming and require qualified operators (20). Recently, alternatives based on the analysis of mycolic acid by high-performance liquid chromatography (HPLC) (2) or electrospray ionization-tandem mass spectrometry analysis (28) have been proposed. However, these methods are still labor-intensive.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid identification of the most frequently isolated bacteria grown on solid medium by the identification of species-specific profiles obtained from isolated colonies (3-5). This technique is now routinely used in a few laboratories (26, 34). Some authors have used MALDI-TOF MS for rapid identification of Mycobacterium species (12, 18, 21). However, the techniques used require several steps, such as 16S rRNA gene-based techniques (18), cell extractions (12), or a statistical analysis (12, 21). In addition, the number of tested strains in these studies is less than 40, encompassing a maximum of 13 species. It was first reported by Hettick et al. that the analysis of mycobacterial whole cells by MALDI-TOF MS could be used for identification. However, the limited number of strains prevented the engineering of a useful database (11).Recently, we engineered a strategy to identify bacteria using MALDI-TOF MS, based on the choice of a limited number of species-specific profiles (3, 5). The aim of the present work is to extend this strategy to the identification of mycobacterial strains without cell extraction. This method will allow us to have a rapid, accurate, and inexpensive identification tool in routine laboratories. The first step was to build a complete database for mycobacterial species isolated in human pathology. This database was then validated by using clinical strains cultivated in solid and liquid media.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens

We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.Of the known species in the genus Mycobacterium, Mycobacterium tuberculosis is the most common and most important pathogen, causing 2 million deaths and over 8 million cases of tuberculosis worldwide annually (2, 3, 4, 7). In addition to M. tuberculosis, infections with nontuberculosis mycobacteria (NTM) can also cause clinical problems. Because of the different pathogenic potentials and susceptibilities of different mycobacterial species, the treatments of mycobacterial infections are different (13, 30, 33, 34). Thus, it is very important to differentiate between mycobacteria at the species level during early-stage diagnostics.Instead of a culture-based identification scheme, which may take 4 to 6 weeks or longer to identify slowly growing mycobacteria, PCR-based protocols (sequencing or PCR-restriction analysis [PRA]) targeting chronometer molecules, such as 16S rRNA (5, 6, 28), hsp65 (17, 19, 25), and rpoB (1, 16, 21), have been widely used to identify mycobacteria. However, in spite of the successful application of these conventional PCR-based methods to culture isolates, there are some drawbacks in their direct application to clinical specimens. This is especially true for sputum samples, which also contain numbers of commensal bacteria from the respiratory tract, producing confusing results by the simultaneous amplification of both commensals and mycobacterial strains. We have recently developed several methods for mycobacterial species identification based on amplification of hsp65 gene sequences directly from sputum samples (15, 27). Limitations due to the intrinsic features of conventional PCR prevented feasible identification of mycobacterial species from sputum samples using this method.The use of the real-time PCR assay in the diagnosis of many infectious diseases has been increasing, as it represents an appealing alternative to conventional PCR. It is an improvement over conventional methods because of its increased sensitivity and specificity, low contamination risk, and ease of performance and speed (8). In particular, fluorescence resonance energy transfer (FRET)-based real-time PCR permits not only the simultaneous identification of multiple target species but also the direct identification of target species from primary specimens such as sputum specimens through melting curve analysis of the amplification product (8). These characteristics of FRET-based real-time PCR provide a useful advantage for the identification of mycobacteria from sputum samples. Recently, several real-time PCR-based methods for mycobacterial detection and identification have been developed and evaluated (9, 22, 23, 26, 29). However, direct application of the real-time PCR-based method to primary specimens was generally limited to M. tuberculosis alone (11, 26). So far, a method which can simultaneously identify several pathogenic NTM as well as M. tuberculosis from primary sputum samples in a single reaction has not been developed.In the present study, we sought to develop a multiprobe real-time PCR targeting the hsp65 gene (HMPRT-PCR) based on melting curve analysis (HybProbes). This enabled the simultaneous identification of several pathogenic mycobacteria, including M. tuberculosis, in a single PCR performed on cultures and sputum samples. The usefulness of these methods was evaluated by blindly applying them to cultured and sputum samples.     

Mycobacterium tuberculosis Synergizes with ATP To Induce Release of Microvesicles and Exosomes Containing Major Histocompatibility Complex Class II Molecules Capable of Antigen Presentation

Major histocompatibility complex class II (MHC-II) molecules are released by murine macrophages upon lipopolysaccharide (LPS) stimulation and ATP signaling through the P2X7 receptor. These studies show that infection of macrophages with Mycobacterium tuberculosis or M. bovis strain BCG enhances MHC-II release in synergy with ATP. Shed MHC-II was contained in two distinct organelles, exosomes and plasma membrane-derived microvesicles, which were both able to present exogenous antigenic peptide to T hybridoma cells. Furthermore, microvesicles from mycobacterium-infected macrophages were able to directly present M. tuberculosis antigen (Ag) 85B(241-256)-I-A^b complexes that were generated by the processing of M. tuberculosis Ag 85B in infected cells to both M. tuberculosis-specific T hybridoma cells and naïve P25 M. tuberculosis T-cell receptor (TCR)-transgenic T cells. In the presence of prefixed macrophages, exosomes from mycobacterium-infected macrophages provided weak stimulation to M. tuberculosis-specific T hybridoma cells but not naïve P25 T cells. Thus, infection with M. tuberculosis primes macrophages for the increased release of exosomes and microvesicles bearing M. tuberculosis peptide-MHC-II complexes that may generate antimicrobial T-cell responses.Exosomes are 50- to 80-nm membrane vesicles that are released by many cell types, including reticulocytes, B cells, and dendritic cells (DCs) (16, 17, 33-35, 40, 42, 49, 53). Invagination of the limiting membrane of late endosomes leads to the formation of intraluminal vesicles in multivesicular endosomes. The intraluminal vesicles are secreted as exosomes upon the fusion of multivesicular endosomes with the plasma membrane.Exosomes from B cells contain major histocompatibility complex class II (MHC-II) molecules and can stimulate CD4⁺ T-cell responses in vitro (40), although they may be more capable of activating primed T cells than naïve CD4⁺ T cells (27). The activation of naïve CD4⁺ T cells by DC exosomes occurs via an indirect pathway in which the exosomes and their constituent peptide-MHC-II molecules are presented in the context of intact antigen (Ag)-presenting cells (APCs) (e.g., DCs that may be MHC-II negative but must bear the costimulatory molecules CD80 and CD86 [48]). The presence of ICAM-1 on exosomes is important for naïve T-cell priming (43).While the shedding of exosomes can be constitutive (27, 40), it can also be significantly enhanced by the stimulation of certain receptors, e.g., Toll-like receptors (TLRs) and the P2X7 purinergic receptor (P2X7R), which trigger inflammatory responses (37, 38). P2X7R can be activated by ATP, which is released into the extracellular milieu following cell death or injury (50). P2X7R signaling induces the assembly of inflammasome signaling complexes (10), which drive the proteolytic activation of caspase-1 and the maturation of interleukin 1b (IL-1b). Another P2X7R-induced response is the rapid extracellular release of MHC-II molecules (38), which was previously observed within 15 min of the addition of ATP and resulted in the release of ∼15% of the total MHC-II pool in macrophages within 90 min (38). Released MHC-II molecules were contained in two membrane fractions: larger (100- to 1,000-nm) plasma membrane-derived microvesicles and smaller (50- to 80-nm) exosomes. The ATP-stimulated release of MHC-II was markedly reduced in macrophages isolated from NLRP3 knockout or ASC knockout mice. Thus, P2X7R activation of the NLRP3 inflammasome induces the biogenesis and release of MHC-II-containing membranes. The precedent of synergy between lipopolysaccharide (LPS) and ATP suggests that MHC-II shedding might be enhanced in the context of bacterial infection, but this hypothesis has not been explored.Mycobacterium tuberculosis is a major human pathogen that infects one-third of the world population. M. tuberculosis and the related organism Mycobacterium bovis strain BCG infect host cells and regulate host cell functions by signaling through innate immune receptors, including TLR2. Cells infected with M. tuberculosis also secrete exosomes containing mycobacterial molecules that function as PAMPs (pathogen-associated molecular patterns) (2-5, 42) and can stimulate proinflammatory responses via TLR2, TLR4, and MyD88 (4, 5). The dissemination of PAMPs by exosomes released from infected cells may induce innate immune responses by a greater number of cells than are directly infected, magnifying host responses. M. tuberculosis and other mycobacteria can activate the ASC/NLRP3/caspase-1 inflammasome in macrophages via a mechanism dependent on the mycobacterial RD1 locus (encoding components of the ESX-1 secretion system, including the ESAT-6 protein) (9, 20, 26). The ability of M. tuberculosis to stimulate inflammasome activity is dependent on increased K⁺ efflux and occurs in macrophages from P2X7 receptor knockout mice (20). Thus, P2X7 receptor activation and M. tuberculosis infection may elicit similar signaling pathways that converge on the NLRP3 inflammasome and, possibly, on the inflammasome-dependent release of MHC-II membranes.In the current study, we demonstrate that infection of macrophages with mycobacteria elicits the shedding of MHC-II-containing membranes. Furthermore, M. tuberculosis increases the ATP-triggered release of exosomes and microvesicles containing MHC-II. In addition, we demonstrate that MHC-II in membranes released from mycobacterium-infected macrophages can present Ag to T cells. These findings suggest that exosomes and microvesicles from mycobacterium-infected cells may broadcast the stimulation of both innate and adaptive immune receptors beyond the directly infected host cells, contributing to the genesis of CD4 T-cell responses to mycobacterial pathogens such as M. tuberculosis.     

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.     

Protection of Mycobacterium tuberculosis from Reactive Oxygen Species Conferred by the mel2 Locus Impacts Persistence and Dissemination

Persistence of Mycobacterium tuberculosis in humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus in M. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of the mel2 locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with the mel2 mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of the mel2 mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox^−/− and iNOS^−/− mice and bone marrow-derived macrophages suggest that the primary mechanism by which mel2 affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role of mel2 in resistance to ROS is important for persistence and dissemination of M. tuberculosis and suggests that homologues in other bacterial species are likely to play a role in pathogenesis.Despite extensive efforts to eradicate tuberculosis, caused by Mycobacterium tuberculosis, worldwide and prevent the spread of antibiotic-resistant strains, tuberculosis remains one of the most frequent causes of death in humans. Currently, one-third of the world''s population is thought to be persistently infected with tuberculosis (6, 19, 20). A better understanding of the mechanisms that lead to persistence in humans is needed before it will be possible to develop rational strategies to prevent establishment of latency and block reactivation from it. Although the mouse model, even when infected by the natural low-dose aerosol route, does not replicate all aspects of pathogenesis by tuberculosis, its cost-effectiveness and the presence of numerous reagents make it an important tool for examination of the acute and persistent stages of infection (26, 50, 51). As a result, much of our knowledge regarding the role of the host in controlling infections as well as the bacterial factors involved has been obtained using the mouse model (26, 31, 34).Initially, tubercle bacilli encounter naïve alveolar macrophages in the mouse lung that produce low levels of reactive oxygen species (ROS) and undergo an oxidative burst in response to infection (8, 52). In the absence of ROS production, there is a modest transient advantage for tuberculosis in the lung, suggesting that ROS initially assist in the control of bacterial growth (16). This innate immune response does little to prevent the growth of the bacteria in mouse lungs, since for the first month after infection, bacterial numbers increase to a million or more. This time point corresponds to the peaks in the numbers of CD4 and CD8 T cells in the lung (38), maximal tumor necrosis factor alpha (TNF-α) levels (5, 32), and the initiation of a strong cell-mediated immune response. The cell-mediated immune response leads to control of bacterial growth, higher gamma interferon (IFN-γ) levels and tuberculosis persistence at stable numbers for between 1 and 2 years (38) before reactivation, most likely the result of a weakened immune system due to the age of the mice (49). TNF-α (44, 57) and IFN-γ (15, 27) levels are important for controlling tuberculosis growth and maintenance of the persistent state in the mouse model of infection. One of the reasons that TNF-α and IFN-γ help to control tuberculosis is that they provide signals for the production of both ROS and reactive nitrogen species (RNS) by macrophages (8, 25, 28, 32, 40, 65). Although M. tuberculosis is considered relatively resistant to ROS, mutations that impact those bacterial pathways involved in resistance can affect virulence (21, 33, 39, 47, 53). In contrast to ROS, RNS, produced by the nitric oxide synthase (iNOS) that is induced by IFN-γ, inhibit the growth of mycobacteria and are critical to maintenance of the persistent state in mice (7, 8, 22, 27, 29, 42). Although the role of RNS in the control of M. tuberculosis growth in mice seems well proven, the involvement of RNS in human (58) and in other animal, including guinea pig (36, 68), tuberculosis models is less clear, possibly because the presence of NO has been difficult to demonstrate (9, 24, 46, 54, 67). These observations suggest that M. tuberculosis genes involved in resistance to either ROS or RNS will play a role in persistence.We recently identified a locus in Mycobacterium marinum, designated mel2, that affects resistance to ROS and RNS (61, 62). Interestingly, this locus has three genes, melF, melG, and melH, with similarity to the luxA, luxG, and luxH bioluminescence genes, respectively, in other bacterial species (23). The luciferase (luxA) genes in other organisms can affect resistance to ROS (41, 55, 66) in addition to their role in bioluminescence. Bacterial luciferases are thought to scavenge H₂O₂ in a catalase-like reaction in which the enzyme-bound flavin mononucleotide (FMN) hydroperoxide and H₂O₂ decompose, releasing water and oxygen, to the enzyme-bound hydroxy-FMN that spontaneously decomposes to water, light, and enzyme-bound FMN (66). These observations suggest the intriguing possibility that the mel2 locus will be important for tuberculosis pathogenesis, since M. tuberculosis must resist ROS produced by the host immune response during infections. In order to test this hypothesis, we isolated an M. tuberculosis mutant in the mel2 locus and confirmed the loss of mel2 activity by an increase in ROS susceptibility. The mel2 mutant was evaluated for its role in virulence using the mouse model and macrophage infection in vitro. Mice and macrophages deficient in ROS and RNS were used to probe the primary host defense mechanisms that the mel2 locus is necessary to protect tuberculosis against. We found that the mel2 mutant displays increased susceptibility to ROS and is defective for growth in activated macrophages. Our data also suggest that the mel2 locus plays an important role during the persistence and dissemination of M. tuberculosis. The primary mechanism by which mel2 affects these events appears to be resistance to ROS. Overall, these studies provide evidence for the role of bioluminescence-related genes, such as mel2, in protecting M. tuberculosis, and most likely other bacterial species that carry similar genes, against ROS.     

Rapid Method for Identification of Six Common Species of Mycobacteria Based on Multiplex SNP Analysis

A multiplex method using the SNaPshot technique was developed to screen for six common mycobacterial species: Mycobacteria tuberculosis, M. avium, M. intracellulare, M. chelonae, M. kansasii, and M. gordonae. A total of 468 mycobacterial clinical isolates were subjected to analysis for the presence of the six mycobacterial species by the multiplex SNaPshot method. Of the 468 mycobacterial isolates, 464 (99.15%) could be correctly identified by this assay. The multiplex SNaPshot technique is a promising discriminatory tool for rapid and accurate identification of frequently encountered clinical mycobacterial species.Even though Mycobacterium tuberculosis continues to be a serious health concern worldwide, it has been increasingly recognized that nontuberculous mycobacteria (NTM) are important human pathogens (4, 16, 23). NTM are ubiquitous organisms, with nearly 100 different species found in soil and water that can act as opportunistic pathogens in humans, causing a wide variety of skin and soft tissue infections, lymphadenitis, and lung disease (6, 15). The early differentiation of M. tuberculosis from NTM and the identification of species among NTM are crucial for immediate implementation of the appropriate therapy because susceptible drugs vary widely among different species (9).Conventionally, identification of mycobacteria is carried out by time-consuming biochemical tests that are not always accurate (5, 17, 25). Chromatographic techniques such as high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC), and thin-layer chromatography (TLC) are labor-intensive, difficult, or expensive (21, 26). DNA sequence analysis of the 16S rRNA gene region is now regarded as the gold standard for the identification of mycobacteria (13, 22, 25, 27). However, equipment and running costs are high. Simple genotypic assays for the identification of mycobacteria, such as Accuprobe (Gen-Probe Inc., San Diego, CA) (1), INNO-LiPA (27), and Genotype Mycobacterium (Hain Diagnostika) (19) are available commercially. Even though these tests are simple, they are often suited for small test volumes and are too expensive for high-throughput laboratories to use in a routine clinical diagnostic setting.In this study, we developed a novel multiplex SNaPshot method using fluorescently labeled terminators and capillary electrophoresis to screen for six common clinically encountered mycobacterial species (M. tuberculosis, M. avium, M. intracellulare, M. chelonae, M. kansasii, and M. gordonae) based on eight single nucleotide polymorphisms (SNPs) located in conserved regions of the 16S rRNA and Hsp65 genes.