Doris Hillemann

Rapid Molecular Detection of Tuberculosis and Rifampin Resistance

BACKGROUND Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect. METHODS We assessed the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), with fully integrated sample processing in 1730 patients with suspected drug-sensitive or multidrug-resistant pulmonary tuberculosis. Eligible patients in Peru, Azerbaijan, South Africa, and India provided three sputum specimens each. Two specimens were processed with N-acetyl-L-cysteine and sodium hydroxide before microscopy, solid and liquid culture, and the MTB/RIF test, and one specimen was used for direct testing with microscopy and the MTB/RIF test. RESULTS Among culture-positive patients, a single, direct MTB/RIF test identified 551 of 561 patients with smear-positive tuberculosis (98.2%) and 124 of 171 with smear-negative tuberculosis (72.5%). The test was specific in 604 of 609 patients without tuberculosis (99.2%). Among patients with smear-negative, culture-positive tuberculosis, the addition of a second MTB/RIF test increased sensitivity by 12.6 percentage points and a third by 5.1 percentage points, to a total of 90.2%. As compared with phenotypic drug-susceptibility testing, MTB/RIF testing correctly identified 200 of 205 patients (97.6%) with rifampin-resistant bacteria and 504 of 514 (98.1%) with rifampin-sensitive bacteria. Sequencing resolved all but two cases in favor of the MTB/RIF assay. CONCLUSIONS The MTB/RIF test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than 2 hours with minimal hands-on time. (Funded by the Foundation for Innovative New Diagnostics.)

Evaluation of the GenoType MTBDRplus Assay for Rifampin and Isoniazid Susceptibility Testing of Mycobacterium tuberculosis Strains and Clinical Specimens

ABSTRACT The new GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany) was tested with 125 clinical isolates and directly with 72 smear-positive sputum specimens for its ability to detect rifampin (RMP) and isoniazid (INH) resistance in Mycobacterium tuberculosis complex (MTBC) strains. In total, 106 RMPr/INHr, 10 RMPs/INHr, and 80 RMPs/INHs MTBC strains were comparatively analyzed with the new and the old MTBDR assays. Besides the detection of mutations within the 81-bp hot spot region of rpoB and katG codon 315, the GenoType MTBDRplus assay is designed to detect mutations in the regulatory region of inhA. The applicability of the new assay directly to specimens was shown, since 71 of 72 results for smear-positive sputa and all 125 results for clinical isolates were interpretable and no discrepancies compared with the results of real-time PCR or DNA sequencing were obtained. In comparison to conventional drug susceptibility testing, both assays were able to identify RMP resistance correctly in 74 of 75 strains (98.7%) and 30 of 31 specimens (96.8%). The misidentification of RMP resistance was obtained for two strains containing rpoB P533L mutations. Compared to the old MTBDR assay, the new GenoType MTBDRplus assay enhanced the rate of detection of INH resistance from 66 (88.0%) to 69 (92.0%) among the 75 INH-resistant strains and 36 (87.8%) to 37 (90.2%) among the 41 specimens containing INH-resistant strains. Thus, the new GenoType MTBDRplus assay represents a reliable and upgraded tool for the detection of INH and RMP resistance in strains or directly from smear-positive specimens.

Rapid Molecular Detection of Extrapulmonary Tuberculosis by the Automated GeneXpert MTB/RIF System

ABSTRACT In total, 521 nonrespiratory specimens (91 urine, 30 gastric aspirate, 245 tissue, 113 pleural fluid, 19 cerebrospinal fluid [CSF], and 23 stool specimens) submitted to the German National Reference Laboratory for Mycobacteria (NRL) from May 2009 to August 2010 were comparatively investigated with the new molecular-based GeneXpert MTB/RIF (Xpert) assay system and conventional liquid and solid culture methods. Twenty (3.8%) of the 521 specimens gave no interpretable result. Whereas the sensitivity of the Xpert assay with tissue specimens was 69.0% (20 out of 29 culture-positive cases detected), 100% sensitivity was found with the urine and stool specimens. The combined sensitivity and specificity of the Xpert assay were calculated to be 77.3% and 98.2%, respectively.

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

ABSTRACT 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.

Use of the Genotype MTBDR Assay for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates

ABSTRACT A commercially available DNA strip assay (Genotype MTBDR; Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect mutations conferring resistance to rifampin (RMP) and isoniazid (INH) in clinical Mycobacterium tuberculosis complex isolates. A total of 103 multidrug-resistant (MDR; i.e., at least resistant to RMP and INH) and 40 fully susceptible strains isolated in Germany in 2001 in which resistance mutations have been previously defined by DNA sequencing and real-time PCR analysis were investigated. The Genotype MTBDR assay identified 102 of the 103 MDR strains with mutations in the rpoB gene (99%) and 91 strains (88.4%) with mutations in codon 315 of katG. All 40 susceptible strains showed a wild-type MTBDR hybridization pattern. The concordance between the MTBDR assay and the DNA sequencing results was 100%. Compared to conventional drug susceptibility testing, the sensitivity and specificity were 99 and 100% for RMP resistance and 88.4 and 100% for INH resistance, respectively. In conclusion, the MTBDR assay is a rapid and easy-to-perform test for the detection of the most common mutations found in MDR M. tuberculosis strains that can readily be included in a routine laboratory work flow.

Molecular Characterization of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis Strains Isolated in Poland

A total of 105 rifampin (RMP)- and/or isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different parts of Poland in 2000 were screened for mutations associated with resistance to these drugs by two molecular methods, namely sequence analysis and real-time PCR technology. Three loci associated with drug resistance were selected for characterization: they were rpoB (RMP), katG, and the regulatory region of inhA (INH). Nineteen different mutations were identified in 64 RMP-resistant strains, and five new alleles were described. The most common point mutations were in codons 531 (41%), 516 (16%), and 526 (9%) of the rpoB gene. Mutations were not found in two (3%) of the isolates. In the case of resistance to INH, six different mutations in the katG gene of 83 resistant strains were detected. Fifty-seven (69%) isolates exhibited nucleotide substitutions at codon 315. One strain harbored a mutation affecting codon 279 (Gly279Thr). Twelve of 26 INH-resistant strains with the wild-type codon 315 (14.5% of all strains tested) had the mutation -15C-->T in the regulatory region of inhA. A full correlation between the DNA sequence analysis and real-time PCR data was obtained. We conclude that the real-time PCR method is fast and reliable for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.ABSTRACT Molecular characterization of the drug resistance of Mycobacterium tuberculosis strains with different origins can generate information that is useful for developing molecular methods. These methods are widely applicable for rapid detection of drug resistance. A total of 166 rifampin (RIF)- and/or isoniazid (INH)-resistant strains of M. tuberculosis have been isolated from different parts of Vietnam; they were screened for mutations associated with resistance to these drugs by sequence analysis investigating genetic mutations associated with RIF and INH resistance. Seventeen different mutations were identified in 74 RIF-resistant strains, 56 of which (approximately 76%) had mutations in the so-called 81-bp “hot-spot” region of the rpoB gene. The most common point mutations were in codons 531 (37.8%), 526 (23%), and 516 (9.46%) of the rpoB gene. Mutations were not found in three strains (4.05%). In the case of INH resistance, five different mutations in the katG genes of 82 resistant strains were detected, among which the nucleotide substitution at codon 315 (76.83%) is the most common mutation. This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis strains from Vietnam, and detection of the katG and rpoB mutations of the INH and RIF-resistant strains should be useful for rapid detection of the INH- and RIF-resistant strains by molecular tests.

Evaluation of the GenoType Mycobacterium Assay for Identification of Mycobacterial Species from Cultures

ABSTRACT A new commercially available DNA strip assay (GenoType Mycobacterium CM/AS; Hain Lifescience, Nehren, Germany) was evaluated for the ability to differentiate mycobacterial species. The test is based on a PCR technique targeting a 23S rRNA gene region, followed by reverse hybridization and line probe technology. The GenoType CM is capable of identifying 23, the GenoType AS a further 14, species either alone or in combination with one or more species. Both tests were evaluated with 156 mycobacterial strains composed of 61 validly published species including different subspecies, 6 not validly published species, and 3 strains other than mycobacterial species. All strains were precharacterized by sequencing of the 5′ region of the 16S rRNA gene and biochemical tests. In total, results for 151 strains were interpretable. Concordant results were obtained for 137 (92.6%) of 148 mycobacterial strains with the CM assay and 133 (89.9%) of 148 mycobacterial strains with the AS assay, and all three non-Mycobacterium species were identified.

First Linezolid-Resistant Clinical Isolates of Mycobacterium tuberculosis

ABSTRACT Linezolid resistance was found in 4 (1.9%) of 210 multidrug-resistant Mycobacterium tuberculosis strains. The MICs of linezolid were 4 μg/ml (one strain) and 8 μg/ml (three strains). Since no mutations were detected in potential target genes, the mechanism of resistance remains unclear.

A Multisite Assessment of the Quantitative Capabilities of the Xpert MTB/RIF Assay

RATIONALE The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. OBJECTIVES Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. METHODS Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. MEASUREMENTS AND MAIN RESULTS Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. CONCLUSIONS Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

In Vitro-Selected Linezolid-Resistant Mycobacterium tuberculosis Mutants

Development of resistance against linezolid, an alternative drug for the therapy of multidrug-resistant tuberculosis (2), was assessed to be rare (5). We aimed to generate linezolid-resistant Mycobacterium tuberculosis strains from 10 different, fully susceptible M. tuberculosis parental strains. Ten linezolid-resistant colonies could be isolated from six different parental strains. The frequency of the in vitro appearance of linezolid-resistant mutants was 2 × 10−8 to 5 × 10−9. Previous investigations with a genetically engineered Mycobacterium smegmatis derivative that harbored only one of the original two copies of the rrn operon revealed a similar rate of 4.5 × 10−9 (6). MIC value determination gave identical results performed with both Bactec 460 TB system and Bactec MGIT 960 (Becton Dickinson Diagnostic Systems, Sparks, MD). MIC values of the linezolid-resistant strains varied from 4 to 32 μg/ml, whereas all parent strains had MIC values of ≤1 μg/ml. Genotypic characterization of the linezolid-resistant strains was performed by sequencing of the 23S rRNA gene (5). Overall, in five strains mutations in the 23S rRNA gene were found. Four strains (derived from three different parental strains) with a MIC value of 32 μg/ml showed an identical G-to-T base pair exchange at position 2061; another strain with a MIC value of 16 μg/ml showed a G-to-T base pair exchange at position 2576 (Fig. ​(Fig.1).1). As a control, the respective parental strains had no alteration in the 23S rRNA gene when aligned with the M. tuberculosis H37 wild-type sequence. The remaining five strains with MIC values from 4 to 8 μg/ml showed no mutations in the 23S rRNA gene. FIG. 1. Secondary structure of the central loop of domain V of M. tuberculosis 23S rRNA (modification of the structures presented in references 6 and 7). The base exchanges determined in the in vitro-selected linezolid-resistant M. tuberculosis strains are indicated ... To investigate whether in vitro-generated linezolid resistance influences the growth rate, suspensions of parental susceptible and resistant strains were inoculated into MGIT tubes (0.1 ml of a 1:100 dilution of a McFarland 0.5 suspension). The average time to detection (TTD) of parental strains was 5.93 ± 1.84 days (mean ± standard deviation). Linezolid-resistant strains without mutation in the 23S rRNA showed a TTD of 5.81 ± 1.81 days. However, linezolid-resistant strains with mutations in the 23S rRNA showed an elevated TTD of 10.10 ± 3.49 days. The isolated M. tuberculosis mutants can be divided into two classes corresponding to in vitro-generated linezolid-resistant M. smegmatis clones (6). In that study, one class showed wild-type growth characteristics in cultures, lower MIC values of 4 to 8 μg/ml, and no mutation in the 23S rRNA, pointing to a nonribosomal mechanism of resistance (6). Mutants of the other class had alterations in domain V of 23S rRNA, high MIC values of ≥64 μg/ml, and a decreased growth rate in culture. In contrary to the study by Sander et al., who found exclusively G2447T mutations in M. smegmatis, different mutations were found in M. tuberculosis and other bacterial species (1, 3, 4). Whereas the G2576T mutation is well known and often described as the predominant mutation in various other gram-positive bacteria, such as Enterococcus faecalis and Staphylococcus aureus (3, 4, 7), to our knowledge the G2061T mutation found in four M. tuberculosis strains is described here for the first time.