Evaluation of the GenoType Mycobacteria Direct assay for detection of Mycobacterium tuberculosis complex and four atypical mycobacterial species in clinical samples

We evaluated GenoType Mycobacteria Direct (GTMD), a novel commercial assay based on nucleic acid sequence-based amplification technology, for the detection of Mycobacterium tuberculosis complex, M. avium, M. intracellulare, M. kansasii, and M. malmoense directly from clinical specimens. A total of 134 respiratory and extrapulmonary samples from 65 patients were processed. Sensitivity, specificity, positive predictive, and negative predictive values for GTMD were 92, 100, 100, and 77%, respectively. The GMTD technique is useful, reliable, and rapid when used during the normal routine of a clinical laboratory.     

GenoType mycobacterium assay for identification of mycobacterial species isolated from human clinical samples by using liquid medium

The GenoType Mycobacterium assay was used to identify 98 mycobacteria isolates by using liquid cultures from positive BACTEC, MGIT, and ESP bottles. This system identifies 16 mycobacteria. There was complete agreement between the GenoType results and the laboratory identifications for Mycobacterium tuberculosis complex and other Mycobacterium spp. GenoType also identified mixed mycobacterial infections.     

Rapid detection of Mycobacterium tuberculosis complex and rifampin resistance in smear-negative clinical samples by use of an integrated real-time PCR method

Sixty-four of 85 (75.3%) smear-negative respiratory (n = 78) and nonrespiratory (n = 7) samples with positive cultures of Mycobacterium tuberculosis complex (MTC) were detected by the GeneXpert system using the Xpert MTB/RIF assay (GX). In addition, GX found rpoB mutations in all six of the rifampin-resistant strains detected. The test was negative in 20 culture-negative and 20 nontuberculous culture-positive samples (100% specificity). GX offers high potential for the diagnosis of tuberculosis due to its capacity for direct detection of MTC, its rapidity, and its simplicity.     

Usefulness of three-channel multiplex real-time PCR and melting curve analysis for simultaneous detection and identification of the Mycobacterium tuberculosis complex and nontuberculous mycobacteria

We attempted to determine the benefits of three-channel multiplex real-time PCR and melting curve analysis not only in detecting and distinguishing between nontuberculous mycobacteria (NTM) and the Mycobacterium tuberculosis complex but also in identifying NTM to the species level.     

Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB)

A novel duplex PCR method that can amplify the 235- and 136-bp rpoB DNAs of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM), respectively, with two different sets of primers was used to differentially identify 44 reference strains and 379 clinical isolates of mycobacteria in a single-step assay. Showing 100% sensitivity and specificity, the duplex PCR method could clearly differentiate M. tuberculosis complex and NTM strains. In addition, restriction fragment length polymorphism analysis and direct sequencing of the amplicon of NTM could be used to supplement species identification.     

Optimisation of acid fast smears for the direct detection of mycobacteria in clinical samples

AIMS: Despite its long history, the acid fast smear remains unstandardised. Technical variations in both the preparation of clinical material and subsequent staining mean that smear sensitivity relative to culture may vary from 50% to over 80%. This study assessed the sensitivity of acid fast microscopy at each of five stages of sample preparation and by both commonly used staining methods. METHODS: Sputum samples thought for varying reasons to be highly likely to be culture positive were used to prepare a series of smears in which the effects of digestion (liquefaction), concentration (centrifugation), and decontamination (sodium hydroxide) could be assessed, together with a comparison of staining by the auramine/phenol and Ziehl-Neelsen techniques. RESULTS: The most effective method for the demonstration of acid fast organisms in sputum was found to be an auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process. CONCLUSIONS: The auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process is the most effective method for the demonstration of acid fast organisms in sputum.     

Molecular strain identification of the Mycobacterium tuberculosis complex in archival tissue samples

AIMS: To investigate the use of different molecular analyses that can identify distinct strains of human pathogenic mycobacteria in formalin fixed and paraffin wax embedded archival tissue samples to see whether it is possible to differentiate between the members of the Mycobacterium tuberculosis complex (M tuberculosis, M bovis, M africanum, M microti, or M canettii) and/or substrains in a high number of samples. This would be of interest for identifying individual infection traits and superinfection by different mycobacterial strains. METHODS: Forty nine archival tissue samples with clinically and/or histologically suspected tuberculosis infection were subjected to molecular DNA analysis. RESULTS: The molecular analysis revealed the presence of M tuberculosis complex DNA in 20 samples, whereas acid fast bacilli could be detected by Ziehl-Neelsen staining in only eight samples. All IS6110 positive samples were further characterised by spoligotyping and seven cases provided M tuberculosis specific signatures, whereas M bovis specific signatures were obtained in four cases. The analysis of mtp40, oxyR, and pncA partial gene sequences confirmed the presence of M tuberculosis in six cases and M bovis in one case. The amplification and sequencing of four further genetic regions (katG, gyrA, TbD1, RD9) characterised six "modern" M tuberculosis strains belonging to genetic groups 2 or 3. CONCLUSION: This study provides clear evidence that archival paraffin wax embedded material can be used for further studies on the strain identification of M tuberculosis complex strains and can therefore unequivocally be used for the study of the epidemiology and evolution of tuberculosis pathogens.     

Molecular method for Bartonella species identification in clinical and environmental samples

A new, efficient molecular method for detection of Bartonella, based on the 16S-23S rRNA intergenic spacer and 16S rRNA amplification by multiplex PCR combined with reverse line blotting, was designed. This assay could simultaneously detect 20 different known species and other Bartonella species not described previously.     

Use of the Gen-Probe amplified mycobacterium tuberculosis direct test for early detection of Mycobacterium tuberculosis in BACTEC 12B medium

To achieve better sensitivity than direct testing and better turnaround time than current culture and identification methods, the Gen-Probe Mycobacterium Tuberculosis Direct method was used to detect Mycobacterium tuberculosis in BACTEC 12B medium cultures when they first gave a growth index (GI) of at least 10 (MTD/BACTEC method). Of 179 acid-fast, smear-positive specimens that were culture positive for M. tuberculosis, all were positive by the MTD/BACTEC method (sensitivity, 100%). Positive results were obtained only with tuberculosis patients. For diagnostic specimens from untreated patients, the mean time to achieve a GI of 10 was 6 days.     

Clinical Evaluation of the Gen-Probe amplified mycobacterium tuberculosis direct test for rapid detection of Mycobacterium tuberculosis in select nonrespiratory specimens

The performance of the Amplified Mycobacterium Tuberculosis Direct Test (MTD; Gen-Probe, Inc., San Diego, Calif.) for rapid diagnosis of extrapulmonary tuberculosis was evaluated by testing 178 nonrespiratory specimens from 158 patients. Criteria for specimen inclusion were (i) a positive smear for acid-fast bacilli (n = 54) and (ii) the source if the smear was negative (tissue biopsies and aspirates and abscess material were tested; n = 124). Results were compared to those of mycobacterial culture; clinical history was reviewed when MTD and culture results disagreed. Forty-eight specimens (27.0%) were positive for mycobacteria, including 23 Mycobacterium tuberculosis complex specimens; of which 21 were smear positive. Twenty-five specimens were MTD positive; 20 of these grew M. tuberculosis complex. All of the five MTD-positive, M. tuberculosis complex culture-negative specimens were considered truly positive, based on review of the medical record. Of the three MTD-negative, M. tuberculosis complex culture-positive specimens, two contained inhibitory substances; one of the two was smear positive. Excluding the latter specimen from analysis, after chart review, the sensitivity, specificity, and positive and negative predictive values of the MTD were 92.6, 100, 100, and 98.7%, respectively, by specimen and 89.5, 100, 100, and 98.6% by patient. Given the few smear-negative samples from patients with extrapulmonary tuberculosis in our study, additional similar studies that include more smear-negative, M. tuberculosis complex culture-positive specimens to confirm our data are desirable.     

Rapid identification of Candida species and other clinically important yeast species by flow cytometry

Two rapid diagnostic assays, utilizing two different Luminex flow cytometry methods, were developed for identification of clinically important ascomycetous yeast species. Direct hybridization and allele-specific primer extension methods were both successful in establishing a DNA-based assay that can rapidly and accurately identify Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis as well as other clinical species. The direct hybridization assay was designed to identify a total of 19 ascomycetous yeast species, and the allele-specific primer extension assay was designed to identify a total of 34 species. Probes were validated against 438 strains representing 303 species. From culture to identification, the allele-specific primer extension method takes 8 h and the direct hybridization method takes less than 5 h to complete. These assays represent comprehensive, rapid tests that are well suited for the clinical laboratory.     

Molecular method for identification of Rickettsia species in clinical and environmental samples

We present a PCR method targeting the 23S-5S internal transcribed spacer coupled with reverse line blotting that allows Rickettsia species detection and identification in a single step. The method is highly sensitive and specific in identifying Rickettsia species from both patient and environmental samples. The generic approach used allowed us to identify new pathogens.     

Molecular methods in the detection and identification of mycobacterial infections.

Nucleic acid amplification (NAA) tests for direct detection of Mycobacterium tuberculosis complex in respiratory specimens have the potential to provide a more rapid diagnosis of pulmonary tuberculosis (TB) than is currently possible by conventional stain, culture, and identification tests. Currently, 2 NAA tests-enhanced Amplified Mycobacterium Tuberculosis Direct (MTD) Test (Gen-Probe, Inc) and Amplicor Mycobacterium tuberculosis Test (Roche Molecular Systems, Inc)-have been approved by the Food and Drug Administration for testing respiratory specimens that are smear positive for acid-fast bacilli (AFB). This restriction to AFB smear-positive specimens was based on data from the initial clinical trials conducted to evaluate these products that showed low sensitivity (ie, 48%-53%) and less-than-optimal specificity (ie, 96%-99%) in AFB smear-negative specimens. Data from the clinical trial for the enhanced MTD test and from 2 subsequent studies, however, suggest that this version of the MTD test is a reliable tool for rapid diagnosis of pulmonary TB, regardless of the AFB smear result. Both NAA tests have been evaluated for diagnosis of extrapulmonary TB, and results were comparable to the results of tests performed with respiratory specimens. The NAA tests also appear to be reliable for rapid identification of M tuberculosis complex in positive broth cultures of all specimen types except blood. The impact of the NAA tests on patient outcome varies based on the AFB smear result. With smear-positive results, public health and hospital infection control resources are predominantly affected. With smear-negative results, however, the potential for affecting patient outcome is much greater. In patients with smear-negative results, the NAA test can result in earlier diagnosis of TB and subsequent initiation of therapy. Use of these tests also may eliminate the need for invasive diagnostic procedures, which are costly and pose an added risk to the patient, and they may allow earlier discharge of hospitalized patients.     

Evaluation of the enhanced amplified Mycobacterium tuberculosis direct test for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

OBJECTIVE: To evaluate the performance of the enhanced Mycobacterium Tuberculosis Direct Test (E-MTD), for the direct detection of M tuberculosis complex (MTBC) in respiratory specimens. DESIGN: Two hundred seventy-four respiratory specimens from 151 patients in respiratory isolation were tested with the E-MTD, and the results were compared with the results of mycobacterial smear, culture, and the earlier form of the test, MTD-1. RESULTS: Forty-one specimens were culture positive for mycobacteria (20 MTBC and 21 nontuberculous mycobacteria), 23 of which were smear positive (16 MTBC, 7 nontuberculous mycobacteria). Twenty-four specimens were positive by E-MTD, and 21 were positive by MTD-1. Of the 20 MTBC culture-positive specimens, 19 were positive by the E-MTD and 19 were positive by the MTD-1. The remaining specimens were MTBC negative by all methods. After resolution of discrepancies, the sensitivity, specificity, and positive and negative predictive values were 95.2%, 100%, 100%, 99.6% for the MTD-1 and 95.2%, 98.8%, 87.0%, and 99.6%, for the E-MTD. For the E-MTD smear-positive and smear-negative specimens, these same values were 93.8%, 100%, 100%, and 87.5% and 100%, 98.8%, 62.5%, and 100%, respectively. CONCLUSION: The results suggest that the E-MTD is a reliable method for the direct detection of MTBC in smear-positive respiratory specimens.     

Simultaneous detection of four human pathogenic microsporidian species from clinical samples by oligonucleotide microarray

Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microarray assay for the simultaneous detection and identification to the species level of four major microsporidian species: Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis. The 18S small-subunit rRNA gene was chosen as the amplification target, labeled with fluorescence dye, and hybridized to a series of species-specific oligonucleotide probes immobilized on a microchip. The specificity and sensitivity of the microarray were clearly demonstrated by the unique hybridization profiles exhibited by each species of microsporidian tested and its ability to detect as few as 10 spores. In order to assess the applicability of this microarray in a clinical setting, we conducted microarray assays of 20 fecal samples from AIDS patients. Twelve of these samples were positive for the presence of microsporidia and could be confidently identified; 11 of them were positive for more than one species. Our results suggested that this microarray-based approach represents an attractive diagnostic tool for high-throughput detection and identification of microsporidian species in clinical and epidemiological investigations.     

Molecular identification of species within the Mycobacterium tuberculosis complex using regions of difference

The differentiation within the Mycobacterium tuberculosis complex (MTBC) based on phenotypic methods is long and does not give an unambiguous result in every case whereas the advance in genetic knowledge leads to new views. Thus, regions of difference (RD), that seem to characterize the species of the MTBC, have been identified. Amplification methods, targeted on these zones, have been developed then. The study of four regions (RD1, RD5, RD9, RD10) has been done on 64 isolates formerly identified thanks to phenotypic methods. Genotypic results confirm phenotypic identifications except in one case. This strain initially identified as M. tuberculosis and isolated from a Gabonese patient, corresponds, according to genotypic identification, to M. africanum. Since phenotypic characterization of M. africanum is difficult, this method would allow to accurately determine the true prevalence of this specie. Moreover, the study of RD10 doesn't seem to be informative. The amplification of only two RD, RD1 and RD9 carries out the identification of all M. tuberculosis and M. bovis BCG isolates. M. bovis and M. africanum will be then identified thanks to RD5. Thus, this easy and rapid method of identification of the major species of MTBC seems to be appropriated for a routine use.     

Effectiveness of an integrated real-time PCR method for detection of the Mycobacterium tuberculosis complex in smear-negative extrapulmonary samples in an area of low tuberculosis prevalence

Early extrapulmonary tuberculosis (EPTB) diagnosis is particularly difficult. Among 108 smear-negative extrapulmonary samples showing a positive culture for Mycobacterium tuberculosis complex (43 body fluids and 65 nonliquid specimens), 63 (58.3%) were positive with the Xpert MTB/RIF assay (GX). GX sensitivity was quite low for samples from sterile locations (especially for pleural fluids: 26.9%) but high for some nonliquid samples, like abscess aspirates (76.5%). In summary, GX may be a useful tool to be considered for EPTB diagnosis.     

Simultaneous identification of Mycobacterium genus and Mycobacterium tuberculosis complex in clinical samples by 5'-exonuclease fluorogenic PCR

Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5'-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosis complex (MTC) and members of the Mycobacterium genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.