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1 Scottish Mycobacteria Reference Laboratory, Department of Laboratory Medicine, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh EH16 4SA, UK
2 Microbiology Department, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK
3 Mast Group Ltd, MAST House, Derby Road, Bootle, Merseyside L20 1EA, UK
Correspondence
A.-Louise Seagar
Louise.Seagar{at}luht.scot.nhs.uk
Received 29 June 2007
Accepted 2 February 2008
Abbreviations: AFB, acid-fast bacilli; CDAT, commercial direct amplification test; GTMD, GenoType Mycobacteria Direct; MTBC, Mycobacterium tuberculosis complex; NTM, non-tuberculous mycobacteria; SMRL, Scottish Mycobacteria Reference Laboratory; TB, tuberculosis.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Historically, laboratory diagnosis of MTBC infection has relied heavily on the time-consuming process of culturing organisms from clinical specimens using solid or liquid media. The advent of fast, reliable and sensitive molecular techniques for the direct detection of MTBC in smear-positive respiratory samples has the potential to revolutionize laboratory and clinical practice. Indications for their use in clinical samples are now included in British tuberculosis guidelines (National Collaborating Centre for Chronic Conditions, 2006). Several commercial direct amplification tests (CDATs) have been developed to allow rapid detection of mycobacteria in pulmonary and extrapulmonary specimens. Such assays work on the basic principle of nucleic acid amplification, although different enzymes and strategies are employed (Table 1
). Species identification is usually limited to MTBC organisms, and an internal amplification control to monitor amplification inhibition is not always included. Nonetheless, they generally have good specificity and sensitivity for smear-positive respiratory specimens when compared with culture and clinical diagnosis and have the potential to substantially reduce the time taken to diagnosis. Non-sputum samples such as pleural fluids, cerebrospinal fluids and smear-negative samples are associated with lower test sensitivities (Piersimoni & Scarparo, 2003).
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Microscopic examination. A loopful of each specimen was placed directly on a microscope slide and stained with auramine phenol using standard procedures. Slides were examined at x400 magnification. The number of AFB present was recorded as + (1–10 bacilli in ten fields), ++ (1–10 bacilli per field) or +++ (10 or more bacilli per field).
Specimen processing and decontamination
In some cases, referred specimens were of small volume (<5 ml) so there was limited material available for additional tests once a sufficient volume had been allocated for routine tests. To assess the effect of specimen volume and the choice of decontamination method on test performance, three different approaches were used for specimen preparation using two different decontamination chemicals.
Group A: N-acetyl-L-cysteine (NALC)-treated ‘split’ specimens. After direct microscopy had been performed at the SMRL, 22 samples containing sufficient volume were ‘split’ into two equal aliquots of at least 1 ml each and decontaminated using NALC/3 % NaOH (referred to as the NALC method). One aliquot was processed for routine mycobacterial culture using solid egg medium (two modified Löwenstein–Jensen slopes, one containing glycerol and one containing pyruvate as growth supplements) and rapid liquid culture (Mycobacterial Growth Indicator Tube; Becton Dickinson). The other aliquot was either decontaminated and tested immediately using the GTMD test (n=2) or the respiratory specimen was stored at –70 °C (n=20) for no longer than 7 months (mean 2.75 months). NALC decontamination was performed on the stored specimen and the resulting pellet was resuspended in 0.5 ml phosphate buffer before starting the cell-disruption stage of the GTMD test.
Group B: NALC-treated ‘whole’ specimens. The number of specimens that could be collected in group A was limited, as only those where excess specimen material was available at SMRL could be divided. Therefore, for 17 sputum samples, the entire specimen volume was decontaminated using the NALC method. The resulting pellet was then resuspended in 1.5 ml phosphate buffer. One millilitre was used to inoculate solid and liquid culture media (as outlined above) and the remaining 0.5 ml volume was stored at 4 °C (for no longer than 3 days).
Group C: NaOH-treated ‘whole’ specimens. A modified Petroff's method using 4 % NaOH (referred to as the NaOH method) was used at the Royal Brompton Hospital for decontamination of the whole volume from 15 samples. After resuspending the pellet in 1.5 ml sterile distilled water, 1 ml was used for mycobacterial culture and the remaining 0.5 ml was stored at 4 °C (for no longer than 3 days) until the cell-disruption stage of the GTMD test was performed. In five cases, the samples were stored at –20 °C (for less than 1 month) until testing.
Identification of cultured isolates. Culture isolates were identified using the GenoType Mycobacterium CM assay (Hain Lifescience), which allows identification of 15 different mycobacterial species (M. avium, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium gordonae, M. intracellulare, Mycobacterium scrofulaceum, Mycobacterium interjectum, M. kansasii, M. malmoense, Mycobacterium peregrinum, Mycobacterium marinum/Mycobacterium ulcerans, Mycobacterium xenopi and MTBC). If identification was not possible with this assay, then the GenoType Mycobacterium AS test (Hain Lifescience) was used for the identification of additional mycobacterial species: Mycobacterium simiae, Mycobacterium mucogenicum, Mycobacterium goodii, Mycobacterium celatum, Mycobacterium smegmatis, Mycobacterium genavense, Mycobacterium lentiflavum, Mycobacterium heckeshornense, Mycobacterium szulgai/Mycobacterium intermedium, Mycobacterium phlei, Mycobacterium haemophilum, M. kansasii, Mycobacterium ulcerans, Mycobacterium gastri, Mycobacterium asiaticum and Mycobacterium shimoidei. Both tests were performed following the manufacturer's instructions as outlined briefly below. Liquid and solid cultures were considered negative if no growth was obtained after 6 or 8 weeks' incubation, respectively.
GTMD method. The complete procedure is divided into four stages. (i) Following heat lysis and sonication of cells, magnetic beads were used to capture the 23S rRNA in a coupling reaction. (ii) After two washing steps, a nucleic acid sequence-based amplification reaction was carried out at 41 °C for 1 h to amplify sequences from the ssRNA template. (iii) The denatured amplified products were subsequently identified by reverse hybridization to immobilized gene probes. (iv) Colorimetric detection of the hybridized amplicons was carried out using streptavidin-conjugated alkaline phosphatase and the appropriate substrate.
Internal control RNA was included with every test sample to check for inhibitory substances. Positive and negative controls were included in each test run. The positive control contained M. avium RNA to assess the performance of the test and the negative control indicated whether sample-to-sample contamination had occurred. RNA contamination was limited by swabbing benches and equipment with bleach before use. To interpret the test results, a template sheet showing the positions of the lines on each strip (provided with the kit) was used. The assay time was approximately 5 h, allowing the results to be generated in one working day.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Microscopy was performed on 53/56 respiratory samples using auramine phenol staining. Microscopy was not performed on three sputum specimens that had been received as part of an external quality assurance programme. Twenty-two samples were found to contain few AFB (+), 11 contained moderate numbers of AFB (++) and 20 contained many AFB (+++).
In group A, 17/20 samples (85 %) in which RNA amplification was successful produced GTMD test results that concurred with the identification of the cultured isolates (Table 2). Eleven samples produced the hybridization pattern of MTBC and two showed that of M. malmoense. The four samples that failed to produce any hybridization signal to the five probes on the strips were shown by culture to contain other atypical mycobacteria (M. xenopi, M. fortuitum, M. simiae and M. celatum). These negative GTMD findings were in agreement with the culture results, as these mycobacterial species are not detectable by the molecular test. Three samples gave negative GTMD results although M. avium and M. kansasii were identified by specimen culture. Microscopic analysis showed that these specimens contained only small numbers of bacilli prior to being divided for decontamination and therefore there may have been too few bacilli for detection.
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In group B, 14/17 GTMD test results (82 %) were concordant with the culture results (Table 3). Seven samples were found to contain MTBC, three samples contained M. kansasii and one contained M. malmoense. Two samples were culture-negative after 8 weeks' incubation and thus did not appear to contain viable mycobacteria. In addition, one smear-positive sample was found to contain M. intracellulare using the GTMD test, but the isolate failed to grow on culture after 8 weeks, indicating a false-negative culture result. Two samples produced growth of M. avium but gave negative GTMD results. A further sample was found to contain M. kansasii using the GTMD test but contained M. abscessus only on culture. This sample had been taken from a patient with cystic fibrosis who had previous isolates of M. kansasii. It is probable that M. kansasii was also present in the sputum but was masked by the rapidly growing M. abscessus on the solid culture medium.
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). Four samples contained MTBC, three samples contained M. kansasii, one contained M. malmoense and three contained other atypical mycobacteria (two M. abscessus and one M. xenopi). One sample was negative by both culture and the molecular test. Three samples gave false-negative GTMD results, as M. avium was obtained by culture.
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Two samples were omitted from the analysis due to partial inhibition or lack of amplification of the internal control RNA. In 52 samples in which amplification occurred successfully, 43 (82.6 %) produced final GTMD results that were confirmed by culture (Table 5). All cases of culture-positive MTBC were detected by the molecular test. Twenty-two samples contained MTBC, seven had M. kansasii, four had M. malmoense, one had M. intracellulare, seven contained other atypical mycobacteria not detectable using the GTMD assay and three specimens contained no viable mycobacteria. Results for eight of the remaining nine samples indicated false-negative GTMD results, as M. avium (n=7) and M. kansasii (n=1) were isolated upon specimen culture. The molecular test indicated M. avium from one smear-positive sample that was culture-negative. Although the number of samples tested was small, the decontamination method used did not appear to have any effect on assay performance, with 82 % of NALC-treated specimens and 80 % of NaOH-treated specimens producing concordant GTMD and culture identification results.
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For optimal assay performance, we recommend that the entire specimen volume is used for processing, as dividing the specimen into two equal volumes prior to NALC decontamination (as for group A) may have accounted for three of the seven false-negative GTMD test results (two M. avium and one M. kansasii). Microscopic examination showed few AFB (+) to be present in these specimens, which were divided further prior to decontamination. This may have resulted in too few bacilli in the aliquot tested to be detected by the GTMD assay. From a laboratory perspective, the processing protocols used for group B and C specimens were found to be more appropriate, as the entire specimen was decontaminated using either NALC/3 % NaOH (group B) or 4 % NaOH (group C) without any adverse effect on the GTMD results. It was possible to detect mycobacterial RNA after storage of specimens at –70 °C for 5 months, but freezing specimens for as long as 7 months is not recommended as RNA degradation may occur.
The GTMD assay is easy to perform and can be completed in one working day. Although it takes longer than some of the other commercially available direct nucleic acid amplification methods, it does provide rapid detection of four additional mycobacterial species rather than MTBC alone. Interpretation of GTMD test results compared with some other commercial nucleic acid assays is also made easier for the user by the inclusion of an internal amplification control to monitor the presence of inhibitors that interfere with nucleic acid amplification. Such inhibitors can lead to false-negative results in 3–15 % of clinical samples (Forbes & Hicks, 1996). In our study, RNA amplification was successful in most cases and thus nucleic acid amplification inhibitors did not appear to be a major problem.
It is difficult to compare the results from this work directly with the large number of other reports published using CDATs. The literature reflects the fact that groups have assessed different CDATs using different specimen types and assessed their outcome using different criteria. Compared with culture, our results show the sensitivity, specificity, and positive and negative predictive values for the detection of MTBC, M. avium, M. intracellulare, M. kansasii and M. malmoense using GTMD to be 80.5, 75, 97 and 27.3 %, respectively. These figures differ from the results of 92, 100, 100 and 77 % from a larger study by Franco-Álvarez de Luna et al. (2006) primarily due to the larger proportion of false-negative GTMD results obtained in our study (17 vs 5.9 %). Nonetheless, we are encouraged by the results from this work and believe that a GTMD-positive result has the potential to significantly improve patient care and public health by providing rapid and specific information to clinicians about the presence of MTBC and some other common atypical mycobacteria in smear-positive samples. Further work is required to assess the sensitivity of the assay for non-respiratory samples and smear-negative samples, although some success has been reported with identification of MTBC in biopsy specimens, bronchial aspirates, an abscess and a faecal specimen (Franco-Álvarez de Luna et al., 2006). In our hands, the GTMD test has identified M. tuberculosis in one tissue specimen. The isolate had failed to grow on solid culture, despite prolonged incubation. From a laboratory perspective, a new version (version 4.0) of the GTMD assay includes a modified protocol to reduce the amount of specimen manipulation required during the RNA isolation stage and to improve sensitivity. Rapid diagnosis and differentiation between MTBC and NTM allows the appropriate drug susceptibility panel to be tested as soon as the isolate has grown. Whilst this can save time in the laboratory, far greater savings are likely across public health due to improved patient care and infection control measures as a result of a speedier identification service.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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