MLVA和Spoligotyping用于西藏地区216株结核分枝杆菌临床分离株的基因分型研究

目的评价间隔区寡核苷酸分型（Spoligotyping）及多位点可变数量串联重复序列分析（MLVA）两种分型方法在西藏地区结核病分子流行病学中的应用。方法收集西藏地区结核分枝杆菌（Mycobacteriumtuberculosis）临床分离株，应用Spoligotyping及MLVA两种分型方法进行比较分析。结果共在西藏地区收集到216株结核分枝杆菌临床分离株，采用Spoligotyping分型方法，216株菌可分为3个基因群13种基因型，其中最大的1个基因群即北京家族（8eijingfamily）含有195株菌，占90．28％。北京家族菌株中，有BCG接种史者占45．64％（89／195），无BCG接种史者占54．36％（106／1195），两者间的差异无统计学意义（X^2=0．059，P〉0．05）。采用MLVA分型方法，216株菌可分成19个基因群108种基因型，其中80种基因型只有1株菌，占37．03％（80／216），另有136株菌表现出28种基因型，成簇数为28，占62．96％（136／216）。在20个VNTR位点的等位基因多态性发现Miru31位点的多态性最高，多态性指数（h）达到0．77，而Mtub29、Mtubl2位点的多态性较差，都低于0．05。其中Mtuh02位点可鉴别北京家族和非北京家族，它鉴别的北京家族与Spoligotyping鉴别的北京家族符合率达到100％。结论西藏地区结核分枝杆菌具有明显的接引多态性，其主要流行型为北京家族。北京家族菌株与BCG接种无相关性。应用Spoligotyping和MLVA两种分型方法进行结核病流行病学研究，将提高结核病的流行病学调查和病原学监测效果。     

Usefulness of Spoligotyping To Discriminate IS6110 Low-Copy-Number Mycobacterium tuberculosis Complex Strains Cultured in Denmark

Mycobacterium tuberculosis complex strains cultured in Denmark have been analyzed by IS6110 restriction fragment length polymorphism (RFLP) on a routine basis from 1992 and onwards. Due to the influx of immigrants with tuberculosis, the number of strains harboring only one to five copies of IS6110 has increased steadily. Since the discriminatory power of IS6110 fingerprinting for such strains is poor, we have performed additional genotyping of all low-copy-number strains by the recently described PCR-based method known as spoligotyping. A total of 311 clinical strains were typed: 14 Mycobacterium bovis BCG, 48 M. bovis, and 249 M. tuberculosis strains. Spoligotyping correctly differentiated M. bovis and M. bovis BCG from M. tuberculosis strains, but it did not differentiate M. bovis from M. bovis BCG. All M. bovis BCG strains exhibited identical spoligotype patterns. The discriminatory power of spoligotyping of low-copy-number M. tuberculosis strains was higher than that of IS6110 fingerprinting. Based on RFLP typing solely, 83% of the low-copy-number M. tuberculosis strains were found to form part of a cluster, and 75% were found to form a cluster on the basis of spoligotyping. When the two techniques were combined, the amount of clustering decreased to 55%. The combination of these two techniques might be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of the IS6110 element.     

Evaluation of Roche Amplicor PCR assay for Mycobacterium tuberculosis.

The Roche Amplicor Mycobacterium tuberculosis PCR test (RMtb-PCR) was compared with mycobacterial culture, with the BACTEC 460 system and inoculation on Lowenstein-Jensen media. Results were interpreted with an adjusted "gold standard" incorporating clinical diagnosis. A total of 1,480 clinical specimens from 1,155 patients, including tissues and fluids, as well as 141 specimens which demonstrated a positive growth index on the BACTEC 460 system were assessed. The sensitivity, specificity, and positive and negative predictive values of RMtb-PCR compared with the adjusted gold standard for clinical specimens were 79, 99, 93, and 98%, respectively. In smear-positive specimens, the sensitivity of RMtb-PCR was 98% versus 53% for smear-negative specimens. When RMtb-PCR was performed two times per week, PCR results were available an average of 21 days before the culture results. For specimens demonstrating a positive growth index on the BACTEC 460 system, RMtb-PCR had a sensitivity and specificity of 98 and 100%, respectively. This study demonstrates the value of a commercial nucleic acid amplification kit for rapid diagnosis of M. tuberculosis, particularly in smear-positive specimens or BACTEC culture-positive specimens.     

Evaluation of Cobas TaqMan MTB PCR for detection of Mycobacterium tuberculosis

Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a real-time PCR assay for M. tuberculosis complex, the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), was introduced. We performed a prospective study to evaluate the diagnostic performance of the Cobas TaqMan MTB test system. A total of 406 specimens collected from 247 patients were simultaneously tested by conventional culture, Cobas Amplicor MTB PCR, and TaqMan MTB PCR. The cross-reactivity with other Mycobacterium species and the detection limit were also evaluated. Among 406 specimens, a total of 24 specimens (5.9%) were culture positive: 14 specimens were positive by both TaqMan and Amplicor MTB PCRs, while 5 specimens were positive by only TaqMan PCR. The remaining five specimens were negative by both PCR methods. Seven specimens with negative culture results were positive by TaqMan PCR, but five of these were negative by Amplicor MTB PCR. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were 79.1%, 98.2%, 73.1%, and 98.7% for TaqMan and 58.3%, 99.5%, 87.5%, and 97.4% for the Amplicor MTB PCR test, respectively. There was no cross-reactivity with M. tuberculosis and nontuberculous mycobacterial species. The detection limit for the Cobas TaqMan MTB PCR test was 4.0 copies/μl. The Cobas TaqMan MTB PCR test showed higher sensitivity for detection of the M. tuberculosis complex without disturbing the specificity and NPV than the Amplicor MTB PCR test.     

Spoligotyping of Mycobacterium tuberculosis isolates from multiple-drug-resistant tuberculosis patients from Bombay,India

Spoligotyping was undertaken in 65 multiple-drug-resistant Mycobacterium tuberculosis isolates from Bombay, India. The spoligotype patterns showed seven closely related clusters, a cluster with 2 Beijing-like isolates, and unique spoligotypes (43%). Of the clusters, one with 29% of all the isolates suggested transmission of a dominant resistant clone.     

Evaluation of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Analysis and Spoligotyping for Genotyping of Mycobacterium bovis Isolates and a Comparison with Restriction Fragment Length Polymorphism Typing

Common strain typing methods for differentiation of Mycobacterium bovis isolates include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. MIRU-VNTR typing and spoligotyping were evaluated in this study, and these typing methods were compared with RFLP typing. A total of 386 M. bovis isolates from cattle, badgers, and deer in the Republic of Ireland that had previously been typed by IS6110, polymorphic GC-rich sequence (PGRS), and direct-repeat (DR) RFLP were included in the study. Spoligotyping and analysis of six VNTR loci (QUB 11a, QUB 11b, ETR A, 4052, MIRU 26, and 1895) were performed on the samples. RFLP analysis was the method that gave the greatest differentiation of strains, with a Hunter-Gaston discriminatory index (HGDI) of 0.927; the HGDI recorded for MIRU-VNTR typing was marginally lower at 0.918, and spoligotyping was the least discriminatory method, with an HGDI of 0.7. Spoligotype SB0140 represented approximately 50% of the isolates. Within the group of isolates represented by SB0140, there was a much lower level of concordance between RFLP and MIRU-VNTR typing than for groups represented by other spoligotypes. A combination of spoligotyping and MIRU-VNTR typing offered advantages over MIRU-VNTR typing alone. In a combined spoligotyping and MIRU-VNTR typing protocol, the number of VNTR loci could be reduced to four (QUB 11a, QUB 11b, ETR A, and 4052) while maintaining a high level of strain differentiation.The development of molecular techniques for differentiation of Mycobacterium bovis isolates has been of considerable benefit in epidemiological studies. Typing methods that have been commonly used include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing (7, 19).RFLP analysis of M. bovis isolates has commonly utilized polymorphism of the insertion sequence IS6110 and repetitive DNA elements such as the polymorphic GC-rich sequence (PGRS) and the direct-repeat (DR) region. Analysis of polymorphism of IS6110, the PGRS, and the DR region in combination has provided a high level of discrimination between strains (7, 19). REA has been widely used in New Zealand and has also given excellent resolution of strains (4). However, both RFLP analysis and REA require relatively large quantities of DNA and are laborious and time-consuming procedures. Complex banding patterns make analysis and interlaboratory comparisons difficult. Spoligotyping is a PCR-based typing method that reveals the presence or absence of unique spacer sequences located between the direct-repeat sequences of the DR region (12). It is a relatively easy procedure to perform, and the results can be expressed in a digital format. However, spoligotyping does not differentiate M. bovis strains to the same extent as RFLP analysis or REA (7, 19). Minisatellite-like loci in the Mycobacterium tuberculosis complex genome, described as mycobacterial interspersed repetitive units, may show polymorphism of the number of tandem repeats. A wide range of M. tuberculosis complex MIRU-VNTR loci have been evaluated, and loci which are informative for M. bovis isolates have been identified (8, 16, 17, 20, 23). Similar to spoligotyping, MIRU-VNTR typing has the advantages of ease of procedure and the generation of results in a digital format.In recent years, genotyping by IS6110, PGRS, and DR RFLP has been used in epidemiological studies of M. bovis infection in the Republic of Ireland (5, 6, 14). While RFLP analysis has given a high level of strain differentiation, its replacement by MIRU-VNTR typing or by a combination of MIRU-VNTR typing and spoligotyping offers potential advantages. The objective of this study was to evaluate MIRU-VNTR typing or a combination of MIRU-VNTR typing and spoligotyping for discrimination of M. bovis strains, to compare the discriminatory powers of the two methods against RFLP analysis, and to investigate the level of concordance between the three typing systems.     

Spoligotyping of Mycobacterium tuberculosis isolates among pulmonary tuberculosis patients in Amhara Region,Ethiopia

The aim of this study was to characterize Mycobacterium tuberculosis isolates circulating in the Amhara Region of Ethiopia. Sputum samples were collected from new pulmonary tuberculosis (TB) patients in the Region. Genotyping of mycobacterial DNA was performed by spoligotyping and isolates were assigned to families using the SpolDB4 and the model‐based program ‘Spotclust’. A high level of diversity was found among the 237 isolates. Sixty‐five different spoligopatterns were obtained. The T (30.8%), Central Asian (CAS; 21.1%) and U (17.7%) families were the predominant isolates comprising 69.6% of the total strains. Eighty‐five per cent of the U lineage belonged to Spoligo‐International‐Type (SIT) 910 and SIT 1729. Only a few of these strains are included in SpolDB4 to date. Of the total strains, 41 (17.3%) were unique and have not been described in SpolDB4 to date. This study indicated that the TB epidemic in Amhara Region, Ethiopia, is characterized by the circulation of numerous M. tuberculosis strain families. The high proportion of SIT 910 and SIT 1729 strains may indicate an increase in the importance of these lineages in the transmission of TB in the study region.     

Spoligotyping of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mumbai, India

Tuberculosis remains a major health problem in India, with 2 million new cases and 421,000 deaths each year. In this paper, we describe the spoligotyping results of 216 Mycobacterium tuberculosis culture isolates from patients with pulmonary tuberculosis in Mumbai, India. As spoligotyping data from India have rarely been described until now, and as there is limited information on the major circulating clades of M. tuberculosis, the data obtained were also compared to an international spoligotype database (SpolDB4) that contained patterns from 22,546 isolates from more than 100 countries. Eighty-four (39%) of the isolates were definitively marked as orphan strains, indicating the paucity of such data from India. The remaining 132 isolates clustered among 59 shared types; among these, 42 shared types were already present in the database, 17 were newly created, and 5 of them were specifically reported from Mumbai. A total of 9 major types in this study clustered 32% of the isolates. At the phylogenetic level, 30% of the isolates belonged to the Central Asian families CAS1 and CAS2, of the major genetic group (MGG) 1, 29% to MGG 2 and 3 families (spacers 33-36 missing) and 17% to the ancestral East African Indian (EAI) family. Finally, nearly 10% of the isolates belonged to the W-Beijing family in a broad sense, also in the MGG 1 group. In conclusion, historic clones of the MGG 1 group of M. tuberculosis are responsible for roughly 60% of all tuberculosis cases in Mumbai. Together with the fact that organisms presumably of European descent (such as the Haarlem family) were only rarely found, our observations suggest that tuberculosis in Mumbai, India is essentially caused by historical clones of tubercle bacilli undergoing active circulation due to uncontrolled demography, high prevalence of the disease, and a paucity of resources.     

Evaluation of a high-throughput repetitive-sequence-based PCR system for DNA fingerprinting of Mycobacterium tuberculosis and Mycobacterium avium complex strains

Repetitive-sequence-based PCR (rep-PCR) is useful for generating DNA fingerprints of diverse bacterial and fungal species. Rep-PCR amplicon fingerprints represent genomic segments lying between repetitive sequences. A commercial system that electrophoretically separates rep-PCR amplicons on microfluidic chips, and provides computer-generated readouts of results has been adapted for use with Mycobacterium species. The ability of this system to type M. tuberculosis and M. avium complex (MAC) isolates was evaluated. M. tuberculosis strains (n = 56) were typed by spoligotyping with rep-PCR as a high-resolution adjunct. Results were compared with those generated by a standard approach of spoligotyping with IS6110-targeted restriction fragment length polymorphism (IS6110-RFLP) as the high-resolution adjunct. The sample included 11 epidemiologically and genotypically linked outbreak isolates and a population-based sample of 45 isolates from recent immigrants to Seattle, Wash., from the African Horn countries of Somalia, Eritrea, and Ethiopia. Twenty isolates exhibited unique spoligotypes and were not analyzed further. Of the 36 outbreak and African Horn isolates with nonunique spoligotypes, 23 fell into four clusters identified by IS6110-RFLP and rep-PCR, with 97% concordance observed between the two methods. Both approaches revealed extensive strain heterogeneity within the African Horn sample, consistent with a predominant pattern of reactivation of latent infections in this immigrant population. Rep-PCR exhibited 89% concordance with IS1245-RFLP typing of 28 M. avium subspecies avium strains. For M. tuberculosis as well as M. avium subspecies avium, the discriminative power of rep-PCR equaled or exceeded that of RFLP. Rep-PCR also generated DNA fingerprints from M. intracellulare (n = 8) and MAC(x) (n = 2) strains. It shows promise as a fast, unified method for high-throughput genotypic fingerprinting of multiple Mycobacterium species.     

Typing of Mycobacterium tuberculosis Strains Resistant to Rifampicin and Isoniazid by Molecular Biological Methods

Mutations in the rpoB, katG, inhA, oxyR/ahpC genes in rifampicin- and isoniazid-resistant M. tuberculosis strains isolated from residents of Moscow, Astrakhan', and Moldova Republic were studied by molecular biological methods (heteroduplex analysis, single strand conformational polymorphism, biochips). Twenty-five combinations of mutations were detected. Some differences in the type distribution of detected mutations were found. The use of biochips is the most perspective method for determining the type of mutation.     

Characterization of Mycobacterium tuberculosis complex isolates from Greek patients with sarcoidosis by Spoligotyping

Spoligotyping was undertaken with 38 Mycobacterium tuberculosis isolates from Greek sarcoidosis patients and 31 isolates from patients with tuberculosis. Fifty percent of the isolates from sarcoidosis patients and 16.13% of the isolates from patients with tuberculosis were represented by a unique pattern, whereas the remaining isolates belonged to seven shared types. Interestingly, half of the isolates from sarcoidosis patients did not resemble the spoligotypes of the isolates from patients with tuberculosis, most of which pertained to shared spoligotypes.     

Enterobacterial Repetitive Intergenic Consensus Sequences as Molecular Targets for Typing of Mycobacterium tuberculosis Strains

The presence of enterobacterial repetitive intergenic consensus (ERIC) sequences was demonstrated for the first time in the genome of Mycobacterium tuberculosis; these sequences have been found in transcribed regions of the chromosomes of gram-negative bacteria. In this study genetic diversity among clinical isolates of M. tuberculosis was determined by PCR with ERIC primers (ERIC-PCR). The study isolates comprised 71 clinical isolates collected from Sardinia, Italy. ERIC-PCR was able to identify 59 distinct profiles. The results obtained were compared with IS6110 and PCR-GTG fingerprinting. We found that the level of differentiation obtained by ERIC-PCR is greater than that obtained by IS6110 fingerprinting and comparable to that obtained by PCR-GTG. This method of fingerprinting is rapid and sensitive and can be applied to the study of the epidemiology of M. tuberculosis infections, especially when IS6110 fingerprinting is not of any help.     

Evaluation of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and PCR for direct detection of Mycobacterium tuberculosis in clinical specimens.

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTD) is a direct specimen assay for the identification of Mycobacterium tuberculosis from respiratory samples. rRNA is amplified, and the product is detected with a specific chemiluminescent probe. We performed a retrospective evaluation of three separate respiratory specimens from each of 250 patients by using the AMTD and compared the results with those of microscopy, culturing, and a patient chart review. From the latter results, 198 patients (594 specimens) were found negative for M. tuberculosis by culturing and clinical criteria. The overall specificity of the AMTD after discrepancy resolution was 98.5% (585 of 594). There were 52 patients with culture-proven and/or clinically diagnosed tuberculosis. Of these 156 specimens, the organism was cultured from 142 (91%), and acid-fast microscopy was positive for 105 (67.3%). The AMTD was positive for 142 (91%) specimens from these patients. Tuberculosis patient samples were tested by a PCR assay which uses primers for amplification of the IS6110 insertion sequence of the M. tuberculosis complex. The PCR assay detected 144 of the 156 (92.3%) specimens. Overall, when three specimens per patient were examined, the AMTD found all 52 patients positive for tuberculosis, while the PCR assay found 51 patients positive by agarose gel analysis and all 52 patients positive by Southern blot hybridization.     

Use of Luminex MagPlex Magnetic Microspheres for High-Throughput Spoligotyping of Mycobacterium tuberculosis Isolates in Port-au-Prince,Haiti

Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in the resource-poor countries where the majority of tuberculosis cases occur. Spoligotyping is a PCR-based M. tuberculosis complex genotyping method with advantages of technical simplicity, numerical output, and high reproducibility. It is based on the presence or absence of 43 distinct “spacers” separating insertion elements in the direct repeat region of the M. tuberculosis genome. The spoligotyping assay involves reverse hybridization of PCR products to the capture spacers attached to nitrocellulose membranes or to microspheres. Here we report modification of the classic 43-spacer method using the new generation of Luminex multiplexing technology with magnetic microspheres. The method was successfully established and validated on strains with known spoligotypes in our laboratory in Haiti. The distribution of spoligotypes determined in a collection of 758 recent M. tuberculosis isolates was in accordance with previous data for Haitian isolates in the SITWITWEB international database, which were obtained with the traditional membrane-based method. In the present form, spoligotyping may be suitable as a high-throughput, first-line tool for genotyping of Mycobacterium tuberculosis in countries with limited resources.     

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.     

Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.     

Comparative Evaluation of the New Gen-Probe Mycobacterium tuberculosis Amplified Direct Test and the Semiautomated Abbott LCx Mycobacterium tuberculosis Assay for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory and Extrapulmonary Specimens

Two commercial assays that detect Mycobacterium tuberculosis complex (MTB) in clinical specimens by rRNA target amplification (AMTDII) and ligase chain reaction (LCx) were evaluated. The tests were applied to 457 respiratory (n = 273) and extrapulmonary (n = 184) specimens collected from 357 patients. The results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered to be the “gold standard.” Seventy specimens were from patients with pulmonary tuberculosis and 28 specimens were from patients with extrapulmonary tuberculosis. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values for respiratory specimens were 92.8, 99.4, 98.5, and 97%, respectively, for AMTDII and 75.7, 98.8, 96.4, and 90.5%, respectively, for LCx. With extrapulmonary specimens, the overall sensitivities, specificities, and positive and negative predictive values were 78.6, 99.3, 95.6, and 96.2%, respectively, for AMTDII and 53.6, 99.3, 93.7, and 92.1%, respectively, for LCx. The level of agreement between AMTDII and LCx assay results was 78.2%. We conclude that although both nucleic acid amplification methods are rapid and specific for the detection of MTB in clinical specimens, AMTDII is significantly more sensitive than LCx with both respiratory (P = 0.005) and extrapulmonary (P = 0.048) specimens.     

Identification of Rifampin-Resistant Mycobacterium tuberculosis Strains by Hybridization, PCR, and Ligase Detection Reaction on Oligonucleotide Microchips

Three new molecular approaches were developed to identify drug-resistant strains of Mycobacterium tuberculosis using biochips with oligonucleotides immobilized in polyacrylamide gel pads. These approaches are significantly faster than traditional bacteriological methods. All three approaches-hybridization, PCR, and ligase detection reaction--were designed to analyze an 81-bp fragment of the gene rpoB encoding the beta-subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides and enabled us to identify 30 mutant variants of the rpoB gene within 24 h. These variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allele-specific on-chip PCR, it was possible to directly identify mutations in clinical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a model mixture containing 1% of resistant and 99% of susceptible bacteria. This level of sensitivity is comparable to that from the determination of M. tuberculosis drug resistance by using standard bacteriological tests.     

PCR Methods for Rapid Identification and Characterization of Actinobacillus seminis Strains

Twenty-four isolates of Actinobacillus seminis were typed by PCR ribotyping, repetitive extragenic palindromic element (REP)-based PCR, and enterobacterial repetitive intergenic consensus (ERIC)-based PCR. Five types were distinguished by REP-PCR, and nine types were distinguished by ERIC-PCR. PCR ribotyping produced the simplest pattern and could be useful for identification of A. seminis and for its differentiation from related species. REP- and ERIC-PCR could be used for strain differentiation in epidemiological studies of A. seminis.