A Simple and Efficient Multiplex PCR Assay for the Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex to the Species Level

Purpose

The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guérin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health.

Materials and Methods

A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates.

Results

All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level.

Conclusion

The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.     

Genetic diversity of Mycobacterium tuberculosis in Mbarara, South Western Uganda

Background

We determined the genetic diversity of mycobacteria isolated from tuberculosis patients in Mbarara Uganda, using region of difference (RD) analysis and spacer oligonucleotide typing (spoligotyping).

Methods

Sputum samples were cultured on Lowenstein Jensen media. The isolates were characterized using RD analysis and spoligotyping.

Results

The majority (92.8%) of the patients were new cases, 60% were males and 44% were HIV positive with a mean age of 33.7 years. All the 125 isolates were identified as M.tuberculosis sensu stricto. Most (92.8%) of the isolates were modern strains. Spoligotyping revealed 79 spoligotype patterns, with an overall diversity of 63.2%. Sixty (48%) isolates formed 16 clusters each consisting of 2–15 isolates. Mst (59.2 %) of the isolates were Uganda genotype strains. The major shared spoligotypes in our sample were SIT 135 (T2-Uganda) with 12 isolates and SIT 128 (T2) with 5 isolates. Sixty nine (87%) patterns had not yet been defined in the SpolDB4.0.database.

Conclusion

The TB epidemic in Mbarara is caused mainly by modern M.tuberculosis strains of the Uganda genotype. The wide diversity of strains may indicate that the majority of the TB cases are reactivation rather than re-infection. However this needs to be ascertained with more discriminative finger printing techniques.     

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

Purpose

Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed.

Materials and Methods

The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains.

Results

The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%).

Conclusion

The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.     

Modern lineages of Mycobacterium tuberculosis in Addis Ababa,Ethiopia: implications for the tuberculosis control programe

Background

The genotyping of Mycobacterium tuberculosis strains is important to have unique insights into the dissemination dynamics and evolutionary genetics of this pathogen and for TB control as it allows the detection of suspected outbreaks and the tracing of transmission chains.

Objective

To characterize M. tuberculois isolates collected from newly diagnosed pulmonary TB patients in Addis Ababa

Methods

One hundred and ninety two sputum samples were cultured on Löwenstein-Jensen (LJ) slants and isolates were heat killed for molecular genotyping. The isolates were characterized using spoligotyping and were compared with the International SpoIDB4 database.

Result

T genotype constitutes the most predominant in our study (95, 49.5%) followed by the CAS genotype (42, 21.9%). Other genotypes found were Haarlem (H) (24, 12.5%), the LAM (3, 1.5%), the Beijing genotype (1, 0.5%); four (2.1%) isolates were designated as Unknown.

Conclusion

All the isolates belong to the modern lineage and there is high clustering in the genotype of isolates which indicated the presence of recent TB transmission. Therefore, the Tuberculosis Control Programme needs to do more in advocating and strengthening the health system for early detection and treatment of active TB cases as delay in treatment is the key factor in disease transmission.     

Comparison of diagnostic performance of three real-time PCR kits for detecting Mycobacterium species

Purpose

PCR is widely used for rapidly and accurately detecting Mycobacterium Species. The purpose of this study was to assess the diagnostic performance of three real-time PCR kits and evaluate the concordance with two older PCR methods.

Materials and Methods

Using 128 samples, the five PCR methods were assessed, including an in-house PCR protocol, the COBAS Amplicor MTB, the COBAS TaqMan MTB, the AdvanSure TB/NTM real-time PCR, and the Real-Q M. tuberculosis kit. The discrepant results were further examined by DNA sequencing and using the AdvanSure Mycobacteria Genotyping Chip for complete analysis.

Results

For Mycobacterium tuberculosis (MTB) detection, all five kits showed 100% matching results (positive; N = 11 and negative; N = 80). In non-tuberculous mycobacterium (NTM) discrimination, the AdvanSure yielded two true-positive outcomes from M. intracellulare and one false positive outcome, while the Real-Q resulted in one true-positive outcome and one false negative outcome for each case and another false negative result using the provided DNA samples.

Conclusion

Real-time PCR, yielded results that were comparable to those of the older PCR methods for detecting MTB. However, there were disagreements among the applied kits in regard to the sample test results for detecting NTM. Therefore, we recommend that additional confirmatory measures such as DNA sequencing should be implemented in such cases, and further research with using a larger numbers of samples is warranted to improve the detection of NTM.     

Outbreaks of Imipenem Resistant Acinetobacter Baumannii Producing OXA-23 ��-Lactamase in a Tertiary Care Hospital in Korea

Purpose

Since November 2006, imipenem-resistant Acinetobacter baumannii isolates have increased in Kyung Hee University Hospital in Seoul, Korea. The purpose of this study was to determine the genetic basis and molecular epidemiology of outbreak isolates.

Materials and Methods

Forty-nine non-repetitive isolates of the 734 IRAB strains were investigated in order to determine their characteristics. The modified Hodge and the ethylenediaminetetraacetic acid (EDTA)-disk synergy test were performed for the screening of carbapenemase and metallo-β-lactamase production. Multiplex polymerase chain reaction (PCR) assays were performed for the detection of genes encoding for OXA-23-like, OXA-24-like, OXA-58-like and OXA-51-like carbapenemase. Pulsed-field gel electrophoresis (PFGE) was performed for strain identification.

Results

All isolates showed 100% resistance to ciprofloxacin and gentamicin, 97.9% resistance to cefepime, piperacillin/tazobactam, aztreonam, ceftazidime and piperacillin, 93.9% resistance to tobramycin and 57.1% resistance to amikacin. All of the 49 isolates (100%) showed positive results in the modified Hodge test and negative results in the EDTA-disk synergy test. They all (100%) possessed the encoding gene for an intrinsic OXA-51-like carbapenemase and an acquired OXA-23-like carbapenemase in the multiplex PCR assay. PFGE patterns revealed that all isolates were clonally related from A1 to A14.

Conclusion

It is concluded that all of the 49 IRAB isolates acquired resistance to imipenem by producing OXA-23 carbapenemase and they might have originated from a common source.     

The prevalence of aminoglycoside-modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in Pseudomonas aeruginosa

INTRODUCTION:

Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an important component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance.

OBJECTIVES:

The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four important modifying enzyme genes (aac (6′)-I, aac (6′)-II, ant (2″)-I, aph (3′)-VI) in P. aeruginosa in Iran.

METHODS:

A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction.

RESULTS:

The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (6′)-II (36%) was the most frequently identified gene in phenotypic resistant isolates, followed by ant (2″)-I, aph (3′)-VI, and aac (6′)-I.

CONCLUSIONS:

Aminoglycoside resistance in P. aeruginosa remains a significant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.     

Direct fluorescent antibody assay and polymerase chain reaction for the detection of Chlamydia trachomatis in patients with vernal keratoconjunctivitis

OBJECTIVES:

To identify Chlamydia trachomatis via polymerase chain reaction and a direct fluorescent antibody assay in patients with vernal keratoconjunctivitis while comparing the efficacies of both tests for detecting Chlamydia trachomatis in these conditions.

METHODS:

Conjunctival scraping samples were obtained from 177 patients who were divided into two groups: a vernal keratoconjunctivitis group (group A) and a control group (group B). The polymerase chain reaction and a direct fluorescent antibody assay were performed. Sensitivity, specificity, receiver operating characteristic curves, and areas under the curve were calculated for both tests in groups A and B. Receiver operating characteristic curves were plotted using a categorical variable with only two possible outcomes (positive and negative).

RESULTS:

Statistical analysis revealed a significant association between vernal keratoconjunctivitis and Chlamydia trachomatis infection detected by a direct fluorescent antibody assay with high sensitivity and specificity. All patients in group A with positive polymerase chain reactions also presented with positive direct fluorescent antibody assays.

CONCLUSION:

The association between vernal keratoconjunctivitis and Chlamydia trachomatis infection was confirmed by positive direct fluorescent antibody assays in 49.4% of vernal keratoconjunctivitis patients and by positive polymerase chain reactions in 20% of these patients. The direct fluorescent antibody assay detected Chlamydia trachomatis in a higher number of patients than did the polymerase chain reaction. Although the diagnosis of trachoma is essentially clinical, the disease may not be detected in vernal keratoconjunctivitis patients. Due to the high frequency of chlamydial infection detected in patients with vernal keratoconjunctivitis, we suggest considering routine laboratory tests to detect Chlamydia trachomatis in patients with severe and refractory allergic disease.     

Metallo-��-Lactamase-Producing Pseudomonas spp. in Korea: High Prevalence of Isolates with VIM-2 Type and Emergence of Isolates with IMP-1 Type

Purpose

Two Korean nationwide studies showed that metallo-β-lactamases (MBLs)-producing-Pseudomonas spp. are not rare. The aim of this study was to assess the trends of MBL-producing isolates among imipenem-resistant isolates of Pseudomonas spp.

Materials and Methods

Imipenem-resistant clinical isolates were collected from 23 hospitals and one commercial laboratory participating in the KONSAR program in 2005. Polymerase chain reaction (PCR) was used to detect MBL genes.

Results

Alleles of MBL genes were detected in 10.8% of 415 Pseudomonas aeruginosa and 66.7% of 12 P. putida isolates from 18 of 24 hospitals/laboratory. Among the 14 IMP-1-like and 39 VIM-2-like MBLs, emergence of IMP-6 was detected for the first time.

Conclusion

Prevalence of MBL-producing P. aeruginosa has not significantly increased, but IMP-6 emerged in P. aeruginosa.     

Diagnostic Yield of Bronchial Washing Fluid Analysis for Hemoptysis in Patients with Bronchiectasis

Purpose

Bronchiectasis is the main cause of hemoptysis. When patients with bronchiectasis develop hemoptysis, clinicians often perform bronchoscopy and bronchial washing to obtain samples for microbiological and cytological examinations. Bronchial washing fluids were analyzed from patients with bronchiectasis who developed hemoptysis, and the clinical impacts of these analyses were examined.

Materials and Methods

A retrospective observational study of patients who underwent fiberoptic bronchoscopy for hemoptysis in Seoul National University Bundang Hospital, a university affiliated tertiary referral hospital, between January 2006 and December 2010 were reviewed. Among them, patients who had bronchiectasis confirmed by computed tomography and had no definite cause of hemoptysis other than bronchiectasis were reviewed. The demographic characteristics, bronchoscopy findings, microbiological data, pathology results and clinical courses of these patients were retrospectively reviewed.

Results

A total of 130 patients were reviewed. Bacteria, non-tuberculous mycobacteria (NTM), and Mycobacterium tuberculosis were isolated from bronchial washing fluids of 29.5%, 21.3%, and 0.8% patients, respectively. Suspected causal bacteria were isolated only from bronchial washing fluid in 19 patients, but this analysis led to antibiotics change in only one patient. Of the 27 patients in whom NTM were isolated from bronchial washing fluid, none of these patients took anti-NTM medication during the median follow-up period of 505 days. Malignant cells were not identified in none of the patients.

Conclusion

Bronchial washing is a useful method to identify microorganisms when patients with bronchiectasis develop hemoptysis. However, these results only minimally affect clinical decisions.     

Epidemiology and Control of an Outbreak of Vancomycin-Resistant Enterococci in the Intensive Care Units

Purpose

This study was aimed to describe a vancomycin-resistant enterococci (VRE) outbreak across three intensive care units (ICUs) of a Korean hospital from September 2006 to January 2007 and the subsequent control strategies.

Materials and Methods

We simultaneously implemented multifaceted interventions to control the outbreak, including establishing a VRE cohort ward, active rectal surveillance cultures, daily extensive cleaning of environmental surfaces and environmental cultures, antibiotic restriction, and education of hospital staff. We measured weekly VRE prevalence and rectal acquisition rates and characterized the VRE isolates by polymerase chain reaction (PCR) of the vanA gene and Sma1-pulsed-field gel electrophoresis (PFGE).

Results

During the outbreak, a total of 50 patients infected with VRE were identified by clinical and surveillance cultures, and 46 had vancomycin-resistant Enterococcus faecium (VREF). PFGE analysis of VREF isolates from initial two months disclosed 6 types and clusters of two major types. The outbreak was terminated 5 months after implementation of the interventions: The weekly prevalence rate decreased from 9.1/100 patients-day in September 2006 to 0.6/100 by the end of January 2007, and the rectal acquisition rates also dropped from 6.9/100 to 0/100 patients-day.

Conclusion

Our study suggests that an aggressive multifaceted control strategy is a rapid, effective approach for controlling a VRE outbreak.     

Polymerase Chain Reaction-Based Clonality Analysis of Cutaneous B-Cell Lymphoproliferative Processes

INTRODUCTION:

The differential diagnosis of B-cell lymphoproliferative processes remains a challenge for pathologists, dermatologists and oncologists, despite advances in histology, immunohistochemistry and molecular biology.

OBJECTIVE:

Evaluate aid and limitations of clonality analysis in the diagnosis of primary cutaneous B-cell lymphomas and B-cell pseudolymphomas.

METHODS:

This study included 29 cases of B-cell lymphoproliferative processes classified as primary cutaneous B-cell lymphomas (13), B-cell pseudolymphomas (6) and inconclusive cases (10) using histology and immunohistochemistry. The clonality analysis was performed by polymerase chain reaction analysis of immunoglobulin light chain and heavy chain rearrangements.

RESULTS:

DNA quality was shown to be generally poor; eight samples were inadequate for polymerase chain reaction analysis. The results showed monoclonality in eight of the primary cutaneous B-cell lymphomas and polyclonality in four of the B-cell pseudolymphomas. In addition, monoclonality was shown in two of the inconclusive cases by histology and immunohistochemistry, demonstrating the utility of polymerase chain reaction as an ancillary diagnostic tool for primary cutaneous B-cell lymphomas.

DISCUSSION:

The low quality DNA extracted from these cases demanded the use of an IgH protocol that yielded small fragments and IgK. Both methods used together improved detection.

CONCLUSION:

Use of the two protocols, immunoglobulin heavy chain FR3-trad and immunoglobulin light chain-Kappa Biomed protocols for clonality analysis improved diagnostic accuracy.     

Microarray analysis of the in vitro granulomatous response to Mycobacterium tuberculosis H37Ra

Background:

The hallmark of tuberculosis is the granuloma, an organized cellular accumulation playing a key role in host defense against Mycobacterium tuberculosis. These structures sequester and contain mycobacterial cells preventing active disease, while long term maintenance of granulomas leads to latent disease. Clear understanding on mechanisms involved in granuloma formation and maintenance is lacking.

Objective:

To monitor granuloma formation and to determine gene expression profiles induced during the granulomatous response to M. tuberculosis (H37Ra).

Methods:

We used a previously characterized in vitro human model. Cellular aggregation was followed daily with microscopy and Wright staining for 5 days. Granulomas were collected at 24 h, RNA extracted and hybridized to Affymetrix human microarrays.

Results:

Daily microscopic examination revealed gradual formation of granulomas in response to mycobacterial infection. Granulomatous structures persisted for 96 h, and then began to disappear.

Conclusions:

Microarray analysis identified genes in the innate immune response and antigen presentation pathways activated during the in vitro granulomatous response to live mycobacterial cells, revealing very early changes in gene expression of the human granulomatous response.     

Co-immunization of plasmid DNA encoding IL-12 and IL-18 with Bacillus Calmette-Guérin vaccine against progressive tuberculosis

Purpose

Bacillus Calmette-Guérin (BCG) vaccine has widely been used to immunize against tuberculosis, but its protective efficacy is variable in adult pulmonary tuberculosis, while it is not efficiently protective against progressive infection of virulent Mycobacterium tuberculosis strains. In this study, the protective effects of plasmid DNA vaccine constructs encoding IL-12 or IL-18 with the BCG vaccine were evaluated against progressive infection of M. tuberculosis, using mouse aerosol challenge model.

Materials and Methods

Plasmid DNA vaccine constructs encoding IL-12 or IL-18 were constructed and mice were immunized with the BCG vaccine or with IL-12 DNA or IL-18 DNA vaccine constructs together with the BCG vaccine.

Results

The BCG vaccine induced high level of interferon gamma (IFN-γ) but co-immunization of IL-12 or IL-18 DNA vaccine constructs with the BCG vaccine induced significantly higher level of IFN-γ than a single BCG vaccine. The BCG vaccine was highly protective at early stage of M. tuberculosis infection, but its protective efficacy was reduced at later stage of infection. The co-immunization of IL-12 DNA vaccine constructs with the BCG vaccine was slightly more protective at early stage of infection and was significantly more protective at later stage infection than a single BCG vaccine.

Conclusion

Co-immunization of IL-12 DNA vaccine with the BCG vaccine induced more protective immunity and was more effective for protection against progressive infection of M. tuberculosis.     

Preventive effects of mycobacteria and their culture supernatants against asthma development in BALB/c mice

Purpose

Live Mycobacterium bovis Bacille Calmette-Guérin (BCG) has a suppressive effect on asthma, but its use in clinical practice may be limited due to adverse reactions. To develop a product that is effective for suppressing asthma with minimal adverse reactions, we investigated whether the heat-killed body or culture supernatants of mycobacteria could also prevent asthma development.

Methods

Female BALB/c mice were treated with live BCG, the heat-killed body, or culture supernatants of BCG or Mycobacterium tuberculosis intraperitoneally, while sensitizing and provoking with ovalbumin. Then they underwent a methacholine bronchoprovocation test, and the peribronchial inflammatory cell numbers and cytokine levels in splenocyte culture supernatants were assessed.

Results

The airway sensitivity to methacholine decreased significantly after treatment with not only live BCG (30.8 versus 10.0 mg/mL, P<0.001) but also with the culture supernatant (BCG, 23.0 mg/mL, P<0.05; M. tuberculosis, 20.5 mg/mL, P<0.05). In contrast, heat-killed mycobacteria did not effectively decrease airway sensitivity. The peribronchial eosinophil counts and the goblet cell proportions in total epithelial cells decreased significantly in most of the groups. The interferon-γ/interleukin-5 ratios increased significantly in most of the treatment groups except for the heat-killed groups, and were significantly related to airway sensitivity (r=0.312, P<0.01) and peribronchial eosinophil counts (r=-0.416, P<0.001). Interleukin-17A level was inversely related to airway sensitivity (r=-0.212, P<0.05) and was significantly lower in the live BCG group than in the control (137±20 versus 308±57 pg/mL, P<0.05).

Conclusions

BCG and mycobacteria culture supernatants may effectively prevent the development of asthma associated with altered Th1/Th2 cytokines and interleukin-17A levels.     

Hereditary haemorrhagic telangiectasia: mutation detection, test sensitivity and novel mutations

Background

Hereditary haemorrhagic telangiectasia (HHT) is a genetic disorder present in 1 in 8000 people and associated with arteriovenous malformations. Genetic testing can identify individuals at risk of developing the disease and is a useful diagnostic tool.

Objective

To present a strategy for mutation detection in families clinically diagnosed with HHT.

Methods

An optimised strategy for detecting mutations that predispose to HHT is presented. The strategy includes quantitative multiplex polymerase chain reaction, sequence analysis, RNA analysis, validation of missense mutations by amino acid conservation analysis for the ENG (endoglin) and ACVRL1 (ALK1) genes, and analysis of an ACVRL1 protein structural model. If no causative ENG or ACVRL1 mutation is found, proband samples are referred for sequence analysis of MADH4 (associated with a combined syndrome of juvenile polyposis and HHT).

Results

Data obtained over the past eight years were summarised and 16 novel mutations described. Mutations were identified in 155 of 194 families with a confirmed clinical diagnosis (80% sensitivity). Of 155 mutations identified, 94 were in ENG (61%), 58 in ACVRL1 (37%), and three in MADH4 (2%).

Conclusions

For most missense variants of ENG and ACVRL1 reported to date, study of amino acid conservation showed good concordance between prediction of altered protein function and disease occurrence. The 39 families (20%) yet to be resolved may carry ENG, ACVRL1, or MADH4 mutations too complex or difficult to detect, or mutations in genes yet to be identified.     

HIV co-infection with tuberculous and non-tuberculous mycobacteria in western Kenya: challenges in the diagnosis and management

Background

Tuberculosis (TB) and HIV co-infections have a global prevalence with devastating morbidity and massive mortality, Sub-Saharan Africa being the worst hit.

Objectives

To evaluate the prevalence of TB-HIV co-infection and demonstrate the confusion caused by NTM and HIV/AIDS co-infection in TB diagnosis and treatment in western Kenya.

Methods

In a cross-sectional study carried out at 10 hospitals in western Kenya, sputa from consenting 872 TB suspects underwent microscopy, and culture on Lowenstein-Jensen and Mycobacteria Growth Index Tube media. Isolates were identified using the Hain''s GenoType^® Mycobacterium CM and GenoType^® Mycobacterium AS kits. A total of 695 participants were screened for HIV using Uni-Gold™ test and positives confirmed with the enzyme linked immunosorbent assay.

Results

A total of 346 (39.7%) participants were diagnosed with TB. Out of the 346 TB cases, 263 (76%) were tested for HIV infection and 110 (41.8%) of these were sero-positive (co-infected). The female to male TB-HIV co-infection prevalence ratio (PR) was 1.35. This study reports isolation of non-tuberculous mycobacteria from TB suspects at a rate of 1.7%.

Conclusion

A high TB-HIV co-infection rate was observed in this study. The NTM disease could be misdiagnosed and treated as TB in western Kenya.     

Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex,Real-Time PCR

The rapid identification of mycobacteria from culture is of primary importance for the administration of empirical antibiotic therapy and for the implementation of public health measures, yet there are few commercially available assays that can easily and accurately identify the mycobacteria in culture in a timely manner. Here we report on the development of a multiplex, real-time PCR assay that can identify 93% of the pathogenic mycobacteria in our laboratory in two parallel reactions. The mycobacteria identified by this assay include the Mycobacterium tuberculosis complex (MTC), the M. avium complex (MAC), the M. chelonae-M. abscessus group (MCAG), the M. fortuitum group (MFG), and M. mucogenicum. The primer targets included the 16S rRNA gene and the internal transcribed spacer. The assay was initially validated with a repository of reference strains and was subsequently tested with 314 clinical cultures identified by the AccuProbe assay or high-performance liquid chromatography. Of the 314 cultures tested, multiplex, real-time PCR produced congruent results for 99.8% of the 1,559 targets evaluated. The sensitivity and the specificity were each 99% or greater for MTC (n = 96), MAC (n = 97), MCAG (n = 68), and M. mucogenicum (n = 9) and 95% and 100%, respectively, for MFG (n = 19). We conclude that this multiplex, real-time PCR assay is a useful diagnostic tool for the rapid and accurate identification of MTC and clinically relevant nontuberculous mycobacteria.Mycobacterium tuberculosis, the causative agent of tuberculosis, is among the leading infectious causes of death in developing nations (23). In resource-rich countries, where tuberculosis is not endemic, nontuberculous mycobacteria (NTM) are responsible for the majority of mycobacterial infections in both immunocompromised and immunocompetent individuals (14). The clinical gravity of mycobacterial infections necessitates rapid isolation by culture and timely identification by the appropriate diagnostic assays. Rapid identification not only serves to focus empirical antibiotic therapy and thus avoid unnecessary drug exposure but also may aid with the appropriate respiratory isolation and prevention of secondary cases (15).Few molecular methods commercially available in the United States easily and rapidly identify mycobacteria in culture. Although the AccuProbe assay by Gen-Probe (San Diego, CA) is sufficient for the identification of isolates of the Mycobacterium tuberculosis complex (MTC) and the M. avium complex (MAC) (37), it lacks probe sets specific for rapidly growing mycobacteria, such as the M. chelonae-M. abscessus group (MCAG) and the M. fortuitum group (MFG), which make up approximately 30% of the pathogenic mycobacteria in the Clinical Microbiology Laboratory of Stanford Hospital (Stanford, CA). MCAG consists of M. chelonae, M. abscessus, and M. immunogenum; MFG consists of M. fortuitum, M. senegalense, M. farcinogenes, M. porcinum, M. septicum, M. peregrinum, and M. alvei (11). For these isolates, other methods, such as biochemical reactions (6, 33), high-performance liquid chromatography (HPLC) (13), and DNA sequencing (17), are required for identification. To address the need for a simple and rapid molecular assay with a broader identification scope, we developed a multiplex, real-time PCR assay that can identify 93% of the pathogenic mycobacterial isolates (both slowly growing and rapidly growing) recovered in the Clinical Microbiology Laboratory of Stanford Hospital. This assay simplifies the identification of MTC and MAC and also accommodates the identification of rapidly growing mycobacteria. Although conventional PCR-based and singleplex, real-time PCR assays for the identification of MTC and NTM have previously been reported (3, 7, 18, 19, 25, 29, 30, 32, 35), multiplex, real-time PCR assays with simple interpretative criteria have not been developed. A multiplex, real-time PCR assay for the identification of 18 mycobacterial species, including MTC and rapidly growing mycobacteria, was recently described (21); however, the complex nature of this assay may preclude its use in the routine laboratory. The only simple application of PCR for the identification of rapidly growing mycobacteria consists of PCR-restriction analysis (8, 36). Multiplex, real-time PCR has several crucial advantages over conventional PCR. Besides the ease of use and rapid availability of results, it also eliminates the need for postamplification handling and thus the potential contamination of the laboratory.Here we describe the development and validation of a multiplex, real-time PCR assay for the simple identification of MTC and clinically relevant NTM.