BACTEC Culture Research Articles

Comparative evaluation of IS6110 PCR via conventional methods in rapid diagnosis of new and previously treated cases of extrapulmonary tuberculosis

In developing countries the diagnosis of extrapulmonary tuberculosis (EPTB) is a major burning challenge. EPTB encounters many problems like pauci-bacillary nature, inadequate specimen volume. All the limitations reflect in the poor contribution of conventional bacteriological technique in the establishment of diagnosis of EPTB. Nucleic acid amplification methods are rapid and sensitive has modified strategies for the detection of mycobacterial DNA. A fragment of DNA of 123 bp belonging to insertion sequence IS6110 based on specific gene of Mycobacterium tuberculosis complex was amplified by polymerase chain reaction (PCR) for the rapid diagnosis of EPTB. The present study was to comparative evaluation of IS6110 PCR via conventional methods in the rapid diagnosis of new and Previously treated cases of extra pulmonary tuberculosis. Four hundred fifty specimens were collected from suspected cases of EPTB were processed for Mycobacteria by Zeihl Neelson (ZN) staining and BACTEC culture for M. tuberculosis. All the specimens were also processed for IS6110 based PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of M. tuberculosis complex. We found significant difference was seen in sensitivities of different tests. Of these 450 specimens, 60 (13.4%) were positive for AFB by ZN staining, 202 (45%) for BACTEC culture and IS6110 PCR were positive for M. tuberculosis complex in 283 (63%) specimens (p< 0.05). However, there was no significant difference (p< 0.05) as far as specificity of different tests. We found that IS6110 PCR has higher sensitivity than smear microscopy and BACTEC culture in both cases of new cases as well as in previously treated cases. IS6110 PCR can be highly useful in diagnosis of new and treated cases of EPTB. It may facilitate therapeutic decisions for those with suspected of EPTB.

Direct inoculation on Phoenix panels for identification and antimicrobial susceptibility from positive BACTEC cultures: First study from India

Purpose: This was a prospective study planned in a super-specialty hospital in Delhi to reduce turnaround times of identification–susceptibility results of positive blood cultures. Materials and Methods: One hundred consecutive single morphology non-duplicate cultures were inoculated on Becton Dickinson Phoenix™ panels by growth recovered directly from liquid BACTEC™ media and after pure growth on solid media. Results: Complete concordance was observed in 72.4% of gram-negative and 45.8% of gram-positive isolates. For gram-negative isolates, categorical agreement (CA) was >83% and essential agreement (EA) was >96% among all antibiotics tested, very major errors (VME) were 0.13%, major errors (ME) 0.54%, and minor errors (MiE) were 3.01%. For gram-positive isolates, VME was 0.73%, 1.10% MiE and no ME. It was observed that average time from receipt of specimen to release of reports was 30:34 h and 32 h for gram-negative and gram-positive isolates if reports of “Direct” panels were to be released. Conclusions: By direct panel inoculation, a decrease of at least 18–20 h in turnaround time was observed compared with the standard method. This helps early change to effective antibiotic therapy and also reduces the expenditure incurred for a patient’s hospital stay by average Rs 20,000 ($443) per day.

The advantage of using IS6110-PCR &lt;i&gt;vs&lt;/i&gt;. BACTEC culture for rapid detection of &lt;i&gt;Mycobacterium tuberculosis&lt;/i&gt; from pleural fluid in northern India

Pleural tuberculosis is an extra-pulmonary disease which poses a diagnostic dilemma. The detection of mycobacterial DNA by IS6110 polymerase chain reaction (PCR) in clinical samples is a promising approach for the rapid diagnosis of pleural tuberculosis infections. The aim of the present study is to evaluate the advantage of using IS6110 PCR for rapid detection of Mycobacterium tuberculosis (M. tuberculosis) from pleural fluid. 102 clinically suspected cases of pleural tuberculosis cases were enrolled from inwards and outwards of the Department of Pulmonary Medicine at Chattrapati Shahuji Maharaj Medical University, Lucknow from April 2007 to April 2010. The pleural fluids were processed at the Mycobacteriology Laboratory of Department of Microbiology at Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Pleural fluid samples were processed and examined by Ziehl Neelsen (ZN) staining for acid fast bacilli and detection of M. tuberculosis by BACTEC culture. We applied IS6110 PCR to detect specific M. tuberculosis complex in pleural fluid samples. We found a significant difference in sensitivity of different tests, acid fast bacilli were detected in 17 (16.6%) samples by ZN Staining , 47 (46.1%) by BACTEC culture and using IS6110 PCR, 62 (60.7%) were positive for IS6110 PCR for M. tuberculosis. We found IS6110 PCR was much more sensitive than ZN staining and BACTEC culture. IS6110 PCR detection of M. tuberculosis may be very useful in cases that are highly suspect as pleural tuberculosis and those that are negative for AFB and culture. IS6110 PCR may gain an immense prospective to better clinicians ability to improve diagnosis of pleural tuberculosis.

Rapid Identification of the Mycobacterium tuberculosis Complex by Combining the ESAT-6/CFP-10 Immunochromatographic Assay and Smear Morphology

Early secretory antigen 6 (ESAT-6) and cell filtrate protein 10 (CFP-10) are two antigens secreted as a complex by the replicating Mycobacterium tuberculosis complex (MTC). Recently, an immunochromatographic assay (ICA) using a monoclonal antibody against the ESAT-6/CFP-10 complex was developed for the purpose of MTC detection. In this study, the efficacy of the assay was tested with 603 BACTEC cultures that were incubated for 3 additional days after positive signals appeared in the BACTEC MGIT 960 system. Bacterial isolates were recovered from these 603 BACTEC cultures, and 332 MTC isolates, 270 nontuberculosis mycobacterial isolates, and 1 Nocardia isolate were identified by using standard biochemical assays. The ESAT-6/CFP-10 assay detected 322 MTC cultures, resulting in a sensitivity of 97% and a specificity of 97.4%. To reduce the false-negative rate and improve the sensitivity, either serpentine cording in an acid-fast bacillus stain of the cultural smear, the ESAT-6/CFP-10 assay, or a combination of both was used for MTC detection. The sensitivity was then increased to 99.1%, and the negative predictive value increased to 98.9%, but the specificity decreased to 94.8% and the positive predictive value decreased to 95.9%. However, a combination of serpentine cording in cultural smears and the positivity of the ICA resulted in the specificity and positive predictive values of 100%. Therefore, BACTEC cultures with both serpentine cording and positivity of the ESAT-6/CFP-10 assay could be reported to contain MTC directly. The ESAT-6/CFP-10 assay may be an alternative of the Capilia assay (MPB64-ICA) as a convenient and cost-effective method for identification of MTC in culture.

Multiple bilateral costo–chondral abscesses due to Mycobacterium tuberculosis

Multiple abscesses of the costo-chondral junctions are very uncommon in practice. In this report we present the case of a 55 year old man who presented to us with chest pain and fever of few months duration. On imaging with ultrasound and CECT we were able to demonstrate multiple abscesses of costo-chondral junctions bilaterally. We confirmed tuberculosis by FNAC and BACTEC cultures from abscesses.

The Thioamides Methimazole and Thiourea Inhibit Growth of M. avium Subspecies paratuberculosis in Culture

BackgroundThyrotoxicosis is conceptualized as an “autoimmune” disease with no accepted infectious etiology. There are increasingly compelling data that another “autoimmune” affliction, Crohn disease, may be caused by Mycobacterium avium subspecies paratuberculosis (MAP). Like M. tb, MAP is systemic. We hypothesized that some cases of thyrotoxicosis may be initiated by a MAP infection. Because other thioamides treat tuberculosis, leprosy and M. avium complex, we hypothesized that a mode of action of some thioamide anti-thyrotoxicosis medications may include MAP growth inhibition.MethodsThe effect of the thioamides, thiourea, methimazole and 6-propo-2-thiouracil (6-PTU) were studied in radiometric Bactec® culture, on ten strains of three mycobacterial species (six of MAP, two of M. avium and two of M. tb. complex). Data are presented as “cumulative growth index,” (cGI) or “percent decrease in cumulative GI” (%-ΔcGI).Principal FindingsMethimazole was the most effective thioamide at inhibiting MAP growth. At 128µg/ml: MAP UCF-4; 65%-ΔcGI & MAP ATCC 19698; 90%-ΔcGI. Thiourea inhibited MAP “Ben” maximally; 70%-ΔcGI. Neither methimazole nor thiourea inhibited M. avium or M. tb. at the doses tested. 6-PTU has no inhibition on any strain studied, although a structurally analogous control, 5-PTU, was the most inhibitory thioamide tested.SignificanceWe show inhibition of MAP growth by the thioamides, thiourea and methimazole in culture. These data are compatible with the hypothesis that these thioamides may have anti-prokaryotic in addition to their well-established eukaryotic actions in thyrotoxic individuals.

The “Starry-Sky” Appearance of Tubercular Meningitis

To the Editors: A 13-year-old previously well Nepali girl from an average socioeconomic background presented with nonpulsatile intermittent severe headache and nonprojectile vomiting for 15 days. She had 1 episode of left focal seizure 1 week before presentation. There was no history of contact with tuberculosis. No meningeal signs or focal neurodeficits were present. The fundus and rest of the systemic examination were normal. Magnetic resonance imaging revealed multiple small (<10 mm) ring-enhancing lesions in the cerebral and cerebellar hemispheres, basal ganglia, thalami, and brainstem with perilesional edema giving a starry-sky appearance (Fig. 1). Routine biochemical investigations, skeletal survey, and chest radiograph were normal. Neurocysticercosis serology, quantiferon, and Mantoux tests were negative. No fever was documented during the hospital stay. She was treated with intravenous steroids, phenytoin, mannitol, and acetazolamide for a presumptive diagnosis of neurocysticercosis. Due to brainstem involvement, cysticidal therapy was not administered. Her headache continued to worsen, hence methylprednisolone therapy was instituted. Lumbar puncture showed cloudy cerebrospinal fluid (CSF) with 460 cells (85% lymphocytes), glucose 2 mg/dL, protein 500 mg/dL, lactate dehydrogenase 888 U/L, and adenosine-deaminase 17.94 U/L. Ehrlich-Ziehl-Neelsen staining of the CSF was positive for acid-fast bacilli (AFB). CSF polymerase chain reaction was also positive for AFB. Bactec culture showed growth of AFB susceptible to isoniazid, rifamipicin, pyrazinamide, and ethambutol. Retroviral serology was also negative. Following the onset of 4 drug anti-tubercular therapy with steroids whereafter headache and fever disappeared.FIGURE 1.: Axial, saggital, and coronal magnetic resonance images of the brain depicting multiple ring-enhancing lesions with perilesional edema.The starry-sky appearance on neuroimaging has been reported in neurocysticercosis,1–3 we emphasize that it can be observed in childhood tubercular meningitis. Routine biochemical and radiologic investigations may not distinguish the 2 entities, and CSF studies may expedite the microbiologic diagnosis and appropriate treatment in such cases. Satvinder Kaur, MD Ketan Kulkarni, MD Vineet Gupta, MD, MRCPH Apollo Centre of Advanced Pediatrics Indraprastha Apollo Hospital New Delhi, India

Isolation of Mycobacterium avium subspecies paratuberculosis from muscle and peripheral lymph nodes using acid-pepsin digest prior to BACTEC culture

Meat has received little attention regarding human exposure to Mycobacterium avium subsp. paratuberculosis, a possible infectious trigger of Crohn's disease. Meat has less contamination with other organisms than gut tissues, facilitating modifications to existing decontamination protocols prior to BACTEC culture that could increase analytical sensitivity. Using spiked meat samples we trialled enzymatic and chemical digestion techniques to concentrate larger starting samples, and modifications to existing clinical mycobacteriological decontamination protocols. An acid-pepsin digestion method using a 20 g sample was considerably more sensitive (detection limit 0.88 log 10 viable organisms per gram) than previous techniques. However, it was cumbersome for routine use, and subject to frequent contamination. Modifications to an existing centrifugation protocol yielded a simple, robust technique with slightly improved sensitivity (detection limit 1.77 log 10 per gram). Use of these sensitive tests in parallel identified M. a. paratuberculosis in the muscle of 59% and peripheral lymph nodes (PLN) of 85% of clinically infected sheep. The numbers of M. a. paratuberculosis in these infected tissues were low (1.67 ± 0.92 log 10 per gram in muscle and 2.06 ± 0.69 log 10 per gram in PLN), such that many would not have been detected by routine methods. Fewer subclinically infected animals with gross lesions harboured M. a. paratuberculosis in meat (4.5%) or PLN (32%), and the numbers of organisms in such infected animals were lower. Because most animals raised specifically for meat production are young and unlikely to be heavily infected, and because meat is usually consumed cooked, the risk of human exposure to viable M. a. paratuberculosis via meat may be small. Measures to prevent heavily infected animals, especially those with clinical signs, from entering the human food chain would further reduce this risk.

Superior detection of pathogens in synovial fluid by the Bactec 9240 Peds Plus/F system compared to the conventional agar-based culture method

To improve culture yield in cases of possible septic arthritis, we compared culture of joint fluid aspirates on conventional agar-based media to culture in Bactec 9240 Peds/Plus F blood culture bottles with and without the addition of fastidious organism supplement (FOS). Over a period of 21 months, we analysed 123 synovial fluid samples and isolated 20 pathogens. The Bactec methods proved superior by yielding more pathogens than the conventional culture method (p=0.074). However, this method also yielded more contaminants within the first three days of incubation (p=0.027). All contaminants detected after three days of incubation were the result of overgrowth on conventional method agar plates. The Bactec methods provided clinicians with a positive pathogen result one day earlier than the conventional counterpart (p=0.001). Four isolates of Neisseria gonorrhoeae were only cultured with the Bactec method. No significant benefit was demonstrated by supplementing blood culture bottles with FOS. We recommend that whenever infection by fastidious organisms is suspected, synovial fluid aspirates should be cultured using automated blood culture systems to increase the culture yield and to decrease the time to detection.

Tuberculous liver abscesses and venous thromboembolism – An unusual association

A rare association of tuberculous liver abscesses with bilateral transudative pleural effusion, ascites and venous thromboembolism in a 22-year-male is reported. While the BACTEC culture and polymerase chain reaction of the aspirate from liver abscess were positive for mycobacterium tuberculosis, all the reports of pleural fluid and ascitic fluid were negative. The bilateral pleural effusion and the ascites may be due to a non-tuberculous cause like hypoalbuminaemia. The finding of venous thromboembolism was incidental.

Pilot study on multidrug resistant tuberculosis in Nigeria

Drug resistant tuberculosis (TB) has lately emerged and it represents a serious public health problem. We set out to determine drug resistance among TB patients. Using automated BACTEC cultures, multidrug resistant-tuberculosis (MDR-TB) was investigated in 117 diagnosed cases in Abuja, Nigeria. Ten (31%) of 32 culture-positive patients were resistant to at least one and four (13%) to all of the four drugs tested. No association between drug resistance and human immunodeficiency virus (HIV) infection was found. MDR-TB is present in Nigeria and larger studies are urgently required. TB clinical management and control efforts should be improved.

Factors Affecting Isolation and Identification of Mycobacterium avium subsp. paratuberculosis from Fecal and Tissue Samples in a Liquid Culture System

Culture of Mycobacterium avium subsp. paratuberculosis is the definitive diagnostic test for Johne's disease, a chronic granulomatous enteropathy of animals. Compared to solid media, the identification of all strains of the organism in liquid media can be more difficult because the appearance of colonies and mycobactin dependence are not observable, and the growth of other organisms needs to be distinguished, commonly by PCR. Factors affecting the isolation rate of S strains and the contamination rate in modified Middlebrook 7H9 broth (Bactec 12B) and 7H10 agar were studied using 11,598 fecal samples and 2,577 tissue samples from sheep from 1,421 farms over 10 years. Minimization of contamination in Bactec cultures required the avoidance of the carryover of fecal particles from the first sedimentation step in the double-incubation centrifugation method, and contamination was reduced significantly by incubating the sample in a solution containing vancomycin, amphotericin B, and nalidixic acid for 3 days compared to 2 days. The growth of irrelevant microorganisms confounded the identification of M. avium subsp. paratuberculosis in liquid culture by inhibiting IS900 PCR and in solid medium culture by inhibiting the growth of M. avium subsp. paratuberculosis or obscuring colonies. The contamination of samples was clustered in certain laboratory submissions and was reduced by including ampicillin in Bactec medium without affecting the odds of isolation of M. avium subsp. paratuberculosis. The long-term contamination rate for fecal cultures was about 7%, and that for tissue cultures was <0.2%. Liquid medium was more sensitive than solid medium culture for M. avium subsp. paratuberculosis. The applicability of these findings for C strains is discussed.

Newer methods over the conventional diagnostic tests for tuberculous meningitis: do they really help?

Rapid diagnosis of tuberculous meningitis (TBM) is crucial as the disease outcome depends on the stage at which the treatment is initiated. The reliability of the available tests has not been established; thus, the present study was conducted to evaluate the conventional diagnostic tests as compared to the newer methods. Cerebrospinal fluid was collected from 100 children, and analyzed for various biochemical and cytological tests. The samples were subjected to Ziehl-Neelsen (Z-N) staining, Lowenstein-Jensen (L-J) culture, BACTEC culture and polymerase chain reaction (PCR). Twenty-two patients could be identified as definitive TBM based on the demonstration of Mycobacterium tuberculosis by BACTEC culture and PCR. Of these 22 cases, Z-N staining was positive in only two and L-J culture in six cases. Both the BACTEC culture and PCR had 100% agreement in the diagnosis of TBM. However, BACTEC culture could be a better diagnostic test as drug sensitivity can also be performed by this method.

Prediction of delayed treatment response in pulmonary tuberculosis: Use of time to positivity values of Bactec cultures

New drugs that can shorten tuberculosis (TB) treatment and target drug resistant strains are urgently needed. A test which could predict patients at risk of a delayed response to treatment would facilitate clinical trials of new anti-tuberculosis drugs. A widely-used test for the assessment of response to treatment is sputum smear examination. Patients who are smear positive after 2 and 3 months of treatment are said to have delayed and significantly delayed treatment responses respectively. Time to positivity (TTP) values of Bactec cultures, from the first 2 weeks of treatment were used to predict delayed and significantly delayed treatment responses in patients with first time pulmonary tuberculosis. Changes in TTP values early in treatment were transformed to a response ratio (r). Values of r that were less than a threshold value (r(c)) indicated patients who were at risk of having delayed or significantly delayed response to treatment. Accuracy of prediction was sensitive to the timing of sputum sampling and adherence to therapy in the first 2 weeks. Based on TTP data from the first 2 weeks of treatment, significantly delayed treatment response could be predicted with a sensitivity of 75% and a specificity of 62% while the positive (PPV) and negative predictive values (NPV) were 14% and 97% respectively. While the high NPV indicates that a large proportion of patients with a satisfactory response to treatment can be reliably identified, the low PPV value underlines the need to use TTP in conjunction with other markers of disease activity to predict unfavourable treatment response in tuberculosis treatment.

First Cultivation and Characterization of Mycobacterium ulcerans from the Environment

Background Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin.Methodology/Principal FindingsOne culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5′ end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3′ end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone.ConclusionStrain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.

Immune parameters as markers of tuberculosis extent of disease and early prediction of anti-tuberculosis chemotherapy response

This study investigates how the extent of pre-treatment radiological disease and early anti-tuberculous treatment response affect levels of selected circulating host immune markers. Twenty HIV-uninfected tuberculosis patients with BACTEC culture positivity for Mycobacterium tuberculosis at diagnosis and treated with directly observed short course anti-tuberculosis chemotherapy and 13 healthy community controls were enrolled. Serum samples were collected throughout treatment. After the intensive phase of treatment, 12 patients remained sputum culture-positive (slow responders) and eight patients were culture negative (fast responders). C-reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1), soluble urokinase plasminogen activator receptor (suPAR), soluble lymphocyte activation gene-3 (sLAG-3), granzyme B, soluble tumour necrosis factor receptor one and two (sTNFR I and sTNFR II) and soluble death receptor 5 (sDR5) concentrations were measured. High levels of CRP at diagnosis were found to be associated (p</=0.05) with the presence of multiple cavities on chest x-rays and high levels of suPAR and sICAM-1 at diagnosis were associated (p</=0.05) with the extent of alveolar disease. Also significant were the associations between the level of granzyme B (p</=0.01) and LAG-3 (p</=0.05) at diagnosis, and the size of the cavities. The combination of diagnosis and week one measurements of selected serological markers in mathematical models was able to identify the fast responders with up to 87.5% accuracy and the slow responders with up to 83.3% accuracy These preliminary results suggest that predictive models for differential early treatment responses using combinations of host markers hold promise.

Cavitary Disease and Quantitative Sputum Bacillary Load in Cases of Pulmonary Tuberculosis

We examined sputum bacterial loads in adults with newly diagnosed tuberculosis using quantitative culture and time-until-positive (DTP) culture in BACTEC 460. Patients with cavitary disease had higher CFU levels than those without cavities and shorter DTPs. Within radiographic strata of moderately and far advanced tuberculosis, higher CFU counts were associated with cavitary disease.

Rapid Identification of Staphylococcus aureus in Blood Cultures by Use of the Direct Tube Coagulase Test

Direct tube coagulase testing for identification of Staphylococcus aureus from BACTEC culture broth showed a sensitivity, a specificity, and positive and negative predicative values of 34%, 100%, 100%, and 80.2% with 2 h of incubation and 65%, 98.7, 99.7%, and 88.6% with 4 h of incubation. Anaerobic blood culture contributed significantly to the detection of S. aureus.

Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture

The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS 900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS 900-like sequences. One copy of IS 900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR ( r = −0.70). In pooled faecal samples, QPCR also agreed with culture ( κ = 0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.

Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of &lt;i&gt; Mycobacterium tuberculosis&lt;/i&gt; in clinical samples

The correlation between the presence of specific gene sequence of M. tuberculosis and specific diagnosis of clinical tuberculosis is not known. This study compared the results of polymerase chain reaction (PCR) amplification of M. tuberculosis specific DNA sequences (IS6110, 65kDa, 38kDa and mRNA coding for 85B protein) from different clinical samples of pulmonary and extrapulmonary tuberculosis. One hundred and seventy-two clinical samples from suspected tuberculosis patients were tested for smear examination, culture (LJ and rapid BACTEC 460 TB system) and PCR. PCR was performed with specific primers for the targets: IS6110, 65 kDa, 38 kDa and 85 B. Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture (P < 0.05). Smear positive samples (56) and the samples (36) showing positive results by conventional methods (smear and LJ medium culture) and BACTEC were found to be positive by all PCR protocols. No significant difference was found between the four PCR protocols (P> 0.05). The primer specific for amplifying the 123bp IS6110 fragment gave the highest positivity (83%), followed by 65kDa, 38kDa and 85B RT-PCR in descending order. These data suggest that the presence of IS6110 correlates more closely with the diagnosis of clinical tuberculosis than that of 65kDa, 38kDa and 85B proteins.