Single-tube nested PCR in the diagnosis of tuberculosis.

Chan, Charles; Yuen, Kwok‐Yung; Chan, K. S.; Yam, Wing-Cheong; Yim, Kan-Hing; Ng, Wai Fu; Mh, Ng

doi:10.1136/jcp.49.4.290

Cited by 49 publications

(31 citation statements)

References 19 publications

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“…The reasons for the false positive results could be cross contamination during the initial handling or an amplicon carry over contamination, which can be overcome by using a single tube nested PCR [18]. It was also found that the use of dUTP/Uracil-N-glycosylase could decrease the amplicon contamination [18].…”

Section: Discussionmentioning

confidence: 99%

“…the reasons for the Lower Sensitivity may be many: Firstly, the presence of PCR inhibitors which are found to be more associated with the extrapulmonary specimens as compared to the pulmonary specimens [18][19]. A better extraction procedure like an immunomagnetic separation technique may be used to capture all the M. tuberculosis DNA, but not the inhibitors [18].…”

Section: Possible Tbm: (I) Clinical Criteria (A)mentioning

confidence: 99%

“…A better extraction procedure like an immunomagnetic separation technique may be used to capture all the M. tuberculosis DNA, but not the inhibitors [18]. Various resin matrixes like 'Gene releaser preparations' which absorb the inhibitors without entailing a further loss of DNA may also be used [18].…”

Section: Possible Tbm: (I) Clinical Criteria (A)mentioning

confidence: 99%

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The Diagnostic Utility of Bact/ALERT and Nested PCR in the Diagnosis of Tuberculous Meningitis

Sastry¹

2013

JCDR

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Objective: The early laboratory diagnosis of Tuberculous Meningitis (TBM) is crucial, to start the antitubercular chemotherapy and to prevent its complications. However, the conventional methods are either less sensitive or time consuming. Hence, the diagnostic potentials of BacT/ALERT and Polymerase Chain Reaction (PCR) was evaluated in this study. Material and Method:The study group comprised of 62 cases and 33 controls. The cases were divided according to Ahuja's criteria into the confirmed (two cases), highly probable (19 cases), probable (26 cases) and the possible (15 cases) subgroups. Ziehl Neelsen's (ZN) and Auramine Phenol (AP) staining, Lowenstein Jensen (LJ) medium culture, BacT/ALERT and nested Polymerase Chain Reaction (PCR) which targeted IS6110 were carried out on all the patients. Observation and Results:The sensitivity of the LJ culture was 3.22%. BacT/ALERT showed a sensitivity and a specificity of 25.80% and 100% and those of nested PCR were found to be 40.32% and 96.97% respectively. The mean detection time of growth of the LJ culture was 31.28 days, whereas that of BacT/ALERT was 20.68 days. The contamination rate in the LJ culture and BacT/ALERT were 7.2% and 5.8% respectively. Conclusion:Nested PCR was found to be more sensitive, followed by BacT/ALERT as compared to the LJ culture and smear microscopy. As both false negative and false positive results have been reported for nested PCR, so it should not be used alone as a criterion for initiating or terminating the therapy, but it should be supported by clinical, radiological, cytological and other microbiological findings.

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Section: Discussionmentioning

confidence: 99%

Section: Possible Tbm: (I) Clinical Criteria (A)mentioning

confidence: 99%

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The Diagnostic Utility of Bact/ALERT and Nested PCR in the Diagnosis of Tuberculous Meningitis

Sastry¹

2013

JCDR

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“…Single colonies were picked and suspended in PBS to reach approximately a 0.5 McFarland standard, and 10-fold dilutions were made. Viable counts were simultaneously made as previously published (9). Sputum samples known to be negative for any of the Burkholderia species tested were pooled and homogenized using 0.1% dithiothreitol in PBS (Sputasol) at room temperature for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Novel Pan-Genomic Analysis Approach in Target Selection for Multiplex PCR Identification and Detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia Complex Species: a Proof-of-Concept Study

Lau

Martelli

et al. 2011

J Clin Microbiol

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Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n ‫؍‬ 6; B. cenocepacia, n ‫؍‬ 3; B. cepacia, n ‫؍‬ 4; B. arboris, n ‫؍‬ 2; B. contaminans, B. anthina, and B. pyrrocinia, n ‫؍‬ 1 each; other unnamed members, n ‫؍‬ 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified.

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“…The few studies which have evaluated PCR for the diagnosis of extrapulmonary TB have included a high percentage of human immunodeficiency virus (HIV)-seropositive patients. The sensitivity of PCR in these studies ranges 42-93% [3][4][5][6]. Previously, we and others showed that PCR can be used for the detection of pulmonary TB cases in routine laboratories [7][8][9].…”

Section: Aamentioning

confidence: 99%

Clinical utility of the polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis

Kolk¹,

Kox²,

Leeuwen³

et al. 1998

Eur Respir J

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aaMicroscopy with the auramine or Ziehl-Neelsen (ZN) stain is frequently negative in cases of extrapulmonary tuberculosis (TB). This means that treatment may be started and continued until the culture results confirm or refute the diagnosis at least 3 weeks later. The polymerase chain reaction (PCR), which permits direct identification of the Mycobacterium tuberculosis complex [1, 2] is more sensitive than microscopy, has a much lower detection limit and gives results within 24 h of receipt of the sample. The few studies which have evaluated PCR for the diagnosis of extrapulmonary TB have included a high percentage of human immunodeficiency virus (HIV)-seropositive patients. The sensitivity of PCR in these studies ranges 42-93% [3][4][5][6]. Previously, we and others showed that PCR can be used for the detection of pulmonary TB cases in routine laboratories [7][8][9]. Using our improved PCR technique [2], we evaluated the contribution of PCR to the diagnosis of TB in patients suspected of having extrapulmonary TB. Patients with suspected tuberculous meningitis were excluded from this analysis and are the subject of a separate publication [10]. Materials and methods PatientsTwo hundred and twenty six specimens were obtained from 156 patients residing in the Netherlands who were suspected of having TB on the basis of symptoms, signs and radiological findings as judged by the treating clinician at the time of presentation. None of the patients had a recent (ð5 yrs) diagnosis of mycobacterial disease. The specimens were taken before antituberculous treatment was started.Comparisons between conventional techniques (microscopy and culture) and PCR were made on samples from a single specimen. The specimens were obtained from various tertiary hospitals in the Netherlands and were sent by regular mail at ambient temperature to the Royal Tropical Institute. They arrived within 48 h and were processed on the same day and stored at 4°C. The specimens included pleural fluid (n=55), tissue biopsies (n=52), blood (n=56), pus from wound (n=32), bone marrow (n=6), ascites (n= 2), synovial fluid (n=5), pericardial fluid (n=3), urine (n= 13) and faeces (n=2). Treatment of specimensOn receipt at the local laboratory, the specimen was split and a portion was sent to us for testing with PCR. The remaining portion was handled as detailed below. Protocols for specimen processing were standardized [11].Clinical utility of the polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis. A.H.J. Kolk, L.F.F. Kox, J. van Leeuwen, S. Kuijper, H.M. Jansen. ©ERS Journals Ltd 1998. ABSTRACT: This study examines the diagnostic utility of the polymerase chain reaction (PCR) in 156 patients (five human immunodeficiency virus (HIV) seropositive) suspected of extrapulmonary tuberculosis.The results of PCR in 226 samples from 11 different sites were compared with the results of microscopy and culture.Positive culture results were predicted in 86% of samples by PCR but in only 31% by microscopy. Specificity of PCR was 92%. In cases with...

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Single-tube nested PCR in the diagnosis of tuberculosis.

Cited by 49 publications

References 19 publications

The Diagnostic Utility of Bact/ALERT and Nested PCR in the Diagnosis of Tuberculous Meningitis

The Diagnostic Utility of Bact/ALERT and Nested PCR in the Diagnosis of Tuberculous Meningitis

Novel Pan-Genomic Analysis Approach in Target Selection for Multiplex PCR Identification and Detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia Complex Species: a Proof-of-Concept Study

Clinical utility of the polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis

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