The Utility of Polymerase Chain Reaction (PCR) in the Diagnosis of Pulmonary Tuberculosis

doi:10.1378/chest.107.6.1631

Chest

Volume 107, Issue 6, June 1995, Pages 1631-1635

https://doi.org/10.1378/chest.107.6.1631 Get rights and content

A fragment of DNA of 123 bp belonging to insertion sequence IS6110, specific of Mycobacterium tuberculosis complex, was amplified by polymerase chain reaction (PCR) of respiratory samples, for the diagnosis of pulmonary tuberculosis. A total of 314 samples (286 sputum and 28 bronchoalveolar lavages) from 242 patients were evaluated by PCR, and the results were compared with the those obtained by acid-fast-stained smears, culture, and clinical diagnosis. Mycobacterium tuberculosis was detected by PCR in 102 of 105 patients with clinical diagnosis of pulmonary tuberculosis. All smear and culture-positive samples were PCR positive. The sensitivity of PCR, culture, and staining was 97%, 88%, and 65%, respectively, and the specificity was 100% in all cases. In ten patients with old residual lesions, but no active disease, M tuberculosis genome was detected by PCR. In our experience, PCR proved to be a useful method for the rapid diagnosis of pulmonary tuberculosis.

Section snippets

Patients

A total of 242 patients were classified by clinical diagnosis: pulmonary tuberculosis, 105; residual tuberculosis, 44; and a control group of 93 patients without TB (Table 1).
Diagnosis of TB was based on the isolation and identification of M tuberculosis in clinical samples of pulmonary origin in 92 patients, while in another 13 cases the diagnosis was established by clinical, epidemiologic, radiologic, and therapeutic criteria. The more relevant clinical characteristics in this group are

Results

Ziehl-Neelsen staining was positive in 68 cases (64.7%) out of 105 patients diagnosed as having pulmonary tuberculosis. Fifty (83%) of 60 patients with cavitary lesions showed positive results by microscopy. In nine patients from this group, we performed fiberoptic bronchoscopy, and the bronchoalveolar lavage obtained proved positive on staining in two cases. In the remaining 37 patients from the same group, 4 samples from each patient were analyzed, all being negative. The culture on LJ was

Discussion

The rapid diagnosis of lung TB continues to be based on the detection of AFB in sputum by ZN staining. The sensitivity of microscopy depends on the clinical presentation, and is greater in cavitary forms with hemoptysis. More than 10,000 bacilli per milliliter of sputum are necessary to secure microscopic positivity.²⁴ The success of microscopy is highly variable (22 to 96%),25, 26, 27 though most authors rate it at around 60%.28, 29, 30, 31 In our study, microscopic sensitivity was 64.7% and

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Cited by (46)

Polymerase Chain Reaction targeting insertion sequence IS6110 for the diagnosis of pulmonary tuberculosis among Sudanese children and young adults
2014, International Journal of Mycobacteriology
Citation Excerpt :
In a review of many published studies regarding the effect of BCG vaccination on TST, only 1% of subjects vaccinated as infants were TST-positive if tested ⩾10 years after being vaccinated by BCG [20]; second, false-positive due to infection by environmental non-TB mycobacteria (due to cross-reactivity). Previous studies showed the success of microscopy is highly variable from 22% to 96% and most authors rate it at round 60% [21]. ZN stain is routinely used in all TB diagnostic centers in Sudan, while Auramine fluorochrome method is only used in the National Reference Laboratory (NRL).
This study was carried out in Khartoum State during the period from January 2011 to December 2013 to improve the rate of detection of Mycobacterium tuberculosis (MTB) in children with symptoms of tuberculosis (TB) infection using different conventional and advanced diagnostic techniques. One hundred and ninety-seven specimens of gastric lavage and sputum were collected from different hospitals in Khartoum State, including Elbolok Hospital, Jafar Ibn Owf Hospital, Elasha’ab Teaching Hospital, Soba University Hospital and Academy Charity Hospital.
All children participating in the study were subjected to the Mantoux test after obtaining appropriate consent injected by 5 tuberculin units of tuberculin purified protein derivative, and the results were recorded after three days. Specimens were decontaminated and inoculated on Lowenstein–Jensen media according to the modified Petroff’s method. Two smears were prepared and stained by Ziehl–Neelsen stain and Auramine fluorescent dye; bacterial DNA was extracted from each specimen by using phenol chloroform method, and then the Polymerase Chain Reaction technique was adopted to detect Insertion Sequence IS6110 gene of MTB in these specimens. This study showed that the positive results for TST, ZN, Auramine, Culture and PCR were 86 (43.7%), 16 (8.1%), 22 (11.2%), 32 (16.2%) and 35(17.8%), respectively. The study concluded that the PCR technique is the most sensitive and specific technique for a quick identification of MTB in gastric lavage and sputum from children who are unable to expectorate a good quality sputum sample or who are diagnosed as negative using conventional diagnostic methods.
Radiological deterioration in a patient with cavitary lung lesion
2011, Enfermedades Infecciosas y Microbiologia Clinica
Endobronchial ultrasound increases the diagnostic yields of polymerase chain reaction and smear for pulmonary tuberculosis
2010, Journal of Thoracic and Cardiovascular Surgery
Citation Excerpt :
Owing to the small sample size in the subgroup with a lesion size of less than 3 cm, the role of EBUS in the diagnostic yield of NAATs deserves further investigation. The value of NAATs in respiratory samples to obtain a rapid diagnosis of pulmonary TB has been described in many studies.10,22 A positive NAAT result may be valuable in the early detection of active TB cases that are smear-negative.23
Our objective was to determine the contribution of endobronchial ultrasound in the diagnostic yields of acid-fast bacillus smear, nucleic acid amplification tests, and culture in bronchoalveolar lavage fluid for pulmonary tuberculosis.
During a 1-year interval, 99 patients who had initial sputum-negative acid-fast bacillus smears or no sputum but were later proven to have a positive culture for Mycobacterium tuberculosis in their sputum or bronchoalveolar lavage fluid were retrospectively studied. Among them, 56 patients underwent bronchoscopy with endobronchial ultrasound (EBUS group) and 43 patients received conventional bronchoscopy for bronchoalveolar lavage (non-EBUS group).
The diagnostic yields of the nucleic acid amplification tests (89.3%, 50/56; P = .006), acid-fast bacillus smear (30.4%, 17/56; P = .013), and M tuberculosis culture in bronchoalveolar lavage fluid (67.9%, 38/56; P = .041) were significantly higher in the EBUS group of patients. The results of those who underwent conventional bronchoscopy were 65.1% (28/43), 9.3% (4/43), and 46.5% (20/43), respectively. Combining bronchoalveolar lavage fluid smear and nucleic acid amplification tests, we made a rapid diagnosis of pulmonary tuberculosis in 51 (91.1%) of the 56 EBUS patients and 29 (67.4%; P = .004) of the 43 non-EBUS patients.
The introduction of endobronchial ultrasound increases the diagnostic yield of the nucleic acid amplification tests, acid-fast bacillus smear, and M tuberculosis culture from bronchioalveolar lavage fluid in patients with pulmonary tuberculosis who have negative sputum smear or no sputum production.
Adequately washed bronchoscope does not induce false-positive amplification tests on bronchial aspirates in the diagnosis of pulmonary tuberculosis
2002, Chest
Citation Excerpt :
However, we have demonstrated that bronchoscopic aspirate can be a useful specimen for the amplification of M tuberculosis DNA in the diagnosis of pulmonary tuberculosis, if each bronchoscopic laboratory maintains high-quality control monitoring during bronchoscopic disinfection. In recent years, PCR and other amplification-based techniques have been widely used in the diagnosis of tuberculosis.15–18 Most studies have used sputum as the specimen from which M tuberculosis DNA has been amplified for the diagnosis of pulmonary tuberculosis.
To investigate the clinical usefulness of amplification (COBAS AMPLICOR; Roche Diagnostics Systems; Branchburg, NJ) on bronchoscopic aspirate specimens in the diagnosis of pulmonary tuberculosis, with particular regard to the possibility of false-positive results in subsequent specimens due to residual Mycobacterium tuberculosis DNA.
A prospective clinical study at a tertiary referral medical center.
Four hundred fiberoptic bronchoscopic procedures were performed, using seven bronchoscopes on 335 consecutive patients, for therapeutic or diagnostic purposes. Serial bronchial aspirates were collected and tested for M tuberculosis, using COBAS AMPLICOR (CA). Bronchoscopes were cleaned and disinfected automatically, between patient use, by the same endoscope washer. The name of each bronchoscope and the sequence of its use were recorded, together with the sequence of washing. The CA results were compared with the bacteriologic and histologic results for M tuberculosis infection. When there was a suspicion of contamination, outward polymerase chain reaction analysis was performed.
Of 392 specimens (332 subjects), excluding the 8 specimens (4 subjects) in which bacteriologic and histologic analyses were omitted, a smear-positive result for acid-fast bacilli (AFB), culture-positive or biopsy-positive results, and CA-positive results were obtained in 16, 49, and 32 specimens, respectively. In AFB smear-positive subjects, the sensitivity, specificity, positive predictive values (PPVs), and negative predictive values (NPVs) were 92%, 67%, 92%, and 67%, respectively. In AFB smear-negative subjects, the sensitivity, specificity, PPV, and NPV values were 38%, 99%, 74%, and 94%, respectively. The CA test was more sensitive than the AFB smears for the diagnosis of pulmonary tuberculosis (53% vs 27%, respectively; p < 0.05). False-positive CA results were seen in only six specimens. Three of these six subjects received a diagnosis of pulmonary tuberculosis on clinical and radiologic grounds, and none of the six results seemed to be associated with bronchoscopic cross-contamination.
Adequately cleaned and disinfected bronchoscopes did not cause false-positive amplification test results for M tuberculosis on bronchial aspirates by cross-contamination. Furthermore, sensitivity was greater with the CA tests. Therefore, CA tests on bronchial aspirates seem to be useful in the diagnosis of pulmonary tuberculosis.
Should we continue to perform medical thoracoscopy in pleural tuberculosis?
2019, Minerva Pneumologica
Comparison of sensitivity and specificity of PCR and sputum smear compared to culture in diagnosis of tuberculosis
2019, Tehran University Medical Journal

View all citing articles on Scopus

Presented in part at the 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif, October 11–14, 1992.

revision accepted October 11.

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Chest

Clinical Investigations: TuberculosisThe Utility of Polymerase Chain Reaction (PCR) in the Diagnosis of Pulmonary Tuberculosis

Section snippets

Patients

Results

Discussion

Tubercle

Lancet

Lancet

Lancet

Tubercle

Chest

Lancet

Epidemiology of tuberculosis

Am Rev Respir Dis

Comparison of a radiometric method (BACTEC) and conventional media for recovery of mycobacteria from smear-negative specimens

J Clin Microbiol

Collaborative feasibility study of a biphasic system (Roche Septi-Check AFB) for rapid detection and isolation of mycobacteria

J Clin Microbiol

Antibody and antigen detection for the immuno-diagnosis of tuberculosis: what not? what more is needed? where do we stand today?

J Infect Dis

Diagnosis of pulmonary tuberculosis by detection of tuberculostearic acid in sputum by using gas chromatography-mass spectrometry with selected ion monitoring

J Infect Dis

Detection and identification of Mycobacterium tuberculosis by DNA amplification

J Clin Microbiol

A comparative study of the polymerase chain reaction and conventional procedures for the diagnosis of tuberculous pleural effusions

Tubercle

Detection and identification of mycobacteria by amplification of mycobacterial DNA

Mol Microbiol

Sequence analysis and amplification by polymerase chain reaction of a cloned DNA fragment for identification of Mycobacterium tuberculosis

J Clin Microbiol

Polymerase chain reaction for detection of Mycobacterium tuberculosis

J Clin Microbiol

Clinical Investigations: Tuberculosis
The Utility of Polymerase Chain Reaction (PCR) in the Diagnosis of Pulmonary Tuberculosis