Rapid and Specific Detection of Mycobacterium tuberculosis from Acid-Fast Bacillus Smear-Positive Respiratory Specimens and BacT/ALERT MP Culture Bottles by Using Fluorogenic Probes and Real-Time PCR

Abstract

A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.Mycobacterium tuberculosis remains a serious public health issue due to its high risk of person-to-person transmission, morbidity, and mortality (15,20). Currently, approximately 8 million new infections and 3 million deaths are attributed to tuberculosis (TB) each year (L. B. Reichman, Letter, Chest 112:855, 1997). Progressive increases in TB infections are expected, and a worldwide annual incidence of 12 million cases by 2005 is predicted by the World Health Organization (20). The resurgence of TB in industrialized countries since the mid-1980s, primarily due to the increased incidence of immunocompromised patients with AIDS, and the emergence of multidrug-resistant strains of M. tuberculosis has accented the need for rapid diagnosis of this disease (15,20). Rapid detection of active TB infection is critical for effective patient management and implementation of infection control measures.Conventional detection of mycobacteria is based on a number of protocols, including microscopic examination of smears stained with the Ziehl-Neelsen stain or auramine fluorescent dye and selective culture techniques (8,17,26,39). The key aspect of TB control is rapid diagnosis, which for many years has been based on the staining of smears for the presence of acid-fast bacilli (AFB). The AFB smear test lacks specificity, so there is a need for a laboratory test for specific detection of the M. tuberculosis complex (MTB) that can be performe...

Rapid detection of Mycobacterium tuberculosis complex by real-time PCR in sputum samples and its use in the routine diagnosis in a reference laboratory

“…Other studies also reported false-negative results by RT-PCR in paucibacillary samples (Miller et al, 2002;Cleary et al, 2003;Lira et al, 2013). These authors observed an inversely proportional relationship between the C t values and the number of AFB detected in microscopy, as also encountered in our study (data not shown).…”

“…The use of RT-PCR for TB diagnosis has increased, as it has proven to be highly sensitive and specific for detection of the Mycobacterium tuberculosis complex (MTBC) directly in clinical samples or from culture isolates (García-Quintanilla et al, 2002;Miller et al, 2002;Broccolo et al, 2003;Cleary et al, 2003;Armand et al, 2011;Lira et al, 2013). Many regions of the mycobacterial genome have been used in molecular diagnosis of TB, such as IS6110, 16S rRNA, hsp65, rpoB, sdaA, devR and mpt64 (Manjunath et al, 1991;Lachnik et al, 2002;Kim et al, 2004;Chakravorty et al, 2005;Negi et al, 2007;Hwang et al, 2013;Nimesh et al, 2013).…”

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

“…Other studies, with different targets, have reported similar results (Shrestha et al, 2003) and many have been designed as multiplex assays to differentiate Mtb complex from other non-tuberculosis mycobacteria based on melting curve analysis (Shrestha et al, 2003). Furthermore, these assays were often shown to be as sensitive and specific as the available commercial assays such as the Cobas Amplicor (Miller et al, 2002;Shrestha et al, 2003).…”

“…Several real-time PCR LDTs have been developed for diagnosis of pulmonary tuberculosis. In one of the earlier studies by Miller et al (Miller et al, 2002), a real-time PCR assay was developed on the LightCycler platform (Roche Diagnostics, Indianapolis, IN) with primers targeting the internal transcribed spacer region of the mycobacterium genome with specific hybridization probes designed for the detection of Mtb complex. The sensitivity and specificity of this assay for smear-positive respiratory specimens was 98.1% and 100% respectively with a turn-around time of less than 5 hours (Miller et al, 2002).…”

“…In one of the earlier studies by Miller et al (Miller et al, 2002), a real-time PCR assay was developed on the LightCycler platform (Roche Diagnostics, Indianapolis, IN) with primers targeting the internal transcribed spacer region of the mycobacterium genome with specific hybridization probes designed for the detection of Mtb complex. The sensitivity and specificity of this assay for smear-positive respiratory specimens was 98.1% and 100% respectively with a turn-around time of less than 5 hours (Miller et al, 2002). Other studies, with different targets, have reported similar results (Shrestha et al, 2003) and many have been designed as multiplex assays to differentiate Mtb complex from other non-tuberculosis mycobacteria based on melting curve analysis (Shrestha et al, 2003).…”