Detection and identification of mycobacteria in sputum from suspected tuberculosis patients
© Hatta et al; licensee BioMed Central Ltd. 2010
Received: 11 November 2009
Accepted: 16 March 2010
Published: 16 March 2010
Detection of Tuberculosis agent like nontuberculous mycobacteria (NTM) species by culture and microscopic methods remains difficult and time consuming. A fast and reliable diagnosis of tuberculosis would greatly improve the control of the disease. The purpose of this study is to compare the conventional multiplex PCR and multiplex PCR reverse cross blot hybridization assay to culture method in terms of mycobacteria species detection.
Among the 117 positively cultured samples, nontuberculous mycobacteria (NTM) species were found in 9 samples of multiplex PCR reverse cross blot hybridization assay; compared to only 3 NTM species found in our conventional multiplex PCR, and 13 NTM species were successfully identified among 162 negatively cultured samples compared to only 5 NTM species identification in conventional multiplex PCR results.
The sensitivity of the multiplex PCR reverse cross blot hybridization assay comparing to culture method was 86.03%, the specificity is 35.46%, the positive predictive value was 41.94% and the negative predictive value was 82.41%. For conventional multiplex PCR these values are 81.62%, 38.65%, 41.89%, 79.51%, respectively. Furthermore, in terms of mycobacteria species detection, the conventional multiplex PCR was relatively equal compared to the multiplex PCR reverse cross blot hybridization assay, and to be particularly having no significant discrepant results on the identification of Mycobacteria tuberculosis in both methods.
Tuberculosis is caused by Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum. Together with Mycobacterium microti and the vaccine strain M. bovis BCG they belong to the Mycobacterium tuberculosis complex. Some other cases caused by nontuberculous mycobacteria (NTM), are mostly mycobacteria belonging to the Mycobacterium avium-Mycobacterium intracellulare complex . Opportunistic mycobacteria commonly associated with the human immunodeficiency virus (HIV) are Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium fortuitum and Mycobacterium scrofulaceum. An estimated 1.7 billion individuals are infected with Mycobacterium tuberculosis. Mortality is highest in developing countries, where over three-quarters of cases occur .
Early detection is of major importance in the control of tuberculosis . The emergence of multidrug resistant strains and its association with outbreaks on community in endemic areas illustrates that rapid diagnosis is essential [5, 6]. A fast and reliable diagnosis of tuberculosis would greatly improve the control of the Tuberculosis . Current conventional diagnosis of tuberculosis or other mycobacteria could be time-consuming, because the culture of mycobacteria may take 4 to 8 weeks. Some mycobacteria are very difficult or almost impossible to grow in vitro such as M. genavense and M. leprae. Direct staining and microscopy of clinical samples lack sensitivity and specifity . In principle, these drawbacks could be solved by an application of PCR, which allows in vitro amplification of target DNA to a detectable level within a matter of hours .
Various researchers have recently described the rapid detection of M. tuberculosis by PCR, and many have reported a high degree of sensitivity in detecting M. tuberculosis in clinical samples by means of DNA amplifications . Recently a nested PCR has been developed in detecting Salmonella typhi in blood, feces and urine from suspect typhoid fever and multiplex PCR to detect M. tuberculosis complex bacteria and other mycobacteria which this technique is based on the amplification of the specific insertion sequence IS6110 and 16S rDNA respectively [9, 10]. This study uses the multiplex PCR reverse cross blot hybridization assay and the conventional multiplex PCR to detect and identify the Mycobacterium species from clinical samples of patients suspected of mycobacterial diseases in comparison with the Conventional methods.
Three hundred and eighty-seven samples of sputum from patients suspected of mycobacterial disease were obtained from the lung hospital in Makassar, Indonesia. Microscopy and culture were performed according to the standard methods at the Department of Medical Microbiology, Molecular Biology and Immunology Laboratory. Ziehl Neelsen staining with some modifications was used for microscopic detection . Sputum samples were decontaminated and cultured on Lowenstein Jensen medium, which is locally produced [12, 13], after being extracted with Boom Method, the PCR assays were performed.
This study was reviewed and approved by Hasanuddin University and informed consent was obtained from all participants or their parents or their guardians.
Multiplex PCR reverse cross blot hybridization
For the amplification of mycobacterial 16S rDNA sequences, the 5'- biotinylated primers pMyc14bio (5'-GAGGTACT CGAGTGGCGAAC-3') and pMyc7bio (5'GGCCGGCTACCCGTCGTC-3') were used. In the PCR mixture, the primer Pt18 (5'GAACCGTGAGGGCATCGAGG-3') and the 5'-biotinylated primer INS2bio (5'-GCGTAGGCGTCGGTGACAAA-3') (Grenner Inc, Japan), were also included, amplifying the M. tuberculosis complex-specific insertion sequence IS6110.
Using AmpliTaq Gold PCR Master Mix (AB Applied Biosystem, USA), samples were incubated for 10 minutes at 40°C, to break down possible contaminating amplicons by Uracil DNA glycosylase (UDG) [12, 14]. Then, incubated at 94°C for 40 seconds, 65°C for 40 seconds and 50 seconds at 72°C, with 40 cycles.
Tailing of oligonucleotide probes with dTTP
Multiplex PCR reverse cross blot hybridization results
M. tuberculosis complex
M. tuberculosis complex
Conventional multiplex PCR 
Primers (HT1: 5'-CCTGCGAGCGTAGGCGTCGG-3'; HT2: 5'-CTCGTCCAGC GCCGCTTCGG-3'; HT3: 5'-CTTGCTGGAGGTGCTCGACG-3'and HT4: 5'-GGAGGTGCCGT GCAGGTAGG-3') 0.5 uM of each, 5 ul DNA template and 47 ul of distilled water (Ultrapure, Invitrogen Co, Japan) were added to a 0.2 microcentrifuge tube containing AmpliTaq Gold. Conditions for thermocycling were as follow: 95°C for 10 minutes, 40 cycles of amplification (94°C for 30 seconds followed by 60°C for 40 seconds and 72°C for 40 seconds) and 72°C for 10 minutes. Using 1.8% agarose gel containing ethidium bromide (Sigma, USA), 5 uL of PCR product were analysed by electrophoresis at 100 V for 30 minutes. PCR Product length for HT1/HT2 and HT3/HT4 are 123 base pairs (bp) for M. Tuberculosis and 322 bp for M. avium.
Difference in the results between positive and negative groups for culture, microscopy, and conventional multiplex PCR in the same sputum samples and for multiplex PCR reverse cross blot hybridization assay results were analyzed using the SPSS (SPSS Inc., Chicago, Il) computer package.
The electrophoresis of conventional multiplex PCR and pattern of multiplex PCR reverse cross blot hybridization in nitrocellulose membrane
The culture, microscopy and multiplex PCR reverse cross blot hybridization assay
Comparison of culture, microscopy and multiplex PCR reverse cross blot hybridization assay
Number of samples
with culture results
Positive (n = 136)
Negative (n = 251)
Positive (n = 115)
Negative (n = 272)
Comparison between conventional multiplex PCR and multiplex PCR reverse cross blot hybridization assay results
Comparison of conventional multiplex PCR and PCR reverse cross blot hybridization assay
Conventional multiplex PCR positive/PCR Cross Blot Hybridization positive
Positive Culture (n = 136)
Negative culture (n = 251)
Moreover, among 162 positive samples by multiplex PCR reverse cross blot hybridization but negative in culture, 149 samples were positive for M. tuberculosis and 5 samples of M. avium. These findings again showed similarity with the conventional multiplex PCR results. Other 3 samples positive for M. chelonae and 1 sample was shown positive M. intracellulare, M. kansasii, M. fortuitum, M. genavense and M. smegmatis, respectively. These last mentioned 8 samples were failed the conventional multiplex PCR detection. (tables 3). The sensitivity of the conventional Multiplex PCR comparing to culture method was 81.62%, the specificity was 38.65%, the positive predictive value was 41.89% and the negative predictive value was 79.51%. No significant difference was found in identification of M. tuberculosis by conventional multiplex PCR and multiplex PCR reverse cross blot hybridization assay results in both culture positive and negative samples.
The Sensitivity and the Specificity of conventional multiplex PCR and PCR reverse cross blot hybridization results
Reverse Cross blot Hybridization
Positive Predictive Value
Negative Predictive value
In terms of mycobacteria species detection, the conventional multiplex PCR was relatively equal compared to the multiplex PCR reverse cross blot hybridization. Conventional multiplex PCR method is easier and simpler in application compared to multiplex PCR reverse blot cross hybridization assay. On the other hand, Multiplex PCR reverse cross blot hybridization is a more complicated method; however it can detect considerably more nontuberculous mycobacteria (NTM) species such as M. avium, M. intracellulare, M. kansasii, M. fortuitum, M. chelonae, M. genavense and M. smegmatis, something that is unidentifiable by our conventional Multiplex PCR.
The conventional multiplex PCR and Multiplex PCR reverse cross blot hybridization assay should be suitable for a rapid and correct diagnosis of patients suspected of having mycobacterial disease. These two methods will help the clinicians significantly in deciding the suitable antimicrobial treatment for their patients within shorter period of time.
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