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Diagnosis of Mycobacterium tuberculosis from Sputum DNA Using Multiplex PCR

R. GOPINATH,1 SUTHANDIRA MUNISAMY,2 M. JEYADEVASENA,1

J. M. JEFFREY,3 and ELANCHEZHIYAN MANICKAN1

1 Department of Microbiology, Dr. ALM PG1BMS, University of Madras, Taramani, Chennai - 600 113, Tamil Nadu, India,

E-mail: This email address is being protected from spam bots, you need Javascript enabled to view it (E. Manickan)

21RT Perundurai Medical College, Perundurai, Erode District,

Tamil Nadu - 638053, India

department of Genetics, Dr. ALM PG 1BMS, University of Madras, Taramani, Chennai - 600 113, Tamil Nadu, India

ABSTRACT

The conventional techniques like the Acid-Fast Bacilli (AFB) staining and smear microscopy employed for the diagnosis of tuberculosis (ТВ) have a significant disadvantage of poor sensitivity and the gold standard diagnostic method employing culture is time-consuming. This study was conducted to isolate and identify Mycobacterium tuberculosis (MTB) from the sputum sample and to evaluate the efficacy of PCR as a modern diagnostic tool, for diagnosis of tuberculosis, especially in the smear-negative cases. The multiplex polymerase chain reaction (MPCR) offers a great promise as a diagnostic tool for ТВ owing to the rapid analysis, significant sensitivity, and specificity. Amplified nucleic acid hybridization assays such as MPCR have shown promising results. The aim of this study was to perform MPCR analysis using MTB genes namely PPE41, MPT53, LPQH, ESAT-6, and CFP-10, and analyze its efficiency in the diagnosis of ТВ. 252 sputum samples were collected from ТВ Hospital and they were subjected to Ziehl-Neelsen, culture analysis employing Lowenstein-Jensen (L-J) medium, and Multiplex PCR (mpcr) for the specific detection of mycobacterium DNA. The MPCR was performed for targeting genes of MTB. Of 252 cases, 102 samples showed positive for AFB, 124 samples were positive for culture method and 196 samples showed positive for MPCR. The overall results showed a sensitivity as well as specificity of the MPCR assay as 97.7% and 100%, respectively. Whereas the sensitivity and specificity in the case of culture was 89.2% and 98.1% and with AFB it was 63.3% and 92%, respectively. Analysis of the positive and negative predictive values of MPCR was found to be 91.1% and 99.3% when compared to the culture (78.2% and 87.1%) and AFB (71.2% and 82%), respectively. MPCR analysis performed by employing specified MTB primers was able to detect more ТВ positive cases when compared to the conventional AFB and L-J culture method for the detection of MTB. MPCR was found to be a more rapid, reliable, more sensitive, and highly specific diagnostic tool for the purpose of detection as well as the differentiation of MTB patients and this can serve as the potential diagnostic tool for the analysis of sputum samples.

INTRODUCTION

Tuberculosis (ТВ) is deemed as one of the leading cause for morbidity and mortality worldwide with an infection rate affecting approximately one-third of the population [1]. However, this is not the case when people predisposed to human immunodeficiency vims (HIV) get infected with ТВ. People with HIV-infection are 26 to 31 times more likely to procure a severe form of ТВ over a particular period of time when compared to HIV-negative persons [2, 3]. In 2015, the world had an estimated 10.4 million new ТВ cases. Over half of these were among men (5.9 million), and women constituted over a third (3.5 million). Ten percent of the cases were among children [1]. The situation is further aggravated due to the increasing occurrence of multi-dmg resistant (MDR) and extremely drug- resistant (XDR) ТВ and with the significant emergence of HIV [4]. The laboratory diagnosis is extensively based on direct microscopy and culture for mycobacterium. Direct microscopic examination for detection of AFB has very low sensitivity and often is less specific [5, 6]. L-J culture test though being regarded as a gold standard technique is a labor intensive and time consuming procedure. Thus, the detection of MTB in clinical samples harboring small numbers of the organism still pose itself as a maj or challenge

[7]. As an alternative to these classical methods, new nucleic acid-based technologies have shown promises as a more rapid, sensitive, and specific means of detection and identification of Mycobacteria [8]. The need for a rapid diagnostic tool has led to the development of molecular methods for the strategic detection of Mycobacterium tuberculosis. The polymerase chain reaction (PCR) in the field of Molecular Biology has proven to be a useful technique in the diagnosis of ТВ infection. The problems associated with PCR method is the possibility of obtaining false-positivity in results due to the cross contamination of clinical specimens with M. tuberculosis DNA product with the PCR laboratory, and the inability of the PCR method to notice the minute difference between viable and nonviable organisms [9, 10]. The single gene target can result in false negativity of the results while more reliable results can be obtained by utilization of more than one target gene for amplification [11]. The multiplex PCR (MPCR) is the PCR in which several target genes are amplified simultaneously and a more sensitive and specific tool of diagnosis for Mycobacterium tuberculosis infection [12]. The aim of the present study was to evaluate the sensitivity and specificity of MPCR by utilizing MTB genes namely PPE41, MPT53, LPQH, ESAT-6, and CFP-10 for the diagnosis of ТВ patients.

MATERIALS AND METHODS

12.2.1 STUDY POPULATION

A total of 252 sputum samples were collected from IRT, Perundurai Medical College, Perundurai, Erode District, Tamil Nadu. Written consents were obtained from each patient after adequate furnishing of information and the approval for the study protocol was obtained horn the Institutional Human Ethical Committee (No: UM/IHEC/16-2013-I).

12.2.2 SMEAR MICROSCOPY (SM)

Two drops of the sample pellet decontaminated by NaOH-NALC method was employed in smear microscopy (ZN staining), in accordance to the standard protocol [13]. The grading of the AFB results was done according to the WHO/Intemational Union Against ТВ and Lung Disease guidelines and scored as “0” for the absence of AFB, scanty 1-9, 1+, 2+, or 3+ [13].

12.2.3 SAMPLE PREPARATION FOR DNA EXTRACTION

Commercially available Norgen Biotek Sputum DNA extraction kit (Canada) was utilized for the extraction of DNA from sputum samples.

12.2.4 DNA AMPLIFICATION OF MYCOBACTERIUM TUBERCULOSIS (ТВ)

The extracted DNA samples were subjected to MPCR amplification using appropriate primers of five MTB genes (Table 12.1). Amplifications were earned out using Eppendorf™ Thermal Cycler (Germany) consisting of initial denaturation at 95°C for 5 min followed by 32 cycles of denaturation at 95°C for 30 sec, annealing at 61°C for 30 sec and extension at 72°C for 45 sec. The amplifications were followed by final extension at 72°C for 5 min. The amplified products were then stored at a temperature of 4°C till the detection by electrophoresis.

TABLE 12.1 Sequences of Primers Used in Multiplex PCR

Gene

Sequence

PCR Product Size (bp)

PPE41

5’-AC GGATCCATGCATTTCGAAGCG-3’ 5’-AG GAATTCAGTGTCTGTACGCG-3’

604

MPT53

5’-ATG GAT CCA TGA GTCT TCG CCT G-3’ 5’-ATG AAT TCG GAC GTC AGC GCA GC-3’

539

LPQH

5’-ATG GAT CCG TGA AGC GTG GAC TG-3’ 5’-ATG AAT TCG GAA CAG GTC ACC TCG-3’

497

ESAT-6

5 ’ -TAAGGATCC ATG AC AGAGC AGGAGTG-3 ’ 5 ’-GCGAATTCTGCGAACATCCCAGTG-3 ’

288

CFP-10

5’-TAA GGA TCC ATG GCA GAG ATG AAG AAC-3’ 5’-GCG AAT TCG AAG CCC ATT TGC GAG GA-3’

303

12.2.5 DETECTION OF AMPLIFIED PRODUCTS

The MPCR products were then analyzed by electrophoresis at 100 V on 2% agarose gels stained with Ethidium bromide (0.5 pg/ml) and visualized under Gel documentation system (GELSTAN, India). The PCR products were compared with H37Rv positive control and PCR grade water and patient negative sample as negative control.

12.2.6 STATISTICAL ANALYSIS

The Sensitivity and specificity along with the positive predictive and negative predictive value of the MPCR were evaluated by utilizing online MedCalc and this data was compared with microbiological tests.

RESULTS AND DISCUSSION

In this study performed, a sample population of 252 sputum samples were collected and enrolled for the study. These samples were subjected to Ziehl- Neelsen staining, LJ culture and mpcr to detect MTB. Out of the sample population, 102 were found to be positive for AFB (Figure 12.1), 124 were positive for LJ culture (Figure 12.2) whereas mpcr amplification showed positive in 196 samples (Figure 12.3). mpcr showed higher number of positive result than compared to other gold standard techniques.

The statistical analysis of AFB smear examination showed a sensitivity of 63.3% and a specificity of 92%. The overall sensitivity and specificity of the culture is 89.2% and 98.1% whereas for MPCR it is 97.7% and 100% respectively.

The positive as well as the negative predictive values were found to be 71.2% and 82% for AFB; 78.2% and 87.1% for culture and 91.1% and 99.3% for MPCR, respectively (Table 12.2).

Ziehl-Neelsen staining

FIGURE 12.1 Ziehl-Neelsen staining.

LJ culture slant

FIGURE 12.2 LJ culture slant.

Multiplex PCR products of five MTB genes

FIGURE 12.3 Multiplex PCR products of five MTB genes. Lane Ll-H37Rv positive control; L2 to L6-patient sample showing the amplification of MTB genes PPE41 at 604bp, MPT53 at 539bp, LPQH at 497bp, CFP-10 at 303bp and ESAT-6 at 288bp; L7-negative control; L8-negative sample of patient.

TABLE 12.2 Comparison of MPCR Results with AFB and Conventional L-J Medium

Method

Sputum Samples from ТВ Patients N = 252

Sensitivity

Specificity’

PPV

NPV

AFB smear Microscopy

63.3%

92%

71.2%

82%

LJ Culture

89.2%

98.1%

78.2%

87.1%

Multiplex PCR

97.7%

100%

91.1%

99.3%

ТВ is a leading threat to the health in both the developing as well as developed countries. In India, the diagnosis of ТВ is primarily based on the clinical features, histopathology, and demonstration of acid-fast bacilli and isolation of MTB in culture. These techniques have several drawbacks, such as lack of rapidity and inadequate sensitivity and specificity [14]. DNA amplification by PCR provides a rapid and sensitive method for the detection of MTB complex from clinical samples and cultures in the Mycobacteria laboratory routine. The conventional method of culture on the LJ media is regarded as the gold standard technique for the detection of Mycobacterium growth [15]. PCR due to higher rapidity and sensitivity can facilitate the earlier and accurate identification of the causative organism of ТВ and thus can very much be feasible for retreatment, prevention, and control of this chronic infection. PCR as a diagnostic tool had been widely used for many years for the purpose of detecting Mycobacterial DNA. However, PCR with one type of primer can cause ambiguity in the detection of MTB-DNA and hence should be verified with other primers specific for MTB [16]. The species-specific PCR assays developed for M. tuberculosis have been invalidated due to false-negative results owing to the absence of specific target sequences such as mtp40 in some MTB strains [17]. The common drawback associated with the PCR assays is the risk of obtaining false positivity in results due to contamination or presumably due to the presence of killed or dormant bacilli in the samples (Beige et al., 1999). The multiplex-PCR proposed in this study was based on the 2-3 amplification of the Mycobacterial genomic fragments [18]. This result was in accordance to study by Negi et al. [12] which reported a sensitivity of 73.6%with MPCR in smear-negative culture-positive which is significantly higher than the majority of studies reporting it at around 60% or even less in the study by Sarmiento et al. [19].

MTB detection by PCR assays is significantly more sensitive and rapid when compared to that of the culture and microscopy with their sensitivities ranging between 36% and 100% and their specificities ranging between 85% and 100% using various PCR targets such as the IS6110, MPB64, hup-B, TRC4, GCRS, Pab gene, etc.. [20-22]. Deshpande et al. [23] stated in their study that the IS6110 PCR assay was feasible in tenns of the sensitivity (82.4%) and specificity (75.9%) as compared to the culture results which revealed 68.6% sensitivity and 79.3% specificity. Therefore, it has been analyzed that the PCR based on IS6110 is a supportive method for the precise diagnosis of clinically diagnosed ТВ. Most reports of studies involving the IS6110-based detection have claimed sensitivities of about 75% and specificities of approximately 100%. Boondireke et al. (2007) has stated in their study that they obtained a sensitivity of 92.1% and specificity of 98.2%in MPCR.

MTB have proved its application as a reliable technique for diagnostic purposes. In our study, we found an overall sensitivity and specificity of 97.7% and 100% respectively in the MPCR analysis. MPCR using MTB specific primers detected more ТВ cases compared to that of conventional culture method. To the best of our knowledge, although MPCR assay has been utilized for ТВ diagnosis. It is obvious that further evaluation is required for further improvisation of the MPCR protocol for routine diagnostic purposes.

CONCLUSION

MPCR using MTB primers was able to pick up more ТВ patients compared to conventional L-J culture methods for the detection of MTB. MPCR using MTB genes is a highly sensitive and specific tool in the diagnosis of ТВ patients. MPCR using five gene primers is a rapid, reliable, and highly sensitive and specific diagnostic tool for the purpose of detecting and differentiating of the MTB patients and will be useful in diagnosing sputum samples. In future, MPCR with more than three target genes may have an important role in strengthening the diagnosis of ТВ.

ACKNOWLEDGMENTS

We thank UGC-UPE Phase II Biomedical Research, the University of Madras for providing the financial support for the accomplishment of this project. GR was a project fellow in this project.

KEYWORDS

  • • extremely drug resistant
  • human immunodeficiency virus
  • multi-drug resistant
  • multiplex PCR
  • Mycobacterium tuberculosis
  • polymerase chain reaction
  • tuberculosis

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