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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 6  |  Issue : 3  |  Page : 146-149

Analysis, comparison, and significance of nested polymerase chain reaction with conventional methods for the diagnosis of pulmonary and extrapulmonary tuberculosis among the rural population of North India


1 Department of Microbiology, Uttar Pradesh University of Medical Sciences, Etawah, Uttar Pradesh, India
2 Department of Pathology, Uttar Pradesh University of Medical Sciences, Etawah, Uttar Pradesh, India

Date of Submission17-Jun-2018
Date of Decision15-Aug-2018
Date of Acceptance11-Nov-2018
Date of Web Publication13-Aug-2019

Correspondence Address:
Seema Dayal
Department of Pathology, Uttar Pradesh University of Medical Sciences, Saifai, Etawah, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjhr.cjhr_87_18

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  Abstract 


Introduction: Tuberculosis (TB) is one of the most common infectious diseases among the Indian population, especially in rural scenario. It affects young people in majority. Living at slum areas and poverty are predisposing etiological factors. The diagnosis of TB is based on microbiology, cytology, and histopathology. Materials and Methods: The present study was carried out in the Department of Microbiology in collaboration with the Department of Pathology, UPUMS, Saifai, Etawah, UP, India, from January 2015 to June 2016. Samples were collected from suspected tubercular patients. A total of 101 samples were collected including tissue, ascitic fluid, pleural fluid, cerebral fluids, bronchoalveolar lavage, tracheal aspirates, urine, sputum, and pus from clinically suspected cases. Ziehl–Neelsen (ZN) staining, fluorescent staining with auramine phenol, culture on Löwenstein–Jensen (LJ) media, mycobacterial growth indicator tube (MGIT), and Middlebrook media were done. Subsequently, biochemical tests such as niacin detection, catalase test, and nitrate reduction test were done for identification of mycobacteria. Nested polymerase chain reaction (PCR) was performed for the detection of Mycobacteria in clinical samples. Results: Of the 101 suspected tubercular patients, males were more, and the maximum age group involved was 21–40 years. ZN staining was found positive in 16 cases which included ten extrapulmonary and six pulmonary, whereas auramine phenol was found positive in 22 cases which included 14 extrapulmonary and 8 pulmonary. Of 101 patients, 35 (34.6%) were found to be LJ culture positive, whereas 49 (48.5%) were found positive with MGIT culture. PCR for TB was found maximum, which was 76 (75.2%). Conclusion: TB is common among the young group with more predilection among males. Smear- and culture-negative specimens could not rule out TB. Nested PCR proved to be highly sensitive in detecting Mycobacterium tuberculosis DNA. Hence, when TB is suspected, a combination of conventional test and newer rapid techniques such as PCR is always required for the early diagnosis and treatment of TB.

Keywords: Histopathology, microbiology, nested polymerase chain reaction, tuberculosis


How to cite this article:
Singh A, Dayal S. Analysis, comparison, and significance of nested polymerase chain reaction with conventional methods for the diagnosis of pulmonary and extrapulmonary tuberculosis among the rural population of North India. CHRISMED J Health Res 2019;6:146-9

How to cite this URL:
Singh A, Dayal S. Analysis, comparison, and significance of nested polymerase chain reaction with conventional methods for the diagnosis of pulmonary and extrapulmonary tuberculosis among the rural population of North India. CHRISMED J Health Res [serial online] 2019 [cited 2019 Oct 17];6:146-9. Available from: http://www.cjhr.org/text.asp?2019/6/3/146/264382




  Introduction Top


TB appears to be a disease as old as human history. Bones of prehistoric man dating back to 8000 BC have shown typical changes of TB.[1] It has been described in India as early as Rigveda dated around 2000 BC. It was described as Yakshma.[2] It is a worldwide health problem with significant incidence and mortality rates, especially in developing countries like India. The six countries that stand out having largest number of incident cases in 2014 were India, Indonesia, China, Nigeria, Pakistan, and South Africa. India, Indonesia, and China alone accounted for a combined total of 43% of global cases in 2014.[3]

TB is an infectious bacterial disease caused by Mycobacterium tuberculosis which commonly affects the lungs. It is transmitted from person to person through droplet from throat and lung of people with active respiratory disease.[4] Primary affects the lungs and can also affect intestine, meninges, bones and joint, lymph node, skin, and other body parts. In healthy person, it often causes no symptoms.

Definitive diagnosis of TB involves demonstration of M. tuberculosis by microbiological, cytopathology, and histopathological techniques.[5] The organisms are demonstrable in multibacillary form by acid-fast staining and by culture. Sputum smear microscopy requires 10,000–100,000 organisms/ml.[6] Among the direct detection methods, microscopy of smear by differential or special staining is already in practice for M. tuberculosis, but has very poor sensitivity because the number of bacteria in extrapulmonary samples is low usually and similar explanation is true for poor sensitivity of M. tuberculosis-specific antigen detection. Although histology of extrapulmonary samples coupled with culture is thought to be most sensitive, it could miss up to 20% of cases as bacterial load in case of extrapulmonary cases is very low.[7]

The conventional method of isolation is inadequate for the early diagnosis as it takes 4–8 weeks apart from being poor in sensitivity. BACTEC method yields culture results in 7–10 days, but has limitation of radioactive waste. Mycobacterial growth indicator tube (MGIT) is a nonradioactive detection system using fluorochromes for the detection and drug screening. A rapid, sensitive, and specific tool like polymerase chain reaction (PCR) is an added windfall, which yields results even with low microbial load.[8] The presence of an extremely small number of bacteria can be detected in 24–48 h. Hence, the present study was conducted to compare nested PCR with conventional microbiological methods for the diagnosis of clinically suspected cases of TB and to evaluate sensitivity and specificity of nested PCR for early and rapid diagnosis of TB.


  Materials and Methods Top


The present study was carried out in the Department of Microbiology in collaboration with the Department of Pathology, UPUMS, Saifai, Etawah, UP, India, from January 2015 to June 2016. Samples were collected from suspected patients depending on their clinical presentation. A total of 101 samples were collected from different parts of body including tissue, ascitic fluid, pleural fluid, cerebral fluids, bronchoalveolar lavage, tracheal aspirates, urine, sputum, and pus from clinically suspected cases, and a control group (patients with no signs and symptoms of TB) of thirty samples was also included in the study. Inclusion criteria were all age groups and clinical and radiological evidence of TB whereas exclusion criteria were patients on antitubercular therapy and unwilling patient. Ziehl–Neelsen (ZN) staining, fluorescent staining with auramine phenol, culture on Löwenstein–Jensen (LJ) media, and MGIT tubes were done as per the standard protocol. Identification of culture isolate were done by biochemical tests.

Nested polymerase chain reaction

DNA extraction was done using QIAamp ® DNA Mini Kit (Qiagen, Germany) and carried out by strictly following the manufacturer's instructions.

  1. The samples (200 μl of the deposit) were lysed by incubation with proteinase K and a special lysis buffer containing guanidine hydrochloride and later treated with ethanol and centrifuged
  2. The sample was applied to the QIAamp mini spin column. DNA was adsorbed onto the QIAamp silica membrane during a brief centrifugation step
  3. DNA bound to the QIAamp membrane was washed in two centrifugation steps to remove any residual contaminants without affecting DNA binding
  4. Purified DNA was eluted from the mini spin column in a concentrated form in the elution buffer containing TE Buffer.


Nested PCR protocol was carried out as per the method described by Miyazaki et al.[9] Primers targeting the gene sequence encoding for the 38 kDa protein (protein antigen b) of M. tuberculosis were used as described by Sjöbring et al.,[10] for the detection of mycobacterial DNA in clinical samples.

The sequence of TB PCR primer pairs for the first and second round of amplifications was as follows:

Forward primer MTB1: 5′-ACCACCGAGCGGTTCGCC TGA-3′.

Reverse primer MTB2: 5′-GATCTGCGGGTCGTCCCA GGT-3′.

Internal forward primer MTB3F: 5′-TGACGTTGGCGGAGA CCG-3′.

Internal reverse primer MTB4: 5′-ATGGTGCCCTGGTAC ATG-3′.

The first amplification reaction mixture consisted of 45 μl of master mix (50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, 0.3 mM each of dNTP, 100 pmol each of primer MTB1 and MTB2, and 2.5 u of Taq polymerase) and 5 μl of target DNA. After the DNA was denatured at 95°C for 10 min, PCR performed under the following cycling conditions, denaturation at 94°C for 1 min, annealing at 63°C for 90 s, and extension at 72°C for 1 min. For the second round of PCR, 5 μl from the first PCR was sampled and mixed with 45 μl of freshly prepared reaction mixture (containing internal primers, MTB3 and MTB4). Cycling parameters were kept same as the first PCR. Both cycles were run for 35 cycles. PCR products were analyzed by electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and documented by gel documentation system (Gel Doc XR + System ®, Bio-Rad, USA). For each amplification, a positive control containing DNA extracted from reference strain M. tuberculosis H37Rv and no template control containing molecular grade water was run. Nested PCR amplicon of 322 bp obtained was specific for M. tuberculosis.

For TB detection in tissue, samples were grossed, processed, and sectioned at 2–4 μm, and H and E stain was applied.


  Results Top


[Table 1] depicts that of 101 suspected cases, 57 were male and 44 were female; the maximum age group involved was 21–40 years.
Table 1: Distribution of suspected cases of tuberculosis according to age and sex

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  Discussion Top


In India, TB remains a major global public health problem. Conventional methods including smear and culture used in the diagnosis of pulmonary and extrapulmonary TB have poor sensitivity in the samples with paucibacillary load. Problem in the diagnosis arises when clinical specimens contain very few Mycobacterium species and their slow growth rate limit detection by the conventional method such as acid-fast staining and bacterial culture, respectively. The early diagnosis of TB helps in the early treatment and thus preventing the possible transmission of the infection. With the development of molecular techniques, delay in the accurate detection of pathogen in acid-fast bacilli (AFB) smear-negative paucibacillary specimens is minimized. To identify TB in suspected patients, nested PCR was performed by amplification of the target nucleic acid regions that coded for 38 kDa MTB protein.

In our study, patients from all age groups were included, and a maximum number of suspected cases were in the age group of 21–40 years, further confirming that TB involves young population. The higher incidence of TB was found among males; similar results were seen by Zhang et al.[11] in their study. Explanation regarding these observations could be on the basis that males have wide social network that would lead to greater exposure to Mycobacterium and simultaneously males attend more OPD than females.

In the present study, we compared results of light microscopy of ZN stain smear with that of fluorescent microscopy of auramine phenol stain smear for the detection of AFB. Of 101 samples, 15.84% showed positivity with ZN staining whereas 21.78% was with auramine phenol stain [Table 2]. Hence, the results showed that auramine phenol stain was more sensitive in the detection of AFB in pulmonary as well as extrapulmonary. Similar results were found by Githui et al.,[12] Ulukanligil et al.,[13] Murray et al.,[14] and Jain et al.[15] They also showed that fluorescent staining is more sensitive as compared to ZN staining for the demonstration of AFB. This may occur because organisms in fluorescent staining offer much better contrast, appearing as brilliant yellow against a darkened background. As low power is used for examination, large number of specimens can be examined in a given time, and colorblind investigators may also use this method without difficulties. These are the additional advantages of fluorescent staining.
Table 2: Smear positivity among suspected tuberculosis pulmonary and extrapulmonary samples by Ziehl- Neelsen staining and auramine-phenol staining

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In the present study, we compared solid media (LJ) and automated BACTEC MGIT 960 system for isolation of M. tuberculosis in clinically suspected cases of TB. MGIT 960 system provided better isolation rate of Mycobacterium (45%) from a variety of clinical samples compared to solid media (34.6%) [Table 3]. Similar findings were seen in a study done by Rishi et al.[16] and Bunger et al.,[17] the reason behind that was because of added growth supplement like OAEDC.
Table 3: LJ positivity, mycobacterial growth indicator tube, and nested polymerase chain reaction positivity among pulmonary and extrapulmonary tuberculosis

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The contamination rate was found be less in case of BACTEC MGIT 960 system (6%) as compared to the solid media (8%), which was not concordant to the rate of contamination that was reported by Chihota et al. (16.7% by MGIT and 9.3% by solid media).[18] The possible reason for reduced contamination rate was use of N-acetyl-l-cysteine-sodium hydroxide method, addition of MGIT PANTA mixture, and use of biosafety cabinet for processing for culture. The time taken for the detection of Mycobacterium growth according to smear positivity was noted to be significantly shorter for BACTEC MGIT 960, i.e. 7–14 days as compared to solid media, i.e. 11–35 days. This was supported by Chihota et al.,[18] Rishi et al.,[16] and Bunger et al.[17] However, for maximum recovery of Mycobacterium, it is important to use both types of media.

We have used the molecular assay which was based on the amplification of genes encoding 38 KDa protein, detected almost solely in M. tuberculosis and specific for M. tuberculosis complex. It is actively secreted and partly attached to the surface of Mycobacterium by lipid tail, which is responsible for binding of protein to protein. Thus, 38 KDa protein gene appears to be ideal for amplification.

In the present study, of 101 clinically suspected tubercular patients, conventional bacteriological technique was positive in 51 (50.4%) whereas PCR was in 76 (75.2%). PCR was found to be positive in 56% culture-negative specimens. Our results were significant for PCR as seen by Negi et al.,[19] Ogusku et al.,[20] and Dwivedi.[21]


  Conclusion Top


TB is common among the young group with more predilection among males. Smear- and culture-negative specimens could not rule out TB. Nested PCR proved to be highly sensitive in detecting M. tuberculosis TB DNA in pulmonary and extrapulmonary TB, especially in extrapulmonary because of its paucibacillary nature. Hence, when TB is suspected, a combination of conventional test and newer rapid technique such as PCR is always required for the early diagnosis and treatment of TB.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Mohan H. Tuberculosis. Textbook of Pathology. 6th ed. New Delhi: Jaypee Brothers Medical Publishers; 2010. p. 149-59.  Back to cited text no. 5
    
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Trajman A, Pai M, Dheda K, van Zyl Smit R, Zwerling AA, Joshi R, et al. Novel tests for diagnosing tuberculous pleural effusion: What works and what does not? Eur Respir J 2008;31:1098-106.  Back to cited text no. 7
    
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Miyazaki Y, Koga H, Kohno S, Kaku M. Nested polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol 1993;31:2228-32.  Back to cited text no. 9
    
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Sjöbring U, Mecklenburg M, Andersen AB, Miörner H. Polymerase chain reaction for detection of Mycobacterium tuberculosis. J Clin Microbiol 1990;28:2200-4.  Back to cited text no. 10
    
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Zhang X, Andersen AB, Lillebaek T, Kamper-Jørgensen Z, Thomsen VØ, Ladefoged K, et al. Effect of sex, age, and race on the clinical presentation of tuberculosis: A 15-year population-based study. Am J Trop Med Hyg 2011;85:285-90.  Back to cited text no. 11
    
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Githui W, Kitui F, Juma ES, Obwana DO, Mwai J, Kwamasga D. A comparative study on the reliability of the fluorescence microscopy and Ziehl – Nelson method in the diagnosis of pulmonary tuberculosis. East Afr Med J 1993;70:263-6.  Back to cited text no. 12
    
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Ulukanligil M, Aslan G, Tasçi S. A comparative study on the different staining methods and number of specimens for the detection of acid fast bacilli. Mem Inst Oswaldo Cruz 2000;95:855-8.  Back to cited text no. 13
    
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Murray SJ, Barrett A, Magee JG, Freeman R. Optimisation of acid fast smears for the direct detection of mycobacteria in clinical samples. J Clin Pathol 2003;56:613-5.  Back to cited text no. 14
    
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Jain A, Bhargawa A, Agarwal SK. A comparative study of two commonly used staining techniques for acid fast bacilli in clinical specimens. Indian J Tuberc 2002;49:161.  Back to cited text no. 15
    
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Rishi S, Sinha P, Malhotra B, Pal N. A comparative study for the detection of mycobacteria by BACTEC MGIT 960, Lowenstein Jensen media and direct AFB smear examination. Indian J Med Microbiol 2007;25:383-6.  Back to cited text no. 16
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Bunger R, Singh VA, Avneet, Mehta S, Pathania D. Evaluation of BACTEC micro MGIT with LJ media for detection of Mycobacterium in clinically suspected patients of extra pulmonary tuberculosis in a tertiary care hospital at Mullana (Ambala). J Med Microb Diagn 2013;2:2.  Back to cited text no. 17
    
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Chihota VN, Grant AD, Fielding K, Ndibongo B, van Zyl A, Muirhead D. Liquid vs. Solid culture for tuberculosis: Performance and cost in a resource-constrained setting. Int J Tuberc Lung Dis 2010;14:1024-31.  Back to cited text no. 18
    
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Negi SS, Anand R, Pasha ST, Gupta S, Basir SF, Khare S, et al. Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples. Indian J Med Microbiol 2007;25:43-9.  Back to cited text no. 19
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Ogusku MM, Sadahiro A, Hirata MH, Hirata DC, Zaitz C, Salem JI. PCR in the diagnosis of cutaneous tuberculosis. Brazillian J Microbiol 2003;34:165-70.  Back to cited text no. 20
    
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Dwivedi A. Optimisation of 38 k Da based PCR assay for detection of Mycobacterium tuberculosis from clinical samples. Indian J Tuberc 2003;50:209.  Back to cited text no. 21
    



 
 
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