|Year : 2016 | Volume
| Issue : 4 | Page : 258-262
Conventional versus molecular methods for diagnosis of tuberculosis in a tertiary care center: A study from Punjab
Shavi Nagpal1, GS Chopra1, Aroma Oberoi1, Navjot Singh2, Shereen R Varghese1
1 Department of Microbiology, Christian Medical College and Hospital, Ludhiana, Punjab, India
2 Department of Medicine, Christian Medical College and Hospital, Ludhiana, Punjab, India
|Date of Web Publication||14-Sep-2016|
Dr. Aroma Oberoi
Department of Microbiology, Christian Medical College and Hospital, Ludhiana, Punjab
Source of Support: None, Conflict of Interest: None
Background: A fast and accurate diagnosis is necessary to control and eliminate tuberculosis (TB). There have been various studies evaluating the efficacy of polymerase chain reaction (PCR) testing in clinical practice. Most of these studies have found PCR as a useful tool to diagnose TB and more so for the extra-pulmonary cases. Aims and Objectives: The aim of the study was to evaluate the results of TB by real-time PCR versus the conventional methods of diagnosis in a tertiary care center in Punjab. Materials and Methods: This study was done in a tertiary care center of Punjab to look for the results of clinical samples tested for TB using PCR, Ziehl–Neelsen staining and culture on Lowenstein–Jensen medium. 16S rRNA gene was used for Mycobacterium tuberculosis detection in PCR. This study included all samples tested for TB from July 2015 to October 2015. Results: A total of 214 samples were tested for TB using smear microscopy, culture and PCR (including 194 [90.6%] extra-pulmonary and 20 [9.3%] pulmonary samples). These included 76 cerebrospinal fluid, 30 pleural fluid, 42 tissues, 17 ascitic fluid, 13 urine, 15 bronchoalveolar lavage (BAL), 16 pus, and 5 sputum samples. Smear microscopy was positive in a total of 4 samples out of 214 (1.8%), which included 2 sputum, 1 tissue, and 1 ascitic fluid samples, while culture was positive for 6 samples (2.8%) which included 2 sputum, 1 pus, 1 BAL, 1 tissue, and 1 ascitic fluid sample. The TB PCR results were positive in a total of 71 (33.1%) samples out of 214. In addition, three samples also tested positive for Mycobacteria other than TB. Conclusion: PCR is a very rapid and accurate diagnostic tool for early detection of TB in particularly for extrapulmonary TB.
Keywords: Lowenstein–Jensen medium, real-time polymerase chain reaction, tuberculosis, Ziehl–Neelsen staining
|How to cite this article:|
Nagpal S, Chopra G S, Oberoi A, Singh N, Varghese SR. Conventional versus molecular methods for diagnosis of tuberculosis in a tertiary care center: A study from Punjab. CHRISMED J Health Res 2016;3:258-62
|How to cite this URL:|
Nagpal S, Chopra G S, Oberoi A, Singh N, Varghese SR. Conventional versus molecular methods for diagnosis of tuberculosis in a tertiary care center: A study from Punjab. CHRISMED J Health Res [serial online] 2016 [cited 2020 Apr 7];3:258-62. Available from: http://www.cjhr.org/text.asp?2016/3/4/258/190572
| Introduction|| |
Mycobacterium tuberculosis (MTB) is an infectious human pathogen causing serious public health problem worldwide. This disease primarily affects lungs but can also affect the intestines, meninges, bones, lymph nodes, skin, and other tissues of the body. It is estimated that about one-third of the current global population is infected asymptomatically with tuberculosis (TB), of whom 5–10% will develop clinical disease during their lifetime. Most new cases and deaths occur in the developing countries where the infection is often acquired in childhood.
India is the highest TB burden country in the world in terms of incident cases that occur each year. The estimated prevalence and incidence rate of all forms of TB in India (2011) were 256 and 185 cases per 100,000 population, respectively. Poverty, economic recession, malnutrition, overcrowding, indoor air pollution, tobacco, alcohol abuse, and diabetes makes people residing in developing countries more vulnerable. To make the situation worse, TB has formed a lethal combination with human immunodeficiency virus (HIV). TB can involve any organ system in the body. While pulmonary TB is the most common presentation, extrapulmonary TB (EPTB) is also an important clinical problem. The term EPTB has been used to describe isolated occurrence of TB at body sites other than the lung.
In the era before the HIV pandemic, and in studies involving immunocompetent adults, it has been observed that EPTB constituted about 15–20% of all cases of TB. In HIV-positive patients, EPTB accounts for more than 50% of all cases of TB. Nontuberculous Mycobacteria (NTM)/Mycobacterium other than TB (MOTT) are found worldwide in soil and in animals. They are also increasingly implicated in various human infections including lung infections, skin infections, and lymphadenopathy, etc.
The most powerful weapon is the combination of case finding and treatment. The important case finding tools for TB are sample microscopic examination, radiography, and culture. Smear examination by direct microscopy for acid-fast bacilli (AFB) is considered the method of choice. The reliability, cost effectiveness and ease of direct microscopic examination has made it number one case finding method worldwide. The Ziehl–Neelsen (ZN) staining is a rapid and cheap method, to detect AFB. Many different culture media have been devised for growing the tubercle bacillus. Egg-based media are among the best known solid media used for isolation of MTB, in particular, Lowenstein–Jensen (LJ) medium as these media tend to yield high number of positives from direct clinical specimens because it is less inhibitory to the Mycobacteria. The culture process is not only difficult, tedious, lengthy (takes at least 6–8 weeks) but also needs special training and expertise. The culture requires time and viable microorganisms, which are difficult to obtain, especially in treated patients. Confirmation is usually done from the biological characteristics of culture growth and with selected molecular or biochemical tests.
Culture remains the gold standard for diagnosing TB. However, the disease most often remains undiagnosed and even worse, untreated. Major difficulty is with extrapulmonary samples, which are associated with low sensitivity of AFB smear and culture. The diagnosis of EPTB is especially challenging for various reasons like lack of adequate sample amount or volumes, paucibacillary nature of specimens yielding very few bacilli and lack of an efficient sample processing technique universally applicable on all types of extrapulmonary samples. The polymerase chain reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Many reports have described the application of PCR to the diagnosis of pulmonary TB from sputum examination, but there are very few reports from India describing its application in extra-pulmonary cases., The molecular-based diagnosis by PCR technique is faster and sensitive. A chronic and rampant disease like TB requires a rapid, sensitive, and specific diagnosis owing to limitations of the traditional microbiological methods (paucibacillary nature of specimens) and the extensive differential diagnosis.
This study was undertaken to compare the molecular method with the conventional diagnostic procedures for TB.
| Materials and Methods|| |
This study was done in the Mycobacteriology and Molecular Laboratory, of a tertiary care hospital in northern India, to look for the results of clinical samples tested for TB using real-time (RT) PCR. These samples were also tested for TB according to standard microbiological techniques of diagnosis such as acid fast (ZN) staining and culture on LJ medium. The study included all samples tested from July 2015 to October 2015.
Clinical samples received from suspected cases of TB were processed for AFB smear and culture, the samples were first homogenized and concentrated using Petroff's method (4% NaOH solution). This mixture was homogenized by allowing it to stand at room temperature for 15–20 min. After this step, phosphate buffer was added and mixed well. The specimen was then centrifuged at 3000 rpm for 15–20 min. After centrifugation, a portion of sediment was directly inoculated onto LJ medium slopes; the other portion was used for preparation of direct smear for ZN staining. The tissue biopsy specimens were minced and homogenized in a sterile homogenizer, and a portion of the homogenate was directly inoculated onto LJ medium slopes, and other portion was used for making smears for ZN staining. The LJ medium bottles were incubated at 37°C in the incubator. Culture readings were taken every week and discarded as negative in case of no growth at the end of 8 weeks.
DNA extraction was performed on all the received samples by Geno-Sen's DNA extraction mini kit (Genome Diagnostics Pvt. Ltd., New Delhi, India). TB DNA RT amplification was done by Geno-Sen's MTB/MOTT (rotor gene) RT-PCR kit (Genome Diagnostics Pvt. Ltd., New Delhi, India). 16S rRNA gene was used for MTB detection in PCR.
| Results|| |
A total of 214 samples were tested for TB using smear microscopy, culture, and RT-PCR (including twenty pulmonary samples [9.3%] and 194 [90.6%] extra-pulmonary). The sample distribution is shown in [Figure 1]. Smear microscopy was positive for a total of 4 samples out of 214 (1.8%), which included 2 sputum, 1 tissue, and 1 ascitic fluid samples, while culture was positive for 6 samples (2.8%) which included 2 sputum, 1 pus, 1 bronchoalveolar lavage (BAL), 1 tissue, and 1 ascitic fluid sample [Table 1]. The TB PCR results were positive in a total of 71 (33.1%) samples out of 214 [Table 2]. The samples detected positive by ZN smear, and LJ culture were all positive by PCR too.
|Table 1: Type of specimens and positivity of polymerase chain reaction, culture, and smear|
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|Table 2: Total number of samples including polymerase chain reaction, culture, and smear positive samples|
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In addition to the above-mentioned results, 3 samples also tested positive for MOTT. These included 1 tissue, 1 pleural fluid, and 1 BAL sample.
| Discussion|| |
In this study, we compared the conventional methods and RT-PCR in diagnosing TB. PCR for TB showed the best diagnostic yield followed by culture and smear preparation. Prompt diagnosis of TB, especially extrapulmonary, remains a challenge always. Rapid and accurate diagnosis of TB is absolutely imperative in developing country like India where LJ medium is still used as the gold standard for its diagnosis. The culture methods are time-consuming, therefore, clinical acumen, and evidence like radiological diagnosis are still the main parameters being used to treat the patient with a full course of anti-tubercular treatment. As the majority of our clinical samples were extra-pulmonary, we can derive from our study that one has to go for methods other than conventional methods like PCR for diagnosis.
In a recent study By Sharma et al., PCR was found to be highly sensitive and specific tool in the diagnosis of paucibacillary conditions like EPTB. In their study, overall sensitivities of microscopy, culture, and PCR were 7.33%, 11.3%, and 74.6%, respectively. In another study by Siddiqui et al., there was 5% positivity rate by ZN staining, 15% positivity rate by LJ culture medium, and 70% positivity rate by PCR. The samples detected positive by ZN smear, and LJ culture were all positive by PCR too. However, in this study, the yield of staining and culture positivity was much less that of a PCR.
In a study by Singh et al., wherein PCR was compared with conventional tests for diagnosis of TB in granulomatous lymphadenopathy, it was concluded that PCR is the most sensitive single technique available to date for the demonstration of MTB in specimens derived from patients with a clinical suspicion of tubercular lymphadenitis. One study from Bangladesh showed that PCR yielded 93 (68.9%) positive results (out of 135 samples) on suspected TB sputum samples, 20 (21.5%) of which were culture-negative sputum specimens. PCR diagnosis of TB was concluded to be a rapid and sensitive method of diagnosis. Our study showed 33% PCR positivity as compared to other studies showing 68–70%,, however, their positivity for smear and culture were also higher as compared to our study. In this study, PCR, smear staining, and culture positivity were less probably because of lesser number of representative samples.
Three of our patient also tested positive for MOTT. This carries a great significance as the NTM are ubiquitous and causes varied diseases, some mimicking those of MTB. However, the treatment given for MOTT is entirely different from the standard antitubercular treatment. PCR is found to be a specific test and easily differentiates between the two.
In many studies, problems with false positive PCR results, at rates ranging from 0.8% to 30% have been reported. Specificity of PCR results varies between laboratories due to procedural differences, differences in cross-contamination rates and the choice of primers. Furthermore, the primary limitation of PCR arises from the absence of suitable gold standard to assess its efficiency. Furthermore, samples containing nonviable Mycobacteria may lead to a false positive PCR thereby misleading physicians. The drawbacks of PCR are its high cost, specific requirement of infrastructure, equipment, and expertise. In our study, all the efforts were taken to overcome these limitations by proper preventive laboratory techniques and use of sterilized methods.
At present, India is experiencing an epidemic of TB. Considering the number of cases diagnosed with TB in India, there is an urgent need to use multiple/newer diagnostic modalities for rapid detection of MTB to control the transmission of TB. Molecular methods such as PCR are underutilized, and it is becoming imperative to have PCR available at as many health centers as possible. The clinical utility of detecting MTB by PCR is its reduction in the time to detection and its accuracy in detecting the pathogen in AFB smear-negative paucibacillary specimens. PCR is suitable as a public health tool in a country like India as the tests is rapid and early diagnosis of TB is crucial for prompt treatment and for the control of disease transmission.
Recently, there have been multiple new modalities of laboratory investigations that have revolutionized the diagnosis of TB. One of them is Xpert (MTB/rifampicin [RIF]) test, a nested RT-PCR-based test. It has been shown to be rapid, with the result for TB and RIF resistance in under 2 h. Other advantages include minimal biosafety facilities required, no risk of cross-contamination and do not require a skilled technician. It only detects RIF resistance or sensitivity and the former is a surrogate marker for multidrug resistance TB. This might lead to overestimation of MDR-TB even in regions where RIF mono-resistance is high. In such cases, line probe assays have an edge over Xpert MTB/RIF assay. Xpert test has been endorsed by the WHO for diagnosis of TB. In a study done at Mumbai, wherein Xpert test was used for EPTB diagnosis, it was seen that sensitivity of this test was 81% with specificity of 99.6%. Sensitivity was found to be higher for the majority of specimen except for cerebrospinal fluid. Furthermore, it correctly identified 98% of phenotypic RIF resistant cases and 94% of phenotypic susceptible cases. Liquid culture, for example, BACTEC, MGIT, needs more sophisticated equipment but detects the growth faster and has higher sensitivity than solid media.
| Conclusion|| |
Conventional methods of smear microscopy and culture remain the gold standard for diagnosing pulmonary TB; however, poor performance of these conventional methods on extrapulmonary specimens demands for more sensitive and specific techniques. PCR is a very rapid and accurate diagnostic tool for early detection of TB, particularly for EPTB. In a country like India with such high burden of TB and limited resources for diagnosing TB, PCR is found to be very rapid and sensitive method to aid in early diagnosis, treatment, and cure of TB, especially EPTB. Newer methods such as Xpert test also holds good potential for diagnosis of EPTB.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]