ORIGINAL ARTICLE Year : 2021  |  Volume : 70  |  Issue : 1  |  Page : 43-47

Clinical usefulness of Mycobacterium tuberculosis/NTM real-time polymerase chain reaction in diagnosis of tuberculosis

Alaa Rashad1, Hebat-Allah G Rashed2, Mohammed Zakaria2, Asmaa Omar2, Amal Hosni2, Hanaa Nafady-Hego3
1 Department of Chest, Qena Faculty of Medicine, South Valley University, Qena,Department of Chest and Tuberculosis, Faculty of Medicine, Assiut University, Assiut, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt
3 Department of Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt

Date of Submission25-Oct-2019Date of Decision26-Dec-2019Date of Acceptance27-Jan-2019Date of Web Publication27-Mar-2021

Correspondence Address:
MD Alaa Rashad
Department of Chest, Qena Faculty of Medicine, South Valley University, Qena, 111-232
Egypt

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ejcdt.ejcdt_201_19

Background Tuberculosis (TB) management and prevention of further spread is crucially based on early and prompt diagnosis. Even though conventional methods are the gold standard for detection of Mycobacterium tuberculosis (MTB), they are time consuming and do not detect nontuberculous mycobacteria in most cases. The present study evaluated Anyplex MTB/NTM real-time (RT)-PCR assay (Seegene) for TB diagnosis in pulmonary and extrapulmonary TB cases in comparison with culture on Lowenstein–Jensen as a gold standard for diagnosis of MTB.
Patients and methods The study included 100 patients with suspected TB: pulmonary TB represented 68 patients and extrapulmonary TB represented 32 patients. All pathological samples were stained by Ziehl–Neelsen staining (ZNS), cultured on Lowenstein–Jensen media and processed for PCR by the Anyplex MTB/NTM RT-PCR.
Results Anyplex MTB/NTM RT-PCR showed a sensitivity and specificity of 100 and 65.5%, respectively, when compared with the sensitivity and specificity of ZNS, which were 87.2 and 79.3%, respectively, in the clinically suspected pulmonary TB group. However, in the clinically suspected extrapulmonary TB group, Anyplex MTB/NTM RT-PCR had sensitivity and specificity of 100 and 79.2%, respectively, when compared with those of ZNS, which were 75 and 100%, respectively.
Conclusion The use of Seegene Anyplex MTB/NTM assay for detection and differentiation of pulmonary and extrapulmonary TB in our area is more convenient and accurate compared with ZNS.

Keywords: Anyplex Mycobacterium tuberculosis/NTM real-time PCR, tuberculosis, Ziehl–Neelsen staining

How to cite this article:
Rashad A, Rashed HAG, Zakaria M, Omar A, Hosni A, Nafady-Hego H. Clinical usefulness of Mycobacterium tuberculosis/NTM real-time polymerase chain reaction in diagnosis of tuberculosis. Egypt J Chest Dis Tuberc 2021;70:43-7
How to cite this URL:
Rashad A, Rashed HAG, Zakaria M, Omar A, Hosni A, Nafady-Hego H. Clinical usefulness of Mycobacterium tuberculosis/NTM real-time polymerase chain reaction in diagnosis of tuberculosis. Egypt J Chest Dis Tuberc [serial online] 2021 [cited 2021 Dec 5];70:43-7. Available from: http://www.ejcdt.eg.net/text.asp?2021/70/1/43/312135   Introduction 

Despite the global efforts to eradicate tuberculosis (TB), its morbidity and mortality represents a big burden worldwide. The WHO reported in 2017 that ∼10 million new cases of TB were detected, and the mortality rate was 1.3 million [1]. Recently, in Egypt, TB incidence per 105 population per year was 13 (12–14), with 13 (12–14) thousand people infected with TB, and the rate of HIV coinfected people was less than 0.1 per 105 population [53 (34–76) patients] [1]. A total of 430 cases died owing to TB in 2017, and 13 of them had HIV coinfection [1]. Multidrug-resistant Mycobacterium tuberculosis (MTB) has made the management of TB and controlling the spread of the disease challenging for health care workers. In Egypt, the rate of multidrug-resistant-TB is 2.1 (1.8–2.7), and the number of infected patients is 2.2 (1.8–2.7) thousand [1].

The decision to start anti-TB therapy depends on an accurate diagnosis of TB [2]. The definite diagnosis for TB is done by identification of acid-fast bacilli (AFB) using routine Ziehl–Neelsen staining (ZNS) if the number of MBT is more than 10 000 bacilli/ml sputum, or cultivation of MTB on culture if the smear is negative [3]. Different culture media have been used for growing Tubercle bacillus. Egg-based media are the best known solid media used for identification of MTB: Lowenstein–Jensen (LJ) medium contains less inhibitory materials for MBT, so it tends to yield more positive results from pathological specimens. Even though the technique is sensitive, as it can identify 10–100 bacilli/ml sputum, it requires incubation period up to 16 weeks [4]. Our hospital is a tertiary one that accepts patients from the whole of Upper Egypt area; therefore, rapid, accurate, and noninvasive technique is mandated. Molecular tests have been introduced to overcome the low sensitivity and delayed diagnosis of conventional methods and have been addressed in many studies in the literature [5]. Of these assays, we think that Anyplace MTB/NTM real-time (RT)-PCR detection assay (Seegene) is suitable in our area with low TB incidence: it can detect and discriminate MTBC and NTM, it is valid for a wide range of specimen, it is automated, and the result interpretation can be done within 3.5 h of sample receipt [6]. The present study evaluated Anyplex MTB/NTM RT-PCR assay (Seegene) for TB diagnosis in pulmonary and extrapulmonary TB in comparison with culture on LJ as a gold standard for diagnosis of MTB.

  Patients and methods 

This is a cross-sectional descriptive study. All samples were taken after approval of the patients. They signed an informed consent form in accordance with the last update of the Declaration of Helsinki and after approval of Assiut University Committee for human research and ethics.

Sample collection

Pathological specimens from 100 patients of clinically suspected TB were collected and analyzed by microbiological tests: direct staining by ZN stain for AFB, culture on LJ media, and molecular test, such as Anyplex MTB/NTM RT-PCR. Samples were either from suspected pulmonary TB [sputum (53 samples) and bronchoalveolar lavage (15 samples)] (group A) or suspected extrapulmonary [pleural fluid (11 samples), pus (six samples), urine (six samples), stool (five samples), ascetic fluid (three samples), and bone tissue (one sample)] (group B). The study was conducted between November 2014 and February 2016 on all patients who were admitted to the Chest Department, TB clinic, and the orthopedic operation room, Assiut University Hospital, Assiut, Egypt, and subjected to full history taking, clinical examination, and chest radiograph. The study protocol was based on the World Medical Association’s Declaration of Helsinki last update and was reviewed and approved by the Assiut university hospital review board.

Acid-fast bacilli and Lowenstein–Jensen culture procedures

Detection of AFB was done by ZNS method as described by Stewart [7]. In summary, decontamination of all samples was done using 4% N-acetyl-L cysteine-sodium hydroxide and then centrifuged at 3000g for 15 min and re-suspended in phosphate buffer then smears were stained and ZNS was analyzed. For mycobacterial cultures, half an milliliters aliquot of decontaminated samples was inoculated onto LJ media (Biomerieux, F-69280 Marcy l’Etoile, France), incubated at 37°C for 8 weeks, and examined weekly for growth. Mycobacterial isolates were identified by the specific morphological characteristics of the isolated colony.

Processing of samples for Seegene Anyplex Mycobacterium tuberculosis/NTM tuberculosis PCR

All samples were investigated by Seegene Anyplex TB PCR test (Seegene, Soul, South Korea). DNA extraction was done as a follow: the sample was mixed with 100 μl of DNA extraction solution by vortexing, then the sample was boiled for 20 min at 100°C at a heat block, and then the mixtures were centrifuged for 5 min at 15 000g. Overall, 5 μl of an aliquot of supernatant was mixed with 15 μl of master mix, which contains 10 μl×2 Anyplex PCR master mix, 2 μl 10×MTB/NTM oligonucleotide mix, and 3 μl 8-methoxypsoralen . CFX96 Touch RT-PCR Detection System (Bio-Rad Laboratories Inc., USA) was used for detection and amplification: MTBC identification detects MPB64 and IS6110 genes, whereas NTM identification detects 16S rRNA gene. For the quality control, the kit contains in-house extraction controls, positive and negative amplification, and internal control in the master mix to detect inhibition, and we processed them in each run. The interpretation of data was done automatically by CFX Maestro software that showed the results to threshold and cutoff values. A positive control and a negative control were used for quality control in each run [8].

Statistical analysis

Statistical Package for Social Sciences (SPSS), version 21 (IBM Inc., Armonk, New York, USA) was used to analyze the data. Data were presented as mean±SD, frequencies, and percentage. LJ culture was used as a gold standard test, whereas ZNS and Anyplex MTB/NTM RT-PCR as a screening test. Specificity, sensitivity, positive predictive, and negative predictive values were measured as per the recommendations of Standards for Reporting of Diagnostic Accuracy Studies [9].

  Results 

Characteristics of the study groups

Our study included two groups of patients: group A comprised 68 patients of clinically suspected pulmonary TB cases, and group B comprised 32 patients of clinically suspected extrapulmonary TB cases. Group A included 44 (64.7%) males, and the age ranged from 17 to 77 years, with mean±SD age of 47.35±14.58 years, whereas group B included 18 (56.2%) males, and the age ranged from 24 to 78 years, with the mean±SD age of 44.81±15.24 years.

Analysis of Ziehl–Neelsen staining, Lowenstein–Jensen culture, and Mycobacterium tuberculosis/NTM real-time PCR results

Data are shown in [Table 1]. AFB revealed that 40 (58.8%) pulmonary samples and six (18.8%) extrapulmonary samples were positive, whereas LJ culture showed 39 (57.4%) pulmonary samples and eight (25%) extrapulmonary samples were positive. Anyplex MTB/NTM RT-PCR showed 49 (72.1%) pulmonary samples [MTB; 48 (70.6%) and NTM; one (1.5%)] and 13 (40.6%) extrapulmonary samples were positive.

Table 1 Ziehl–Neelsen stain, Lowenstein–Jensen culture, and Anyplex Mycobacterium tuberculosis/NTM real-time PCR methods results among studied cases

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Comparison of Ziehl–Neelsen stain with Lowenstein–Jensen culture

LJ culture was used as the gold standard, definite diagnosis. Smear microscopy of pulmonary and extrapulmonary samples demonstrated overall specificities, sensitivities, negative predictive, and positive predictive values (with 95% confidence intervals) as follows: pulmonary: 79.3, 87.2, 82.1, and 85%, respectively, and extrapulmonary: 100, 75, 92.3, and 100%, respectively. Data are shown in [Table 2].

Table 2 Comparison of Ziehl–Neelsen stain with Lowenstein–Jensen culture

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Comparison of the Anyplex Mycobacterium tuberculosis/NTM assay with Lowenstein–Jensen culture

The Anyplex MTB/NTM assay when compared with LJ culture demonstrated overall specificities, sensitivities, negative predictive, and positive predictive values (with 95% confidence intervals) as follows: pulmonary: 65.5, 100, 100, and 79.6%, respectively, and extrapulmonary: 79.2, 100, 100, and 61.5%, respectively. Data are shown in [Table 3].

All culture-positive samples were correctly identified by the Anyplex MTB/NTM assay.

  Discussion 

Owing to the simplicity and low cost, ZNS is widely used as a conventional method for laboratory diagnosis of TB in developing countries. However, the ZN smear technique has several limitations, such as low sensitivity, particularly in extrapulmonary cases as ∼5000 to 10 000 AFB/ml is needed for positive smear result by AFB smear microscopy [8],[10]. Herein, ZNS sensitivity and specificity in suspected pulmonary TB specimens were 87.2 and 79.3%, respectively, and in suspected extrapulmonary TB specimens were 75 and 100%, respectively. These results are in agreement with Ulukanligil et al. [11], who reported that the ZNS microscopy had sensitivity of 80% when the culture was employed as a reference assay, and with Khatib et al. [12] and Muddaiah et al. [13], who reported that sensitivity was 93.45% and specificity was 88.10% in patients with pulmonary TB, and with Singh et al. [14] who reported using the culture as a reference diagnosis, the sensitivity, specificity, positive predictive value, and negative predictive value for smear were 78.6, 87.5, 89, and 76%, respectively, in patients with pulmonary TB, and the sensitivity, specificity, positive predictive value, and negative predictive value of smear examination were 56.3, 89, 72, and 80.3%, respectively, in extrapulmonary TB samples, and with Kelamane and Mispah [15], who reported that the sensitivity and specificity of ZN microscopy to culture was 62.7 and 98.5%, respectively.

Our results are contrary to Bilgin et al. [16] who reported that with a comparison of ZN staining with culture, results revealed sensitivity of 32.3% for nonrespiratory samples, which is lower than our results, and with Chaware et al. [17], who showed that examination of smears made from sputum gives a sensitivity of 55% compared with the culture, as our samples were from patients highly suspected to have TB.

PCR is a molecular-based assay for diagnosis, which is faster and sensitive than other conventional diagnostic method [17],[18],[19]. The novel Seegene Anyplex plus MTB/NTM Detection kit uses Dual Priming Oligonucleotide technology to prevent nonspecific priming. Anyplex MTB/NTM detection assay is used for identification of MTB and NTM infections [5]. PCR results can be obtained within 6–8 h, in addition to rapid diagnosis. PCR improved the detection of pulmonary and extrapulmonary TB [20]. In this study, the results of PCR in suspected pulmonary TB cases showed 49 (72.1%) were positive cases, comprising 48 (70.6%) MTB cases and one (1.5%) NTM cases. Sensitivity and specificity were 100 and 65.5%, respectively, and in suspected extrapulmonary TB, it was found that 13 (40.6%) were positive, comprising eight (25%) MTB cases and five (15.6%) NTM cases. Sensitivity and specificity were 100 and 79.2%, respectively. These results are in agreement with Johar et al. [20] who reported that for pulmonary specimens, sensitivity and specificity of PCR were 89 and 71%, respectively. For extrapulmonary specimens, sensitivity and the specificity of PCR were 92 and 67%, respectively. Chakravorty et al. [21] reported that the sensitivity and specificity of PCR were 99.1% and 71.2%, respectively, when compared with JM culture as the gold standard. Raveendran and Wattal [22] reported that PCR can be used as an alternative assay in the diagnosis of extrapulmonary TB, and compared with LJ culture, it showed sensitivities ranging from 42 to 100%, and the range of specificities was from 85 to 100%.

Lim et al. [5] reported the Anyplex TB PCR had sensitivity and specificity of 87.5 and 98.2%, respectively, but specificity in our results was lower. Variability between laboratories regarding sensitivity and specificity of PCR could be explained by various reasons starting from procedural differences between laboratories passing through cross-contamination and finally the usage of different primers. In addition, detection of nonviable mycobacteria by PCR is considered a major drawback when compared with conventional culture methods [23]. PCR is found to be a very rapid and sensitive method to aid in early diagnosis, treatment, and cure of TB, especially extrapulmonary, but the drawbacks of PCR are its high cost; the specific requirement of infrastructure, equipment, and expertise; and lack of treatment monitoring by PCR [19]. Our results are contrary to Wang et al. [24] who found that although TB RT-PCR is highly specific (up to 100%), its sensitivity varied from 42.8 to 96.7%, based on the type of clinical specimen.

  Conclusion 

Seegene Anyplex MTB/NTM assay for TB detection is rapid, reliable, convenient, and suitable for MTB and NTM infection detection and differentiation in pulmonary and extrapulmonary samples, with high specificity in comparison with conventional methods.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

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  [Table 1], [Table 2], [Table 3]