|Year : 2018 | Volume
| Issue : 3 | Page : 191-195
Assessment of diagnostic accuracy of GeneXpert Mycobacterium tuberculosis/rifampicin in diagnosis of pulmonary tuberculosis in Kosovo
Rrezarta Bajrami1, Gjyle Mulliqi1, Arsim Kurti2, Greta Lila1, Lul Raka1
1 National Institute of Public Health of Kosovo; University of Prishtina “Hasan Prishtina”, Pristina, Kosovo
2 National Institute of Public Health of Kosovo, Pristina, Kosovo
|Date of Web Publication||6-Sep-2018|
Dr. Lul Raka
National Institute of Public Health of Kosovo and University of Prishtina, Rrethi i Spitalit, p.n. 10000, Pristina
Source of Support: None, Conflict of Interest: None
Background: Despite control program for Mycobacterium tuberculosis (MTB) and anti-tuberculosis (TB) drugs, MTB continues to be a major threat to global health; furthermore, it is the second leading cause of death worldwide. The distribution of TB in Kosovo has the highest rate in Europe. This study was designed to assess the performance of single-sputum GeneXpert MTB/ rifampicin (RIF) assay against the Ziehl-Neelsen smear, solid culture in diagnosing suspected pulmonary cases, and RIF resistance. Methods: A cross-sectional study was conducted to examine various diagnostic methods between 2014 and 2016 in the Department of Microbiology, the National Institute of Public Health, Kosovo, and the Lung Clinic. The present study compares diagnostic methods of MTBC and RIF resistance using Xpert MTB/RIF test in 813 samples from 760 patients suspected of TB by conventional smear microscopy and culture method. Results: Three hundred and ninety-six patients (52.1%) were male and the mean age was 48.6 ± 18.1 years. A total of 261 patients (32.6%) had underlying lung diseases. Of the 801 specimens investigated, 165 (20.5%) were MTBC-positive by culture, while 199 (28.3%) were positive by Xpert assay. Among the examined specimens, Two specimens with false negatives were shown in the Xpert method. Compared with culture, the Xpert assay achieved 82.3% (95% confidence interval [CI]: 65.5%–93.2%) sensitivity and 97.6% (95% CI: 91.5%–99.7%) specificity. GeneXpert could detect 11.7% and 50% additional positive cases as compared to Lowenstein–Jensen culture and smear microscopy, respectively. Three cases with resistance to RIF were detected from clinical isolates. Conclusions: According to the results obtained in this study, GeneXpert MTB/RIF assay can be used as a useful tool for rapid and efficient diagnosis of TB.
Keywords: Diagnosis, molecular, tuberculosis
|How to cite this article:|
Bajrami R, Mulliqi G, Kurti A, Lila G, Raka L. Assessment of diagnostic accuracy of GeneXpert Mycobacterium tuberculosis/rifampicin in diagnosis of pulmonary tuberculosis in Kosovo. Biomed Biotechnol Res J 2018;2:191-5
|How to cite this URL:|
Bajrami R, Mulliqi G, Kurti A, Lila G, Raka L. Assessment of diagnostic accuracy of GeneXpert Mycobacterium tuberculosis/rifampicin in diagnosis of pulmonary tuberculosis in Kosovo. Biomed Biotechnol Res J [serial online] 2018 [cited 2021 Dec 6];2:191-5. Available from: https://www.bmbtrj.org/text.asp?2018/2/3/191/240712
| Introduction|| |
Tuberculosis (TB) presents as a serious public health problem, globally with an estimated 10.4 million new TB cases in 2015. It is a leading cause of deaths from infectious diseases (with worldwide estimated 1.8 million deaths in 2015), large numbers of deaths mainly occurring in low- and middle-income countries.,, Early diagnosis of diseased individuals can reduce the treatment period and transmission and so it can decrease the burden of TB also rapid diagnosis and detection of rifampicin (RIF) resistance is necessary for TB control, as transmission and emergence of multidrug-resistant TB (MDR-TB) cause serious health problems. GeneXpert MTB/RIF and GenoType MTBDRplus were approved by the WHO in 2011 and recommended for diagnosis of TB and MDR-TB in developing and high prevalence countries.,,,,,
Kosovo is located in Southeastern Europe, with a population of about two million. In the past decades, Kosovo's health-care system has been a major reform and has had many problems and obstacles, most notably political commitment and scarce resources. One of the big problems is that no health insurance system has yet been established, which is the main barrier to any attempt to improve the level of health care at all sectors.,, Since 1999, TB has once again emerged as a public health problem in Kosovo. The prevalence of TB in Europe is very high and the rate of informing people in 2012 was 46/100,000. There has been a decreasing trend in notified cases until 2006 when the number of cases remained more or less stable.,
For many decades, conventional methods such as smear microscopy and culture techniques have been used for the diagnosis of pulmonary TB. Smear microscopy has the disadvantages of low sensitivity and poor quality control. The culture techniques developed for mycobacterial growth are slower, taking 2–6 weeks to yield results and require a sophisticated infrastructure and technical expertise.
Such diagnostic delays predispose to the emergence of resistant strains and transmission of these strains, thus increasing the overall morbidity and mortality due to TB. Currently available tools to diagnose TB in endemic areas are not sensitive enough to halt the spread of disease. At present, only 28% of expected incident cases of TB are detected as smear positive. In developed countries, tools such as molecular detection, drug susceptibility testing, and culture are available. These techniques are sensitive but require a sophisticated infrastructure and take long time to provide results, thus resulting in diagnostic delays.
A pivotal barrier to the control of TB is the deficiency of an accurate and expeditious diagnostic test. At present, we do not have a rapid accurate diagnostic test that provides timely diagnosis of active TB, for this reason, most of the patients with symptoms and signs of TB either remain undiagnosed or are treated based on clinical suspicion., There is an urgent requirement of a rapid, safe, and a precise point-of-care TB diagnostic test. In recent years, some nucleic acid amplification (NAA) tests have been proposed to overcome the delay in the diagnosis of TB by conventional methods. NAA tests have the advantage of accurate identification of MTB directly from clinical specimens, providing significant advantage for disease management and infection control. NAA tests are approximately 25%–30% more sensitive than acid–fast bacilli (AFB) smear and thus can be of use in the setting of moderate/high clinical suspicion of TB when smear microscopy is negative.,, Only two Food and Drug Administration approved NAA tests have been available for use on AFB smear-positive respiratory specimens: the polymerase chain reaction (PCR) and transcription-mediated amplification.,, Cepheid has introduced the GeneXpert MTB/RIF assay for the diagnosis of active cases of TB. The GeneXpert MTB/RIF assay utilizes real-time PCR to simultaneously identify MTBC bacteria and detect RIF resistance in both smear-positive and smear-negative sputum samples. The test provides results within 2 h. It is technically simple to conduct and is safe as it produces no culturable aerosols. From December 2010, the WHO has endorsed the utilization of Xpert MTB/RIF assay as an early test for the diagnosis of TB in patients with suspected MDR-TB or HIV-associated., The aim of the present study was to investigate the GeneXpert MTB/RIF assay diagnostic method for direct detection of Mycobacterium tuberculosis (MTB) resistance to RIF and its comparison with conventional methods.
| Methods|| |
The present study was conducted between 2014 and 2016 in the TB Laboratories of the Lung Clinic at University Clinical Center of Kosovo and in the Department of Microbiology within the National Institute of Public Health of Kosovo, Prishtina, Kosovo. The samples used in this study were collected from among suspected cases of MTB infection based on clinical criteria. A total of 801 samples were enrolled in the study. Among respiratory specimens collected, 643 samples were sputum, 75 were thoracentesis fluid, and 15 were bronchoalveolar lavage. Nonrespiratory samples included 40 urine samples and 28 cerebrospinal fluid.
Both respiratory and nonrespiratory specimens were examined by the standard N-acetyl-L-cysteine and sodium hydroxide method. Freshly prepared Mycoprep at room temperature in NALC-NaOH solution (Becton Dickinson, Sparks, USA) was added to the specimens at equal volume, mixed on vortex, and left for 15 min for digestion. Afterward, the sterilized phosphate buffer with pH 6.8 (twice the amount of mixture) (Becton Dickinson, Sparks, USA) was added to the mixture and centrifuged at 300 rpm for 20 min. Sedimentation in 2.5 ml sterile phosphate buffer was solved for further study, supernatant was also removed from the mixture. Lowenstein–Jensen (LJ) medium (Liofilchem Diagnostici, Roseto d'Abruzzi, Italy) was used to inoculate with 0.5 mL of dissolved samples solution. The LJ medium was then incubated for 8 weeks at 37°C for a weekly review. Furthermore, 0.5 ml of the sample solution was added to the liquid of the Mycobacterium Growth Indicator Tubes (MGIT) (Becton Dickinson, Sparks, USA). After that, the MGIT tubes were incubated in an automated MGIT 960 system (Becton Dickinson, Sparks, USA) at 37°C for 6 weeks. The residual solution left was used for PCR investigation using Xpert MTB/RIF assay (Cepheid, Maurens-Scopont, France). From the clinical specimens, add 1 ml of the remainder to a screw-capped tube containing 2 ml of the sample reaction in the ratio 1–2, this reagent disables the samples with NaOH and isopropanol. The mixture was incubated for 15 min at room temperature and stirred every 5 min to remove any collimated in liquid. The resulting mixture was transferred to an Xpert MTB/RIF cartridge using sterile pipettes. Xpert MTB/RIF cartridges contain internal controls for sample processing (DNA extraction and for PCR presence inhibitors). The cartridge containing a specimen was placed on the GeneXpert instrument (GX). The test results were available for <2 h and were interpreted automatically by the GX system as follows: positive or negative results are related to the presence of MTB DNA, while the false results are due to the presence of PCR inhibitors.
Sterile specimens were digested and processed directly and without contamination, then centrifuged for 20 min at a speed of 3000 rpm. Sediments were reconstituted with 2.5 ml of sterile phosphate buffer and used for AFB microscopy, culture, and Xpert PCR. The presence of AFB was routinely observed for all samples. The smears were fixed and stained with Ziehl-Neelsen stain (Liofilchem Diagnostic, Roseto d'Abruzzi, Italy) and then examined using a 400 × magnification Olympus fluorescence microscope (Olympus, New York, USA)., Positive cultures of Mycobacteria were studied by smear test, pigment production, and biochemical tests such as nitrate reduction and niacin accumulation (Becton Dickinson, Sparks, USA). After growing and identifying different species, in cases where MTBC strains have been identified, DST is performed on the LJ medium.,
The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of Xpert assay and sputum acid–fast smear were compared to TB culture as a reference standard using contingency two-by-two tables. P < 0.05 were considered statistically significant. All statistical analyses of this paper were performed using MedCalc programs version 16.2.,
| Results|| |
A total of 801 samples, 733 respiratory samples and 68 nonrespiratory samples from 760 patients with suspected TB infections, were used to detect TB using Xpert gene and other common methods. 396 patients (52.1%) were male and the mean age was 48.6 ± 18.1 years. On the other hand, 261 patients (32.6%) had underlying lung disease. None of the patients was infected with HIV.
In addition, the MTBC-positive culture was used as a reference standard. Of the 801 samples in this study, 165 samples (20.5%) with MTBC culture and 199 samples (28.3%) with Xpert assays were positive. However, 92 of the samples (11.4%) were positive for Ziehl–Neelsen staining. A comparison of molecular and routine diagnostic methods is presented in [Table 1]. Seven of the positive smears in AFB were negative in the Xpert assay because these specimens were nontuberculous mycobacteria. Furthermore, 15 samples were false-negative Xpert methods compared with the reference method.
|Table 1: Two-by-two contingency tables comparing Xpert assay and sputum acid-fast smear with the reference standard|
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[Table 1] shows a two-by-two contingency table comparing sputum acid–fast smear and sputum culture. The sputum culture was used as a known reference standard method, the sensitivity, specificity, PPV, and NPV for sputum acid–fast smear were 53.3%, 98.8%, 94.1%, and 85.8%, respectively.
The sensitivity, specificity, PPV, and NPV and their corresponding 95% CIs are shown in [Table 2]. The sensitivity, specificity, PPV, and NPV of Xpert MTB/RIF test were estimated as 93.3%, 93.0%, 82.3%, and 97.5%, respectively. Xpert assay had statistically significant higher sensitivity than the sputum acid–fast smear (P < 0.001).
|Table 2: Sensitivity, specificity, positive predictive value, and negative predictive value of Xpert assay and sputum acid-fast smear comparing to sputum culture|
Click here to view
In the present study, three cases of resistance to RIF were identified from clinical isolates. GeneXpert could detect 11.7% and 50% additional positive cases as compared with LJ culture and smear microscopy, respectively.
Due to gender, age, and underlying lung disease, in this cohort study, there is no meaningful relationship with MTB detection methods.
| Discussion|| |
Commonly used techniques for detecting MTBC are low sensitivity in clinical specimens., Molecular techniques, such as GeneXpert systems, have changed the field of diagnosis of TB. These tests contain high-sensitivity and specificity results. In December 2010, the World Health Organization approved the Xpert MTB/RIF Diagnostic Test for rapid diagnosis of TB and MDR-TB., The GX is a simple, rapid technique for real-time PCR and has increased the sensitivity of detection of MTB complex. It must be part of the diagnostic arsenal of TB without replacing conventional microbiological tools and allow early diagnosis and appropriate treatment.,
Many studies have been done on molecular techniques for the detection of MTBC, indicating that PCR is a useful and convenient method for rapid diagnosis of tuberculous from clinical specimens.
It is also remarkable that this method also has limitations, including the extraction method and PCR inhibitors are associated in some clinical samples. Steingart et al. in a systematic review, among the 27 studies showed that Xpert test of respiratory samples in the diagnosis of pulmonary TB had a sensitivity of 89% (95% CI: 85%–92%) and specificity of 99% (95% CI: 98%–99%)., Different results from different studies are available. These differences in the results and sensitivity of the Xpert assay in the diagnosis of TB can be associated with entry criteria and methods used to obtain sputum samples. The results obtained in this study were consistent with previous studies, indicating a sensitivity of 96.4% of the Xpert assay for diagnosis of TB in acid–fast-positive sputum specimens., Similar results were obtained in Saudi Arabia, Pakistan, Thailand, and Turkey.,,, The GX is a simple, rapid technique for real-time PCR and has increased the sensitivity of detection of MTB complex. It must be part of the diagnostic arsenal of TB without replacing conventional microbiological tools and allow early diagnosis and appropriate treatment. Our study and previous studies have shown that positive acid-fast smears are completely correlated with Xpert assay and TB culture. Incorrect and error results from Xpert assay occurred in (3/116; 2.6%) specimens, while contaminated culture in the samples was (5/116; 4.3%). False-negative Xpert PCR results identified in two samples. The possible reason for this is the presence of PCR inhibitors or inadequate nucleic acid content in these specimens.
The main limitation of our study was the relatively low number of samples as well as the inadequate number of positive samples for assessing the performance of Xpert in the detection of resistance to RIF.
In Kosovo, the diagnostic method of pulmonary TB is based on clinical signs or X-ray of the chest without confirmation of bacteriological methods. Even when sputum smear request, only few of them are confirmed by culture. Recently, ruptured molecular tests in Kosovo are being conducted for possible cases of pulmonary TB. Xpert MTB/RIF is only used on request from a doctor. Furthermore, the preference for testing with extrapulmonary samples and examples of children with TB.
One of the major barriers to the TB control program can be the high cost of Xpert assay. A very limited number of tests are due to economic difficulties: In Kosovo, a single Xpert MTB/RIF test costs 90 euro, which is unbearable. This is while the international price for each test is around 10 euro per test.
| Conclusions|| |
The present study emphasizes the sensitivity and specificity of Xpert assay in the diagnosis of MTB. An important feature of this test is the rapid diagnosis for the rapid treatment of TB, especially in patients with negative sputum acid–fast smear.
Due to problems with the use of microscopy examination or AFB sputum test in patients with HIV, patients with low bacterial load and children, the use of GeneXpert, which has high sensitivity and specificity, is very important in the diagnosis of TB. As a result, the use of the diagnostic method of GeneXpert MTB assay can be very helpful in patients infected with HIV.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]