|Year : 2023 | Volume
| Issue : 2 | Page : 143-148
Cartridge-based nucleic acid amplification test in drug-resistant Mycobacterium tuberculosis as compared to solid cultures: Is it time to look beyond cartridge-based nucleic acid amplification test?
Ashok Dhakar1, Anuj Singhal1, Kiran Kumar Kompella2, Arun Kumar Yadav3, Vishal Mangal1
1 Department of Internal Medicine, Armed Forces Medical College, Pune, Maharashtra, India
2 Department of Internal Medicine, RR Hospital, Pune, Maharashtra, India
3 Department of Community Medicine, Armed Forces Medical College, Pune, Maharashtra, India
|Date of Submission||13-Jul-2022|
|Date of Decision||22-Aug-2022|
|Date of Acceptance||15-Sep-2022|
|Date of Web Publication||27-Oct-2022|
Arun Kumar Yadav
Department of Community Medicine, Armed Forces Medical College, Wanowrie, Pune - 411 040, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: There is a need to study the performance, validity, and accuracy of cartridge-based nucleic acid amplification test (CB-NAAT) for accessing drug resistance among pulmonary tuberculosis (TB) compared with the solid culture drug susceptibility test (DST).
Methods: Patients with symptoms of cough for more than 2 weeks with anyone symptoms such as night sweats, fever, and unintentional weight loss were studied. Cases with previously diagnosed drug-resistant pulmonary TB by sputum CB-NAAT having constitutional symptoms but not on any ATT for a minimum of 2 months were also included in the study. The patient's information, including age, immune surveillance status, clinical features, and chest X-rays, were recorded. Each sputum sample was divided into three aliquots and tested for smear microscopy, liquid culture (LC), and genotypic DST. Results of all three diagnostic modalities were compared with CB-NAAT reports.
Results: Of 236 patients with sputum-positive CB-NAAT (n = 236), 49.4% (n = 117) were rifampicin resistant, while 50. 4% (n = 119) were rifampicin sensitive. The genotypic DST assays carried out of all enrolled patients showed that 76.3% (n = 181) patients were resistant to one or more first-line antitubercular drugs (FL ATTs) or second-line (SL) ATTs, while 23.7% (n = 55) patients were sensitive to all ATTs. Among all the study participants, 56.4% (n = 133) of patients had sputum smear-positive by ZN stain, while 88.6% (n = 209) showed growth on LC (BACTEC) media. On concordant analysis of CB-NAAT with DST assays, we found that among 119 CB-NAAT rifampicin-sensitive patients, 66 patients were drug-resistant (DR) TB to any of the FL or SL ATTs. The sensitivity, specificity, positive predictive value, and negative predictive value of CB-NAAT for detecting rifampicin resistance on sputum for pulmonary TB when compared with the gold-standard DST assays were 97.67%, 76.67%, 70.59%, and 98.29%, respectively.
Conclusions: This study found that the use of rapid molecular technique (CB-NAAT) in screening DRTB at the community level is suboptimal compared to the gold-standard solid culture method. Although CB-NAAT's sensitivity in detecting DR pulmonary TB is significantly higher, the specificity is lower in that population who have received ATT earlier.
Keywords: Cartridge-based nucleic acid amplification test, line probe assays, liquid cultures, multidrug-resistant tuberculosis, solid cultures, sputum microscopy
|How to cite this article:|
Dhakar A, Singhal A, Kompella KK, Yadav AK, Mangal V. Cartridge-based nucleic acid amplification test in drug-resistant Mycobacterium tuberculosis as compared to solid cultures: Is it time to look beyond cartridge-based nucleic acid amplification test?. MRIMS J Health Sci 2023;11:143-8
|How to cite this URL:|
Dhakar A, Singhal A, Kompella KK, Yadav AK, Mangal V. Cartridge-based nucleic acid amplification test in drug-resistant Mycobacterium tuberculosis as compared to solid cultures: Is it time to look beyond cartridge-based nucleic acid amplification test?. MRIMS J Health Sci [serial online] 2023 [cited 2023 Jun 7];11:143-8. Available from: http://www.mrimsjournal.com/text.asp?2023/11/2/143/359953
| Introduction|| |
Drug-resistant tuberculosis (DR TB) remains a significant public health concern. There is a trend for increase in multidrug-resistant TB (MDR TB) cases as a proportion of all notified TB cases globally. The incidence rate of MDR TB in India is 9.6%.
Cartridge-based nucleic acid amplification test (CB-NAAT) is an automated cartridge-based molecular technique based on polymerase chain reaction, which can detect Mycobacterium tuberculosis (MTB) from any of the pulmonary or extrapulmonary specimens with varying sensitivity and specificity. The test carries the advantage that it gives rapid results and can detect rifampicin resistance (rifampicin resistance determining region [RRDR] of rpo B5 allele) within 2 h. GeneXpert is the only rapid test for diagnosis of TB currently recommended by the WHO, for the detection of M. tuberculosis and reporting of rifampicin resistance directly from sputum samples. The reported sensitivity ranges between 72.5% and 98.2%, with a specificity of 90%–95%. However, the drug susceptibility test (DST) remains the gold standard for the detection of drug resistance, as it can detect drug-resistant against any of the first-line antitubercular therapy (FL ATT) or second-line antitubercular therapy (SL ATT).
Rifampicin resistance (RR) was considered a predictor of MDR-TB, because 78% of rifampicin (Rif)-resistant TB (RR-TB) were MDR-TB. Genotypic studies have shown that KatG P Ser315Thr-mediated isoniazid resistance is among the earliest to evolve even before rifampicin resistance and is related to the virulence of mycobacterium. CB-NAAT can quickly detect common mutations in the 81 base pair RRDR. The rpoB mutations outside the RRDR are undetectable by CB-NAAT, the most common being Ile 491 Phe mutation in the rpo B gene present in 56% of rifampicin-resistant (RR) TB cases. It can detect rifampicin resistance only if rpo B allele responsible for Rif resistance is present in at least 65% of DNA present in the sample. Similarly, CB-NAAT has a low sensitivity of detection of Leu 533 CCG, 572 Phe and 531 CTG mutations, which is responsible for up to 5% of all Rif resistance.,
We studied the performance, validity, and accuracy of CB-NAAT for accessing drug resistance among pulmonary TB compared with the solid culture DST.
| Methods|| |
The study was conducted at the directly observed therapy, short course (DOTS) nodal center affiliated with a tertiary care governmental hospital of western Maharashtra. During the study, all presumptive adult pulmonary TB patients reporting at DOTS Center, from October 2018 to September 2020, were enrolled in the study. The inclusion criteria included CB-NAAT-positive patients aged between 15 and 70 years not on any antitubercular drug (ATT) for more than 2 months. Exclusion criteria included any patient on ATT for more than 2 months.
A patient case record form was designed with the help of TB experts, epidemiologist, and biostatistician. The case record form was pilot-tested and included patient's information such as age, immune surveillance status, clinical features, and chest X-rays (CXR) reports. Each sputum sample was divided into three aliquots namely for smear microscopy, liquid culture (LC) by BACTEC (Becton Dickinson Microbiology Systems, Cockeysville, Md.), and genotypic DST. Results of all three diagnostic modalities were compared with CB-NAAT reports. The study was approved by the Institutional Ethics Committee vide letter no: 1 IEC/Oct/2018 dt October 22, 18.
The sample size was calculated a priori to estimate the 95% confidence interval for sensitivity and specificity of CB-NAAT compared to DST with 5% absolute error of margin. Using the test sensitivity as 92% and specificity 95%, the calculated highest sample was 186.
Data were collated in Excel sheet. Continuous variable was described as mean and standard deviation, and categorical variables were defined as number and percentages. Sensitivity and specificity were calculated. P <0.05 was considered statistically significant. All data were analyzed using SPSS statistical software version 25.0 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.).
| Results|| |
In the study, 496 patients were screened. Two hundred and thirty-six patients were included in the study, who had a CB-NAAT report (rifampicin-sensitive or rifampicin resistant [RR]). Among them, majority of the patients were middle aged, with 33.1% of patients (n = 78) in their fourth decade and 24.6% of patients (n = 58) in their fifth decade. Of total enrolled patients, 61.4% (n = 145) were male.
Of 236 patients, 30 patients were found to be HIV-positive, while 141 were HIV-negative; data about the rest of the patients were unknown. Further evaluation of HIV-positive patients revealed that 80% (n = 24) were MDR TB cases detected either by DST or CB-NAAT.
CXR of all the patients enrolled were studied. Variations in the pattern of CXR among drug-sensitive and DR pulmonary TB cases are depicted in [Figure 1]. Of 180 DR TB by DST assays, 54.4% (n = 98) of patients had cavitary lesions on CXR. However, of 56 drug-sensitive cases 5% (n = 3) had lung collapse, 17% (n = 10) consolidation, 12% (n = 7) pleural effusion, and 5% (n = 3) had cavitation.
|Figure 1: CXR pattern among drug-sensitive and DR Pulmonary TB. CXR: Chest X-ray, DR: Drug resistant, TB: tuberculosis|
Click here to view
Concordant analysis between DST and sputum smear microscopy revealed that microscopy positivity was more among drug-resistant cases (67%; 121 among 180 samples) compared to drug-sensitive cases (21.4%; 12 among 56 samples). In our study, the overall sensitivity of sputum microscopy to detect M. tuberculosis was 56.4% (133 among 236).
Of these 236 patients with sputum-positive CB-NAAT (n = 236), 49.4% (n = 117) were RR, while 50. 4% (n = 119) were rifampicin-sensitive. The genotypic DST assays carried out of all enrolled patients showed that 76.3% (n = 181) of patients were resistant to one or more FL ATTs or SL ATTs, while 23.7% (n = 55) of patients were sensitive to all ATTs. Among all the study participants, 56.4% (n = 133) of patients had sputum smear-positive by ZN stain, while 88.6% (n = 209) showed growth on liquid culture (BACTEC) media, as shown in [Table 1]a and [Table 1]b.
Absolute number of DR TB identified by CB-NAAT and DST with simultaneous analysis of sputum smear and LC reports is depicted in [Table 2]a and [Table 2]b, respectively. The most of the discordance among results of CB-NAAT and DST was found in the subgroup of patients showing positive reports on both sputum smear microscopy and LC.
A concordant analysis of CB-NAAT results was done with DST assays. Among 119 CB-NAAT rifampicin-sensitive patients, 66 (55.5%) patients were found to be DR TB to any of the FL or SL ATTs [Table 3]a. Thirty-six percent (n = 24) were relapse cases, while rest 64% (n = 42) were newly detected. Further analysis showed that of these 66 DR TB patients, 71.2% (n = 47) were resistant to one or more SL ATTs (maximum resistance to moxifloxacin 40.9%), while only 28.8% (n = 19) were resistant to only FL ATTs. Isolated isoniazid resistance with rifampicin susceptible (Hr TB) was found among 15 patients, and isolated rifampicin resistance (RR TB) for FL ATTs was detected among 27 patients. In the second group, among 117 CB-NAAT RR patients, 115 were DR TB on DST to various FL and SL ATTs, of which 27.8% (n = 32) were relapse cases, while the rest 72.2% (n = 83) were newly detected [Table 3]b. Patients who were resistant to one or more SL ATTs in this group were only 33.3% (n = 39). None of the cases was found to be Hr TB or RR TB in this group. The proportion of DR to various FL and SL ATTs on DST found among CB-NAAT-sensitive and resistance is shown in [Figure 2].
|Figure 2: Comparison of drug resistance by DST among CB-NAAT-sensitive and resistant samples. DST: Drug susceptibility test, CB-NAAT: Cartridge-based nucleic acid amplification test|
Click here to view
The sensitivity, specificity, PPV, and NPV of CB-NAAT for detecting rifampicin resistance on sputum for pulmonary TB when compared with the gold-standard DST assays were 97.67%, 76.67%, 70.59%, and 98.29%, respectively.
Drug resistance patterns for various FL and SL ATTs were compared among newly detected pulmonary TB and relapse cases. In our study, 75% (n = 177) of patients were newly detected pulmonary TB who were recruited before initiation of treatment, while the remaining 25% (n = 59) of patients were cases of pulmonary TB with more than 2 months of ATT default or relapse TB. In the first group of 177 newly detected pulmonary TB cases, DST assays revealed that 70.6% (n = 125) of patients were DR TB, while the remaining 29.4% (n = 52) were sensitive to all ATTs. Among newly detected pulmonary TB cases who were found to be DR TB, 65.6% (n = 82) of patients were isolated from RR TB, and 28% (n = 35) were resistant to one or more SL ATTs. Most of the resistance to SL ATTs was attributed to moxifloxacin (n = 25). In the other group comprising 59 relapse pulmonary TB cases, 94.9% (n = 56) were DR TB, which was statistically significant (P < 0.001). Among this group, only one patient was isolated from RR TB, and 91% (n = 51) were resistant to SL ATTs.
| Discussion|| |
The main gap in our fight against TB is the lack of availability of a fast, accurate, and economical test for drug sensitivity that can be used in a resource-limited setting. The development of nucleic acid-based tests like CB-NAAT, though has provided novel avenues for the generation of highly sensitive point-of-care tests, still has significant limitations in terms of diagnostic accuracy for DR TB.
Our study found that DR TB was more common among relapse cases. These findings corroborated with earlier studies, which also substantiated that drug-resistant TB was more common among relapse cases compared to freshly detected TB cases., This observation was likely due to the development of antibiotic resistance caused by low serum concentration of antitubercular drugs and partly due to cross-resistance among antitubercular drugs. Poor drug compliance among patients on long-term treatment also comes as contributing factor. The study also found difference among drug resistance patterns in newly detected and relapsed cases. While the newly detected drug-resistant pulmonary TB cases were mostly isolated RR TB cases, in contrast, most of the relapsed drug-resistant TB cases were resistant to SL ATTs.
In our study, we found that the specificity of CB-NAAT to detect drug-resistant cases of pulmonary TB was less compared to previous studies. Interestingly, a significant proportion of drug-resistant TB missed by CB-NAAT (Rif-sensitive) were relapsed cases. Our study recommends that a high suspicion of MDR TB should be placed in relapse pulmonary TB cases. The study also found that the resistance to SL ATTs was more common among rifampicin-sensitive CB-NAAT compared to rifampicin resistance CB-NAAT. This was likely as patients were resistant to other SL ATTs without concurrent Rif resistance like HR TB. The mechanism for the same may be studied in detail.
Majority of HIV-seropositive were found to be MDR TB cases. This corroborated with the findings of a previous study done by Sethi et al., which stated that there was a significantly higher association of MDR TB with HIV-seropositive patients compared to HIV-seronegative. The proportion of similar cases was higher in our study, which could have been due to referral bias, as our study was conducted at a tertiary care referral center. Findings of our study were also analogous to the study conducted by Lu et al., which showed that a high proportion of cavitary lesions were observed among MDR TB cases. Cavitary lesions on CXR may be used as a soft indicator of MDR TB.
Previous studies have demonstrated that the global average of isoniazid-resistant TB was 11.6% in new TB cases and 7.2% in previously treated TB cases. In this study, also 15 had isolated isoniazid resistance without rifampicin resistance (Hr TB) on DST. The findings were in concurrence with the previous study.
Similarly, resistance pattern to other SL ATTs was also studied. Previous studies have also verified that the prevalence of resistance to levofloxacin was comparatively more in South Asian countries. This could be due to nonjudicious use of FQ in the subcontinent.
The limitations of this study were mainly due to the lack of sufficient clinical data available on patients and their follow-up during the course of treatment, which would have helped us in identifying other predictors of the emergence of DR TB. Future work should include clinical data, including atypical presentations in high-risk groups like children and the elderly together with longitudinal studies.
CB-NAAT cannot quantify TB resistance, which is a valuable tool in TB endemic settings. There were a significant number of cases which were rifampicin-sensitive as per CB-NAAT, were found to have either isolated isoniazid monoresistance or resistance to SL ATT on DST assays. We need some alternate tests to overcome these fallacies of CB-NAAT. One of the ways is to include specific mutant probes in the CB-NAAT design. One of the ways is to include specific mutant probes in the CB-NAAT design and further, by more introduction of MDR-TB diagnostic tests like line-probe assay at primary health-care setups and utilizing whole genome sequencing for mutational analysis of drug resistant strains.
| Conclusions|| |
This study found that the rapid molecular technique in screening DRTB at the community level is suboptimal compared to the gold-standard solid cultures method. This study also highlights the high prevalence of MDR-TB cases in patients visiting governmental TB institutes with relapse TB.
We acknowledge the help rendered by Dr. Sanjay Darade, District Tuberculosis Officer, Pune, for unstinted support during this study. This study was conducted as part of the MUHS MD degree award in the subject of General Medicine. The authors thank all study participants, TB diagnostic laboratory staff and TB Clinic staff of Nodal Centre at our institute.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 2], [Figure 1]
[Table 1], [Table 2], [Table 3]