Introduction
Tuberculosis (TB) caused by Mycobacterium TB complex (MTB) primarily infects lungs that are known as pulmonary TB. However, infection of other body sites such as skin, bones, tissue, meninges, and lymph nodes is also common which comes under the heading extrapulmonary TB. Incidence of TB in India was 26.4 Lakh in 2019. During 2019, 66,255 multidrug-resistant/rifampicin (RIF)-resistant (MDR/RR) TB cases were diagnosed in India.[ 1 ]
For the past decades, the burden of diagnosis of TB was solely on the shoulders of smear microscopy and conventional culture. However, both the techniques had their drawbacks such as low sensitivity and observational bias of microscopy and very long turnaround time of weeks to months in solid culture. Fluorescent microscopy (FM) improved the sensitivity, but the specificity was compromised. The mean diagnostic turnaround time was decreased to 21 days with the introduction of liquid culture techniques.[ 2 ] Nevertheless, to limit the transmission of disease effectively, this is still a significant amount of turnaround time.
Early diagnosis and treatment remain the cornerstone of TB control. RIF RR, MDR, and extremely drug-resistant strains of MTB are notoriously infectious and difficult to treat if not diagnosed and treated in time. With no major advances in therapeutic options, rapid diagnosis with newer diagnostic tests utilizing nucleic acid amplification methods such as line probe assay (LPA) and CB-NAAT (Xpert MTB/RIF or TrueNat) with simultaneous detection of MTB complex and resistance has helped in reducing the transmission rates by significantly shortening the time in required to initiate the treatment and also minimizing the risk of further resistance.[ 3 ]
World Health Organization has recommended the use of the LPA and Xpert MTB/RIF (or GeneXpert) assay. Both these molecular methods target the 81-blood pressure hot spot region called the RIF RR-determining region of the rpoB gene. In addition to this, the LPA (MTBDRplus) detects mutations associated with katG genes and the inhA regulatory region gene.[ 4 , 5 ]
Cephaid GeneXpert MTB/RIF (Xpert) assay employs heminested polymerase chain reaction and molecular beacon technology to detect MTB complex directly from the clinical sample and also providing RIF RR results concurrently in 2 h with minimum requirement of training and manual handling.[ 6 ]
Nonsputum samples are frequently being tested by cartridge-based nucleic acid amplification test (CB-NAAT) assay due to the diversity of extrapulmonary samples, their procurement, and extraction of MTB DNA from them compared to sputum samples.[ 7 ] Therefore, the present study was aimed at assessing the use of GeneXpert-MTB/RIF assay in detecting MTB and RIF RR among extrapulmonary and nonsputum respiratory samples.
Materials and Methods
Study subjects
The present study was conducted on extrapulmonary and nonsputum pulmonary samples received of TB suspected patients (according to PMDT guidelines) from outpatient and inpatient departments of a tertiary care hospital and from various TB units in and around Aligarh region from December 2018 to June 2020.
Specimen collection and processing
Both aseptically collected sterile specimens that are usually free from other microorganisms – fluids such as pleural, ascitic, cerebrospinal fluid (CSF), and fine-needle aspirates and specimens contaminated by normal flora or specimens not collected aseptically (not sterile) – gastric lavage, bronchioalveolar lavage, and pus were included in the study. Decontamination of nonsterile samples was performed by N-acetyl-L-cysteine and sodium hydroxide method in a Class II biosafety cabinet.
Cartridge-based nucleic acid amplification test and line probe assay
All the samples collected were subjected to smear examination by Ziehl–Neelsen (ZN) staining technique and light-emitting diode (LED)-FM, culture on solid Lowenstein–Jensen (LJ) media and GeneXpert MTB/RIF assay. GeneXpert MTB/RIF assay was performed according to the manufacturer’s instructions (Cepheid, Sunnyvale, CA). The test was repeated for samples showing invalid results.[ 8 ] Samples which showed growth on LJ media were subjected to LPA and solid culture drug susceptibility testing (DST) by 1% proportion method. LPA assay (Hain Lifesciences, Nehren, Germany) was performed in adherence to GenoType MTBDRplus version 2.0 manufacturer’s protocol available at http://www.hain-lifescience.de/en/instructions-for-use.html
Statistical analysis
Data were analyzed using MedCalc developed by MedCalc software (Acacialaan 22, 8400 Ostend, Belgium). Data were presented as frequency (percentage). Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) with 95% confidence intervals were calculated. P < 0.05 was considered statistically significant (https://www.medcalc.org/calc/diagnostic_test.php
Ethics committee approval
The study was approved by the Institutional Ethics Committee, Faculty of Medicine, Aligarh Muslim University, Aligarh (D.No. 239/FM dated on May 11, 2019).
Results
Total 642 samples of purulent discharge 37.6% (242), pleural fluid 25.8% (165), CSF 22.6% (145), gastric aspirate 6.9% (44), bronchoalveolar lavage (BAL) 3.9% (25), and ascitic fluid 2% (13), and lymph node aspirate 1.2% (8), respectively, were collected from patients suspected of MDR-TB, in which males were 339 (52.8%) and females were 303 (47.2%). All samples were subjected to ZN staining technique, FM, CBNAAT (GeneXpert), and solid culture on LJ media. Culture-positive samples were subjected to LPA (Genotype MTBDRplus assay).
About 6.9% (44) of samples were FM positive as compared to ZN staining which is 5.8% (37). MTB was detected by GeneXpert in 25.9% (166) and the rest were negative. GeneXpert MTB/RIF assay detected RIF RR in 15.7% (26) samples and 84.3% (140) samples were RIF sensitive. Culture on LJ medium showed growth in 24.6% (158), whereas 72.12% (463) had no growth and 3.3% (21) were contaminated.
Considering mycobacterial culture as the gold standard sensitivity, specificity, PPV, and NPV were calculated for ZN staining technique and FM. Sensitivity, specificity, PPV, and NPV of ZN staining technique and LED-FM were 21.52%, 99.35%, 91.89%, and 78.77% and 27.22%, 99.78%, 97.73%, and 80.07%, respectively, as shown in Tables 1 and 2 .
Table 1: Sensitivity and specificity of Ziehl-Neelsen staining method
Table 2: Sensitivity and specificity of fluorescent microscopy light emitting diode-fluorescent microscopy
When GeneXpert MTB/RIF was compared to solid LJ culture and DST sensitivity, specificity, PPV, and NPV of GeneXpert MTB/RIF in the detection of MTB were 93.75%, 96.53%, 90.36%, and 97.80%, and RIF RR was 92.0%, 97.74%, 88.46%, and 98.48%, respectively, as shown in Tables 3 and 4 .
Table 3: Sensitivity and specificity of GeneXpert Mycobacterium tuberculosis /rifampicin assay
Table 4: Comparison of rifampicin susceptibility testing by GeneXpert Mycobacterium tuberculosis /rifampicin assay and phenotypic drug susceptibility testing
Positive cultures were also subjected to solid culture DST by 1% proportion method which revealed resistance to both isoniazid (INH) and RIF as 15.82% (25), monoresistance to RIF was not detected in any sample, whereas monoresistance to INH was detected in 19.62% (31). About 64.56% (102) of samples were sensitive to both RIF and INH.
LPA was also performed on the culture-positive samples which detected the exact same result as by DST on LJ medium showing 100% sensitivity and specificity [Table 5 ].
Table 5: Comparison of rifampicin susceptibility testing by GenoType Mycobacterium tuberculosis and drug resistance plus line probe assay and phenotypic drug susceptibility testing
Since LPA and solid culture DST results were 100% concordant. On comparing GeneXpert with LPA, GeneXpert had same sensitivity and specificity, as shown in Table 4 .
In rpoB gene, the most common mutation was seen in the 530–533 region as S531 L (56%). In kat G, the most common mutation seen was S315T1 (92%) and in inh A , the most common mutation seen was in C15T (64%) region as detected by LPA.
Discussion
Rapid diagnosis leading to early patient management is the key to successful patient outcomes and breaking the chain of transmission. Diagnosis of TB in developing countries through their control programs relies largely on smear microscopy which has poor sensitivity furthermore in extrapulmonary cases leading to false negative and misdiagnosed cases. However, many developing nations have included CB-NAAT, TrueNat, and LPA in their TB control programs for diagnosis. In the present study, only 5.8% (37) samples were acid-fast bacilli (AFB) positive by ZN staining technique which was similar to other Indian studies 5.55%, 5.41%, and higher than 2.26%.[ 9–11 ] However, Kasat et al . 2018 showed a greater positivity of 10.2%.[ 12 ] LED-FM 6.9% (44) had a slight better positivity than ZN staining technique in our study but a lower positivity rate of 3.9% by LED-FM on suspected extrapulmonary TB (EPTB) cases were also reported from India.[ 13 ] From the above discussion, we can see that both ZN staining technique and FM are not very effective methods in the diagnosis of EPTB and in nonsputum respiratory samples.
A greater 25.9% (166/642) TB cases were detected by Xpert MTB/RIF in our study. Comparable positivity rate of 22.5% was shown by Iram et al .[ 14 ] However, a comparatively low 15.1%, 18.4%, and 9.54% positivity rate was shown by other studies.[ 12 , 15 , 16 ] A higher positivity rate in our study may be due inclusion of nonsputum respiratory samples like BAL. Solid culture on LJ media showed only 24.6% (158/642) growth which was >9.6% as depicted by Chakraborty et al . 2016.[ 10 ] Similarly, a lower culture positivity rate of 20.35% for pulmonary samples and 12.82% for extrapulmonary samples was shown by Mechal et al .[ 15 ]
Sensitivity, specificity, PPV, and NPV of AFB smear by ZN staining and LED-FM came as 21.52%, 99.35%, 91.89%, and 78.77% and 27.22%, 99.78%, 97.73%, and 80.07%, respectively. A higher sensitivity of 65.7% and a lower specificity of 95.7% for ZN staining and sensitivity of 88.0% and specificity of 95.6% by LED-FM were shown by Arora and Dhanashree 2020 in combined pulmonary and extrapulmonary samples.[ 17 ]
In the present study, sensitivity, specificity, PPV, and NPV of GeneXpert MTB/RIF assay on extrapulmonary samples and nonsputum respiratory samples were found to be 93.75%, 96.53%, 90.36%, and 97.80%. Mechal et al . have also shown a similar NPV among pulmonary (94%) and extrapulmonary (97%) samples.[ 15 ] Such high NPV helps in the diagnostic elimination of TB in extrapulmonary samples by GeneXpert MTB/RIF. Several studies from India and around the world have shown a variable sensitivity and specificity 89%, 95%, 85.71%, 97.36%, 100%, 87.5%, 100%, 99.62%, 77.3%, 98.2%, 76.5%, 95.9% 79.3%, 90.3%, 83.3%, 98.9%.[ 15 , 18–24 ] This disparity in the overall performance of GeneXpert MTB/RIF might be due to variations in the proportion of sample types.
A systematic review showed a 69% and 95% performance sensitivity of GeneXpert MTB/RIF assay for the diagnosis of smear-negative and smear-positive nonrespiratory samples, respectively.[ 25 ] Therefore, GeneXpert MTB/RIF assay may be considered the first-line test for improved case detection in smear-negative EPTB suspects and nonsputum respiratory samples even in low-income settings like India.
In the present study, GeneXpert MTB/RIF assay detected RIF RR in 15.7% (26) samples. Overall, sensitivity, specificity, PPV, and NPV of GeneXpert in the detection of RIF RR in extrapulmonary and nonsputum respiratory samples were 92.0%, 97.74%, 88.46%, and 98.48%, respectively. Various studies from around India have shown variable sensitivity and specificity in the detection of RIF RR such as 97%, 95%,[ 26 ] 81.82%, 100%,[ 21 ] 96.3%, and 98.6%.[ 27 ] High specificity of GeneXpert indicates that these cases are less likely to be false positive. Rapid detection to MDR-TB will help in the initiation of early treatment thus significantly reducing the risk of transmission of MDR-TB.
In the current study, culture-positive samples were also subjected to solid culture DST (1% proportion method) and LPA (Genotype MTBDRplus assay). Both tests were 100% concordant. RIF and INH were resistance in 15.82% (25) and sensitive in 64.56% (102) of samples, monoresistance to INH was detected in 19.62% (31) Monoresistance to RIF was not detected in any sample. Dave et al . have also shown a concordance of 98% between culture DST and LPA for drug resistance patterns.[ 28 ]
Sensitivity (92%) and specificity (97.79%) of GeneXpert MTB/RIF assay in the detection of RIF RR came out to be lower when compared to sensitivity (100%) and specificity (100%) of Genotype MTBDRplus LPA keeping solid culture DST as the gold standard. However, it has to be noted that LPA was performed only on culture-positive growth. Similar results were seen from Delhi, India (64.4% agreement with Xpert MTB/RIF; 100% agreement with LPA).[ 29 ] Although in the detection of RIF RR Xpert MTB/RIF assay had slightly lower sensitivity and specificity as compared to LPA, Xpert MTB/RIF assay scores point over LPA that it simultaneously detects MTB bacterium and also RIF RR in nonsputum samples, whereas LPA needs culture growth for these samples.
The most common mutation detected by LPA among nonsputum samples in rpoB , kat G, and inh A genes were S531 L (56%), S315T1 (92%), and C15T (64%), respectively. A study from the same tertiary care center on sputum samples had shown the most common mutation as rpoB S531 L 69.94%, kat G S315T1 80.81%, and inh A C15T 10.10% in respective genes.[ 30 ]
GeneXpert MTB/RIF has provided diagnostic relief to both clinician and patient by providing a clear status of RIF RR simultaneously with the diagnosis of TB at the very first time. This is particularly helpful in the detection of RIF RR EPTB. GeneXpert MTB/RIF may have taken the center stage among all molecular diagnostic tests due to its high sensitivity, specificity, and less turnaround time but it has its own limitations. It only detects RIF RR which if taken as a surrogate marker of MDR-TB and INH resistance is not determined by LPA or other methods can lead to overestimation of MDR-TB. Such cases may not need full-line MDR-TB therapy. Requirement of stable electrical power supply, annual calibration of the instrument, and temperature control are few other cons which hamper its utilization in rural areas of resource-limited countries. However, despite these limitations, Xpert MTB/RIF assay is quite an asset in the diagnosis of pulmonary TB and EPTB with its rapid result and early detection of RIF RR also it is relatively easier to perform, less human errors, and minimal biosafety requirement.
Conclusion
This study further strengthens the point that GeneXpert MTB/RIF assay has high sensitivity and specificity even in nonsputum samples making the diagnosis of EPTB easier and rapid. RIF RR detection rates are also in good concordance to LPA. Establishment of culture and DST laboratories in resource-limited settings is always not possible. For the time being, it can be considered the best available point-of-care test for the detection of EPTB. Among molecular methods, Xpert MTB/RIF provides a judicious option for early detection of MDR-TB in extrapulmonary and nonsputum respiratory samples.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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