https://orcid.org/0000-0002-9047-9136
Abstract
Background
Tuberculosis is a global health concern and has become more complex to diagnose due to mutations in the causative agent, Mycobacterium tuberculosis. In a setting with high TB prevalence, having a rapid and accurate diagnosis may reduce the rate of infections. The study aimed to evaluate the diagnostic performance of GeneXpert MTB/RIF assay in sputum samples from suspected tuberculosis patients.
Methods
A total of 1 328 sputum samples were collected from patients, across 12 clinics in the Shiselweni region, Eswatini. One thousand one hundred and ten (1110; 84%) samples were simultaneously processed on GeneXpert MTB/RIF assay and MGIT culture.
Results
Two hundred and ninety-seven (297) samples tested GeneXpert positive and 813 GeneXpert negatives, while 310 samples tested positive and 800 tested negative on the MGIT culture method. The positive predictive value on GeneXpert MTB/RIF assay was 83% while the negative predictive value was 97.80%. At p-value = .796, sputum quality did not affect the positivity of the GeneXpert MTB/RIF results. Sputum volume had a significant impact on the performance of the GeneXpert MTB/RIF with increased sensitivity in 4 ml and 6 ml samples.
Conclusions
Although detection of tuberculosis using the GeneXpert MTB/RIF assay in sputum samples is not limited to one specific characteristic, sputum volume assessment should be considered as an integral part of routine laboratory diagnosis of tuberculosis especially in high tuberculosis prevalent settings. However, the ability of the GeneXpert MTB/RIF to provide rapid TB diagnosis is not dependent on sputum quality.
Introduction
Tuberculosis (TB) is one of the major causes of global health concerns [Citation1]. The significance of providing quality health care to TB diagnosed individuals with the assurance of quality laboratory diagnosis is very significant [Citation2]. Thus, accurate detection of Mycobacterium tuberculosis from the sputum samples is highly required [Citation3]. Furthermore, obtaining a reliable laboratory test result for TB diagnosis mainly depends on the integrity of the sputum sample obtained to be analysed [Citation3,Citation4].
Eswatini is one of the Southern African countries with the world’s highest TB prevalence and TB occurrence rate per capita of 300 cases per 100,000 populations [Citation5,Citation6]. Eswatini is divided into four provinces namely: Hhohho, Lubombo, Manzini, and Shiselweni [Citation6]. Shiselweni is the most disadvantaged province when compared to the country’s other provinces and statistically, it has the most reported TB cases [Citation6]. Hence there is a high volume of sputum samples collected from presumed TB burden patients in Shiselweni [Citation7]. Although most of the people in Shiselweni live within ten kilometres of a health clinic, all these clinics do not provide services for TB diagnosis [Citation6]. This means for diagnosis purposes, patients' sputum samples have to be referred to Nhlangano TB laboratory for diagnosis at Nhlangano health centre which is located at least twelve kilometres from the nearest referring clinic [Citation6]. However, the referrals are not always done in time due to the remoteness of the province. Therefore, there are transportation delays that could have negative effects on the sputum integrity [Citation2,Citation6]. A lack of guidance or assistance during sputum sample collection since patients carry the sputum sample containers at home for collection before submission to the TB laboratory compromises the sample suitability for TB diagnosis since the submitted samples have varying characteristics such as volume and quality [Citation6]. Hence the purpose of the study was to evaluate the GeneXpert MTB/RIF(Cepheid, Sunnyvale, CA, USA), performance using sputum samples with various characteristics from suspected pulmonary tuberculosis patients in the Shiselweni region, Eswatini. Developing such an understanding can prove to be very crucial in curbing the TB epidemic and could assist in changing the approach on how sputum samples are collected and accepted for TB diagnosis [Citation8].
GeneXpert MTB/RIF assay is endorsed by WHO and it is considered to be the first-line diagnosis technique for TB diagnosis, particularly in high TB burden countries like Eswatini [Citation9]. This assay is described as a unique, rapid, automated, and cartridge-based nucleic acid amplification (NAA) test [Citation9]. GeneXpert MTB/RIF assay can identify TB along with rifampicin resistance directly from the sputum within two hours of analysis [Citation5,Citation10]. Implementation of the assay on a large scale particularly in developing countries will provide a better diagnosis of TB in high-risk populations. Hence in this study, the purpose was to evaluate the GeneXpert MTB/RIF assay performance in sputum samples with various characteristics from presumed TB patients of all ages in the Shiselweni region, Eswatini.
Materials and method
Study design
The study was conducted at the Nhlangano TB laboratory in the Shiselweni region, Eswatini. The target population involved patients presenting with TB clinical signs and symptoms whose samples were referred to Nhlangano TB laboratory for routine diagnosis. The sputum samples were collected from the 12 clinics operating in the Shiselweni region of Eswatini, which were referred to as Nhlangano TB diagnosis. shows the map of Eswatini showing the location of the Shiselweni region.
Figure 1. Shows a map of Eswatini showing the location of Shiselweni in green [Citation11].
![Figure 1. Shows a map of Eswatini showing the location of Shiselweni in green [Citation11].](/cms/asset/07ffaf42-6965-4114-a48f-a46b4393922a/infd_a_1992005_f0001_c.jpg)
Sputum sample collection
The collection of sputum samples from TB suspected patients was carried out from August 2018 to May 2019. At least 1 mL of sputum samples were collected following the assay manufacturer’s instructions which require not less than 1 mL of sputum samples for GeneXpert assay and MGIT (BD Diagnostics, MD, USA) culture respectively. The sputum samples were collected in wide-mouthed, leak-proof, and screw-capped 50 mL translucent plastic containers following World Health Organisation guidelines and Eswatini National Tuberculosis Program sputum collection policies. The containers used were made of a translucent material to observe specimen volume and quality without opening the container. The sputum containers were appropriately labelled with patient identifiers and the date of sputum collection. Thereafter, the sputum samples were transported, using the national sample transport system to Nhlangano TB laboratory for diagnosis in cooler boxes filled with ice blocks to maintain a cold chain before processing.
Sputum sample processing
Sputum sample quality and volume were macroscopically determined according to Ho et al. [Citation12]. The sputum quality was recorded based on the colour and viscosity, and the volume was also recorded. The three main categories identified were mucopurulent, salivary and blood stained (bloody). The obtained sputum samples were processed on both GeneXpert MTB/RIF assay and MGIT culture, this was done following the manufacturer’s instructions and following MGIT procedure manual respectively [Citation9].
Statistical analysis
All the statistical analyses were performed using the statistics and data analysis software version 14 (STATA 14). The Pearson chi-square test and a Fisher’s test were performed to compare the association between GeneXpert MTB/RIF positive samples by sputum characteristics namely; sputum volume, sputum quality, patient’s gender, patient age group, and the number of repeated tests (test runs).
Statistical descriptions
All samples tested on GeneXpert MTB/RIF assay were also processed on MGIT culture. Statistical analysis was performed to determine sensitivity and specificity as well as positive and negative predictive values on both GeneXpert MTB/RIF and MGIT culture. The sensitivity was defined as the ability of the test to correctly identify those patients (or samples) with the disease. Specificity was defined as the test’s ability to correctly identify those patients (or sputum samples without the disease. The positive predictive value was described as the probability that subjects with a positive screening test truly have the disease while the negative predictive value is the probability that subjects with a negative screening test truly don't have the disease. The likelihood ratio was defined as how much more likely was it that a patient (or sample obtained which tests positive has the disease compared with one that tests negative. To measure the effects of each characteristic on the GeneXpert MTB/RIF positive results, a univariate and multivariate analysis was performed using simple logistic regression and multiple linear regression respectively. The difference was declared as statistically significant if P-value was less than .05. P-value is the probability of obtaining results as extreme as the observed results of a statistical hypothesis assuming that the null hypothesis is correct.
Ethical considerations
Ethical clearance was sought and obtained before conducting the study from the Eswatini Health Research Review and Ethics Board and the University of South Africa.
Results
Of the 1328 samples, 297 tested GeneXpert positive and 813 GeneXpert negatives, while 310 samples tested positive and 800 tested negative on the MGIT culture method. GeneXpert MTB/RIF test was done on 1 110 sputum samples and simultaneously the sputum samples were cultured on MGIT culture as a gold standard. The majority of the samples from male patients were slightly associated with positive GeneXpert MTB/RIF results at (P chi2: 2.9021) and p-value = .088 (), while on MGIT culture the sputum samples from males were more associated with positive results at (P chi2: 4.2554) and p-value= .039 (). Sputum volume had a significant impact on the positive results of both the GeneXpert MTB/RIF and MGIT culture at p-value = .036 and p-value = .071 respectively (). When disaggregating the results by age groups, the 61–75 years age group had the highest percentage of GeneXpert MTB/RIF positive results and third-highest overall positive results on MGIT culture (). However, there was not enough association between the GeneXpert MTB/RIF positive results and age groups at p-value = .608 as well as MGIT (BD Diagnostics, MD, USA) culture results at p-value = .787 ( and Citation2). It has to be noted that this age group (61–75) submitted the fewest sputum samples (n = 15). The sputum samples which were repeated for the second and third time did not have any association with both GeneXpert MTB RIF and MGIT culture results as depicted by at (F exact: 7.5723) and p-value = .122 and p-value = .863 respectively. Sputum quality did not have any significant impact on the performance of the GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) at p-value = .796, and MGIT (BD Diagnostics, MD, USA) culture at p-value = .766 as presented in .
Table 1. Sputum GeneXpert MTB/RIF and MGIT culture Positive by exposure variables.
Measure of effect of sputum sample characteristics on affecting GeneXpert MTB/RIF assay and MGIT culture performance
The sputum sample volume with the most positive results from GeneXpert MTB/RIF assay was 6 mL at an odds ratio of 1.36668 (). The same positive effect was also observed in MGIT culture-positive sputum’s at 1.26982 odds ratio (). Gender also was observed to have an effect on GeneXpert results at odds ratios 0.79065 and 0.74529 for MGIT culture. The odd ratios were preferred at the prevalence of 20% and 95% confidence interval (CI).
Table 2. GeneXpert MTB/RIF and MGIT positive samples adjusted for multivariable covariates.
GeneXpert sensitivity and specificity against MGIT culture
Sensitivity was determined by the proportion of all positive samples on both GeneXpert MTB/RIF assay and MGIT culture tests overall positive in culture while specificity was the proportion of all negative samples on both GeneXpert MTB/RIF assay and MGIT (BD Diagnostics, MD, USA) tests overall negative samples on culture. shows that the sensitivity of the GeneXpert MTB/RIF test was 91.6% and specificity was 95.3%. The positive predictive value (PPV) on GeneXpert MTB/RIF was 83% while the negative predictive value (NPV) was 97.80% ().
Table 3. Sensitivity and specificity of GeneXpert MTB/RIF assay compared to MGIT culture.
Discussion
In this study, it was observed that there was no significant difference in diagnostic yield of GeneXpert MTB/RIF between salivary and non-salivary sputum samples such as mucopurulent and bloody samples (). Despite showing a higher sensitivity in mucopurulent samples (31.1%), this analysis showed that GeneXpert MTB/RIF had the second-highest diagnostic sensitivity on salivary samples (26.65%) when compared with the referent sputum category, mucopurulent samples, while positive blood stained (bloody) sputum samples were very few (14/1110) to be of any statistical significance. These differences were confirmed by a secondary comparison of diagnostic accuracy about mycobacterial culture using MGIT culture. The findings of this study revealed that the enhanced yield of positive GeneXpert MTB/RIF results from salivary samples, in addition to the mucopurulent samples (obvious specimen of choice), maybe of additional importance in high TB burden settings because more samples will be processed on GeneXpert MTB/RIF and as a result, more patients will be treated timely. Potential reasons as to why salivary sputum in this study showed a relatively good yield of positivity on GeneXpert MTB/RIF could include; the ability of GeneXpert MTB/RIF to easily detect Mycobacterium DNA in samples with low bacillary load given the molecular nature of the assay; or more efficient amplification of Mycobacterium DNA in salivary samples than in other complicated sample matrices like blood-stained sputum which could inhibit DNA amplification. The high sensitivity of GeneXpert MTB/RIF in salivary samples was observed by Zimba et al. [Citation13].
Recent studies from Kenya and Uganda also reported on the proportion of salivary sputa. In Kenya 44% of the samples were salivary and the study showed that salivary sputa had lower diagnostic yield than mucopurulent and mucoid sputa using GeneXpert MTB/RIF testing [Citation14]. The study in Uganda examined presumptive TB patients screened with > 2 weeks of cough; the proportion of salivary sputa was at 16% [Citation8]. In Uganda, GeneXpert MTB/RIF test was conducted only among smear-negative patients, and the diagnostic sensitivity, in contrast to our findings and those from Kenya, was significantly higher on salivary samples than mucoid sputa [Citation8].
The Uganda findings on salivary sputum seem contrary to biological plausibility, showing higher diagnostic yield with lower quality sputa [Citation8]. However, the report from Uganda was consistent after a comparison of diagnostic accuracy about mycobacterial culture (higher culture-positive among salivary than non-salivary). Given the higher positivity among salivary sputum samples was confirmed by culture, we agree with Meyer et al. that further study is essential in exploring the possibility of potential dynamics affecting the GeneXpert MTB/RIF amplification in salivary sputum [Citation8].
At least four reasons support why salivary sputum should be considered and not rejected: (i) a high percentage of salivary sputum were still being collected in clinical practice by health workers [Citation14]; (ii) sputum specimen may appear to be a poor ‘negative predictor’ of MTB in sputum but when tested the results varied [Citation12]; (iii) the study in Uganda on the effect of sputum quality on GeneXpert MTB/RIF performance demonstrated higher sensitivity of GeneXpert MTB/RIF in salivary samples than mucoid [Citation8] and (iv) to minimise any missed opportunities for TB diagnosis, sputum rejection criteria that consider the salivary sample as unsuitable for testing should be reconsidered, particularly when using GeneXpert MTB/RIF for TB diagnosis [Citation10,Citation12]. According to this study, 30% of TB cases detected by GeneXpert MTB/RIF would have been missed as bacteriologically-confirmed TB cases had salivary samples not been processed and this would have led to a significant proportion of patients remaining undiagnosed and could have continued being infectious agents of TB. It is for this reason that Ho et al. emphasised that assessment of sputum quality is a neglected aspect of accurate TB diagnostics [Citation12].
In this study, sputum volume of 4 mL and 6 mL warranted a high probability of a positive GeneXpert MTB/RIF result when compared to fewer volumes of less than 4 mL despite the few samples which were 7 mL and had low M. tuberculosis detection. This could be attributed to the fact that as the volume increased the bacillary load per mL of the sputum also increased [15]. The findings of the study concur with reports presented by Fløe et al. [Citation15]. For this reason, sputum specimen volume assessment should be considered as an integral part of routine laboratory diagnosis of TB.
When the sputum samples were stratified according to the age of patients who submitted sputum, there was not enough evidence of the association between age groups and GeneXpert MTB/RIF positive results. However, it was noted that sputum samples from the older age group between 61 and 75 years, even though the number of specimens was very small (25), yielded more GeneXpert MTB/RIF positives. This could be attributed to the fact that the elderly population among all ethnic groups and both genders represent the largest reservoir of TB infection, particularly in developing countries. These findings were in agreement with Rajagopalan et al. [Citation16]. Clinical features of TB in older adults may be atypical, non-specific, and confused with concomitant age-related diseases [Citation16].
The study revealed that sputum samples from males were associated with GeneXpert MTB/RIF positivity than sputum samples from females. The lack of association between GeneXpert MTB/RIF positivity and sputum samples from females in this study could be attributed to biological reasons which make women less proficient at expectoration than men [Citation17]. Another contributing factor could be occupationally related which is that most men in the Shiselweni region work or have worked before in the mining sector in South Africa. Eswatini, Lesotho, and Mozambique are among the main mineworker-sending countries to South Africa [Citation18]. In a study by Ventura [Citation19] it was observed that every year, nearly half a million men travel from across the Southern African region to work in South Africa’s mines and in doing so, may contract TB due to exposure to multiple risk factors as a result of their working and living conditions and their migrant lifestyles [Citation17]. Crowded dormitory-style living conditions and poor housing in informal settlements increase the risk of contracting the airborne disease and regular movements across borders provide a route for transmission of TB infections to families and communities in Shiselweni [Citation19].
At the time of this study, there was no data that elaborated on the effect that repeated GeneXpert MTB/RIF test had on the results of a TB suspected sample. There was no significant effect of the repeated tests also referred to as test runs on the GeneXpert MTB/RIF. Most of the sputum samples that were processed on the GeneXpert MTB/RIF analyser and produced results on the first run were 1 087 (98%), 22 (2%) sputum samples were processed for a second time and only one sample had results on the third run. Therefore the study revealed that a single repeat (second run) is sufficient to cover for any samples that might have needed a rerun, therefore, a third run (second repeat) may not be necessary.
The findings of this study suggest that despite mucopurulent samples still being the outstanding sample of choice for TB diagnosis, assumptions that salivary sputum is of low quality and is unsuitable for TB diagnosis do not apply for GeneXpert MTB/RIF. Therefore, there is a need for testing algorithms and sample acceptance and rejection criteria to be revised to accommodate the findings of this study, and this concurs with the study by Zimba et al. [12]. It was observed from this study that the volume had an effect on the outcome of GeneXpert MTB/RIF results with the majority of positives coming from sputum samples between 4 mL and 6 mL (). According to the obtained results on repeated tests (test run), repeating the test only once (2nd run) in cases where the initial test required to be repeated did not have any significance on the study outcomes as 98% of samples produced reliable results on the initial test (first run).
In conclusion, this study demonstrated that the ability of the GeneXpert MTB/RIF to provide rapid TB diagnosis is not dependent on sputum quality especially in a high TB prevalence setting but on the presence of M. tuberculosis DNA in the sputum sample regardless of the quality. In addition, GeneXpert MTB/RIF requires only five genome copies of purified M. tuberculosis DNA per reaction or 131 colony-forming units per millilitre (mL) of M. tuberculosis to be detected in a sputum sample. In other words, this is the lowest number of colony-forming units per sputum sample that can be reproducibly distinguished from negative samples with 98% confidence when processed on the GeneXpert MTB/RIF. It is for this reason that GeneXpert MTB/RIF assay, a highly sensitive assay, was able to detect TB even in salivary sputum samples for this study which, for a long time, have been traditionally excluded from TB diagnosis on the assumption that they are not suitable to give a positive TB result. Our study was carried out in a relevant, low-income country with a high TB burder, it is, therefore, conceivable that our findings can be generalisable to many other developing countries where GeneXpert MTB/RIF assay can be used in resource-limited settings.
Author contributions
DLM conceptualised the research project, collected data and analysed it, and wrote the manuscript. MCM supervised the project, analysed data, corrected the manuscript and gave advice. SLL supervised the research project, analysed data, critically reviewed the manuscript and endorsed final approval of the version, and he is the corresponding author.
Acknowledgements
Sincerest gratitudes go to the Nhlangano TB laboratory staff and all the clinicians at the local clinics who were screening the TB suspects and referring them to the laboratory for diagnosis. Thank you so much for all your support and scientific contributions to ensure that this work comes to completion. To Mr. Charlie Mamba for your statistical and data analysis input. A special word of appreciation goes to Ms. Nomzamo Ngwenya for taking time out of her busy schedule to proofread the manuscript.
Disclosure statement
No potential conflict of interest was reported by the author(s).
References
- Forbes BA, Hall GS, Miller MB, et al. Practice guidelines for clinical microbiology laboratories: mycobacteria. Clin Microb Rev. 2018; 31:e00038–17.
- Storla DG, Yimer S, Bjune GA. A systematic review of delay in the diagnosis and treatment of tuberculosis. BMC Pub Health. 2008;8:15.
- Shinnick TM, Iademarco MF, Ridderhof JC. National plan for reliable tuberculosis laboratory services using a systems approach. Recommendations from CDC and the association of public health laboratories task force on tuberculosis laboratory services. The Morbidity and Mortality Weekly Report. 2005;54(RR-6):1–12.
- Alisjahbana B, van Crevel R, Danusantoso H, et al. Better patient instruction for sputum sampling can improve microscopic tuberculosis diagnosis. Int J Tubercul Lung Dis. 2011;9:814–817.
- Haumba S, Dlamini T, Calnan M, et al. Declining tuberculosis notification trend associated with strengthened TB and expanded HIV care in Swaziland. Public Health Action. 2015;5(2):103–105.
- Sibbald B. Responding to Swaziland's dual epidemic. CMAJ. 2013;185(1):E13–E14.
- Sanchez-Padilla E, Merker M, Beckert P, et al. Detection of drug-resistant tuberculosis by Xpert MTB/RIF in Swaziland. N Engl J Med. 2015;372(12):1181–1182.
- Meyer AJ, Atuheire C, Worodria W, et al. Sputum quality and diagnostic performance of GeneXpert MTB/RIF among smear-negative adults with presumed tuberculosis in Uganda. Pub Lib Sci Med. 2017;12(7):e0180572.
- Cepheid. 2017. Launch of new TB test Ultra backed by WHO recommendation. [cited 2020 Sept 05]. Available from: www.cepheid.com/us/about-us/news-events/press-releases
- Steingart KR, Schiller I, Horne DJ, et al. GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev. 2014;1:CD009593.
- Central statistics and population’s office. 2019. Ministry of Home Affairs, Eswatini. [cited 2020 Sept 8].
- Ho J, Nguyen PTB, Nguyen TA, et al. The role of macroscopic sputum quality assessments to optimise sputum testing for tuberculosis. Int J Tuberc Lung Dis. 2016;20(3):319–322.
- Zimba O, Tamuhla T, Basotli J, et al. The effect of sputum quality and volume on the yield of bacteriologically-confirmed TB by xpert MTB/RIF and smear. Pan Afr Med J. 2019;33:110.
- Orina F, Mwangi M, Githui W, et al. Effect of sputum quality on GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) results in the detection of Mycobacterium tuberculosis from persons presumed to have tuberculosis in EAPHLN project operational research study sites in Kenya. Afr J Health Sci. 2014;27(4):446–456.
- Fløe A, Hilberg O, Thomsen VØ, et al. Shortening isolation of patients with suspected tuberculosis by using polymerase chain reaction analysis: a nationwide cross-sectional study. Clin Infect Dis. 2015;61(9):1365–1373.
- Rajagopalan S, Yoshikawa T, Nahimo N. Tuberculosis in the elderly. Z Gerontol Geriatr. 2000;33(5):374–380.
- Karigeni T. Southern Africa Tackles Tuberculosis in the Mining Industry. World bank report; 2014.
- Uplekar M, Wells WA, Pai M. Achieving systemic and scalable private sector engagement in tuberculosis care and prevention in Asia. Pub Lib Sci Med. 2015;12(6):e1001842.
- Ventura E. United Nations Migration Health. TB and migration in Southern Africa; 2016. p. 1–4.