AJLM African Journal of Laboratory Medicine 2225-2002 2225-2010 AOSIS AJLM-11-1671 10.4102/ajlm.v11i1.1671 Original Research Effect of sputum quality and role of Xpert® MTB/RIF assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in Addis Ababa, Ethiopia https://orcid.org/0000-0002-6212-4055 Sinshaw Waganeh 1 2 3 https://orcid.org/0000-0002-6154-7275 Kebede Abebaw 1 4 https://orcid.org/0000-0002-5518-1342 Bitew Adane 3 https://orcid.org/0000-0001-9390-0355 Tadesse Mengistu 1 https://orcid.org/0000-0002-4404-8968 Mehamed Zemedu 1 https://orcid.org/0000-0001-5307-8774 Alemu Ayinalem 1 https://orcid.org/0000-0002-8505-7000 Yenew Bazezew 1 https://orcid.org/0000-0001-6840-4229 Amare Misikir 1 https://orcid.org/0000-0002-5930-2690 Dagne Biniyam 1 https://orcid.org/0000-0002-0166-682X Diriba Getu 1 https://orcid.org/0000-0001-8859-1906 Tesfaye Ephrem 1 https://orcid.org/0000-0002-6261-541X Gamtesa Dinka F. 1 https://orcid.org/0000-0001-6039-3430 Abebaw Yeshiwork 1 https://orcid.org/0000-0002-1918-0668 Mollalign Helina 1 https://orcid.org/0000-0002-8708-386X Seid Getachew 1 https://orcid.org/0000-0003-3407-983X Getahun Muluwork 1 Tuberculosis Research Unit/National Tuberculosis Reference Laboratory, Ethiopian Public Health Institute, Addis Ababa, Ethiopia Department of Microbiology, Immunology and Parasitology, School of Medicine, Addis Ababa University, Addis Ababa, Ethiopia Department of Medical Laboratory Science, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia Department of Microbial, Cellular, and Molecular Biology, College of Natural and Computational Sciences, Addis Ababa University, Addis Ababa, Ethiopia Corresponding author: Waganeh Sinshaw, wagamels@gmail.com 31082022 2022 11 1 1671 08072021 24052022 © 2022. The Authors 2022 Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Background

There is limited information on the performance of the Xpert® MTB/RIF test for diagnosis of smear-negative pulmonary tuberculosis (SNPT) and rifampicin resistance (RR) in the same-day diagnosis approach. The effects of sputum quality and other factors affecting the Xpert performance are also under-investigated.

 Objective

This study aimed to determine the performance of the Xpert® MTB/RIF test for detection of SNPT and RR in the same-day diagnosis strategy and the effect of sputum quality and other factors on its performance.

 Methods

A cross-sectional study was conducted from August 2017 to January 2018 across 16 health facilities in Addis Ababa, Ethiopia. Two spot sputum samples were collected from 418 presumptive SNPT patients, tested with Xpert® MTB/RIF, then compared to tuberculosis culture. Additionally, culture isolates were tested for RR by BACTEC MGIT™ 960 drug susceptibility testing (DST) and MTBDRplus version 2.

 Results

The Xpert® MTB/RIF test detected 24 (5.7%) SNPT cases, with a sensitivity of 92.3% (75.9% – 97.9%) and specificity of 99.2% (97.8% – 99.7%) compared with tuberculosis culture. Xpert® MTB/RIF also detected three (11.58%) RR strains with 100.0% concordance with BACTEC MGIT™ 960 DST and MTBDRplus results. Three blood-stained SNPT samples were positive by Xpert (30.0%), which was 6.9 times higher compared to salivary sputum (odds ratio: 6.9, 95% confidence interval: 1.36–34.96, p = 0.020).

 Conclusion

The performance of the Xpert® MTB/RIF to detect SNPT and RR in same-day diagnosis is high. However, SNPT positivity varies among sputum qualities, and good sample collection is necessary for better test performance.

 smear-negative pulmonary tuberculosis Xpert® MTB/RIF test sputum quality tuberculosis culture diagnostic performance Introduction

Smear-negative pulmonary tuberculosis (SNPT) occurs when a presumptive pulmonary tuberculosis (PTB) patient tests negative by acid-fast bacilli microscopy but tests positive by more accurate diagnostic techniques.1 It is one of the most problematic issues in tuberculosis diagnosis.2 Patients with SNPT contributed 17.3% – 41.0% of community tuberculosis transmission in Vancouver, Canada, from January 1995 to March 19993 and 13.0% in the Netherlands from 1996 to 2004.4 Also, 55.4% of Belarus tuberculosis cases were smear-negative but culture-positive in 2012.5 In Ethiopia, bacteriologically confirmed SNPT prevalence was reported as high as 23.9% in 2007.6

Although SNPT is assumed to be less contagious and have lower mortality compared to smear-positive tuberculosis, 50.0% – 71.0% of SNPT patients develop active tuberculosis disease.3,4 These SNPT patients also harbour a high proportion of drug-resistant tuberculosis strains. The prevalence of smear-negative multidrug-resistant (MDR) tuberculosis among presumptive PTB patients enrolled with other patients was 47.0% in Belarus in 20125 and 11.5% in Ethiopia August 2017 to January 2018.7 Mortality due to SNPT is also substantial, especially among the immunocompromised. In Mozambique, the mortality of HIV co-infected patients reached 55.8%.8 Thus, early detection with sensitive diagnostic tools that simultaneously detect drug-resistant tuberculosis is critical.

However, diagnosis of SNPT and smear-negative drug-resistant tuberculosis is challenging in low- and middle-income countries (LMICs) like Ethiopia,9 mainly due to a lack of sensitive diagnostic tools. In most LMICs, smear microscopy, which has low sensitivity, is still the first line of diagnosis. The Xpert® MTB/RIF test is a cartridge-based, fully automated DNA testing platform that, in less than 2 h, simultaneously detects tuberculosis and mutations conferring rifampicin resistance (RR).10 The technology detects RR using five probes targeting mutations in the rpoB region of the Mycobacterium tuberculosis genome11,12: rpoB mutations are responsible for RR in over 99.5% of RR strains.10

The Xpert® MTB/RIF test currently resolves the challenges of initial smear microscopy in tuberculosis case detection, which improves the clinical management of tuberculosis cases.13,14 In addition, the Xpert® MTB/RIF is advantageous over smear microscopy and M. tuberculosis culture by its higher sensitivity, simultaneous detection of RR, shorter turnaround time (TAT) (2 h), and a minimal safety requirement.15 By comparison, culture-based RR confirmation takes more than two weeks to get results.16

Nevertheless, various factors impact the Xpert® MTB/RIF’s performance, such as the type of specimen,17 sputum quality,18,19,20 sample collection and diagnosis strategy,21,22,23 and clinical characteristics of patients.17,24,25 Since 2012, more than 314 GeneXpert® instruments have been installed in Ethiopia. Also, the diagnostic strategy changed from spot-morning-spot, which required three samples collected over two days, to same-day diagnosis (spot-spot diagnosis), which requires two samples collected on the same day.26

However, information is lacking on the performance of the Xpert® MTB/RIF test to diagnose SNPT, smear-negative RR tuberculosis, the effects of sputum quality and other factors in the same-day tuberculosis diagnosis approach in Addis Ababa, Ethiopia. Therefore, this study aimed to determine the performance of the Xpert® MTB/RIF test to diagnose SNPT and RR against conventional tuberculosis culture and drug susceptibility testing (DST) in the spot-spot diagnostic strategy. It also intended to determine the SNPT positivity in sputum samples with varying quality and the effect of sputum quality and other factors on Xpert® MTB/RIF test performance.

 Methods Ethical considerations

The study was reviewed and approved by the Institutional Review Board of the Ethiopian Public Health Institute (ref. no. EPHI/613/535) and the Departmental Research and Ethics Review Committee of the Department of Medical Laboratory Science, Addis Ababa University (ref. no. MLS/364/17). Written informed consent from the participants and assent from the guardians of participants less than 18 years of age were obtained. Permission was requested with a legal letter and received from participanting health facilities. Unique patient identifiers were used instead of patient names to conceal patient identity. The laboratory testing results and other patient information were locked. Laboratory results were reported to the clinicians who ordered the test for further patient management.

 Study setting and design

This cross-sectional study was conducted from August 2017 to January 2018 in Addis Ababa, Ethiopia. Addis Ababa is the federal capital of Ethiopia and has a population of more than three million.27 The study was conducted in 16 systematically selected governmental and private directly observed treatment short-course tuberculosis sites. These sites participate in blind rechecking, and most participate in the on-site supervision programme by the Addis Ababa City Administration health research and laboratory service.

 Data collection

Socio-demographic information, clinical presentations, comorbidities, and other factors were collected using a structured questionnaire by trained clinicians through interviews. The questionnaire was piloted following the on-site training of the data collectors.

 Patient enrollment

A total of 418 presumptive SNPT patients who were negative for two consecutive spot sputum smears were enrolled. Adults and children with presumptive PTB who visited the sites during the study period were eligible. However, those taking anti-tuberculosis drugs for more than one week or unable to submit two spot sputum samples were excluded.

Trained laboratorians gave patients detailed instructions on sputum collection. Each presumptive SNPT patient collected two sputum spot ≥ 3 mL samples into sterile 50 mL falcon tubes. Sputum samples were transported with a triple packaging system to the National Tuberculosis Reference Laboratory (NTRL) of the Ethiopian Public Health Institute (EPHI) for laboratory investigation. Sputum samples were stored at 2 °C – 8 °C until transported by triple packaging system for safety, with a thermometer inside for temperature monitoring. The transportation from study sites to NTRL takes less than one hour since it is within the city. Samples were submitted to NTRL within a maximum of two days. Macroscopic sputum quality was evaluated and categorised as blood-stained, purulent, mucoid, and salivary based on the Global Laboratory Initiative (GLI) guidance.28

 Laboratory investigations

The two consecutive spot sputum samples collected from each patient were pooled into one and homogenised. Afterwards, each sputum pool was split into two; one for Xpert® MTB/RIF test (Cepheid, Sunnyvale, California, United States) and the other for culture testing on BACTEC™ MGIT™ 960 System (Becton-Dickinson and Company, Sparks, Maryland, United States) and in laboratory-made Lowenstein Jensen (LJ). Drug susceptibility testing (phenotypic and genotypic) was performed in the NTRL of EPHI, as explained in Sinshaw et al., 2019.7

 Quality assurance and quality control

The sterility and performance of LJ media-manufactured in-house were verified before use for testing using controls by randomly selecting some prepared LJ medium, putting it in the LJ incubator and monitoring for any contaminant growth. If no growth was observed within 56 days, it was considered as sterile or safe for use. Likewise, a new lot or batch of BACTEC™ MGIT™ 960 media was also verified. Each of the culture and identification procedures was performed in a certified Class II biosafety cabine. Preventive maintenance of the equipment, temperature monitoring, and instrument operation checks were performed. In each batch of sample cultured, reagent sterility and process contamination check control were incorporated based on the NTRL standard procedures. A proficiency testing scheme continuously monitored all study test methods. Also, the NTRL is International Organization for Standardization 15189 accredited by the Ethiopian National Accreditation Office for Xpert® MTB/RIF.

 Data entry and analysis

Double data entry was perfomed on EpiData statistical software version 3.0 (EpiData Association, Odense, Denmark). The clean data were transferred to and analysed using IBM Statistical Package for Social Sciences software version 20.0 (Chicago, Illinois, United States). Data entry and cleaning were performed by the NTRL data manager. The characteristics of the study participants were analysed using descriptive statistics. The sensitivity and specificity of the Xpert® MTB/RIF test were calculated at 95% confidence intervals (CI). Pearson chi-square, Fisher’s exact test, or binary logistic regression was used to determine the associations between dependent and independent variables; variables with a p ≤ 0.2 were selected for multivariable analysis. The strength of associations was measured by odds ratios, and a p < 0.05 was taken as statistically significant.

 Results Socio-demographic and clinical characteristics of study participants

Most of the participants (231; 55.3%) were female. The average participant age was 36 years (standard deviation [s.d.] ± 18). Most of the participants (257, 61.8%) had completed some schooling (Grades 1–12), while 112 (26.9%) were uneducated, and more than half of the participants (218; 52.3%) were married. Cough was the leading symptom (413; 98.8%), followed by fever (255; 61.2%). Of the 225 participants interviewed or laboratory tested, 57 (25.3%) were positive for HIV (Table 1 and Table 2).

Socio-demographic characteristics of the participants and their association with SNPT detection by Xpert MTB/RIF test in Addis Ababa, Ethiopia, from August 2017 to January 2018.

Variables Positive
 Negative
 p Adjusted odds ratio
 p
n % n % Odds ratio 95% confidence interval
Sex
 Male 19 10.20 168 89.80 0.006 3.850 1.290–11.44 0.015
 Female 8 3.50 223 96.50 Ref - -
 Total 27 6.50 391 93.50 - - -
Age group
 < 14 0 0.00 20 100. 00 0.225 - - -
 15–24 7 7.40 87 92.60 - - -
 25–34 12 9.60 113 90.40 - - -
 35–44 5 8.20 56 91.80 - - -
 45–54 1 1.90 52 98.10 - - -
 > 54 2 3.10 63 96.90 - - -
 Total 27 6.50 391 93.50 - - -
Education
 No formal education 7 6.20 105 93.80 0.050 Ref - -
 Grade 1–7 4 3.40 112 96.60 0.006 0.000–0.19 0.003
 Grade 8–12 10 7.10 131 92.90 0.011 0.000–0.30 0.008
 > Grade 12 5 10.60 42 89.40 0.013 0.000–0.40 0.012
 Total‡ 26 6.25 390 93.75 - -
Marital status†
 Single 13 8.20 146 91.80 0.530 - - -
 Married 12 5.50 206 94.50 - - -
 Divorced/widowed/separated 2 5.00 38 95.00 - - -
 Total‡ 27 6.50 390 93.50 - - -
Work†
 Farmer 1 3.20 30 96.80 0.006 Ref - -
 Daily laborer 6 7.90 70 92.10 3.310 0.355–30.96 0.293
 Housewife 1 1.10 86 98.90 0.610 0.026–14.42 0.760
 Driver 0 0.00 10 100.00 - - -
 Teacher 0 0.00 7 100.00 - - -
 Student 0 0.00 46 100.00 - - -
 Merchant 0 0.00 14 100.00 - - -
 Health professional 1 33.30 2 66.70 38.300 1.077–1368 0.045
 Government employee 2 5.70 33 94.30 1.551 0.100–23.40 0.750
 Self-employed 7 11.70 53 88.30 5.250 0.563–48.90 0.150
 Other 7 16.30 36 83.70 7.331 0.770–69.85 0.083
 Total‡ 25 6.10 387 93.90 - - -

, One participant did not respond;

, Six participants did not respond.

Clinical manufestations and behavioural characteristics of the presumptive SNPT patients in Addis Ababa, Ethiopia, from August 2017 to January 2018.

Variables Yes/no Positive % Negative %
Clinical and behavioural characteristics
 Cough Yes 27 6.5 386 93.5
No 0 0.0 5 100.0
 Fever Yes 20 7.8 235 92.2
No 7 4.3 155 95.7
 Chest pain Yes 21 8.3 231 91.7
No 6 3.6 160 96.4
 Loss of appetite Yes 22 9.1 221 90.9
No 5 2.9 170 97.1
 Weight loss Yes 20 12.4 141 87.6
No 7 2.7 249 97.3
 Shortness of breath Yes 14 2.7 120 89.6
No 13 4.6 271 95.4
 Joint pain Yes 10 9.0 101 91.0
No 17 5.5 290 94.5
 Abdominal pain Yes 2 4.0 48 96.0
No 25 6.8 343 93.2
 Swelling Yes 1 5.6 17 94.4
No 26 6.5 374 93.5
 Night sweating Yes 4 6.9 58 93.1
No 23 6.1 355 93.9
 Hemoptysis Yes 4 13.8 25 86.2
No 23 5.9 365 94.1
 Shifting of trachea Yes 0 0.0 5 100.0
No 27 6.6 385 93.4
 Abnormal breathing sound Yes 6 6.2 90 93.8
No 21 6.5 301 93.5
 Dullness during percussion Yes 4 11.8 30 88.2
No 23 6.0 359 94.0
 Tenderness Yes 4 15.4 22 84.6
No 23 5.9 367 94.1
 HIV infection Yes 3 5.3 54 94.7
No 14 8.3 154 91.7
 Migrant Yes 3 27.3 8 72.7
No 24 5.9 380 94.1
 MDR TB contact Yes 1 12.5 7 87.5
No 26 6.4 381 93.6
 Drug-susceptible TB contact Yes 2 7.4 25 92.6
No 25 6.4 363 93.6
 Alcohol consumption Yes, currently 9 13.2 59 86.8
Yes, previously 2 3.6 53 96.4
Not at all 16 5.4 279 94.6
 Smokers Yes, currently 2 15.4 11 84.6
Yes, previously 2 15.4 11 84.6
Not at all 23 6.0 361 94.0
 Chewing chat Yes, currently 3 12.0 22 88.0
Yes, previously 5 15.6 27 84.4
Not at all 19 5.3 342 94.7
Treatment history†
 New - 24 6.5 345 93.5
 Previously treated - 3 6.1 46 93.9

MDR, multidrug-resistant.

, Previously treated means patients who have taken anti-TB treatment in their life for 1 month or more.

 Sputum quality and performance characteristics of Xpert<sup>®</sup> MTB/RIF test

Salivary sputum was the leading sample quality (147, 35.2%), while blood-stained sputum was the least (10; 2.4%) sputum quality submitted (Table 3). The majority of the sputum submitted (310; 74.3%) had 3 mL – 5 mL volume, while 16.8% had 6 mL – 7 mL and 8.9% had 8 mL – 9 mL volume.

Positivity of Xpert MTB/RIF assay in different sputum sample qualities in Addis Ababa, Ethiopia, from August 2017 to January 2018.

Variables Xpert MTB/RIF result
 Chi-square (p) Adjusted odds ratio 95% confidence interval p
Negative
 Positive
n % n %
Sputum quality
 Purulent 108 92.3 9 7.7 0.013 1.39 0.500–3.88 0.520
 Mucoid 137 95.8 6 4.2 0.71 0.232–15.00 0.540
 Saliva 138 93.9 9 6.1 Reference - -
 Blood-stained 7 70.0 3 30.0 6.90 1.360–34.96 0.020
Total† 390 93.5 27 6.5 - - -

, Difference in expected total is because one sputum sample was not evaluated for macroscopic appearance.

The majority of the positive Xpert® MTB/RIF tests, 14 (51.9%), were low and very low in bacillary load. There were 20 (4.8%) unsuccessful Xpert MTB/RIF results (sum of errors, invalids and no results). The most unsuccessful results were errors (15; 3.6%) (Table 4).

Semi-quantitative bacilli DNA quantification and unsuccessful Xpert MTB/RIF test results in Addis Ababa, Ethiopia, from August 2017 to January 2018.

Variables Frequency %
Bacilli DNA quantification by Xpert MTB/RIF test
 High 5 18.52
 Medium 8 29.63
 Low 9 33.30
 Very low 5 18.52
 Total 27 100.00
Unsuccessful Xpert MTB/RIF results
 Error 15 3.60
 Invalid 2 0.50
 No result 3 0.70
 Total 20 4.80

The error code 5007 was the most common (Table 5). Mucoid sputum accounted for seven (46.7%) errors, while purulent sputum accounted for five (33.3%). The other three (20.0%) errors were from salivary sputum.

Xpert MTB/RIF test post-run analysis error results and possible causes in Addis Ababa, Ethiopia, from August 2017 to January 2018.

Error code Error message (taken from cepheid user manual) n % Origin of the error (taken from cepheid user manual)
5007 Probe check control failed and the test was stopped before amplification 12 2.9 Sputum viscosity or wrong sample volume, or cartridge reaction tube improperly filled, contains bubbles, or probe integrity issue detected
5017 Quality control 1 and quality control 2 probe check failed 1 0.24 Cartridge-related issue (based on cepheid information)
5011 Signal loss detected in the amplification curve for analyte (specimen processing control) 2 0.48 Loss of tube pressure because the cartridge tube is not airtight, or cartridge valve is not working correctly

Source: Adapted from World Health Organization. Xpert MTB/RIF implementation manual. Geneva: WHO, 2014; p. 1–35.

The Xpert® MTB/RIF test detected three RR cases. Two of the RR cases were caused by probe E (codons 529–533) missing mutations and one by probe B (codons 511–518) missing mutation.

 Sputum <italic>M. tuberculosis</italic> positivity rate

Mycobacterium tuberculosis positivity rate was 6.1% for salivary sputum and 4.2% for mucoid sputum. However, the M. tuberculosis positivity rate was higher (30.0%) in blood-stained sputum. Blood-stained sputum was 6.9 times more M. tuberculosis positive than salivary sputum (95% CI: 1.36–34.96; odds ratio [OR]: 6.9; p = 0.02), while purulent sputum was the second most positive (7.7%) (Table 3).

 Detection of SNPT and RR by Xpert<sup>®</sup> MTB/RIF test

Of the 418 smear-negative presumptive PTB patients enrolled in this study, 27 (6.5%; 95% CI: 4.10–8.81) were M. tuberculosis positive by Xpert® MTB/RIF. On the other hand, 26 (6.4%; 95% CI: 4.00–8.74) were culture-positive. Twenty-four (5.7%; 95% CI: 3.51% – 7.97%) sputum samples were positive by both Xpert® MTB/RIF and tuberculosis culture (LJ and MGIT) (Figure 1).

Xpert® MTB/RIF assay performance reference to the MGIT and LJ Mycobacterium tuberculosis culture in Addis Ababa, Ethiopia, from August 2017 to January 2018.

Five samples were discordantly positive by both methods; one sample was LJ culture-positive but MGIT culture-negative. On the other hand, four samples were MGIT culture-positive, two of which were LJ culture-negative, while the other two were LJ culture contaminated. Besides, of the two Xpert® MTB/RIF negative culture-positive samples, one was MGIT negative and the other MGIT positive, but both were positive by LJ solid culture. Moreover, three sputum samples were Xpert® MTB/RIF positive but culture-negative. Thus, the diagnostic sensitivity and specificity of the Xpert® MTB/RIF test relative to tuberculosis culture was 92.3% (75.9% – 97.9%) and 99.2% (97.8% – 99.7%). The overall diagnostic accuracy was 98.8% (97.2% – 99.5%) (Figure 1).

Three (11.54%) RR SNPT cases were detected by the Xpert® MTB/RIF test; two of the RR detected were from a new case and one from previously treated cases. These strains were later confirmed as MDR by genotype MTBDRplus version 2 and BACTEC™ MGIT™ 960 DST.

Most of the SNPT cases were detected from men (95% CI: 1.29–11.44; OR: 3.85; p = 0.015]) (Table 1). Patients with weight loss had 4.05 times more risk of being diagnosed with SNPT, in 95% CI (p = 0.007) compared to those without weight loss (Supplementary Table 1).

 Discussion

Smear microscopy is still a front-line tuberculosis diagnostic tool in developing countries like Ethiopia.1,21,29 However, because of its low sensitivity, smear microscopy misses many tuberculosis cases, resulting in delayed diagnosis and treatment initiation.15,30,31 Therefore, this study evaluated the effect of sputum quality and Xpert® MTB/RIF performance for SNPT detection in same-day diagnosis.

Sputum quality has effects on the detection of PTB.19,20,32,33 However, many studies do not consider attributes of sputum quality in MTB testing by the Xpert® MTB/RIF test.34,35 In the present study, the M. tuberculosis positivity by Xpert® MTB/RIF significantly varied across sputum qualities. The majority of sputum, 147 (35.2%), submitted was salivary with a positivity rate of 6.1%. Although the blood-stained sample was the least submitted, the positivity was very high at 30.0%. In 95% CI, blood-stained samples showed 6.9 (p = 0.020) times more positive than salivary.

Many laboratories reject blood-stained sputum, assuming that it brings unreliable Xpert results due to polymerase chain reaction inhibition; however, this study revealed the highest SNPT positivity in blood-stained sputum. Contrary to the present study, a study showed that the Xpert result is only valid at less than 2.0% blood contamination of the sputa. If sputum contamination with blood is beyond 5.0%, the result will be unreliable and absolute inhibition occurs at 20.0% blood contamination.18 However, studies found that patients who are MTB positive in a blood sample have a higher risk of death.36 Therefore, we are missing the most important sputum quality. The current finding is different from a study in Uganda, where SNPT was more in salivary sputum and lowest in blood-stained sputum.20 This might be as a result of the fact that the majority of the Ugandan study participants were HIV positive and most of them had a very low CD4 count (≤ 200 cells/µL). The second highest SNPT positivity, 7.7%, was diagnosed from purulent sputum. Although the difference is not statistically significant, blood-stained sputum showed greater positivity than purulent sputum, whereas purulent sputum is considered the best sputum for MTB detection. The difference in macroscopic sputum appearance significantly varied for MTB positivity by Xpert® MTB/RIF test, implying a need for proper sputum collection, similar to other reports.20,33,34

In Ethiopia, the sputum sample collection strategy for MTB diagnosis was changed from spot-morning-spot to spot-spot (same-day diagnosis) in 2017.26 Same-day diagnosis stops multiple visits to the health facilities by the patient to submit sputum and receive a result. However, it is 2.8% less sensitive with a lower dropout rate than the conventional -spot-morning-spot strategy.21 In the spot-morning-spot strategy, three smear slides are made. The first slide made from the first spot sputum, the second slide made from the morning sputum and the third slide from the second spot sputum.22 This change increases the possibilities of being smear-negative. In the spot-spot approach, in the current study, Xpert® MTB/RIF assay detected an extra 24 (5.7%) SNPT and three RR strains in comparison with smear microscopy. The ability of the Xpert® MTB/RIF test to detect SNPT and RR is high in this study which might be because the missed morning sputum increased the SNPT cases. The Xpert® MTB/RIF testing was performed on the direct sputum before culture to evaluate the performance of the Xpert® MTB/RIF in the peripheral health facilities where direct sputum only is used for Xpert® MTB/RIF testing. Sputum processing, including decontamination, neutralisation, and pellet concentration before culture, is only possible in the tuberculosis culture reference laboratories.

The diagnostic sensitivity of the Xpert® MTB/RIF test in this study was 92.3%, while specificity was 99.2% reference to tuberculosis culture. The present study revealed higher sensitivity and specificity than the Uganda report20 because most of Uganda’s study participants were HIV-positive and used a spot-morning-spot diagnostic approach. Another justification might be tuberculosis prevalence is higher in Ethiopia. Similarly, the present study revealed a very high sensitivity compared to a study in Jigjiga, Ethiopia: 48.5% for smear-negative PTB.37 Likewise, the current study showed higher sensitivity and specificity relative to a review conducted in Liverpool, United Kingdom, in 2013, which was 67.0% – 74.0% pooled sensitivity and 99.0% pooled specificity.38 However, the sensitivity of the Xpert® to diagnose SNPT showed considerable variability in different studies.39,40,41 The possible reasons for the variabilities in sensitivity and specificity might be differences in study design, study population, sample collection strategy, study period, study area or location, tuberculosis prevalence, comorbidity and laboratory performance. The three (11.54%) RR strains detected by the Xpert® MTB/RIF test in this study concords 100.0% with Genotype MTBDR plus version 2 and phenotypic BACTEC™ MGIT™ 960 DST results. In addition, studies reported high Xpert® MTB/RIF detection performance: 95.0% – 97.0% sensitivity and 98.0% – 99.0% specificity, for culture positives.38

The findings in this study are commendable since they facilitate early tuberculosis diagnosis and universal access to DST,42 which are pivotal to the endTB strategy. Furthermore, the World Health Organization approved that tuberculosis diagnostic technologies such as Xpert® MTB/RIF need to be scaled up to become the first line of diagnosis. With the scale-up, patients will benefit from early diagnosis and initiation of treatment.

More than half of the positive Xpert® MTB/RIF results (14; 51.9%) had a low or very low bacillary load, implying that SNPT patients have a paucibacillary load. Twenty (4.8%) Xpert® MTB/RIF results were unsuccessful (the sum of errors, invalids and no results), which is lower than a report from Nigeria.43 The Nigeria study did not include only presumptive smear-negative SNPT cases. Most of our unsuccessful results were due to error results (15; 3.6%), but in the Nigerian study, these were considered invalid results.43 The error rate in the present study was higher than the GLI recommendation (< 3.0%),44 but lower than a report from Addis Ababa, which reported 8.9%.45 The discrepancy in the study reports might result from the difference in the testers’ expertise and experience, study population, sample type, and study period. The error code 5007 was the most common (12; 2.9%), often caused by viscous sputum or wrong sample volume, improper filling of cartridge reaction tube, bubbles, or probe integrity issues. Errors are a loss of valuable time and money for the patients and the laboratory. Thus, patient training on quality sputum collection and laboratory staff refresher training may minimise these errors. Of the three RR cases detected by the Xpert® MTB/RIF test, two were due to probe E (codons 529–533) missing, which is the most frequent type of mutation in the rpoB region of the mycobacteria.46,47 The other was probe B missing (codons 511–518; mutation).

Limitations

The current study did not explain the performance of the Xpert® MTB/RIF test in the same-day diagnosis to diagnose smear-negative RR as it did not include enough RR cases, which is a limitation of the study.

 Conclusion

The performance of the Xpert® MTB/RIF test to detect SNPT in spot-spot samples and rapid detection of RR were high. However, the diagnostic performance of the test significantly varied across different sputum qualities. Thus, good patient instruction or training and close follow-up on sputum sample collection are essential for getting quality sputum for better Xpert® MTB/RIF yield. We recommend testing sputum samples by Xpert® MTB/RIF irrespective of sputum quality.

 Acknowledgements

We acknowledge the Ethiopian Public Health Institute for their material and technical support of the study. We are also thankful for the study site staff and study participants.

Competing interests

The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.

 Authors’ contributions

W.S. designed and conceptualised the study, performed the data analysis and interpretation of the results, and drafted the manuscript. A.K., A.A. and A.B. reviewed and edited the manuscript. A.K. was also involved in the study’s conceptualisation. Z.M. performed the research methods, data analysis, and interpretation of results. E.T., M.T., B.Y., M.A., B.D., G.D., A.A., H.M., Y.A., G.S., M.G. and W.S. performed the laboratory analysis and reporting, site supervision, data collection, and data entry. D.F.G was responsible for data entry, data curation and analysis.

 Sources of support

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

 Data availability

All of the data are available from the corresponding author, W.S.

 Disclaimer

The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated organisations of the authors.

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How to cite this article: Sinshaw W, Kebede A, Bitew A, et al. Effect of sputum quality and role of Xpert® MTB/RIF assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in Addis Ababa, Ethiopia. Afr J Lab Med. 2022;11(1), a1671. https://doi.org/10.4102/ajlm.v11i1.1671

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