|ORIGINAL RESEARCH REPORT
|Year : 2022 | Volume
| Issue : 2 | Page : 62-66
Pus cell as an indicator for Mycobacterium tuberculosis diagnostic yield by GeneXpert MTB/RIF in South-South Nigeria: A prospective study
Ekom Ndifreke Edem1, Anthony Nathaniel Umo2, Olajide Joseph Akinjogunla3, Unwana Ezekiel Akereuke4
1 Department of Medical Microbiology and Parasitology, University of Uyo Teaching Hospital, Uyo, Nigeria
2 Department of Medical Microbiology and Parasitology, College of Health Sciences, University of Uyo, Uyo, Nigeria
3 Department of Microbiology, Faculty of Science, University of Uyo, Uyo, Nigeria
4 Department of Medical Microbiology and Parasitology, Faculty of Basic Clinical Sciences, University of Uyo, Nigeria
|Date of Submission||13-Jan-2022|
|Date of Acceptance||01-Mar-2022|
|Date of Web Publication||13-Jun-2022|
Mr. Ekom Ndifreke Edem
Department of Medical Microbiology, University of Uyo Teaching Hospital, Uyo
Source of Support: None, Conflict of Interest: None
Background: The common problem in tuberculosis (TB) management is mis-diagnosis or under diagnosis of cases leading to high morbidity and mortality. In order to reverse this, new diagnostic tools for detection of Mycobacterium tuberculosis (MTB) have been developed. However, in the evaluation process of these tools many studies have not considered attributes of sputum quality in their testing algorithm. The performance of laboratory tests to diagnose pulmonary tuberculosis is dependent on the quality of the sputum sample tested and quality is measured by the number of pus and epithelial cells present in sputum. Aims and Objectives: this study aimed at investigating the association between pus cell and MTB positivity. Methods: Sputum samples from 140 suspected TB participants were screened for sputum quality and MTB using macroscopy, Ziehl-Neelson staining and GeneXpert techniques. Results: Of the 140 sputum samples subjected to AFB microscopy and GeneXpert MTB/RIF assay, GeneXpert assay had 32 (22.9%) MTB positives while AFB smear had 27 (19.3%) MTB positives and there was no significant relationship between sputum type and MTB yield by AFB compared to Xpert that showed significant relationship between sputum type and MTB yield with P<0.005. Out of the 37 sputum samples with pus cells ≥25, 29 (90.6%) were MTB positive by Xpert and 25 (92.6%) were MTB positive by AFB smear. Conclusion: In conclusion, our study shows a high significant association between pus cells presence and the methods and therefore suggests that presence of pus cells in sputum is predictive for possible positivity to MTB by Xpert MTB/RIF.
Keywords: GeneXpert assay, Mycobacterium tuberculosis, polymorphonuclear leukocyte, sputum, sputum smear microscopy
|How to cite this article:|
Edem EN, Umo AN, Akinjogunla OJ, Akereuke UE. Pus cell as an indicator for Mycobacterium tuberculosis diagnostic yield by GeneXpert MTB/RIF in South-South Nigeria: A prospective study. J Clin Sci 2022;19:62-6
|How to cite this URL:|
Edem EN, Umo AN, Akinjogunla OJ, Akereuke UE. Pus cell as an indicator for Mycobacterium tuberculosis diagnostic yield by GeneXpert MTB/RIF in South-South Nigeria: A prospective study. J Clin Sci [serial online] 2022 [cited 2022 Jun 29];19:62-6. Available from: https://www.jcsjournal.org/text.asp?2022/19/2/62/347244
| Introduction|| |
Mycobacterium tuberculosis (MTB) remains one of the most significant causes of death from an infectious agent. Nigeria has the fourth highest burden of tuberculosis (TB) in the world, with an annual incidence of 311 cases/100,000 population and a mortality rate of 81/100,000 population in 2006. TB can remain dormant for years without showing symptoms or spreading to other people. The most common method for TB diagnosis worldwide is sputum smear microscopy or direct microscopy, which has limited sensitivity, especially among HIV-infected patients. Lack of prompt diagnosis and appropriate treatment of TB increases the risk of transmission, drug resistance, and case fatality. An accurate and timely diagnosis of TB is key to effective treatment and management. The quality of the sputum is measured by the presence or absence of squamous epithelial cell (SEC) found in the upper respiratory tract, which is an indicator of oropharyngeal contamination and polymorphonuclear leukocyte, also known as pus cell, which is an indicator of acute inflammation. The quality of sputum submitted for diagnosis is a key determinant of diagnostic performance. The presence of a few or rare pus cells in sputum is normal; however, when there is an increase in the pus cells, it gives an indication of infection. Pus cells are soldiers, and they are being recruited to the infection site to fight off the infection. Hence, if there are lots of white blood cells being called into active duty, it is a pretty good indication of infection. Several microscopic sputum quality assessment criteria have been developed since the 1970s, aimed primarily at assessing sputum quality. Sputum of good quality has count of more than 25 pus cells regardless of the number of epithelial cells present.
Microscopy has a lesser sensitivity than the Xpert assay and the conventional culture technique. However, culture and Xpert tests, which are more expensive than microscopy, can only diagnose TB in samples containing sufficient concentrations of M. tuberculosis. GeneXpert MTB/RIF (Xpert) assay has constituted a major breakthrough for TB diagnostics, providing a rapid and accurate way of identifying TB patients in high TB-burden, low-income countries. The Xpert assay has a higher sensitivity than microscopy. Nevertheless, the quality of sputum predicts the performance of both microscopy and the Xpert test. Thus, poor quality sputum samples can lead to missed TB diagnosis. Therefore, this study seeks to determine the presence of pus cells as a predictory base for GeneXpert MTB/RIF (Xpert) assay positivity in suspected pulmonary TB patients in South–South Nigeria.
| Methods|| |
Study design and population
This study was a prospective, observational, cross-sectional study to determine the presence of pus cells as a predictor factor for GeneXpert positivity. This study was conducted among TB suspects attending TB Clinic in St. Lukes Hospital, Anua, Uyo, from May 2019 to August 2019. All microbiological testings for the study were carried out in the TB laboratory, St. Lukes Hospital, Uyo, and TB laboratory, University of Port Harcourt Teaching Hospital, Port Harcourt, Rivers State.
Estimated minimum sample size
This study calculated the minimum sample size using a formula developed by Godden for epidemiological studies as shown below to derive its estimated sample size;
where, n = minimum sample size
Z = Z statistic for a level of confidence
P = Expected prevalence or proportion
d = Precision
Given that MDR-TB had 7.33% prevalence in a study by Halilu et al. and assuming a precision, d, which is 0.05, and Z is 1.96 (95% confidence interval [CI]), the estimated minimum sample size was calculated as shown below;
mSs = 103
Assuming an attrition of 40%, then 140 study participants were used.
Inclusion and exclusion criteria
Participants of all ages, ability to produce sputum without inducement, suspected TB patients based on clinical symptoms, and invalid/error results from GeneXpert were included in the study. Participants with sputum collected through induction, unknown HIV status, extrapulmonary TB, nonrespiratory specimen, and sputum sample <3 ml were all excluded from the study.
Collection of samples
An estimated minimum sample size of 140 was included in the study. Following written informed consent and proper education on sputum production, participants provided spot sputum samples.
Laboratory analysis of samples
Sputum specimens submitted were first assessed macroscopically for appropriate quantity and type, and then processed using the Ziehl–Neelsen (ZN) staining technique to determine their smear acid-fast bacillus (AFB) status before conducting the Xpert MTB/RIF (Cepheid, Sunnyvale, US) assay.
The macroscopy of each sputum sample was assessed and graded accordingly. The results were reported as blood stained, mucoid, purulent, or salivary, using standardized photomicrographs from the International Union against TB and Lung Disease guidelines.
Slides were prepared using the ZN staining technique and examined for the presence of AFB. Each specimen was also examined under ×100 oil immersion and given scores for the number of SECs and pus cells per low power field (LPF). Slides were examined under the microscope for 3–5 min. Specimens were processed and examined within 2–3 h of collection to avoid cellular changes in sputum.
Acid-fast bacillus smear microscopy
Using a sterile application stick, 30 μl of raw sputum was placed on a grease-free glass slide and evenly spread, dried, and heat fixed. Thereafter, ZN staining was done as per the standard protocol and then observed under the microscope (Revised National TB Control Programme, 2015). All smears were scored according to the International Union Against Tuberculosis and Lung Disease/World Health Organization guidelines, as AFB negative, scanty (1–9 AFB/100 fields), 1+ (10–99 AFB/100 fields), 2+ (1–10 AFB/field), or 3+ (more than 10 AFB/field). Smears for quality control prepared from TB culture-positive samples were stained and reviewed at the start of each workday.
Sputum quality microscopy
The decision to either classify sputum sample as quality sputum or not was dependent on the total score of both pus cells and SECs.
The screening method applied was that described by Geckler et al. because it takes epithelial cells as criteria for judging sputum quality. Geckler et al. regarded sputum samples with more than 25 epithelial cells per (×100) field as poor quality sputum regardless of the number of pus cells present.
GeneXpert test was performed according to the manufacturer's instruction. Briefly, with the aid of a sterile pipette, 2 ml of GeneXpert reagent was added to 1 ml of sputum sample and incubated at room temperature for 15 min. The mixture was agitated twice at 5 min intervals. The liquefied mixture was transferred into the GeneXpert cartridge using a sterile pipette and loaded into GeneXpert instrument. The result was available within 2 h.
Recorded data were analyzed using the IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp. IBM Corp. Released 2015. Qualitative data were expressed as frequency and percentage. The Chi-square test of significance was used to compare proportions between two qualitative parameters and to also investigate the association between selected background characteristics and the concordance of TB test report and the test report. The CI was set to 95% and the margin of error accepted was set to 5%. Hence, P value was considered significant as the following:
P < 0.05 was considered statistically significant, <0.001 was considered as highly significant, and >0.05 was considered insignificant.
Ethical approval for this study was obtained from the Akwa Ibom State Ministry of Health, Akwa Ibom State, Nigeria, with Ref: MH/PRS/99/Vol.V/631. All clinical isolates were collected for routine diagnosis by the hospital. No patient information was used in this study; informed consent from participants was not required.
| Results|| |
Detection of Mycobacterium tuberculosis using acid-fast bacillus, GeneXpert techniques according to sputum quality
Of 140 sputum samples subjected to AFB microscopy and GeneXpert MTB/RIF assay, GeneXpert assay had 32 (22.9%) MTB positive, 7 (5%) “error,” and 101 (72.1%) MTB negatives, while AFB smear had 27 (19.3%) MTB positives and 113 (80.7%) MTB negatives [Table 1]. Using Xpert and AFB technique, mucoid sputum samples had substantially higher proportion of MTB positives 26(81.2%) and 25(92.6%) respectively, followed by salivary samples with 4(12.5%) and 0 respectively, and blood stained samples with 2(6.2%) and 2(7.4%) respectively [Table 1]. There was no significant relationship between sputum type and MTB yield by AFB compared to Xpert which showed a significant relationship between sputum type and MTB yield with P < 0.005 [Table 1].
Out of the 37 sputum samples with pus cells ≥25, 29 (90.6%) were MTB positive by Xpert and 25 (92.6%) were MTB positive by AFB smear. Of the 103 samples with pus cells <25, 3 (9.4%) were MTB positives by Xpert and 2 (7.4%) by AFB smear. Statistical analysis showed that there was a significant relationship between pus cells and MTB positivity with P < 0.0001. Among the six samples with SEC ≥25, 1 (3.7%) and 1 (3.2%) were MTB positive by AFB and Xpert, respectively, as against 134 samples with SEC <25, which had 31 (96.9%) and 26 (96.3%) MTB positivity by Xpert and AFB, respectively [Table 2].
Statistical analysis indicated that there was no significant relationship between SEC and MTB positivity by AFB method compared to Xpert that showed high significant relationship with P < 0.0001 [Table 2].
| Discussion|| |
In this study, we observed that Xpert and AFB smear microscopy in the detection of MTB was actually dependent on the quality of sputum. This can be seen in the seven “error/invalid” results by Genexpert in [Table 1], which were mostly the inappropriate sputum for the diagnostic assay. Consistent with our report, a recent study from Acuña-Villaorduña et al. had demonstrated that discordant culture-negative/Xpert-positive results are associated with mucoid/salivary sputum, and our results further confirm that these inconsistencies are majorly contributed to inappropriate sputum. Therefore, the low proportion of salivary sputum among all sputum samples detected in this study may be important for explaining the “error” and no MTB detection by Xpert and AFB smear. However, our finding is different from that done by Meyer et al., which has it that salivary sputum does not have lower but perhaps higher diagnostic yield when tested for TB with Xpert. Thus, the conventional assumptions that salivary sputum is of lower quality and bloody sputum is of higher quality for smear microscopy do not apply to samples tested with Xpert.
The reasons are that the AFB smear method requires 5 × 103 to 1 × 104 bacilli/ml of the specimen to generate a positive result (Blakemore et al., 2010), and GeneXpert assay only detects genetic materials of MTB.
Determining the quality of the specimen is also based on the numbers of polymorphonuclear leucocytes and SECs present. Sputum sample carries a few or rare pus cells but when found in large number points to an infection. Geckler et al. regard sputum samples with more than 25 epithelial cells per field as poor quality sputum regardless of the number of pus cells present so as to be able to predict the MTB positivity in the presence of pus cells. Wong et al. had used the presence of pus cells as an indication that the specimen was from a site of active infection and is therefore of acceptable quality. However, since this study used the criteria by Geckler et al., it can be seen that all MTB positivity by the two techniques were from sputum samples with <25/LPF epithelial cells and one from sputum samples with ≥25/LPF epithelial cells. If we were to use the criteria by Van Scoy, who concluded that specimens with ≥25/LPF pus cells are regarded as good sputum quality regardless of the SEC count, 76.4% of the total sputum samples would have been discarded. However, this study does not suggest that sputum specimens with SEC ≥25/LPF should be disregarded on diagnosing TB as this is seen in the present study that 3.7% of sputum samples with SEC ≥25 was positive to TB. A study by Edem et al., which recorded AFB positivity when SEC ≥25/LPF, is in agreement that sputum samples with SEC ≥25/LPF should not be discarded. Our results echo previous findings by Acuña-Villaorduña et al., Shi et al., and Yoon et al. that sputum quality exhibits a strong association with the presence or absence of MTB in sputum tested with GeneXpert and AFB smear.
This study suggests that pus cells could be predictory criteria for MTB positivity which is shown seen in [Table 2] that most MTB/AFB-positive samples were from sputum samples with ≥25 pus cells.
Our study also suggests that screening for the presence of pus cells is more relevant in the context of MTB-positivity than the presence of epithelial cells, as they indicate whether there is a recent infection or not, which is not bound to the number of epithelial cells.
| Conclusion|| |
Good-quality sputum aids in a better diagnosis of sputum samples, as it gives definite assertion on the presence of MTB in the sample. However, since the quality of sputum is measured by the number of pus or SEC cells present in a sputum sample, it is very vital to educate suspected TB patients on appropriate sputum production. Pus cells are inflammatory cells recruited to site of infection to fight against the infection. Therefore, in sputum with a high number of pus cells, it is suspected that there is a respiratory infection, not minding the number of SEC cells (respiratory contaminants) present. In general, our study shows a high significant association between pus cells presence and MTB positivity by the methods and therefore suggests that the presence of pus cells in sputum is predictive for possible positivity to MTB by Xpert MTB/RIF.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al
. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010;363:1005-15.
Wong LK, Barry AL, Horgan SM. Comparison of six different criteria for judging the acceptability of sputum specimens. J Clin Microbiol 1982;16:627-31.
Edem EN, Onwuezobe IA, Ochang EA, Samuel A. Correlating the presence of pus cells in sputum to AFB positivity, the University of Uyo Teaching Hospital (UUTH) experience. Landmark Res J Med Med Sci 2016;3:034-6.
Ramsay A, Bonnet M, Gagnidze L, Githui W, Varaine F, Guérin PJ. Sputum, sex and scanty smears: New case definition may reduce sex disparities in smear-positive tuberculosis. Int J Tuberc Lung Dis 2009;13:613-9.
Khan MS, Osman D, Sabira T, Peter GF. Judging respiratory specimen acceptability for AFB microscopy: Visual vs. microscopic screening. Trop Med Int Health 2009;14:571-5.
Van Scoy RE. Bacterial sputum cultures – A clinician's viewpoint. Mayo Clin Proc 1977;52:39-41.
Datta S, Shah L, Gilman RH, Evans CA. Comparison of sputum collection methods for tuberculosis diagnosis: A systematic review and pairwise and network meta-analysis. Lancet Glob Health 2017;5:e760-71.
Vassall A, van Kampen S, Sohn H, Michael JS, John KR, den Boon S, et al.
Rapid diagnosis of tuberculosis with the Xpert MTB/RIF assay in high burden countries: A cost-effectiveness analysis. PLoS Med 2011;8:e1001120.
Yoon SH, Lee NK, Yim JJ. Impact of sputum gross appearance and volume on smear positivity of pulmonary tuberculosis: A prospective cohort study. BMC Infect Dis 2012;12:172.
Toman K. Toman's Tuberculosis; Case Detection, Treatment and Monitoring – Questions and Answers. 2nd
ed. Geneva: World Health Organization; 2004.
Godden B. Sample size formulas. J Stat 2004;3:66.
Halilu TB, Bala Z, Florence S, Yerima IB. Multi-drug resistance tuberculosis (MDR-TB) survey in North East Nigeria. J Pharm Cosmet Sci 2013;1:45-52.
International Union against Tuberculosis and Lung Disease (IUATLD). Technical Guide: Sputum Examination for Tuberculosis by Direct Microscopy in Low Income Countries. 5th
ed. Paris, France: International Union against Tuberculosis and Lung Disease; 2000.
Geckler RW, Gremillion DH, McAllister CK, Ellenbogen C. Microscopic and bacteriological comparison of paired sputa and transtracheal aspirates. J Clin Microbiol 1977;6:396-9.
Acuña-Villaorduña C, Orikiriza P, Nyehangane D, White LF, Mwanga-Amumpaire J, Kim S, et al.
Effect of previous treatment and sputum quality on diagnostic accuracy of Xpert®
MTB/RIF. Int J Tuberc Lung Dis 2017;21:389-97.
Meyer AJ, Atuheire C, Worodria W, Kizito S, Katamba A, Sanyu I, et al.
Sputum quality and diagnostic performance of GeneXpert MTB/RIF among smear-negative adults with presumed tuberculosis in Uganda. PLoS One 2017;12:e0180572.
Blakemore R, Story E, Helb D, Kop J, Banada P, Owens MR, et al
. Evaluation of the analytical performance of the Xpert MTB/RIF assay. J Clin Microbiol. 2010;48:2495-501.
Marlowe EM, Novak-Weekley SM, Cumpio J, Sharp SE, Momeny MA, Babst A, et al
. “Evaluation of the cepheid xpert MTB/RIF assay for direct detection of mycobacterium tuberculosis complex in respiratory specimens,” Journal of Clinical Microbiology, 2011;49:1621-3.
Shi J, Dong W, Ma Y, Liang Q, Shang Y, Wang F, et al
. GeneXpert MTB/RIF Outperforms Mycobacterial Culture in Detecting Mycobacterium tuberculosis from Salivary Sputum. BioMed Research International, 2018 Article ID 1514381.
[Table 1], [Table 2]