Back
 OJMM  Vol.11 No.4 , December 2021
Pulmonary Tuberculosis among Suspected Sudanese Patients in Wad Madani Tuberculosis Center
Abstract: Background: Tuberculosis is a health problem in Sudan and may become a greater challenge in the future due to the weakness in infection prevention measures, increase in cases of drug-resistant and the difficulty of diagnosis. Objective: The aim of this study was to detect Mycobacterium tuberculosis (MTB) from sputum of clinically suspected patients using the three available techniques. Methods: Three hundred participants referred to Wad Madani Tuberculosis Center during 2017-2019 were included. Early morning fresh sputum samples were subjected to Mycobacterium tuberculosis examination by Ziehl-Neelsen (ZN) stain without concentration, ZN stain with centrifugation and geneXpert assay. Results: Of the 300 suspected cases; Mycobacterium tuberculosis detected in 17% (51/300) by ZN stain without concentration, 20% (59/300) by ZN stain with centrifugation and 34% (103/300) by geneXpert. The two techniques of ZN stains possessed 100% specificity and relative differences in sensitivity when compared to geneXpert assay. The significant association observed between ZN stains and geneXpert results indicated validity of ZN techniques for detection. Conclusions: The study confirmed that geneXpert is better for identification of Mycobacterium tuberculosis when compared to ZN techniques which are also important for diagnosis in poor places and where the geneXpert assay is not available.

1. Introduction

Tuberculosis (TB) is an infectious disease of lungs, abdomen and skin caused by Mycobacterium tuberculosis. Diagnosis of tuberculosis can be achieved using different approaches; direct smears from sputum for microscopy, culture-based testing, serology and nucleic acid amplification techniques (NAATs), in addition, chest radiography plays an important role in the diagnosis as well as disease prognosis and treatment follow-up [1] [2]. At last time, newly discovered diagnostic tools have been appeared, such as interferon-gamma release assays and geneXpert assay [3]. Successful elimination of TB needs selection of appropriate method for rabid and accurate detection, and this reduces mortality and transmission rates [4]. For several reasons, such as ease and low cost, ZN stain is used for diagnosis of tuberculosis in most developing countries where as culturing which has been considered as the golden standard method is not routinely used [1] [5] [6] [7] [8]. Major errors from ZN technique include false negativity and to a less degree false positivity [6]. In order to control the disease, it is important to use tests that increase the sensitivity of identification and help in the selection of treatment [9]. GeneXpert is a nucleic acid amplification technique that has been modified to specifically identify the species MTB and differentiate between the rpob resistance gene producers [4] [10] [11]. Globally, the test was endorsed by the WHO as the first choice for detection of drug-resistant tuberculosis [7] [12] [13], however, rpob resistance does not generalize to all types of resistance [14]. In Sudan TB considered as public health priority, through the national TB program, the Government has been striving to detect cases, give care to patients and involve other sectors in control efforts. GeneXpert assay has become an important part of tuberculosis diagnostic algorithms in many low- and middle-income countries [15] [16]. However, despite this effectiveness, smear for microscopy remains the primary diagnostic method for tuberculosis. In the context of negative tuberculosis detection, sensitivity and specificity of smear microscopy have been recorded to be 30% - 89% and 93% - 100% [17] [18], noted that, lower specificity has been observed in tuberculosis prevalence surveys. Cross positivity may be due presence of other acid fast bacilli like actinomycetes and nocardia [19]. In addition, laboratory errors such as analytic errors or sample mix-ups may lead to false positive smear microscopy results. Knowing that, smear microscopy enables to differentiate between Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) [20]. This study was conducted to assess ZN stain, ZN stain with concentrated sputum and geneXpert assay for detection of MBT.

2. Methods

2.1. Study Design and Setting

A cross-sectional laboratory based was followed in the period from September 2017 to September 2018 in Wad Madani Tuberculosis Center. Most admitted patients came from Gezira State which is the biggest and well populated State in Sudan.

2.2. Study Population

This study included all patients attending Wad Madani Tuberculosis Center during study period; suffer from chronic pulmonary symptoms and had tuberculosis suspected chest X-ray report. Patients under anti-tuberculous treatment, extra-pulmonary tuberculosis and known multi-drug resistance cases were excluded.

2.3. Sampling

Participants were constructed to collect early morning sputum into a clean, sterile, leak proof, wide mouth containers. Collected specimens were sent immediately to the Medical Laboratory of Wad Madani Tuberculosis Center, Ministry of Health, Gezira State.

Sample size was calculated by the following formula:

n = Z 2 P ( 1 P ) e 2

n = sample size;

Z = confidence interval (=1.96);

P = prevalence of TB infection (=0.19);

e = marginal error (=0.05);

n = 1.96 2 × 0.19 × ( 1 0.19 ) 0.05 2 = 300 patients

Bio-safety considerations were followed for sputum processing including wearing of personal protective equipments and sodium hydroxide (NaOH) decontamination. From each sample; two ml were taken for geneXpert analysis, then smear for ZN staining without concentration was prepared. The remnant of sputum subjected for smearing after concentration method.

2.4. Concentration of Sputum Samples

Suitable amount of sputum specimens were transferred into sterile centrifugal tubes with cotton stopper. Tubes were spun at 5000 round per minute (rpm) for 5 minutes. Then supernatant were discharged and sediments used for smear preparation.

2.5. Procedure of ZN Stain

Smears were fixed over the glass slide by heating. Carbol fuschin was poured over smear and heated gently until appearing of fumes. After standing for 5 minutes, water washing was one. De-staining was accomplished using 20% sulphuric acid. After water washing methylene blue was added for two minutes. Finally dried smears were examined under oil immersion lens. Degree of positivity was determined by 1 to 9 acid fast bacilli (AFB) per 100 high power field, 1 + (10 - 99 AFB/100 field), 2+ (1 - 10 AFB/ 50 field) and 3 + (more than 10 AFB/20 field) according to the WHO recommendations [21].

2.6. GeneXpert Assay Procedure

To achieve liquefaction and deactivation of specimens; using screw-capped tube two ml of sample reagent were added to one ml of sputum. After shaking for 20 times tubes were incubated at room temperature for 15 min as the follows; 10 minutes firstly then additional 5 minutes. Two ml of liquefied sputum samples were loaded into MTB\RIF cartridges and inserted into the geneXpert chamber. Programmed machine was adjusted to finish after 1 hour and 52 minutes. Lastly the results were read and interpreted according the load of bacilli and rifampicin resistance gene detection [22].

2.7. Ethical Consideration

This study was approved by Ministry of Health, Gezira State and Faulty of Medical Laboratory Sciences, University of Gezira, Sudan.

2.8. Statistics

Data were analyzed by Statistical Package for Social Program (SPSS, V21.0. IBM Chicago). Chi-square used as significance test for the association at level of 0.05. Sensitivity and specificity were calculated according to geneXpert as standard diagnostic method:

Sensitivity = True positive rue positive + False negative × 100

Specificity = True negative True negative + False positive × 100

3. Results

A total of 300 sputum specimens from pulmonary tuberculosis suspected subjects were included, most of participants came from Gezira State. Socio-demographic and clinical characteristics of enrolled patients shown in (Table 1). Positive ZN staining without concentration for acid fast bacilli obtained from 17% (51/300) while centrifugation technique revealed positivity of 20% (59/300). Degree of ZN stain positivity for staining without concentration shown in (Table 2) and with concentration in (Table 3). Sensitivity of 57.28% and 49.51% recorded for ZN stains with and without centrifugation respectively (Table 4). Mycobacterium tuberculosis was detected in 34% (103/300) by geneXpert assay. The relationship between geneXpert result levels and ZN stain with and without centrifugation expressed a significant association (Chi square 0.000) at most levels while the very low level result recorded in 9 by geneXpert and at the same time showed negative by ZN stains, as shown in Table 5. The rpob gene was documented with frequency of 9.7% (10/103) (data not shown).

Table 1. Socio-demographics and clinical characteristics of study subject (No 300).

Table 2. Results of sputum examination using ZN stain without centrifugation among tuberculosis suspected subject (N = 300).

Table 3. Results of sputum examination using ZN stain with centrifugation among tuberculosis suspected subject (N = 300).

Table 4. Sensitivity and specificity of ZN stains with and without centrifugation according to geneXpert among study subjects (No. 300).

Table 5. The association of the levels of geneXpert results with ZN stains (with and without centrifugation) (No. 300).

4. Discussion

Low level of income in Sudan, increase in poverty and insecurity especially in the places of boarder disputes led to an increase in the spread of diseases such as tuberculosis, in addition to the poor quality and continuation of health care services provided to citizens. Despite the endemicity of tuberculosis in Sudan, information like the actual prevalence, diagnostic data and monitoring of drug-resistance situations are limited. Early diagnosis of tuberculosis cases achieves cure and reduces the areas of spread of drug resistant cases and thus, enables control [23].

In view of results of participants whose were positive in the tests used in the present study, the small proportions (17%, 20% and 34%) that showed positive results necessitates a re-evaluation of the criteria for expectation of pulmonary tuberculosis. From the results obtained ZN stain without concentration revealed lowest sensitivity for detecting MTB from sputum; false negative results may occur due to sputum contamination with saliva diluents, personnel competence and or poor quality of equipment and reagents used. Thus, in the current study 49.5% (51/103) of geneXpert positive cases were negatively detected by ZN stain without concentration.

The findings revealed sensitivity and specificity of ZN stain without centrifugation of 49% and 100% for detecting MBT, near results were concluded in France [24] and Pakistan [25]. ZN stain with centrifugation gave better sensitivity when compared to that without, thus false negative results were 42.7% (44/103). By looking to the given time required to complete centrifugation method, which is approximately 15 minutes, and simplicity of this technique, negative results of ZN satin without concentration should be repeated using a concentration method especially in case of no geneXpert availability. In line, other study recommended concentration approaches for examine pre-bronchoscope sputum in order to enhance sensitivity [26]. Since ZN stain without concentration can detect MTB amount of 5000 - 10,000 CFU/ sputum ml [27], centrifugation method actually detect less than 5000 CFU/ml.

So far, the geneXpert assay has the best results for rabid pulmonary tuberculosis diagnosis but, beside possibility of contamination during sample collection and processing the high infrastructures requirements remain major factors that affect diagnosis by this method [21]. Approximately two thirds of tuberculosis suspected participants gave negative results for sputum test, which proves the hypothesis of re-examination and follow-up, moreover, those patients could be checked to exclude other causes of non-infectious and infectious chronic pulmonary diseases such as aspergillosis, actinomycosis and nocardiosis [28] [29] [30].

5. Conclusion

As conclusion, sputum concentration for suspected tuberculosis cases is strongly recommended to increase detection sensitivity when direct smears negatively interpret.

Acknowledgements

We would like to thank the patients for their consent to participate in this study, and we are grateful to those who are working in Wad Medsani Tuberculosis Center for their valuable support and our thanks extend to Mr. Ayman Hamid and his colleagues in the ART center in Wad Medani Teaching Hospital for their technical assistance.

Limitation of Study

The study did not include follow-up of patients who showed negative results for TB by the three used tests, such as taking additional investigations; culture and lung biopsy, as well as investigations for other chronic respiratory infections.

Cite this paper: Elfaham, Z. , Ibrahim, E. , Ahmed, E. , Mohammed, A. and Nour, B. (2021) Pulmonary Tuberculosis among Suspected Sudanese Patients in Wad Madani Tuberculosis Center. Open Journal of Medical Microbiology, 11, 298-307. doi: 10.4236/ojmm.2021.114019.
References

[1]   Palomino, J.C. (2005) Nonconventional and New Methods in the Diagnosis of Tuberculosis: Feasibility and Applicability in the Field. European Respiratory Journal, 26, 339-350. https://doi.org/10.1183/09031936.05.00050305

[2]   Banada, P.P. and Koshy, R. (2013) All and D. Detection of Mycobacterium tuberculosis in Blood by Use of the Xpert MTB/RIF Assay. Journal of Clinical Microbiology, 51, 2317-2322. https://doi.org/10.1128/JCM.00332-13

[3]   Dorman, S.E. (2010) New Diagnostic Test for Tuberculosis: Bench, Bedside and Beyond. Clinical Infectious Diseases, 50, S173-S177. https://doi.org/10.1086/651488

[4]   Piatek, S.A., van Cleeff, M., Alexander, H., Coggin, L.W., Rehr, M., van Kampen, S., et al. (2013) GeneXpert for TB Diagnosis: Planned and Purposeful Implementation. Global Health: Science and Practice, 1, 18-23.
https://doi.org/10.9745/GHSP-D-12-00004

[5]   Van Rie, A., Fitzgerald, D., Kabuya, G., Van Deun, A., Tabala, M., Jarret, N., et al. (2008) Sputum Smear Microscopy: Evaluation of Impact of Training, Microscopedistribution, and Use of External Quality Assessment. Guidelines for Resource Poor Settings. Journal of Clinical Microbiology, 46, 897-901.
https://doi.org/10.1128/JCM.01553-07

[6]   Nour, E.M.M., Saeed, E.M.A., Zaki, A.Z.S.A. and Saeed, E.N.S. (2011) Specificity of Sputum Smear Compare to Culture Diagnosis of Pulmonary Tuberculosis. World Journal of Medical Sciences, 6, 121-125.

[7]   Lawn, D.S., Nicol, P.M. (2011) Xpert MTB/RIF Assay: Development, Evaluation and Implementation of a New Rapid Molecular Diagnostic for Tuberculosis Andrifampicin Resistance. Future Microbiology, 6, 1067-1082.
https://doi.org/10.2217/fmb.11.84

[8]   Walusimbi, S., Bwabga, F., De Costa, A., Haile, M., Joba, M. and Hoffner S. (2013) Meta Analysisto Compare the Accuracy of GeneXpert, MODS and the WHO 2007 Algorithm for the Diagnosis of Smear Negative Pulmonary Tuberculosis. BMC Infectious Diseases, 13, Article No. 507. https://doi.org/10.1186/1471-2334-13-507

[9]   Uddin, M.N., Chowd hury, M.R., Ahmed, S., Rahman, M.T., Khatum, R., van Leth, F.R., et al. (2013) Comparison of Direct versus Concentrated Smear Microscopy in Detection of Pulmonary Tuberculosis. BMC Research Notes, 6, Article No. 291.
https://doi.org/10.1186/1756-0500-6-291

[10]   Weyer, K., Mirzayev, F., Migliori, G.B., Van Gemert, W., D’Ambrosio, L., Zignol, M., et al. (2013) Rapid Molecular TB Diagnosis: Evidence, Policy Making and Global Implementation of Xpert MTB/RIF. European Respiratory Journal, 42, 252-271.
https://doi.org/10.1183/09031936.00157212

[11]   Zeka, A.N., Tasbakan, S. and Cavusoglu, C. (2011) Evaluation of the GeneXpert MTB/RIF Pulmonary and Extrapulmonary Specimens and Detection of Rifampinresistance in Assay for Rapid Diagnosis of Tuberculosis. Journal of Clinical Microbiology, 49, 4138-4141. https://doi.org/10.1128/JCM.05434-11

[12]   Raj, A., Singh, N. and Mehta, P.K. (2012) Gene Xpert MTB/RIF Assay: A New Hope for Extrapulmonary Tuberculosis. IOSR Journal of Pharmacy, 2, 83-89.

[13]   Abed Al-Darraji, H.A., AbdRazak, H.A., Ng, K.P., Altic, F.I. and Kamarulzaman, A. (2013) Thediagnostic performance of a Single GeneXpert MTB/RIF Assay in Anintensified Tuberculosis Case Finding Survey among HIV-Infected Prisonersin Malaysia. PLoS ONE, 8, Article No. e73717.
https://doi.org/10.1371/journal.pone.0073717

[14]   World Health Organization (2009) TB Diagnostics and Laboratory Services.
http://www.who.int/tb/dots/lab.pdf

[15]   Sikhondze, W., Dlamini, T., Khumalo, D., Maphalala, G., Dlamini, S., Zikalala, T., et al. (2015) Countrywide Roll-Out of Xpert5 MTB/RIF in Swaziland: The First Three Years of Implementation. Public Health Action, 5, 140-146.
https://doi.org/10.5588/pha.15.0001

[16]   Van Kampen, S.C., Susanto, N.H., Simon, S., Astiti, S.D., Chandra, R., Burhan, E., et al. (2015) Effects of Introducing xpert MTB/RIF on Diagnosis and Treatment of Drug-Resistant Tuberculosis Patients in Indonesia: A Prepos Intervention Study. PLoS ONE, 10, Article ID e0123536. https://doi.org/10.1371/journal.pone.0123536

[17]   World Health Organization (2009) Approaches to Improve Sputum Smear Microscopy for Tuberculosis Diagnosis Expert Group Meeting. World Health Organization, Geneva.

[18]   Cattamanchi, A., Davis, J.L., Pai, M., Huang, L., Hopewell, P.C. and Steingart, K.R. (2010) Does Bleach Processing Increase the Accuracy of Sputum Smear Microscopy for Diagnosing Pulmonary Tuberculosis? Journal of Clinical Microbiology, 48, 2433-2439. https://doi.org/10.1128/JCM.00208-10

[19]   van’tHoog, A.H., Meme, H.K., Laserson, K.F., Agaya, J.A., Muchiri, B.G., Githui, W.A., et al. (2012) Screening Strategies for Tuberculosis Prevalence Surveys: The Value of Chest Radiography and Symptoms. PLoS ONE, 7, Article ID: e38691.
https://doi.org/10.1371/journal.pone.0038691

[20]   World Health Organization (2003) Quality Assurance of Sputum Microscopy in DOTS Programmes: Regional Guidelines for Countries in the Western Pacific. Manila, Philippines. World Health Organization, Geneva.

[21]   Sudan, Federal Ministry of Health, Directorate of Communicable and Non Communicable Diseases (2018) Sudan National TB Management Guideline.
https://www.humanitarianresponse.info

[22]   Hakeem, A., Shahbaz Hussain, M. and Imran, M. (2013) Sarwar Gene Xpert MTB/RIF—A Novel Diagnostic Tool for Tuberculosis in Pulmonary Samples. IOSR Journal of Dental and Medical Sciences, 8, 1-3.

[23]   Mostaza, J.L., Garcia, N., Fernandez, S., Bahamonde, A., Fuentes, M.I. and Palomo, M.J. (2007) Analysis and Predictor of Delay in Suspicion and Treatment among Hospitalized Patients with Pulmonary Tuberculosis. Anales de medicina interna, 24, 478-483.

[24]   Pierre Le, Palud, P., Cattoir, V., Malbruny, B., Magnier, R., Campbell, K., Oulkhouir, Y., et al. (2014) Retrospective Observational Study of Diagnostic Accuracy of the Xpert? MTB/RIF Assay on Fiberoptic Bronchoscopy Sampling for Early Diagnosis of Smear-Negative or Sputum-Scarce Patients with Suspected Tuberculosis. BMC Pulmonary Medicine, 14, Article No. 137.
https://doi.org/10.1186/1471-2466-14-137

[25]   Kanwal, K. and Butt, T. (2015) Diagnostic Yield of Bronchoalveolar Lavage Gene Xpert in Smear-Negative and Sputum-Scarce Pulmonary Tuberculosis. Journal of the College of Physicians and Surgeons Pakistan, 25, 115-118.

[26]   Aderaye, G.G., Egziabher, H., Aseffa, A., Worku, A. and Lindquist, L. (2007) Comparison of Acid-Fast Stain and Culture for Mycobacterium Tuberculosis in Pre and Post-Bronchoscopy Sputum and Bronchoalveolar Lavage in HIV-Infected Patients with Atypical Chest X-Ray in Ethiopia. Annuals of Thoracic Medicine, 2, 154-157.
https://doi.org/10.4103/1817-1737.36549

[27]   Parsons, M.L., Somoskovi, A., Gutierrez, C., Evan, L., Paramasivan, C.N., et al. (2011) Laboratory Diagnosis of Tuberculosis in Resource-Poor Countries. Challenges and Opportunities. Clinical Microbiology Reviews, 24, 314-350.
https://doi.org/10.1128/CMR.00059-10

[28]   Naaraayan, A., Kavian, R., Lederman, J., Basak, P. and Jesmajian, S. (2015) Invasive Pulmonary Aspergillosis—Case Report and Review of Literature. Journal of Community Hospital Internal Medicine Perspectives, 5, Article ID: 26322.
https://doi.org/10.3402/jchimp.v5.26322

[29]   Han, J.Y., Lee, K.N., Lee, J.K., Kim, Y.H., Choi, S.J., Jeong, Y.J., et al. (2013) An Overview of Thoracic Actinomycosis: CT Features. Insights into Imaging, 4, 245-252.
https://doi.org/10.1007/s13244-012-0205-9

[30]   Chen, J., Zhou, H., Xu, P., Zhang, P., Ma, S. and Zhou, J. (2014) Clinical and Radiographic Characteristics of Pulmonary Nocardiosis: Clues to Earlier Diagnosis. PLoS ONE, 9, Article ID: e90724. https://doi.org/10.1371/journal.pone.0090724

 
 
Top