Background

Tuberculosis is a major communicable disease that can be transmitted though close contact with infected patient (Etim and Briyai, 2017; 2018). Inhalation of droplet nuclei aerosolized (Okorie et al., 2016; Jaleta et al., 2017) through coughing, sneezing and spitting of an infected patient is the major spread measure (Etim and Briyai, 2017). Tuberculosis is caused by a bacteria agent called Mycobacterium tuberculosis (Okorie et al., 2016; Etim and Briyai, 2017; 2018). Tuberculosis is a major cause of morbidity and mortality globally (Raviglione et al., 2012; Zhao et al., 2012; Range et al., 2012; Desikan, 2013; Bhuju et al., 2013; Nasiri et al., 2014; WHO, 2017; Etim and Briyai, 2017, 2018). WHO (2017) reported that tuberculosis is among the 10^th leading cause of death in the world. Desikan (2013) reported that about 2 billion people are infected with tuberculosis causing about 2 million deaths per year. The author further reported about 8.8 million incident cases of tuberculosis occurred in 2010, which is equivalent to about 178 cases per 100,000 populations. Furthermore, WHO (2017) reported that 10.4 million contacted tuberculosis in 2015 causing about 1.8 million death. This further shows that global prevalence of tuberculosis appears to be on the increase (Etim and Briyai, 2018).

Tuberculosis pandemic is intense in low and middle income countries. According to WHO (2017), Etim and Briyai (2017), about 60% of global tuberculosis cases occur in Nigeria, China, South Africa, India, Pakistan and Indonesia. Generally tuberculosis affects all age grade (children, adolescent and adult) irrespective of sex (Etim and Briyai, 2017). Tuberculosis are known to occur with other diseases such as HIV (Anochei et al., 2013; Oluwaseun et al., 2013; Abiodun et al., 2015; Okorie et al., 2016; WHO, 2017; Etim and Briyai, 2018). Cases of multi drug resistance tuberculosis have been severally reported in literature (Traore et al., 2000; Nwachukwu and Peter, 2010; Bazira et al., 2011; Dinic et al., 2012; Aliyu et al., 2013; Ryu, 2015; Sani et al., 2015; Abiodun et al., 2015; Okorie et al., 2016; Etim and Briyai, 2017; 2018), and it has been on the increasing trend (Sharma et al., 2011; Range et al., 2012).

Several methods have been adopted for the diagnosis of tuberculosis. Sputum smear-microscopy is one of the widely used approaches especially in developing countries (Cattamanchi et al., 2010; Desikan, 2013; Rye, 2015). This typically involves smearing sputum on a slide, stained appropriately and viewed under high power microscopy to detect the causative acid-fast bacillus M, tuberculosis (Chew et al., 2011). Fluorescence microscopy, Nucleic acid amplification tests are other methods of tuberculosis diagnosis (Desikan, 2013). Gene xpert machine have also been adopted for rapid diagnosis of tuberculosis in many hospitals worldwide. Possible infection of laboratory technicians in poorly equipped laboratory is a major problem associated with smear microscopy method of tuberculosis diagnosis (Nyirenda et al., 1998; Chew et al., 2011). Joshi et al. (2006) reported a higher incidence of tuberculosis disease risk among laboratory technicians compared to the general public. Several methods are used for sterilization during diagnosis of Mycobacterium tuberculosis.

During diagnosis of tuberculosis, chemicals are used in many middle- and high-income countries to enhance the diagnostic sensitivity (Cattamanchi et al., 2010). Sodium hypochlorite popularly called bleach is ideal processing chemical agent widely used in many low-income nations (Cattamanchi et al., 2010; Chew et al., 2011). It’s used as disinfectant. Best et al. (1990), Cattamanchi et al. (2010) reported that bleach may be useful in infection control in laboratories lacking biosafety facilities. Authors have reported that it could be used for the diagnosis of Mycobacterium tuberculosis (Bonnet et al., 2008; Ongkhammy et al., 2009; Cattamanchi et al., 2010; Chew et al., 2011). Bonnet et al. (2008), Cattamanchi et al. (2010), Van Deun et al. (2000) reported that it could improve microscopy field clarity through digestion of mucus and debris, and concentrating bacilli through centrifugation or sedimentation during diagnostic sensitivity of sputum. This study aimed at assessing the effect of bleach on the diagnosis of Mycobacterium tuberculosis.

1 Materials and Methods

1.1 Study area

This study was carried out in Central Hospital Benin City, Edo State. Edo state lies between longitude 06^o04^’E and 06^o43^’E and latitude 05^o44^’N and 07^o34^’N with a land mass of 17, 450 sq.km located. Edo state is one of the Niger Delta state with a population of over 3.1 million. The study area has personnel’s in civil services probably due to the presence of higher institutions and other government agencies. Farming is a major occupation of indigenes of the area. Petty trading is also a source of livelihood to several families in the area.

1.2 Participants and selection criteria

Participants were drawn from Direct Observation Therapy (DOT) centre in Central Hospital Benin City. Informed consent(s) of the participant(s) from the DOT clinics of the hospital were obtained. The age of the participants was > 15 years. The inclusion criteria include individuals that have coughed for more than two weeks, weight loss, night sweat, swelling at the neck, hand or armpit and fever. While individuals with diabetes and known cases of cardiovascular diseases were excluded.

1.3 Sample collection

Sixty sputum (60) samples were collected from DOT centre between March and July, 2014. Triplicate sputum samples were collected over two consecutive days for the new case patients, while for the follow up cases, patients submitted two sputum samples. The initial sputum sample was collected in the laboratory at first visit to the clinic while the second sputum samples were in the morning before mouth cleaning at the participant’s home, and the last samples were collected at the laboratory. The participants were educated on the method to produce quality sputum and method of collection especially at the second sputum collection that was done at home. Samples were collected in in well-ventilated area.

1.4 Sample preparation and analysis

The smear method previously described by Chew et al. (2011) was employed in this study. Approximately 40 µl of the sputa were used to make smears for unprocessed or bleached samples and was applied over a single area of about 1 to 2 cm. The sputum slides were allowed to air-dried. They were then heat-fixed and subsequently stained with Ziehl-Neelsen techniques. Each of the sputum smear was flooded with 0.3% strong carbon fuchsin, and it was heated and allowed to stand for 10 minutes before being dewashed with distilled water. Thereafter, an acid-alcohol was added for 2 minutes, before being washed off with water and counter-stained with methylene blue, and then rinsed with water and allowed to dry (Kent and Kubica, 1985).

Furthermore, remaining sputum samples which are approximately 1 to 2 ml were placed in a 15 ml tube with screw cap containing the same quantity of undiluted household bleach. The mixture of sputum and bleach was incubated at room temperature undisturbed for half an hour. Then after, a drop of the bleach digested sputum was transferred to a slide. Again, the bleach smear of about 1 to 2 cm was made, air-dried, heat-fixed and stained with Ziehl-Neelsen techniques. Both smears (bleached and unbleached) were viewed under light microscope using oil immersion lens. The resultant characteristics (positive and negative smears) were defined according to the National Tuberculosis and Leprosy Control Program (NTBLCP) Acid Fast Bacilli (AFB) grading (1978).

2 Results and Discussion

Table 1 presents the effect of bleach on diagnosis of Mycobacterium tuberculosis using smear microscopy method. Sixty (60) sputum samples were analyzed and results revealed that 32 (53.3%) were positive for acid-fast bacilli using direct microscopy techniques. The sputum that was bleached had same positive results i.e. 32 (53.3%). The results clearly showed that all sputum smears that were positive to AFB under direct microscopy techniques were also positive when bleach was applied in the microscopy method. Furthermore, a decrease in the mean number of AFB observed in a given field when viewed under the microscope. 7 (21.9%) smears graded as + by direct microscopy appeared scanty and 10 (31.3%) graded as ++ decreased to +. Also, 15 (46.9%) smears graded as +++ decreased to ++.

The study validates the findings of previous authors indicating that bleach improves diagnostic smear microcopy method of tuberculosis (Cattamanchi et al., 2010; Van Deun et al., 2010; Chew et al., 2011). In addition, due to the disinfectant potentials of bleach, it could be useful in control infection in laboratories lacking biosafety facilities. Furthermore, bleach can easily disintegrate the bacilli if allowed to act for 60 minutes (Wah et al., 2001; Yassin et al., 2003; James et al., 2014). Based on this, bleach method of Mycobacterium tuberculosis diagnosis is meant for only microscopy and not sputum intended for culture (James et al., 2014). The bleach smear microscopy showed a reducing potential on Mycobacterium tuberculosis (James et al., 2014). The bleach method is characterized by high density of AFB in the fields, and decline in debris present in the sample thereby allowing a clear field for bacteria detection (Wah et al., 2001; James et al., 2001). This lead to reduction in the time needed for microscopy to be carried out (James et al., 2014).

3 Conclusions

This study assessed the effect of bleach on the diagnosis of Mycobacterium tuberculosis in a hospital. The results revealed that the use of bleach smear microscopy for the diagnosis of Mycobacterium tuberculosis is practicable and could be useful essentially in hospital setting that direct microscopy is not carried out routinely. Therefore, we recommend that further studies should be carried out to determine other means of harnessing bleach for the proper diagnosis of tuberculosis.

Authors’ contributions

The manuscript was carried out by all the authors. Author EJ Uhunmwangho conceived the idea and carried out the laboratory analysis and Authors AO Iyamu, BO Eledo and SC Izah wrote the initial draft. Author SC Izah managed correspondence. All authors read and approved the final manuscript.

Reference

Abiodun I.S., Olanrewaju A.I., Ladipo O.A., and Oluberu A.O., 2015, Incidence of HIV and pulmonary tuberculosis co-infection among patients attending out-patient clinic in a Nigerian hospital, International Journal of Biomedical Research, 6(09): 669-673

https://doi.org/10.7439/ijbr.v6i9.2465

Aliyu G., El-Kamary S.S., Abimiku A., Ezati N., Mosunmola I., Hungerford L., Brown C., Tracy K.J., Obasanya J., and Blattner W., 2013, Mycobacterial etiology of pulmonary tuberculosis and association with HIV infection and multidrug resistance in Northern Nigeria, Tuber Res Trea. 2013: 1-9

https://doi.org/10.1155/2013/650561

PMid:23970967 PMCid:PMC3730141

Anochie P.I., Onyeneke E.C., Onyeneke C.N., Ogu A.C., Onyeozirila A.C., 2013, Tuberculosis and Human Immunodeficiency Virus Co-Infection in Rural Eastern Nigeria, J Med Diagn Meth 2: 118

http://doi:10.4172/2168-9784.1000118

Best M., Sattar S.A., Springthorpe V.S., and Kennedy M.E., 1990, Efficacies of selected disinfectants against Mycobacterium tuberculosis. Journal of Clinical Microbiology, 28(10), 2234-2239

PMid:2121783 PMCid:PMC268154

Bhuju S., Fonseca S.D.S., Marsico A.G., Vieira G.D.B.O., Sobral L.S., Stehr M., Singh M., and Saad M.H.F., 2013, Mycobacterium tuberculosis isolates from Rio de Janeiro reveal unusually low correlation between pyrazinamide resistance and mutations in the pncA gene. Infection, Genetics and Evolution 19, 1-6

https://doi.org/10.1016/j.meegid.2013.06.008

PMid:23770140

Bonnet M., Ramsay A., Githui W., Gagnidze L., Varaine F., and Guerin P.J., 2008, Bleach sedimentation: an opportunity to optimize smear microscopy for tuberculosis diagnosis in settings of high prevalence of HIV, Clinical Infectious Diseases, 46(11): 1710-1716

https://doi.org/10.1086/587891

PMid:18444789

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? J Clin Microbiol., 48: 2433-2439

https://doi.org/10.1128/JCM.00208-10

PMid:20421442 PMCid:PMC2897477

Chew R., Calderon C., Schumacher S.G., Sherman J.M., Caviedes L., Fuentes P., Coronel J., Valencia T., Hererra B., Zimic M., Huaroto L., Sabogal I., Escombe A.R., Gilman R.H., and Evans C.A., 2011, Evaluation of bleach-sedimentation for sterilising and concentrating Mycobacterium tuberculosis in sputum specimens, BMC Infectious Diseases, 11: 269

https://doi.org/10.1186/1471-2334-11-269

PMid:21985457 PMCid:PMC3213181

Desikan P., 2013, Sputum smear microscopy in tuberculosis: Is it still relevant? The Indian Journal of Medical Research, 137(3), 442-444

Dinic L., Akande P., Idigbe E.O., Ani A., Onwujekwe D., Agbaji O., Akanbi M., Nwosu R., Adeniyi B., Wahab M., Lekuk C., Kunle-Ope C., Nwokoye N., and Kankia P., 2012, Genetic determinants of drug-resistant tuberculosis among HIV infected patients in Nigeria, J Clin Microbiol., 50(9): 2905-2909

https://doi.org/10.1128/JCM.00982-12

PMid:22740709 PMCid:PMC3421781

Etim N.G., and Briyai F.O., 2017, Prevalence of Pulmonary and Rifampicin-resistant tuberculosis among patients attending Federal Medical Centre, Yenagoa, Bayelsa state, Nigeria, International Journal of Healthcare and Medical Sciences, 3(11): 85-92

Etim N.G., and Briyai F.O., 2018, Pulmonary and Rifampicin-Resistant Tuberculosis Associated with Human Immunodeficiency Virus Infection Prevalence in Bayelsa State, Nigeria: A Case of Patients Attending Federal Medical Centre Yenagoa, American Journal of Laboratory Medicine, 3(1): 20-24

International Union against Tuberculosis and Lung Disease, Technical guide for sputum examination for tuberculosis by direct smear microscopy, 3rd ed. Paris: IUATLD, 1978

Jaleta K.N., Gizachew M., Gelaw B., Tesfa H., Getaneh A., and Biadgo B., 2017, Rifampicin-resistant Mycobacterium tuberculosis among tuberculosis-presumptive cases at University of Gondar Hospital, northwest Ethiopia Infection and Drug Resistance, 10: 185-192

https://doi.org/10.2147/IDR.S135935

PMid:28652786 PMCid:PMC5476602

James A., Abba S.U., Ibrahim A., Mbah H., Musuluma H., Ochei K., Darboe S., and Torpey K., 2014, Improving the case detection of pulmonary tuberculosis by bleach microscopy method in the North West of Nigeria. Journal of Medical Laboratory and Diagnosis, 4(3): 34-37

https://doi.org/10.5897/JMLD2013-0066

Joshi R., Reingold A.L., Menzies D., and Pai M., 2006, Tuberculosis among health-care workers in low- and middle-income countries: a systematic review, PLoS Med., 3: e494

https://doi.org/10.1371/journal.pmed.0030494

Kent P.T., and Kubica G.P., 1985, Public health mycobacteriology: A guide for the level III laboratory, Atlanta: Centers for Disease Control

Nasiri M.J., Rezaei F., Zamani S., Darban-Sarokhalil D., Fooladi A.A.I., Shojaei H., and Feizabadi M.M., 2014, Drug resistance pattern of Mycobacterium tuberculosis isolates from patients of five provinces of Iran, Asian Pacific Journal of Tropical Medicine, No volume, 193-196

Nwachukwu E., and Peter G.A., 2010, Prevalence of Mycobacteria tuberculosis and human immunodeficiency virus (HIV) infections in Umuahia, Abia state, Nigeria, African Journal Microbiology Research, 4(14): 1486-1490

Okorie O., John A., Gidado M., Akang G., Emperor U., Rupert E., Vivian I, Meribole E., and Osakwe P., 2016, The Prevalence of Drug-Resistant Tuberculosis among People Living with HIV (PLHIV) in Abia State, Advances in Infectious Diseases, 06(2)

https://doi.org/10.4236/aid.2016.62009

Oluwaseun E., Akinduti P., and Oluwadum A., 2013, Primary multidrug resistant tuberculosis among HIV seropositive and HIV seronegative patients in Abeokuta, South Western Nigeria, Journal of Research Communication, 1(10): 224-237

Ongkhammy S., Amstutz V., Barennes H., and Buisson Y., 2009, The bleach method improves the detection of pulmonary tuberculosis in Laos, The International Journal of Tuberculosis and Lung Disease, 13(9): 1124-1129

Range N., Friis H., Mfaume S., Magnussen P., Changalucha J, Kilale A., Mugomela A., and Andersen A.B., 2012, Anti-tuberculosis drug resistance pattern among pulmonary tuberculosis patients with or without HIV infection in Mwanza, Tanzania, Tanzania Journal of Health Research, 14(4): 1-9

Raviglione M., Marais B., and Floyd K., 2012, Scaling up interventions to achieve global tuberculosis control: progress and new developments, Lancet, 379: 1902-1913

https://doi.org/10.1016/S0140-6736(12)60727-2

Ryu Y.J., 2015, Diagnosis of Pulmonary Tuberculosis: Recent Advances and Diagnostic Algorithms, Tuberculosis and Respiratory Diseases, 78(2): 64-71

https://doi.org/10.4046/trd.2015.78.2.64

PMid:25861338 PMCid:PMC4388902

Sani R.A., Garba S.A., Oyeleke S.B., and Abalaka M.E., 2015, Prevalence of Pulmonary Tuberculosis (PTB) in Minna and Suleja Niger State, Nigeria, American Journal of Medicine and Medical Sciences, Vol. 5 No. 6, 2015, pp.287-291

Sharma S.K., Kaushik G., Jha B., George N., Arora S.K., Gupta D., Singh U., Hanif M., and Vashisht R.P., 2011, Prevalence of multidrug-resistant tuberculosis among newly diagnosed cases of sputum-positive pulmonary tuberculosis. Indian J Med Res 133: 308-311

PMid:21441685 PMCid:PMC3103156

Traore H., Fissette K., and Bastian I., 2000, Detection of rifampicin resistance in Mycobacterium tuberculosis isolates from diverse countries by commercial line probe assay as an initial indicator of multidrug resistance, International Journal of Tuberculosis and Lung Disease, 4: 481-484

PMid:10815743

Van Deun A., Maug A.K., Cooreman E., Hossain M.A., Chambuganj N., Rema V., Marandi H., Kawria A., and Portaels F., 2000, Bleach-sedimentation method for increased sensitivity of sputum smear microscopy: does it work? Int J Tuberc Lung Dis, 4: 371-376

PMid:10777088

Wah W.A., Mar M.N., Ti T., and Win M., 2001, Improved method of direct microscopy for detection of acid-fast bacilli in sputum, Southeast Asian J. Trop. Med. Public Health 32(2): 390-393

WHO, 2017, Tuberculosis, Fact sheet, http://www.who.int/mediacentre/factsheets/fs104/en/, August 16^th, 2017

Yassin M.A., Cuevas L.E., Gebrexabher H., and Squire S.B., 2003, Efficacy and safety of short term bleach digestion of sputum in case finding for pulmonary tuberculosis in Ethiopia. Int. J. Tuberc, Lung. Dis. 7:678-683

PMid:12870690

Zhao Y., Xu S., and Wang L., 2012, National survey of drug-resistant tuberculosis in China, New England Journal of Medicine, 366: 2161-2170

https://doi.org/10.1056/NEJMoa1108789

PMid:22670902