Molecular Detection of Buruli ulcer among Patients with Various Degrees of Skin Ulcerative Lesions Revealed Zero Prevalence in Parts of Jigawa State, Northern Nigeria

Molecular Detection of Buruli ulcer among Patients with Various Degrees of Skin Ulcerative Lesions Revealed Zero Prevalence in Parts of Jigawa State, Northern Nigeria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular Detection of Buruli ulcer among Patients with Various Degrees of Skin Ulcerative Lesions Revealed Zero Prevalence in Parts of Jigawa State, Northern Nigeria Yusuf Adeiza Kashim, Mustatpha Musa Dogara, Bashir Sajo Mienda, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4555246/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract The indolent, necrotizing Buruli ulcer disease is a chronic condition caused by Mycobacterium ulcerans , an environmental bacteria that can damage the skin, tissues, and bones. It is now recognized as one of the 20 Neglected Tropical Diseases targeted for elimination by the year 2030. Public health continues to face difficulties due to its mechanism of transmission that is yet to be completely unravel. From December 2019 to September 2023, a hospital-based study was conducted to ascertain the prevalence of Buruli ulcer in parts of Jigawa State, Nigeria. A purposive sampling technique was adopted in selecting and diagnosing 382 samples from patients with various degrees of skin ulcerative lesions across all age groups and gender. The diagnosis of all the samples initially involved the use of Ziehel Nelseen staining procedure. Thirty-five (35) samples with symptoms suspected to be Buruli ulcer were diagnosed using Real-time Quantitative Polymerase Chain Reaction (qPCR) method. The results were negative for the 382 samples diagnosed using Ziehel Nelseen staining procedure as well as the 35 using real-Time PCR. This study concludes that despite testing samples with obvious ulcerative lesions suspected to be Buruli ulcer from patients, both Ziehel Nelseen staining procedure and Real-Time PCR revealed negative results. It is recommended that more samples suspected to have symptoms of Buruli ulcers should be diagnosed using Real-Time PCR and also make use of both IS2404 and IS2606. Public Health Buruli ulcer Mycobacterium ulcerans Neglected Tropical Disease Figures Figure 1 Figure 2 INTRODUCTION The bacteria Mycobacterium ulcerans is the aetiologic agent that causes Buruli ulcer (BU), a chronic, indolent, necrotizing cutaneous illness that is categorized by the World Health Organization (WHO) as one of the skin-related Neglected Tropical Diseases (NTDS) (Mitja et al. , 2017). After leprosy and tuberculosis, it is the most prevalent mycobacterial illness (Keragala et al., 2020 ). Large ulcers, typically on the limbs, are the result of the infection's devastation of soft tissue and skin (Peetermans et al., 2020 ). Clinically speaking, a BU begins as a papule, nodule, plaque, or oedematous lesion that gradually spreads to a large area of skin ulceration (Yotsu et al., 2018 ). Surprisingly, considering the degree of tissue loss, the lesion typically causes little to no pain. Buruli ulcer is distinct from other mycobacterial infections in that the pathophysiology of the illness can be attributed to mycolactone, a lipid-like and diffusible exotoxin, rather than the bacterium (Yotsu et al., 2018 ). In the endemic areas, Buruli ulcer is highly feared and stigmatized due to the unsightly abnormalities it leaves behind. It is also often linked to witchcraft and curses (Osei & Duker, 2015 ). At least 27 nations in Africa, Asia, South America, and the western Pacific region have patchy foci of Buruli ulcer. Most of the cases that have been recorded are from West and Central Africa (Singh, 2019 ). Earlier research had shown that Buruli ulcer is endemic in Nigeria, particularly among those who live close to bodies of water. However, most investigations were all conducted in Nigeria's southern regions. There is no documented study on Buruli ulcer in Jigawa State, Northern Nigeria, particularly among the people that live near water features. It is against this background that this study was conducted to determine the prevalence of Buruli ulcer in parts of Jigawa State. MATERIALS AND METHODS Study Area Jigawa State was created on 28th August, 1991from the old Kano State. The state is situated between latitudes 11.00 o N and longitudes 8.00 o E to 10.15 o E, the state lies in the northwest part of Nigeria. It is bordered on the west by Katsina and Kano States, on the east by Yobe and Bauchi States, and on the north by the international boundary with the Niger Republic. As shown in Figure, 1 the state is divided into three senatorial Districts namely: Northwest, Central and Northeast. The study was conducted in secondary health facilities in Northwest and Central Senatorial districts. The state has two distinct seasons: the dry season, which runs from October to May, and the rainy season, which runs from June to September. The hot temperature during the rainy season is approximately 42°C, while the low temperature is 10°C (Okereke et al. , 2015). The state ranks eighth in terms of population according to the 2006 census. The state has three types of health facilities: the primary health care which is under the purview of the local government, secondary health care is overseen by the state government (the state has sixteen general hospitals and one specialty hospital), and tertiary health care is under the purview of the federal government and there is only one Federal Medical Center (Dogara & Ocheje, 2016 ; Makinde et al., 2018 ). State-level health policy development and execution, as well as translation of federal health policies for implementation, fall under the purview of the Ministry of Health and the Agency for Primary Health Care. A primary health center is intended to be available in every ward of the state, as the state currently functions under a single roof system (Makinde et al., 2018 ). Patients identified with various degrees of ulcerative lesions are admitted into general hospitals for care. Six secondary healthcare facilities were selected, they include General Hospital Dutse, General Hospital Birnin Kudu, General Hospital Jahun, General Hospital Ringim, General Hospital Gumel and General Hospital Kazaure, as shown in Fig. 1 . Study Design A cross-sectional hospital-based study was conducted. Two out of the three senatorial districts were selected for the study. The samples were collected from selected secondary healthcare facilities within the two senatorial districts of Northwest and Central. Sample Size Determination The study adopted a purposive or judgmental sampling technique in the selection of the patients whose samples were collected from the study population. A total of three hundred and eighty-two (382) skin related ulcer patients were screened. Specifically, thirty-five (35) samples were collected from patients who presented symptoms mimicking Buruli ulcer for the molecular analyses. Samples were collected from the patients between December 2019 and September 2023. SCREENING OF SAMPLES FOR THE DETECTION OF MYCOBACTERIUM ULCERANS Microscopic examination Microscopic diagnoses by direct smear examination with Ziehl-Neelsen staining to detect the presence of acid-fast bacilli (AFB) was done using the quantification of smears by the method locally used for the diagnosis of tuberculosis (Portaels, 2001). The positive control sample was collected from the tuberculosis (Tb) positive stock at the Tb Unit of Dutse General Hospital, Jigawa State. Self-sputum was collected and smeared, stained, and subsequently used as the Negative Control. Using a sterile swab stick and fine needle aspirate (FNA), ulcerative lesions, papules and nodules were collected respectively from suspected patients and then smeared on a clean grease free slide using sterile technique. The smear was air dried, and heat fixed by passing through an open flame. The smear was covered with the carbol fuchsin stain. The stain was heated until vapour rose for 5 minutes. The smear was washed with clean water and 3% v/v acid alcohol was applied to the smear for 5 minutes to decolorize the smear to pale pink colour. The smear was rinsed with clean water. Malachite green stain was applied to the smear and allowed to stain for 2 minutes. The stain was rinsed with clean water. Back of the slide was wiped to clean and then placed in the rack for the smear to air-dry. The smear was examined microscopically, using the ×100 oil immersion objective. Molecular analyses The analyses were performed at the Molecular Parasitology Research Laboratory of the Nigerian Institute of Medical Research (NIMR), Yaba Lagos State, Nigeria, a reference laboratory for Buruli ulcer diagnosis and research. Sample collection and processing Thirty-three (33) swab and two (2) Fine Needle Aspiration (FNA) samples were aseptically collected into collection tubes. Each tube was labelled for ease of identification. Subsequently each swab sample collected from lesion on the patients’ limbs (upper or lower) was transferred into 15 ml conical tube, was rehydrated with 2 ml of sterile water and vortexed. This was left to stand for 5 minutes and re-vortexed to increase the concentration of the sample. The swabs were gently discarded from the 15 ml tube leaving the content. Thereafter, 400 µl content obtained from above was measured and transferred into a 1.5ml tube with screw cap for DNA extraction and purification. Regarding the FNA samples, they were vortexed and a 400 µl each was taken and transferred into a 1.5 ml tube with screw cap for DNA extraction. DNA extraction and purification procedure DNA extraction was performed using the Genolysis kit (Hains Lifescience, Nehren, Germany). A 400 µl of specimen suspension was placed into 1.5 ml tubes with screw cap for each sample, Thereafter, tubes were centrifuged at 12000 g for 15 minutes at room temperature. The supernatants were carefully discarded using a P1000 filter tip leaving the pellets at the bottom of the conical tubes. The pellets were re-suspended in 400 µl of distilled water and centrifuged at 12000 g for 15 minutes at room temperature. The supernatant was carefully discarded, and the pellets were re-suspended in 50 µl of A-LYS buffer and incubated for 10 minutes at 95°C. The tubes were centrifuged for 10 seconds to pellet the suspension. The above, 50 µl of buffer A-NB was added and the tubes were kept at 4°C prior to PCR/amplification procedure. Real-time quantitative PCR (RT qPCR) RT qPCR was performed as described by Fyfe & Lavender ( 2022 ). The method is recommended by the WHO for M. ulcerans detection (Bretzel & Beissner, 2018 ) and is based on primers IS2404TF and IS2404TR. The primers set employed for quantification is depicted in Table 1 . Quantitative PCR (qPCR)/amplification condition/procedure The qPCR/amplification reaction was performed in a total volume of 20 µl reaction in Bio-Rad thermal cycler machine (Bio-Rad Laboratories, Marnes- la- Coquette, France). 5 µl of 5x Hot fire Pol probe qPCR mix (Master mix) was added to 11.5 µl water (PPI), followed by 1.5 µl of forward primer and 1.5 µl of reverse primer including 0.5 µl template DNA. The qPCR amplification procedure is presented as follows: The sample was heated at initial denaturation temperature of 95°C for 10 minutes at 1 cycle, followed by final heating at 95°C for 15 seconds at 60°C for 1 minutes at 40 cycles. The detection was made through Taqman probe. The constitution of total reaction volume for the qPCR reaction is shown in Table 2 . Table 1 Primer Set Information for IS2404 Gene Primer Primer Sequence (5′–3′) Primer Length Fragment Size (bp) IS2404TF (5′) AAAGCACCACGCAGCATCT 19 276 IS2404TR (3′) AGCGACCCCAGTGGATTG 18 330 Fyfe & Lavender ( 2022 ). Table 2 Constitution of total reaction volume for the qPCR reaction Component Volume Master mix (5x) 5 µl F Primer 1.5 µl Reverse Primer 1.5 µl DNA template 0.5 µl Water 11.5 µl Total volume 20 µl Statistical Analysis Results of the analysis were converted to percentages and presented on tables. Results Prevalence of Buruli Ulcer in Parts of Jigawa State The results from both microscopic examination (Ziehel Nelseen staining procedure) and molecular analyses (Real-Time PCR) were all negative as shown in the Tables 3 and Table 4 below. Table 3 Prevalence of Buruli Ulcer in Relation to Age Group, Gender and Health Facility Variable Number Tested Number of Positive Percentage Positive (%) Age Group (Years) 0–5 13 0 0 6–11 48 0 0 12–17 127 0 0 18 and above 194 0 0 Gender Male 265 0 0 Female 117 0 0 Health Facility Birnin Kudu GH 67 0 0 Dutse GH 60 0 0 Gumel GH 60 0 0 Jahun GH 65 0 0 Kazaure GH 65 0 0 Ringim GH 65 0 0 Key : GH = General Hospital Table 4 qPCR Showing Quantification Data S/N SAMPLE ID Type of Specimen Target Gene C q C q Mean PCR Status 1 3449 SWAB IS2404 N/A 0.00 Negative 2 3450 SWAB IS2404 N/A 0.00 Negative 3 3451 SWAB IS2404 N/A 0.00 Negative 4 3452 SWAB IS2404 N/A 0.00 Negative 5 3453 SWAB IS2404 N/A 0.00 Negative 6 3454 SWAB IS2404 N/A 0.00 Negative 7 3455 SWAB IS2404 N/A 0.00 Negative 8 3456 SWAB IS2404 N/A 0.00 Negative 9 3457 SWAB IS2404 N/A 0.00 Negative 10 3458 SWAB IS2404 N/A 0.00 Negative 11 3459 SWAB IS2404 N/A 0.00 Negative 12 3460 SWAB IS2404 N/A 0.00 Negative 13 3461 SWAB IS2404 N/A 0.00 Negative 14 3462 SWAB IS2404 N/A 0.00 Negative 15 3463 SWAB IS2404 N/A 0.00 Negative 16 3464 SWAB IS2404 N/A 0.00 Negative 17 3465 SWAB IS2404 N/A 0.00 Negative 18 3466 FNA IS2404 N/A 0.00 Negative 19 3467 SWAB IS2404 N/A 0.00 Negative 20 3468 FNA IS2404 N/A 0.00 Negative 21 3469 SWAB IS2404 N/A 0.00 Negative 22 3470 SWAB IS2404 N/A 0.00 Negative 23 3471 SWAB IS2404 N/A 0.00 Negative 24 3472 SWAB IS2404 N/A 0.00 Negative 25 3473 SWAB IS2404 N/A 0.00 Negative 26 3474 SWAB IS2404 N/A 0.00 Negative 27 3475 SWAB IS2404 N/A 0.00 Negative 28 3476 SWAB IS2404 N/A 0.00 Negative 29 3477 SWAB IS2404 N/A 0.00 Negative 30 3478 SWAB IS2404 N/A 0.00 Negative 31 3479 SWAB IS2404 N/A 0.00 Negative 32 3480 SWAB IS2404 N/A 0.00 Negative 33 3481 SWAB IS2404 N/A 0.00 Negative 34 3482 SWAB IS2404 N/A 0.00 Negative 35 3483 SWAB IS2404 N/A 0.00 Negative 36 Water N/A IS2404 N/A 0.00 Negative 37 Negative Control N/A IS2404 N/A 0.00 Negative 38 Negative Control N/A IS2404 N/A 0.00 Negative 39 Positive Control N/A IS2404 26.75 26.75 Positive Key : N/A = Not Applicable; FNA = Fine Needle Aspirates; C q = Cycle of quantification; ID = Identity; PCR = Polymerase Chain Reaction Samples of Patients with Skin ulcerative lesions Plate 1: Sample of Patient from General Hospital Dutse Plate 2: Sample of Patient from General Hospital Ringim Plate 3: Sample of Patient from General Hospital Kazaure Plate 4: Sample of Patient from General Hospital Gumel Plate 5: Sample of Patient from General Hospital Birnin Kudu Plate 6: Sample of Patient from General Hospital Jahun Discussion Finding from this study is different from that of Ukwaja et al. ( 2016 ) who reported the crude prevalence of 18.7 per 100,000 in their study communities and much higher rate 41.4 per 100,000 in Ogoja. However, this study agrees with one of the commonly held notions by heath policymakers in Nigeria that Buruli ulcer disease is not endemic in the country anymore (Ukwaja et al., 2016 ). The results from this study also differs from the number of cases from the following regions, Nkpo Hamida village, Igbo-Eze North Local Government Area of Enugu State (1 case); Iburu village, Ohaozora Local Government Area of Ebonyi State (1 case), Akoju village, Ikwo Local Government Area of Ebonyi State (1 case); Amazunze village, Nkanu East Local Government Area of Enugu State (1 case); Okro Mbokho village, Eastern obolo, Akwa Ibom State (1 case); Oron village, oron Local Government Area of Akwa Ibom State. (1 case); and (1 case) in Ugwu Tank, Awka South Local Government Area of Anambra State (Okechukwu et al. , 2007). Orujyan et al. ( 2022 ), reported the protective effect of neonatal BCG vaccination against severe forms of BU disease, although there are conflicting reports on its effectiveness. The zero prevalence of BU in parts of Jigawa state may be attributed to the administration of BCG vaccine as a component of routine immunization schedule. This result also revealed that Buruli ulcer is not endemic in Jigawa state, Northern Nigeria, despite the availability of various degrees of skin ulcerative lesions in the study facilities. Conclusion This study diagnosed Buruli ulcer using molecular and microscopic approaches, but nonetheless, the results show that there is no Buruli ulcer in the study area. Recommendation Sampling in this study was hospital-based, future researcher should focus on community sampling. More samples suspected to have symptoms of Buruli ulcers should be diagnosed using Real-Time PCR and make use of both IS2404 and IS2606. Declarations Ethical Approval The study was conducted under human right ethical approval sought from Jigawa State Ministry of Health with reference number: MOH/SEC.3/S/819/1, prior to commencement. Study protocol was submitted for approval by the ministry. Permission was sought from the Medical Directors of all the six (6) hospitals where this research was conducted. Sample collection from the participants was conducted following verbal and documented informed consent by each of the participant. Consent for publication Permission to publish the work under the Creative Commons Attribution-Non-Commercial 4.0 license was granted by each author. If not, we thus assign all copyright ownership, including all incidental rights thereto, to the journal exclusively upon publication. Availability of data and materials The authors are willing to provide the data from this study upon a legitimate request. Competing interests The authors affirm that they have no competing interests. Funding No external funding was obtained for this study. Acknowledgements We value the contributions made by the medical professionals working in each of the participating hospitals in our investigation. Author Contributions Yusuf Adeiza Kashim, conceived, designed, and developed the methodology of the study and as well drafted the manuscript. Mustapha Musa Dogara and Bashir Sajo Mienda, both served as advisor to the research. Additionally, Mustapha Musa Dogara revised the manuscript. Timothy. E. Nwafor, was very instrumental at the molecular findings, including technical support during the real-time quantitative polymerace chain reactions. Olaoluwa Pheabian Akinwale gave lots of technical and frame work support including proofreading of wordings. Kamoru Abdulazeez Adeniyi gave technical advices on figures, plates and tables. Hamza Haruna Abubakar gave technical support during staining and microscopic procedures. References Bretzel, G., & Beissner, M. (2018). PCR detection of Mycobacterium ulcerans—significance for clinical practice and epidemiology. Expert Review of Molecular Diagnostics , 18 (12), 1063-1074. Dogara, M. M., & Ocheje, A. (2016). Prevalence of malaria and risk factors among patients attending Dutse General Hospital, Jigawa State, Nigeria. International Journal of Public and Environmental Health, 11, 270-277. Fyfe, J. A., & Lavender, C. J. (2022). Detection of Mycobacterium ulcerans DNA using real-time PCR. Mycobacterium ulcerans: Methods and Protocols , 71-80. Keragala, B. S. D. P., Herath, H. M. M. T. B., Janapriya, G. H. D. C., Vanitha, S., Balendran, T., Janani, T., ... & Gunasekera, C. N. (2020). Coexistence of mycobacterial infections–Mycobacterium tuberculosis and Mycobacterium leprae–in Sri Lanka: a case series. Journal of Medical Case Reports , 14 , 1-8. Makinde, O. A., Sule, A., Ayankogbe, O., & Boone, D. (2018). Distribution of health facilities in Nigeria: implications and options for universal health coverage. The International journal of health planning and management, 33(4), e1179-e1192. Mitjà, O., Marks, M., Bertran, L., Kollie, K., Argaw, D., Fahal, A. H., … Asiedu, K. (2017). Integrated control and management of neglected tropical skin diseases. PLoS Neglected Tropical Diseases; 11(1):e0005136. Okechuckwu, C., Edwin A., Ghislain, S., Ange, D., Alexandre, T., Lola, S., … Kingsley A. (2007). Orujyan, D., Narinyan, W., Rangarajan, S., Rangchaikul, P., Prasad, C., Saviola, B., & Venketaraman, V. (2022). Protective efficacy of BCG vaccine against mycobacterium leprae and non-tuberculous mycobacterial infections. Vaccines , 10 (3), 390. Osei, F. B., & Duker, A. A. (2015). Analysis of Buruli ulcer prevalence in Amansie West District: a geostatistical approach. Austin Biom Biostat , 2 , 1011. Peetermans, M., de Prost, N., Eckmann, C., Norrby-Teglund, A., Skrede, S., & De Waele, J. J. (2020). Necrotizing skin and soft-tissue infections in the intensive care unit. Clinical Microbiology and Infection , 26 (1), 8-17. Singh, A. (2019). Investigation of potential environmental reservoirs of mycobacterium ulcerans in North Queensland . Doctoral dissertation, James Cook University. Ukwaja, K. N., Meka, A. O., Chukwuka, A., Asiedu, K. B., Huber, K. L., Eddyani, M., … Ntana, K. (2016). Buruli ulcer in Nigeria: results of a pilot case study in three rural districts. Infectious Disease of Poverty; 5: 39. https://doi.org/10.1186/s40249-016-0119-8. Yotsu, R. R., Suzuki, K., Simmonds, R. E., Bedimo, R., Ablordey, A., Yeboah-Manu, D., ... & Asiedu, K. (2018). Buruli ulcer: a review of the current knowledge. Current tropical medicine reports , 5 , 247-256. Plates 1 To 6 Plates 1 to 6 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4555246","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":316660298,"identity":"08a24252-67a4-4c86-9487-7d2cf0c11ae4","order_by":0,"name":"Yusuf Adeiza Kashim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACCRCRwMDA2MAMZHwAYjZ2UrQwzgBpYSZGCwNIC5Bg5gExCWmRbD/++MPDHDvZfnb2h59tfm2T52NmYPzwMQe3FmmeHDOJxG3JxjObGZKlc/tuG7YxMzBLztyGW4scQw4bQ+I25sQNhxkOSOf23GYEamFj5sWnhf/54w+J2+oT9x9mbP5t2XPbnqAWaYkEA6DDDiduYGZmk2b4cTuRoBbJGW9AfjluPOMwG5tlb8Pt5DZmxma8fpE4n/74489t1bL9/ccf3/jx57bt/Pbmgx8+4tGCChjbwGQDsepB4A8pikfBKBgFo2CkAABWt0+l66OSCgAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University Dutse, Ibrahim Aliyu By- Pass","correspondingAuthor":true,"prefix":"","firstName":"Yusuf","middleName":"Adeiza","lastName":"Kashim","suffix":""},{"id":316660299,"identity":"e3073912-9fb0-41c4-ad49-b0383b8887df","order_by":1,"name":"Mustatpha Musa Dogara","email":"","orcid":"","institution":"Federal University Dutse, Ibrahim Aliyu By- Pass","correspondingAuthor":false,"prefix":"","firstName":"Mustatpha","middleName":"Musa","lastName":"Dogara","suffix":""},{"id":316660300,"identity":"cfcef9f1-a7e2-47a0-b41b-02bc2d28d2e3","order_by":2,"name":"Bashir Sajo Mienda","email":"","orcid":"","institution":"Federal University Dutse, Ibrahim Aliyu By- Pass","correspondingAuthor":false,"prefix":"","firstName":"Bashir","middleName":"Sajo","lastName":"Mienda","suffix":""},{"id":316660301,"identity":"9bd2b9b4-263f-40f4-907e-80c83a3dfa43","order_by":3,"name":"Timothy. E Nwafor","email":"","orcid":"","institution":"Nigerian Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Timothy.","middleName":"E","lastName":"Nwafor","suffix":""},{"id":316660302,"identity":"a5477fd9-8616-491b-adde-26c4e32dbbb7","order_by":4,"name":"Olaoluwa Pheabian Akinwale","email":"","orcid":"","institution":"Nigerian Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Olaoluwa","middleName":"Pheabian","lastName":"Akinwale","suffix":""},{"id":316660303,"identity":"eb969eff-8548-4016-9961-1affe88fe88c","order_by":5,"name":"Kamoru Abdulazeez Adeniyi","email":"","orcid":"","institution":"Federal University Dutse, Ibrahim Aliyu By- Pass","correspondingAuthor":false,"prefix":"","firstName":"Kamoru","middleName":"Abdulazeez","lastName":"Adeniyi","suffix":""},{"id":316660304,"identity":"9460b710-72bd-4875-a3e6-0f47e64ead8f","order_by":6,"name":"Hamza Haruna Abubakar","email":"","orcid":"","institution":"General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hamza","middleName":"Haruna","lastName":"Abubakar","suffix":""}],"badges":[],"createdAt":"2024-06-10 00:24:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4555246/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4555246/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60447772,"identity":"6a7bbca5-f32f-4670-9f39-453a2f9b4f11","added_by":"auto","created_at":"2024-07-16 22:09:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":354180,"visible":true,"origin":"","legend":"\u003cp\u003eMap Showing the Study Area\u003c/p\u003e\n\u003cp\u003eSource: Geographic Information System (GIS) Unit, Department of Environmental Management and Toxicology, Federal University Dutse\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4555246/v1/89100ebfe701c5df25ada63c.png"},{"id":60447352,"identity":"e7ba46a4-bb31-46cc-b404-32e06559628d","added_by":"auto","created_at":"2024-07-16 22:01:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":494527,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR Amplification Plot\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4555246/v1/814ecfb117b62dac2b2a4e0f.png"},{"id":60448843,"identity":"96ff4f45-da18-4e8d-8192-34a1d7e176d3","added_by":"auto","created_at":"2024-07-16 22:17:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1715749,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4555246/v1/dc380ced-ced0-462d-85d2-963a22f535a3.pdf"},{"id":60447350,"identity":"8a9ac67c-5aa0-449d-a7f8-748a2f4fa651","added_by":"auto","created_at":"2024-07-16 22:01:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":362001,"visible":true,"origin":"","legend":"","description":"","filename":"Plate1t6.docx","url":"https://assets-eu.researchsquare.com/files/rs-4555246/v1/e32e2f299afaf88df27fe137.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Detection of Buruli ulcer among Patients with Various Degrees of Skin Ulcerative Lesions Revealed Zero Prevalence in Parts of Jigawa State, Northern Nigeria","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe bacteria \u003cem\u003eMycobacterium ulcerans\u003c/em\u003e is the aetiologic agent that causes Buruli ulcer (BU), a chronic, indolent, necrotizing cutaneous illness that is categorized by the World Health Organization (WHO) as one of the skin-related Neglected Tropical Diseases (NTDS) (Mitja \u003cem\u003eet al.\u003c/em\u003e, 2017). After leprosy and tuberculosis, it is the most prevalent mycobacterial illness (Keragala et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Large ulcers, typically on the limbs, are the result of the infection's devastation of soft tissue and skin (Peetermans et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eClinically speaking, a BU begins as a papule, nodule, plaque, or oedematous lesion that gradually spreads to a large area of skin ulceration (Yotsu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Surprisingly, considering the degree of tissue loss, the lesion typically causes little to no pain. Buruli ulcer is distinct from other mycobacterial infections in that the pathophysiology of the illness can be attributed to mycolactone, a lipid-like and diffusible exotoxin, rather than the bacterium (Yotsu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the endemic areas, Buruli ulcer is highly feared and stigmatized due to the unsightly abnormalities it leaves behind. It is also often linked to witchcraft and curses (Osei \u0026amp; Duker, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt least 27 nations in Africa, Asia, South America, and the western Pacific region have patchy foci of Buruli ulcer. Most of the cases that have been recorded are from West and Central Africa (Singh, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Earlier research had shown that Buruli ulcer is endemic in Nigeria, particularly among those who live close to bodies of water. However, most investigations were all conducted in Nigeria's southern regions. There is no documented study on Buruli ulcer in Jigawa State, Northern Nigeria, particularly among the people that live near water features. It is against this background that this study was conducted to determine the prevalence of Buruli ulcer in parts of Jigawa State.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eStudy Area\u003c/h2\u003e\n \u003cp\u003eJigawa State was created on 28th August, 1991from the old Kano State. The state is situated between latitudes 11.00\u003csup\u003eo\u003c/sup\u003eN and longitudes 8.00\u003csup\u003eo\u003c/sup\u003eE to 10.15\u003csup\u003eo\u003c/sup\u003eE, the state lies in the northwest part of Nigeria. It is bordered on the west by Katsina and Kano States, on the east by Yobe and Bauchi States, and on the north by the international boundary with the Niger Republic. As shown in Figure, 1 the state is divided into three senatorial Districts namely: Northwest, Central and Northeast. The study was conducted in secondary health facilities in Northwest and Central Senatorial districts. The state has two distinct seasons: the dry season, which runs from October to May, and the rainy season, which runs from June to September. The hot temperature during the rainy season is approximately 42\u0026deg;C, while the low temperature is 10\u0026deg;C (Okereke \u003cem\u003eet al.\u003c/em\u003e, 2015). The state ranks eighth in terms of population according to the 2006 census.\u003c/p\u003e\n \u003cp\u003eThe state has three types of health facilities: the primary health care which is under the purview of the local government, secondary health care is overseen by the state government (the state has sixteen general hospitals and one specialty hospital), and tertiary health care is under the purview of the federal government and there is only one Federal Medical Center (Dogara \u0026amp; Ocheje, \u003cspan\u003e2016\u003c/span\u003e; Makinde et al., \u003cspan\u003e2018\u003c/span\u003e). State-level health policy development and execution, as well as translation of federal health policies for implementation, fall under the purview of the Ministry of Health and the Agency for Primary Health Care. A primary health center is intended to be available in every ward of the state, as the state currently functions under a single roof system (Makinde et al., \u003cspan\u003e2018\u003c/span\u003e). Patients identified with various degrees of ulcerative lesions are admitted into general hospitals for care.\u003c/p\u003e\n \u003cp\u003eSix secondary healthcare facilities were selected, they include General Hospital Dutse, General Hospital Birnin Kudu, General Hospital Jahun, General Hospital Ringim, General Hospital Gumel and General Hospital Kazaure, as shown in Fig. \u003cspan\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003eStudy Design\u003c/h2\u003e\n \u003cp\u003eA cross-sectional hospital-based study was conducted. Two out of the three senatorial districts were selected for the study. The samples were collected from selected secondary healthcare facilities within the two senatorial districts of Northwest and Central.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003eSample Size Determination\u003c/h2\u003e\n \u003cp\u003eThe study adopted a purposive or judgmental sampling technique in the selection of the patients whose samples were collected from the study population. A total of three hundred and eighty-two (382) skin related ulcer patients were screened. Specifically, thirty-five (35) samples were collected from patients who presented symptoms mimicking Buruli ulcer for the molecular analyses. Samples were collected from the patients between December 2019 and September 2023.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSCREENING OF SAMPLES FOR THE DETECTION OF\u003c/strong\u003e \u003cstrong\u003eMYCOBACTERIUM ULCERANS\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003eMicroscopic examination\u003c/h2\u003e\n \u003cp\u003eMicroscopic diagnoses by direct smear examination with Ziehl-Neelsen staining to detect the presence of acid-fast bacilli (AFB) was done using the quantification of smears by the method locally used for the diagnosis of tuberculosis (Portaels, 2001). The positive control sample was collected from the tuberculosis (Tb) positive stock at the Tb Unit of Dutse General Hospital, Jigawa State. Self-sputum was collected and smeared, stained, and subsequently used as the Negative Control. Using a sterile swab stick and fine needle aspirate (FNA), ulcerative lesions, papules and nodules were collected respectively from suspected patients and then smeared on a clean grease free slide using sterile technique. The smear was air dried, and heat fixed by passing through an open flame. The smear was covered with the carbol fuchsin stain. The stain was heated until vapour rose for 5 minutes. The smear was washed with clean water and 3% v/v acid alcohol was applied to the smear for 5 minutes to decolorize the smear to pale pink colour. The smear was rinsed with clean water. Malachite green stain was applied to the smear and allowed to stain for 2 minutes. The stain was rinsed with clean water. Back of the slide was wiped to clean and then placed in the rack for the smear to air-dry. The smear was examined microscopically, using the \u0026times;100 oil immersion objective.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003eMolecular analyses\u003c/h2\u003e\n \u003cp\u003eThe analyses were performed at the Molecular Parasitology Research Laboratory of the Nigerian Institute of Medical Research (NIMR), Yaba Lagos State, Nigeria, a reference laboratory for Buruli ulcer diagnosis and research.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eSample collection and processing\u003c/h2\u003e\n \u003cp\u003eThirty-three (33) swab and two (2) Fine Needle Aspiration (FNA) samples were aseptically collected into collection tubes. Each tube was labelled for ease of identification. Subsequently each swab sample collected from lesion on the patients\u0026rsquo; limbs (upper or lower) was transferred into 15 ml conical tube, was rehydrated with 2 ml of sterile water and vortexed. This was left to stand for 5 minutes and re-vortexed to increase the concentration of the sample. The swabs were gently discarded from the 15 ml tube leaving the content. Thereafter, 400 \u0026micro;l content obtained from above was measured and transferred into a 1.5ml tube with screw cap for DNA extraction and purification. Regarding the FNA samples, they were vortexed and a 400 \u0026micro;l each was taken and transferred into a 1.5 ml tube with screw cap for DNA extraction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003eDNA extraction and purification procedure\u003c/h2\u003e\n \u003cp\u003eDNA extraction was performed using the Genolysis kit (Hains Lifescience, Nehren, Germany). A 400 \u0026micro;l of specimen suspension was placed into 1.5 ml tubes with screw cap for each sample, Thereafter, tubes were centrifuged at 12000 g for 15 minutes at room temperature. The supernatants were carefully discarded using a P1000 filter tip leaving the pellets at the bottom of the conical tubes. The pellets were re-suspended in 400 \u0026micro;l of distilled water and centrifuged at 12000 g for 15 minutes at room temperature. The supernatant was carefully discarded, and the pellets were re-suspended in 50 \u0026micro;l of A-LYS buffer and incubated for 10 minutes at 95\u0026deg;C. The tubes were centrifuged for 10 seconds to pellet the suspension. The above, 50 \u0026micro;l of buffer A-NB was added and the tubes were kept at 4\u0026deg;C prior to PCR/amplification procedure.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003eReal-time quantitative PCR (RT qPCR)\u003c/h2\u003e\n \u003cp\u003eRT qPCR was performed as described by Fyfe \u0026amp; Lavender (\u003cspan\u003e2022\u003c/span\u003e). The method is recommended by the WHO for \u003cem\u003eM. ulcerans\u003c/em\u003e detection (Bretzel \u0026amp; Beissner, \u003cspan\u003e2018\u003c/span\u003e) and is based on primers IS2404TF and IS2404TR. The primers set employed for quantification is depicted in Table \u003cspan\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eQuantitative PCR (qPCR)/amplification condition/procedure\u003c/h2\u003e\n \u003cp\u003eThe qPCR/amplification reaction was performed in a total volume of 20 \u0026micro;l reaction in Bio-Rad thermal cycler machine (Bio-Rad Laboratories, Marnes- la- Coquette, France). 5 \u0026micro;l of 5x Hot fire Pol probe qPCR mix (Master mix) was added to 11.5 \u0026micro;l water (PPI), followed by 1.5 \u0026micro;l of forward primer and 1.5 \u0026micro;l of reverse primer including 0.5 \u0026micro;l template DNA. The qPCR amplification procedure is presented as follows: The sample was heated at initial denaturation temperature of 95\u0026deg;C for 10 minutes at 1 cycle, followed by final heating at 95\u0026deg;C for 15 seconds at 60\u0026deg;C for 1 minutes at 40 cycles. The detection was made through Taqman probe. The constitution of total reaction volume for the qPCR reaction is shown in Table \u003cspan\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003ePrimer Set Information for IS2404 Gene\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimer Sequence (5\u0026prime;\u0026ndash;3\u0026prime;)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimer Length\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFragment Size (bp)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIS2404TF (5\u0026prime;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAAAGCACCACGCAGCATCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e276\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIS2404TR (3\u0026prime;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAGCGACCCCAGTGGATTG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e330\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eFyfe \u0026amp; Lavender (\u003cspan\u003e2022\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eConstitution of total reaction volume for the qPCR reaction\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eComponent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVolume\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMaster mix (5x)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF Primer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eReverse Primer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDNA template\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.5 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal volume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eResults of the analysis were converted to percentages and presented on tables.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePrevalence of Buruli Ulcer in Parts of Jigawa State\u003c/h2\u003e \u003cp\u003eThe results from both microscopic examination (Ziehel Nelseen staining procedure) and molecular analyses (Real-Time PCR) were all negative as shown in the Tables \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e below.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrevalence of Buruli Ulcer in Relation to Age Group, Gender and Health Facility\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber Tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eNumber of Positive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003ePercentage Positive (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge Group (Years)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u0026ndash;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u0026ndash;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u0026ndash;17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18 and above\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHealth Facility\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirnin Kudu GH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDutse GH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGumel GH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJahun GH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKazaure GH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRingim GH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003eKey\u003c/b\u003e: GH\u0026thinsp;=\u0026thinsp;General Hospital\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eqPCR Showing Quantification Data\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS/N\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSAMPLE ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType of Specimen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTarget Gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003eq\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003csub\u003eq\u003c/sub\u003e Mean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePCR Status\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3456\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3457\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3458\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3459\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3463\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3466\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3468\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3472\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3476\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3477\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3481\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3482\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSWAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS2404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cb\u003eKey\u003c/b\u003e: N/A\u0026thinsp;=\u0026thinsp;Not Applicable; FNA\u0026thinsp;=\u0026thinsp;Fine Needle Aspirates; C\u003csub\u003eq\u003c/sub\u003e = Cycle of quantification; ID\u0026thinsp;=\u0026thinsp;Identity; PCR\u0026thinsp;=\u0026thinsp;Polymerase Chain Reaction\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSamples of Patients with Skin ulcerative lesions\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlate 1: Sample of Patient from General Hospital Dutse\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlate 2: Sample of Patient from General Hospital Ringim\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlate 3: Sample of Patient from General Hospital Kazaure\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlate 4: Sample of Patient from General Hospital Gumel\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlate 5: Sample of Patient from General Hospital Birnin Kudu\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlate 6: Sample of Patient from General Hospital Jahun\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFinding from this study is different from that of Ukwaja et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) who reported the crude prevalence of 18.7 per 100,000 in their study communities and much higher rate 41.4 per 100,000 in Ogoja. However, this study agrees with one of the commonly held notions by heath policymakers in Nigeria that Buruli ulcer disease is not endemic in the country anymore (Ukwaja et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results from this study also differs from the number of cases from the following regions, Nkpo Hamida village, Igbo-Eze North Local Government Area of Enugu State (1 case); Iburu village, Ohaozora Local Government Area of Ebonyi State (1 case), Akoju village, Ikwo Local Government Area of Ebonyi State (1 case); Amazunze village, Nkanu East Local Government Area of Enugu State (1 case); Okro Mbokho village, Eastern obolo, Akwa Ibom State (1 case); Oron village, oron Local Government Area of Akwa Ibom State. (1 case); and (1 case) in Ugwu Tank, Awka South Local Government Area of Anambra State (Okechukwu \u003cem\u003eet al.\u003c/em\u003e, 2007).\u003c/p\u003e \u003cp\u003eOrujyan et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), reported the protective effect of neonatal BCG vaccination against severe forms of BU disease, although there are conflicting reports on its effectiveness. The zero prevalence of BU in parts of Jigawa state may be attributed to the administration of BCG vaccine as a component of routine immunization schedule. This result also revealed that Buruli ulcer is not endemic in Jigawa state, Northern Nigeria, despite the availability of various degrees of skin ulcerative lesions in the study facilities.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study diagnosed Buruli ulcer using molecular and microscopic approaches, but nonetheless, the results show that there is no Buruli ulcer in the study area.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRecommendation\u003c/h2\u003e \u003cp\u003eSampling in this study was hospital-based, future researcher should focus on community sampling. More samples suspected to have symptoms of Buruli ulcers should be diagnosed using Real-Time PCR and make use of both IS2404 and IS2606.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical Approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted under human right ethical approval sought from Jigawa State Ministry of Health with reference number: \u003cstrong\u003eMOH/SEC.3/S/819/1,\u0026nbsp;\u003c/strong\u003eprior to commencement. Study protocol was submitted for approval by the ministry. Permission was sought from the Medical Directors of all the six (6) hospitals where this research was conducted. Sample collection from the participants was conducted following verbal and documented informed consent by each of the participant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePermission to publish the work under the Creative Commons Attribution-Non-Commercial 4.0 license was granted by each author. If not, we thus assign all copyright ownership, including all incidental rights thereto, to the journal exclusively upon publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are willing to provide the data from this study upon a legitimate request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo external funding was obtained for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe value the contributions made by the medical professionals working in each of the participating hospitals in our investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYusuf Adeiza Kashim, conceived, designed, and developed the methodology of the study and as well drafted the manuscript. Mustapha Musa Dogara and Bashir Sajo Mienda, both served as advisor to the research. Additionally, Mustapha Musa Dogara revised the manuscript. Timothy. E. Nwafor, was very instrumental at the molecular findings, including technical support during the real-time quantitative polymerace chain reactions. Olaoluwa Pheabian Akinwale gave lots of technical and frame work support including proofreading of wordings. Kamoru Abdulazeez Adeniyi gave technical advices on figures, plates and tables. Hamza Haruna Abubakar gave technical support during staining and microscopic procedures.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBretzel, G., \u0026amp; Beissner, M. (2018). PCR detection of Mycobacterium ulcerans\u0026mdash;significance for clinical practice and epidemiology. \u003cem\u003eExpert Review of Molecular Diagnostics\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(12), 1063-1074.\u003c/li\u003e\n \u003cli\u003eDogara, M. M., \u0026amp; Ocheje, A. (2016). Prevalence of malaria and risk factors among patients attending Dutse General Hospital, Jigawa State, Nigeria. \u003cem\u003eInternational Journal of Public and Environmental Health,\u003c/em\u003e 11, 270-277.\u003c/li\u003e\n \u003cli\u003eFyfe, J. A., \u0026amp; Lavender, C. J. (2022). Detection of Mycobacterium ulcerans DNA using real-time PCR. \u003cem\u003eMycobacterium ulcerans: Methods and Protocols\u003c/em\u003e, 71-80.\u003c/li\u003e\n \u003cli\u003eKeragala, B. S. D. P., Herath, H. M. M. T. B., Janapriya, G. H. D. C., Vanitha, S., Balendran, T., Janani, T., ... \u0026amp; Gunasekera, C. N. (2020). Coexistence of mycobacterial infections\u0026ndash;Mycobacterium tuberculosis and Mycobacterium leprae\u0026ndash;in Sri Lanka: a case series. \u003cem\u003eJournal of Medical Case Reports\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e, 1-8.\u003c/li\u003e\n \u003cli\u003eMakinde, O. A., Sule, A., Ayankogbe, O., \u0026amp; Boone, D. (2018). Distribution of health facilities in Nigeria: implications and options for universal health coverage. \u003cem\u003eThe International journal of health planning and management,\u003c/em\u003e 33(4), e1179-e1192.\u003c/li\u003e\n \u003cli\u003eMitj\u0026agrave;, O., Marks, M., Bertran, L., Kollie, K., Argaw, D., Fahal, A. H., \u0026hellip; Asiedu, K. (2017). Integrated control and management of neglected tropical skin diseases. \u003cem\u003ePLoS Neglected Tropical Diseases;\u003c/em\u003e 11(1):e0005136.\u003c/li\u003e\n \u003cli\u003eOkechuckwu, C., Edwin A., Ghislain, S., Ange, D., Alexandre, T., Lola, S., \u0026hellip; Kingsley A. (2007).\u003c/li\u003e\n \u003cli\u003eOrujyan, D., Narinyan, W., Rangarajan, S., Rangchaikul, P., Prasad, C., Saviola, B., \u0026amp; Venketaraman, V. (2022). Protective efficacy of BCG vaccine against mycobacterium leprae and non-tuberculous mycobacterial infections. \u003cem\u003eVaccines\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(3), 390.\u003c/li\u003e\n \u003cli\u003eOsei, F. B., \u0026amp; Duker, A. A. (2015). Analysis of Buruli ulcer prevalence in Amansie West District: a geostatistical approach. \u003cem\u003eAustin Biom Biostat\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, 1011.\u003c/li\u003e\n \u003cli\u003ePeetermans, M., de Prost, N., Eckmann, C., Norrby-Teglund, A., Skrede, S., \u0026amp; De Waele, J. J. (2020). Necrotizing skin and soft-tissue infections in the intensive care unit. \u003cem\u003eClinical Microbiology and Infection\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e(1), 8-17.\u003c/li\u003e\n \u003cli\u003eSingh, A. (2019). \u003cem\u003eInvestigation of potential environmental reservoirs of mycobacterium ulcerans in North Queensland\u003c/em\u003e. Doctoral dissertation, James Cook University.\u003c/li\u003e\n \u003cli\u003eUkwaja, K. N., Meka, A. O., Chukwuka, A., Asiedu, K. B., Huber, K. L., Eddyani, M., \u0026hellip; Ntana, K. (2016). Buruli ulcer in Nigeria: results of a pilot case study in three rural districts. \u003cem\u003eInfectious Disease of Poverty;\u003c/em\u003e 5: 39. https://doi.org/10.1186/s40249-016-0119-8. \u003c/li\u003e\n \u003cli\u003eYotsu, R. R., Suzuki, K., Simmonds, R. E., Bedimo, R., Ablordey, A., Yeboah-Manu, D., ... \u0026amp; Asiedu, K. (2018). Buruli ulcer: a review of the current knowledge. \u003cem\u003eCurrent tropical medicine reports\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e, 247-256.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Plates 1 To 6","content":"\u003cp\u003ePlates 1 to 6 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Public Health, Buruli ulcer, Mycobacterium ulcerans, Neglected Tropical Disease","lastPublishedDoi":"10.21203/rs.3.rs-4555246/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4555246/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe indolent, necrotizing Buruli ulcer disease is a chronic condition caused by \u003cem\u003eMycobacterium ulcerans\u003c/em\u003e, an environmental bacteria that can damage the skin, tissues, and bones. It is now recognized as one of the 20 Neglected Tropical Diseases targeted for elimination by the year 2030. Public health continues to face difficulties due to its mechanism of transmission that is yet to be completely unravel. From December 2019 to September 2023, a hospital-based study was conducted to ascertain the prevalence of Buruli ulcer in parts of Jigawa State, Nigeria. A purposive sampling technique was adopted in selecting and diagnosing 382 samples from patients with various degrees of skin ulcerative lesions across all age groups and gender. The diagnosis of all the samples initially involved the use of Ziehel Nelseen staining procedure. Thirty-five (35) samples with symptoms suspected to be Buruli ulcer were diagnosed using Real-time Quantitative Polymerase Chain Reaction (qPCR) method. The results were negative for the 382 samples diagnosed using Ziehel Nelseen staining procedure as well as the 35 using real-Time PCR. This study concludes that despite testing samples with obvious ulcerative lesions suspected to be Buruli ulcer from patients, both Ziehel Nelseen staining procedure and Real-Time PCR revealed negative results. It is recommended that more samples suspected to have symptoms of Buruli ulcers should be diagnosed using Real-Time PCR and also make use of both IS2404 and IS2606.\u003c/p\u003e","manuscriptTitle":"Molecular Detection of Buruli ulcer among Patients with Various Degrees of Skin Ulcerative Lesions Revealed Zero Prevalence in Parts of Jigawa State, Northern Nigeria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-16 22:01:26","doi":"10.21203/rs.3.rs-4555246/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-06-20T04:25:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-19T10:08:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2024-06-10T00:23:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cf5ed629-c898-4636-8712-52c2cf2f5c86","owner":[],"postedDate":"July 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-16T22:01:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-16 22:01:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4555246","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4555246","identity":"rs-4555246","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}