Medical Microbiology

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Browsing Medical Microbiology by Author "Abebe Tamrat"

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    Epidemiology and Molecular Characterization of Mycobacterium Bovis in Humans and Cattle and Assessment of its Zoonotic Importance in Central Ethiopia
    (Addis Ababa University, 2021-11) Gizat Almaw; Mihret Adane ; Abebe Tamrat
    Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis complex (MTBC) and causes tuberculosis in humans (zoonotic tuberculosis-zTB) and animals, mainly in cattle (bovine tuberculosis-bTB). There were limited studies on zTB in Ethiopia but also a reliable estimate of bTB prevalence in cattle in central Ethiopia is missing. In addition no whole genome sequencing (WGS) based M .bovis studies had been performed in Ethiopia before this study. Also, there has been a limited effort in search for an alternative diagnostic method to culture to detect M. bovis in clinical specimens. Therefore, due to these research gaps, this study, which combined bTB and zTB, was conducted from 2018 to 2021 in central Ethiopia (Addis Ababa, Sebeta, Holeta, Sululta, Sendafa and Bishoftu) with the objective of generating epidemiological and molecular data to update our understanding on bTB/zTB. The bTB study in cattle involved a cross sectional one-stage cluster sampling survey of dairy farms in central Ethiopia using tuberculin skin testing and the collection of additional data by questionnaire to estimate the prevalence of bTB and identify potential risk factors contributing to bTB transmission. For the zTB part, surveillance of TB in humans was carried out among individuals working in bTB infected dairy farms, patients presented at selected health centers, and exposure to risk factors was assessed using questionnaire. From consenting TB suspected individuals, demographic and clinical information was collected by questionnaire. Sputum and Fine Needle Aspirates (FNA) samples were collected from suspected cases. In addition, isolation of M. bovis was done from raw milk collected from tuberculin skin test positive cows and from cattle tissue lesions. The genetic diversity of M. bovis isolates was examined using spoligotyping and whole genome sequencing (WGS) analysis. Furthermore, in this study the performance of a TaqMan real time PCR (RT-PCR) assay as an alternative diagnostic method to culture was evaluated. Two hundred ninety-nine (n=299) dairy herds in the six study areas were randomly selected, from which 5,675 cattle were tested. The overall prevalence of bTB after standardisation for herd-size in the population was 54.4% (95% CI 48.7-60%) at the herd level, and it was 24.5% (95% CI 23.3-25.8) at the individual animal level. A Generalized Linear Mixed Model (GLMM) was used to explore risk factors association with bTB status. We found that herd size, animal age, bTB history at farm, and breed were significant risk factors. With regard to zTB, among 110 DFWs in 73 bTB infected dairy farms, 41 had at least one of the symptoms that are typical for TB. Three DFWs had swollen nodes at their neck, a symptom typical for TB lymphadenitis. In assessment of risk factors: raw milk consumption was practiced by more than two thirds of theDFWs with symptoms of TB (68.2%) and over half of DFWs with symptoms did not think TBcould be transmitted via raw milk consumption. Overall in the surveillance of zTB (active and passive), a total of 167 specimens (sputum=131; FNA=36) were collected from 161 TB suspected individuals for the isolation of M. bovis. Of these processed specimens, three samples with M. bovis were detected in total (1.8%, n=167). And of these three, one M. bovis isolate was sequenced and the genotype was spoligotype SB1476 which was previously reported from cattlein Ethiopia suggesting possible zoonotic transmission. With regard to isolation and characterization of M. bovis from cattle, out of 827 cattle (abattoirs and dairy farms), 76 of them(9.2%) had tuberculous lesion. From these tuberculous lesions, 62 isolates (n=137 samples) from42 animals were confirmed to be M. bovis. Similarly out of 975 milking cows which were tuberculin skin test positive (37.8%, n=2582), 490 composite raw milk samples were collectedand of these 11 (2.2%) yield M. bovis isolates showing evidence that raw milk is not safe and can be a source of infection for human TB due to M. bovis. The genetic diversity of 74 M. bovisisolates (one being a human isolate) was assessed and ten different spoligotypes were recorded. In cattle spoligotype SB1176 was the most prevalent type (n=31, 44.3%) followed by SB0133(n=11, 15.7%). Our WGS analysis with a total of 55 M. bovis isolates sequenced (one being ahuman isolate) showed three clonal complexes clearly segregating in the phylogeny: African 2(Af2; n=47), European 3 (Eu3; n=7) and Unknown8 (n=1). In addition, the present study reported for the first time clonal complex European 3 (Eu3) from Ethiopia. With regard toTaqMan assay performance evaluation - the assay performance on 440 clinical samples was variable for different specimens and overall performed well for sputum samples for all targets. In conclusion, this study recorded high prevalence of bTB in dairy cattle in central Ethiopia. M.bovis prevalence in humans in central Ethiopia was low; however, further investigation is neededin all regions at national level given bTB is endemic in cattle in Ethiopia. Knowledge gap onbTB and M. bovis isolation from milk, showed that there is a clear potential for zoonotic transmission and needs further investigation. The TaqMan RT-PCR assay is a promising methodology for the diagnosis of zTB and bTB, but with further validation works needed usingdifferent targets and specimens.
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    Human papillomavirus in Women with Pre-Cancerous Lesion and Cervical Cancer: the Use of Urine as an Alternative Specimen
    (Addis Ababa University, 2021-05) Firdawoke Ededia; Abebe Tamrat; Teka Brhanu
    Background: In a country where the coverage cervical cancer screening is low optimization of the uptake is critical. The implementation of high precision test is advocated by WHO. To augment the implementation human papillomavirus (HPV) based screening in Ethiopia we compared the performance urine HPV DNA test with cervical swab. Methods: Paired samples (n=103) of first void urine and cervical swab were collected from patients Gynecology Clinic of Tikur Anbessa Specialized Hospital (TASH). After extraction of DNA using QIAamp® DNA Mini Kit (Qiagen) the HPV infection, coinfection and type-specific HPV distribution was determined using the Anyplex HPV28 DNA genotyping kit (Seegene, Seoul, Korea) and CFX96 IVD (In Vitro Diagnostic) Real-Time PCR System. The kit simultaneously detects, differentiate, and semi-quantify 28 HPV genotypes 19 high risk (Hr)-HPV types; HPVs 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73 and 82 and 9 LR-HPV types; HPVs 6, 11, 40 ,42, 43, 44, 54, 61 and 70. Additionally, blood sample was collected to detect HPV16 L1 anti-capsid antibody using Prevo-check ® (Abvirus Germany GmbH). It is immunologic rapid test that directs against a protein that is produced by HPV 16 infected cells which interferes with cell division. Pap smear was done by a pathologist and histology results was collected from the chart of the patients and a clinical form was used to collect basic information from the patients by the attending midwife nurses. Result: Of the 103 paired samples, HPV infection prevalence was 83.5% in cervical and 77.7% in urine samples. HPV 16 is the most prevalent in both samples with 56.8% in cervical swab and 54.6% in urine sample followed by HPV 18 (5.8%) in cervical swab and HPV 18 and HPV 39 (6.2%) in urine samples. Multiple infection rate (infection more than one type of HPV) was 22.4% in urine samples and 32.0% in cervical swab. The agreement in the detection of HR-HPV between urine and cervical samples was moderate with a kappa value of 0.57 at 95% CI. Using the cervical HPV results as a reference, the anaylitical sensitivity of urine HPV testing was 88.4% (76/86) and specificity of 76.5% (13/17) and ROC area of 0.82 with (0.7-0.9) 95% CI. The Prevo-check HPV16 L1 antibody test has detected antibody from seven patients have a low clinical sensitivity but specificity of 100%. Of 93 histology result; 69.9% of the participants were diagnosed with SCC. HR-HPV detected in 76.2% and 79.7% from cervical and urine samples. Conclusion: In a country with low cervical cancer screening uptake collection of urine specimen can be considered as an alternative sample since the sample is easy to obtain, showed good diagnostic performance and may increases uptake of cervical cancer screening in Ethiopia. HPV16 and 18 were the predominant HPV detected from women with CIN2+ and above patients.