Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia.

BACKGROUND: Ethiopia is one of the high multidrug-resistant tuberculosis (MDR-TB) burden countries. However, phenotypic drug susceptibility testing can take several weeks due to the slow growth of Mycobacterium tuberculosis complex (MTBC) strains. In this study, we assessed the performance of a Sanger sequencing approach to predict resistance against five anti-tuberculosis drugs and the pattern of resistance mediating mutations. METHODS: We enrolled 226 MTBC culture-positive MDR-TB suspects and collected sputum specimens and socio-demographic and TB related data from each suspect between June 2015 and December 2016 in Addis Ababa, Ethiopia. Phenotypic drug susceptibility testing (pDST) for rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin using BACTEC MGIT 960 was compared with the results of a Sanger sequencing analysis of seven resistance determining regions in the genes rpoB, katG, fabG-inhA, pncA, embB, rpsL, and rrs. RESULT: DNA isolation for Sanger sequencing was successfully extracted from 92.5% (209/226) of the MTBC positive cultures, and the remaining 7.5% (17/226) strains were excluded from the final analysis. Based on pDST results, drug resistance proportions were as follows: isoniazid: 109/209 (52.2%), streptomycin: 93/209 (44.5%), rifampicin: 88/209 (42.1%), ethambutol: 74/209 (35.4%), and pyrazinamide: 69/209 (33.0%). Resistance against isoniazid was mainly mediated by the mutation katG S315T (97/209, 46.4%) and resistance against rifampicin by rpoB S531L (58/209, 27.8%). The dominating resistance-conferring mutations for ethambutol, streptomycin, and pyrazinamide affected codon 306 in embB (48/209, 21.1%), codon 88 in rpsL (43/209, 20.6%), and codon 65 in pncA (19/209, 9.1%), respectively. We observed a high agreement between phenotypic and genotypic DST, such as 89.9% (at 95% confidence interval [CI], 84.2%-95.8%) for isoniazid, 95.5% (95% CI, 91.2%-99.8%) for rifampicin, 98.6% (95% CI, 95.9-100%) for ethambutol, 91.3% (95% CI, 84.6-98.1%) for pyrazinamide and 57.0% (95% CI, 46.9%-67.1%) for streptomycin. CONCLUSION: We detected canonical mutations implicated in resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin. High agreement with phenotypic DST results for all drugs renders Sanger sequencing promising to be performed as a complementary measure to routine phenotypic DST in Ethiopia. Sanger sequencing directly from sputum may accelerate accurate clinical decision-making in the future.

Prevalence and Molecular Characterization of Mycobacterium bovis in Slaughtered Cattle Carcasses in Burkina Faso; West Africa.

This cross-sectional study was conducted at the slaughterhouses/slabs of Oudalan and Ouagadougou in Burkina Faso, between August and September 2013. It aimed at determining the prevalence of bovine tuberculosis (bTB) suggestive lesions in slaughtered cattle carcasses and to identify and characterize the mycobacteria isolated from these lesions. A thorough postmortem examination was conducted on carcasses of a total of 2165 randomly selected cattle. The overall prevalence of bTB suggestive lesions was 2.7% (58/2165; 95% CI 2.1-3.5%). Due to the low number of positive samples, data were descriptively presented. The lesions were either observed localized in one or a few organs or generalized (i.e., miliary bTB) in 96.6% (n = 57) and 3.4% (n = 2), respectively. The identified mycobacteria were M. bovis (44.4%, n = 20), M. fortuitum (8.9%, n = 4), M. elephantis (6.7%, n = 3), M. brumae (4.4%, n = 2), M. avium (2.2%, n = 1), M. asiaticum (2.2%, n = 1), M. terrae (2.2%, n = 1), and unknown non-tuberculous mycobacteria (NTM) (11.1%, n = 5). Moreover, eight mixed cultures with more than one Mycobacterium species growing were also observed, of which three were M. bovis and M. fortuitum and three were M. bovis and M. elephantis. In conclusion, M. bovis is the predominant causative agent of mycobacterial infections in the study area. Our study has identified a base to broaden the epidemiological knowledge on zoonotic transmission of mycobacteria in Burkina Faso by future studies investigating further samples from humans and animals, including wild animals employing molecular techniques.

Molecular Epidemiology of Mycobacterium tuberculosis Complex Strains in Urban and Slum Settings of Nairobi, Kenya.

Kenya is a country with a high tuberculosis (TB) burden. However, knowledge on the genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains and their transmission dynamics is sparsely available. Hence, we used whole-genome sequencing (WGS) to depict the genetic diversity, molecular markers of drug resistance, and possible transmission clusters among MTBC strains in urban and slum settings of Nairobi. We analyzed 385 clinical MTBC isolates collected between 2010 and 2015 in combination with patients' demographics. We showed that the MTBC population mainly comprises strains of four lineages (L1-L4). The two dominating lineages were L4 with 55.8% (n = 215) and L3 with 25.7% (n = 99) of all strains, respectively. Genome-based cluster analysis showed that 30.4% (117/385) of the strains were clustered using a ≤5 single-nucleotide polymorphism (SNP) threshold as a surrogate marker for direct patient-to-patient MTBC transmission. Moreover, 5.2% (20/385) of the strains were multidrug-resistant (MDR), and 50.0% (n = 10) were part of a genome-based cluster (i.e., direct MDR MTBC transmission). Notably, 30.0% (6/20) of the MDR strains were resistant to all first-line drugs and are part of one molecular cluster. Moreover, TB patients in urban living setting had 3.8 times the odds of being infected with a drug-resistant strain as compared to patients from slums (p-value = 0.002). Our results show that L4 strains are the main causative agent of TB in Nairobi and MDR strain transmission is an emerging concern in urban settings. This emphasizes the need for more focused infection control measures and contact tracing of patients with MDR TB to break the transmission chains.

High Prevalence and New Genotype of Coxiella burnetii in Ticks Infesting Camels in Somalia.

Coxiella burnetii is the causative agent of Q fever. It can infect animals, humans, and birds, as well as ticks, and it has a worldwide geographical distribution. To better understand the epidemiology of C. burnetii in Somalia, ticks infesting camels were collected from five different regions, including Bari, Nugaal, Mudug, Sool, and Sanaag, between January and March 2018. Collected ticks were tested for C. burnetii and Coxiella-like endosymbiont DNA by using IS1111, icd, and Com1-target PCR assays. Moreover, sequencing of the 16S-rRNA was conducted. Molecular characterization and typing were done by adaA-gene analysis and plasmid-type identification. Further typing was carried out by 14-marker Multi-Locus Variable-Number Tandem Repeats (MLVA/VNTR) analysis. The investigated ticks (n = 237) were identified as Hyalomma spp. (n = 227, 95.8%), Amblyomma spp. (n = 8, 3.4%), and Ripicephalus spp. (n = 2, 0.8%), and 59.1% (140/237) of them were positive for Coxiella spp. While Sanger sequencing and plasmid-type identification revealed a C. burnetii that harbours the QpRS-plasmid, MLVA/VNTR genotyping showed a new genotype which was initially named D21. In conclusion, this is the first report of C. burnetii in ticks in Somalia. The findings denote the possibility that C. burnetii is endemic in Somalia. Further epidemiological studies investigating samples from humans, animals, and ticks within the context of "One Health" are warranted.

Identification of Risk Factors Associated with Resistant Escherichia coli Isolates from Poultry Farms in the East Coast of Peninsular Malaysia: A Cross Sectional Study.

Antimicrobial resistance is of concern to global health security worldwide. We aimed to identify the prevalence, resistance patterns, and risk factors associated with Escherichia coli (E. coli) resistance from poultry farms in Kelantan, Terengganu, and Pahang states of east coast peninsular Malaysia. Between 8 February 2019 and 23 February 2020, a total of 371 samples (cloacal swabs = 259; faecal = 84; Sewage = 14, Tap water = 14) were collected. Characteristics of the sampled farms including management type, biosecurity, and history of disease were obtained using semi-structured questionnaire. Presumptive E. coli isolates were identified based on colony morphology with subsequent biochemical and PCR confirmation. Susceptibility of isolates was tested against a panel of 12 antimicrobials and interpreted alongside risk factor data obtained from the surveys. We isolated 717 E. coli samples from poultry and environmental samples. Our findings revealed that cloacal (17.8%, 46/259), faecal (22.6%, 19/84), sewage (14.3%, 2/14) and tap water (7.1%, 1/14) were significantly (p < 0.003) resistant to at least three classes of antimicrobials. Resistance to tetracycline class were predominantly observed in faecal samples (69%, 58/84), followed by cloacal (64.1%, 166/259), sewage (35.7%, 5/14), and tap water (7.1%, 1/84), respectively. Sewage water (OR = 7.22, 95% CI = 0.95-151.21) had significant association with antimicrobial resistance (AMR) acquisition. Multivariate regression analysis identified that the risk factors including sewage samples (OR = 7.43, 95% CI = 0.96-156.87) and farm size are leading drivers of E. coli antimicrobial resistance in the participating states of east coast peninsular Malaysia. We observed that the resistance patterns of E. coli isolates against 12 panel antimicrobials are generally similar in all selected states of east coast peninsular Malaysia. The highest prevalence of resistance was recorded in tetracycline (91.2%), oxytetracycline (89.1%), sulfamethoxazole/trimethoprim (73.1%), doxycycline (63%), and sulfamethoxazole (63%). A close association between different risk factors and the high prevalence of antimicrobial-resistant E. coli strains reflects increased exposure to resistant bacteria and suggests a concern over rising misuse of veterinary antimicrobials that may contribute to the future threat of emergence of multidrug-resistant pathogen isolates. Public health interventions to limit antimicrobial resistance need to be tailored to local poultry farm practices that affect bacterial transmission.

Tick Fauna and Associated Rickettsia, Theileria, and Babesia spp. in Domestic Animals in Sudan (North Kordofan and Kassala States).

Ticks and tick-borne diseases (TBDs) have a major economic impact on animal production worldwide. In the present study, 2410 ticks were collected from January to August 2017 from livestock and other domestic animals in North Kordofan and Kassala, Sudan, for species identification and investigation of Rickettsia spp. and piroplasms, either individually or as pools containing up to 10 ticks by molecular methods. In total, 13 tick species were identified by morphology and 16S rDNA sequencing. The most frequent tick species were Hyalomma impeltatum (24.90%), Rhipicephalus evertsi evertsi (18.84%), Amblyomma lepidum (16.06%), and Rhipicephalus camicasi (12.49%). A pan-Rickettsia real-time PCR revealed an overall minimum infection rate (MIR) with Rickettsia spp. of 5.64% (136 positive tick pools/2410 total ticks). Rickettsia africae and Rickettsia aeschlimannii were the most frequently identified species by sequencing. Furthermore, the following highly pathogenic livestock parasites were detected: Theileria annulata, Theileria lestoquardi, Theileria equi, and Babesia caballi. The present study documented Rhipicephalus afranicus as well as Rickettsia conorii israelensis, Rickettsia massiliae, and Babesia pecorum for the first time in Sudan. These findings are significant for the animal production sector as well as in terms of One Health, as the detected Rickettsia spp. can cause serious illness in humans.

Morphological abnormalities in ticks (Acari: Ixodidae) collected from domestic animal species in Sudan.

Morphological abnormalities in ticks have rarely been reported in nature. The existing knowledge about anomalies in ticks collected in Africa is very sparse. In this paper, we describe abnormalities in Amblyomma, Hyalomma, and Rhipicephalus ticks collected from cattle, sheep, goats, camels, and horses in Kassala and North Kordofan states, Sudan, between January and August 2017. A number of 15 adult ticks displayed one or several local anomalies, such as ectromely, abnormalities of the ventral plates, and body deformities, besides newly described multiple cuticula scars. This study presents the first report of local anomalies in ticks belonging to three genera in Sudan and highlights the need to investigate the association between such morphological abnormalities and tick biology.

Ixodid tick species and two tick-borne pathogens in three areas in the Sudan.

Ticks are important parasites from economic and public health points of view because of their ability to reduce farm animals' productivity and transmit zoonotic diseases. We conducted this cross-sectional study between January and March 2016 and between March and April 2017 to identify tick species in West Darfur, Al-Jazeera, and the River Nile states in the Sudan and to investigate whether these ticks carry Rickettsia spp. and Crimean-Congo hemorrhagic fever (CCHF) virus. In total, 1593 ticks were collected from 207 animals and identified based on morphology or 16S rRNA gene and tested for Rickettsia spp. and CCHF virus either individually or as pools containing 2 to 10 pooled ticks using molecular methods. Overall, 14 tick species belonging to three genera, namely Amblyomma, Hyalomma, and Rhipicephalus, were identified. Hyalomma anatolicum and Rhipicephalus evertsi evertsi were the most frequent ticks. A total of 561 tests comprised of individual or pooled ticks were conducted and 13.7% (77/561) were positive for Rickettsia spp. which were mostly Rickettsia aeschlimannii and R. africae. The highest positivity was noticed among H. rufipes collected from cattle and camels in West Darfur. However, none of the screened Hyalomma ticks harbored CCHF viral RNA. These findings suggest that there might be a risk of zoonotic transmission of Rickettsia spp. by ticks but zoonotic transmission of CCHF virus is apparently doubtful. An in-depth and a country-wide epidemiological study is needed to better understand the dynamic of Rickettsia spp. and CCHF virus in the Sudan.

Crimean-Congo haemorrhagic fever virus in Hyalomma impeltatum ticks from North Kordofan, the Sudan.

An evidence for Crimean-Congo hemorrhagic fever virus (CCHFV) was found in Hyalomma impeltatum ticks collected from sheep in North Kordofan in the Sudan. Based on sequencing of the partial segment S, the detected virus belongs to lineage I with closest similarity to CCHFV strains from Senegal. So far, this lineage is unknown in the Sudan.

Seroprevalence and risk factors of caprine brucellosis in Khartoum state, Sudan.

AIM: This cross-sectional study was conducted from April to July 2012 in Khartoum state, Sudan, to determine the seroprevalence of brucellosis in goats and to investigate potential risk factors associated with this disease. MATERIALS AND METHODS: A total of 307 serum samples were collected from both sexes of goats in four different localities and were subjected to testing for brucellosis using rose bengal plate test (RBPT), serum agglutination test (SAT), and competitive enzyme-linked immunosorbent assay (cELISA). RESULTS: The overall seroprevalence was 11.4% (n=35) with a 95% confidence interval (CI) ranging from 7.80 to 15.0. Out of these 35 RBPT-positive samples, the positivity of 18 and 17 were confirmed by SAT and cELISA, respectively. A significant statistical variation was observed between brucellosis seroprevalences in goats purchased from local animal markets and goats that were raised at the farm. Conversely, such significant variations were not observed among the categories of other risk factors with seroprevalences ranging from 3.0% (95% CI between 0.40 and 7.20) to 16.3% (95% CI between 10.4 and 22.3). Location (χ2=9.33, df=3, p=0.02), breed (χ2=3.52, df=1, p=0.05), herd size (χ2=6.59, df=2, p=0.03), and herd expansion (χ2=5.39, df=1, p=0.02) were associated with RBPT-positive status for brucella in the two-tailed Chi-square test. In addition, Sharq an-Nil locality and goats raised at the farm had increased odds of being RBPT positive. CONCLUSION: Brucellosis was detected in goats in all surveyed localities. An effort should be made to educate goat owners/herders about brucellosis as well as about the importance of vaccination.

A Study on the Nature of Association between Demodex Mites and Bacteria Involved in Skin and Meibomian Gland Lesions of Demodectic Mange in Cattle.

The nature of association between Demodex mites and bacteria involved in bovine demodectic mange lesions and the normal flora inhabiting the skin of noninfected animals was investigated. Demodex bovis and D. ghanensis mites were isolated from the infected purulent material extracted from skin and meibomian gland lesions, respectively. The mites could not be demonstrated in skin brushings or impression smears from the eyes of noninfected cattle. Pathogenic bacteria (Staphylococcus aureus and Streptococcus pyogenes (Group A)) and opportunistic organisms (Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Trueperella pyogenes) were isolated from skin lesions of demodectic mange, and Moraxella bovis and Staphylococcus aureus were isolated from meibomian gland lesions. Bacillus subtilis, Escherichia coli, Proteus vulgaris, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes (Group A) were isolated from skin brushings from noninfected cattle. The nature of association between Demodex mites and bacteria in demodectic mange lesions is synergistic and of equal significance. Pathogenic and opportunistic bacteria facilitated the establishment of Demodex mites in the lesions produced and provided an excellent microclimate for the mites to propagate and reproduce, resulting in severe and progressive disease. The "high-turnover" granulomatous reaction which characterized the histopathological changes proved that Demodex mites and associated bacteria were persistent and immunogenic.