Tuberculosis is a major public health problem in Ethiopia. The aims of this study were (i) to investigate the recovery rate of M. tuberculosis from smear positive single morning sputum specimens subjected to long-term storage at -20°C, (ii) to assess the level and risk factors for first- and second-line anti-TB drug resistance, (iii) to evaluate the performance of the GenoType®MTBDRplus and GenoType®MTBDRsl assays for drug susceptibility testing compared to the BacT/ALERT 3D system as reference method, (iv) to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among M. tuberculosis isolates, and (v) to study the population structure and transmission dynamics of M. tuberculosis isolates from patients in Amhara region, Northwest Ethiopia. The median specimen storage time was 132 days. Of 319 specimens, 90.0% were culture positive. The length of time of sputum storage had no significant effect on the recovery rate of M. tuberculosis. Of 260 M. tuberculosis isolates, 15.8% were resistant to at least one first-line drug, 5.0% were multidrug resistant (MDR) and 3.5% were resistant to all first-line drugs. Any resistance to INH, RMP, streptomycin (STM), EMB and pyrazinamide (PZA) was 13.8%, 5.8%, 10.0%, 7.3% and 4.6%, respectively. All isolates were susceptible to second-line drugs. The GenoType®MTBDRplus assay had a sensitivity of 92% and specificity of 99% to detect INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType®MTBDRsl assay had a sensitivity of 42% and specificity of 100% to detect EMB resistance. According to the molecular methods, mutations conferring resistance to INH, RMP, or EMB were detected in 13.5%, 5.8%, and 3.1% of the isolates, respectively, while mutation conferring MDR was present in 5.0% of the isolates. Of 244 M. tuberculosis isolates, 59.0% were classified as known lineages; Dehli/CAS (38.9%), Haarlem (8.6%), Ural (3.3%), LAM (3.3%), TUR (2.0%), X-type (1.2%), S-type (0.8%), Beijing (0.4%) and Uganda II (0.4%) lineage. Interestingly, 31.6% of the isolates were grouped in to four previously undefined phylogenetic lineages and were named as Ethiopia_3 (13.1%), Ethiopia_1 (7.8%), Ethiopia_H37Rv like (7.0%) and Ethiopia_2 (3.7%) lineages. The remaining 9.4% of the isolates could not be assigned to the known or new lineages. Overall, 45.1% of the isolates were grouped in clusters, indicating high rate of recent transmission. Similarly, 66.7% of MDR strains were grouped in clusters.

The Enterovirus genus within the family Picornaviridae contains over 100 serotypes, of which sixty-four are known to be human pathogens. Infection with this group of RNA viruses produces a myriad of clinical conditions including poliomyelitis, meningitis, encephalitis, respiratory illnesses, and hand-foot-and-mouth disease. Outbreaks have been documented worldwide; significant morbidity and mortality exist to warrant laboratory surveillance. Traditionally, enteroviruses have been identified to the level of serotype by the serum neutralization assay. However, numerous problems are associated with this assay. The serum neutralization assay is labor intensive, results are often ambiguous, and reagents are becoming difficult to obtain. Recently, molecular-based typing protocols have been described that are cost effective and produce results that are more reliable. The overall objective of this thesis was to implement a molecular-based typing protocol to replace the serum neutralization method currently used. Three specific aims were identified to reach this objective. First, a database cataloging all enteroviruses isolated at the Florida Department of Health - Tampa Branch Laboratory from 1981 through 2002 was created. Serotype prevalence, specimen submission rates, and temporal trends were analyzed to demonstrate the public health importance of enterovirus surveillance. Next, five oligonucleotide primer sets were compared with respect to sensitivity, specificity, and overall utility in molecular typing protocols developed to accurately determine enterovirus type. Finally, the most effective molecular assay was used to conduct two basic molecular epidemiological analyses of intratypic variation of Coxsackievirus B5 isolates, and of intratypic variation of successive Echovirus 9 passages. The results from this study show that implementation of a molecular-based typing system for enteroviruses would be an improvement over current enterovirus serotyping methods. Results are obtained more rapidly and are more reliable. The implementation of such a system would improve the surveillance capabilities of the State of Florida Department of Health.

An investigation of the distribution of porcine reproduction and respiratory syndrome virus (PRRSV) and factors associated with the presence of PRRSV in Ontario from 2004 – 2007 was conducted. Surveys on the presence of clinical signs associated with PRRS, management practices, animal suppliers, and herd location were administered to the managers of 458 PRRSV positive herds and 61 PRRSV negative herds. Open reading frame (ORF) 5 of the PRRSV genome was sequenced from herds with PRRSV. PRRSV positive herds were compared to PRRSV negative herds. Management practices associated with being PRRSV positive were: not washing animal- and feed-delivery vehicles, feed-delivery and animal-transport vehicles visiting multiple herds at one time, allowing a truck driver to enter the barn, not requiring visitors to shower prior to farm entry, and not utilizing all-in all-out flow in gilt and finisher barns. Specific PRRSV restriction fragment length polymorphism (RFLP) genotypes of the ORF5 gene were compared with clinical signs. Herds with RFLP type ‘1-undetermined-4’, ‘1-undetermined-2’ and 1-3-4 were associated with clinical signs in sows and 2-6-2 was associated with finisher mortality compared to herds with vaccine virus. Additionally, genotypes 1-3-4 and 1-8-4 increased in frequency during this study. The between-herd PRRSV similarity of genome and clinical signs were compared. Abortions and stillbirths were associated with similarity in genetic sequences between herds. This relationship did not extend to those herds where vaccine virus was identified. Patterns in space and time of herds with different RFLP types of PRRSV were investigated after accounting for ownership. There was weak evidence to suggest local spread the genotype 1-3-4. The association between genetic similarity and proximity in space, time, ownership, animal, and semen suppliers was tested. Significant correlation was detected for distances up to 30 km. After controlling for ownership, only small associations between breeding stock and semen suppliers and genetic similarity of PRRSV were found. The spread of PRRSV among herds in Ontario cannot be attributed to any one factor. However, similarity in ownership between herds was a key variable indicating that movement of animals, personnel, and vehicles among herds must be measured in future investigations of PRRSV dynamics. / Ontario Pork and the Canada-Ontario Research and Development (CORD) Program and the OMAFRA/University of Guelph agreement

This thesis reports the development of Comparative Genomic Fingerprinting (CGF), a rapid genotyping method for Campylobacter jejuni that assesses the conservation status of 20 genes previously described as having high intraspecies variability based on comparative genomics studies. This novel method for genotyping C. jejuni, CGF was validated two-fold. First, by comparison to flaA restriction fragment length polymorphism analysis, and second a subset of isolates was validated using two higher resolution CGF assays assessing 35 and 119 genes. CGF was then tested in a molecular epidemiological study of C. jejuni isolated from environmental, animal and human clinical samples from southern Alberta. Reservoirs of infection, subtypes associated with higher incidence of human infection, and the persistence of prevalent subtypes in animal/environmental reservoirs were identified. This thesis demonstrates that CGF analysis is robust and can be used to rapidly assess genetic similarity of C. jejuni isolates and to detect epidemiologically relevant clonal groups. / xii, 184 leaves : ill. ; 29 cm

Gonorrhea is caused by the human pathogen Neisseria gonorrhoeae. More than 106 million new cases of N. gonorrhoeae infections occur each year worldwide. There is no vaccine available against gonococcal infections and treatment of gonorrhea with antibiotics is the only way to eradicate infection. The high prevalence of antibiotic resistance (AMR) in this microorganism makes the effective treatment of gonococcal infections increasingly problematic. The emergence of AMR, especially to extended spectrum cephalosporins (i.e. cefixime and ceftriaxone) which are the last possibilities for single dose treatment options for gonococcal infections, is a serious concern. Gonorrhea may become an untreatable infection in the near future. Saskatchewan (SK) has one of the highest rates of gonorrhea in Canada. In order to better characterize the gonorrhea epidemic in SK, the objectives of the present research were to determine the prevalence and trends of AMR and emerging AMR mechanisms in N. gonorrhoeae isolates. AMR mechanisms were ascertained for the first time in SK in order to identify genetic causes of resistance. This was completed by determining and analyzing the DNA sequences of various genes - penA, mtrR, porB ponA, gyrA, parC mtrR, 23S rRNA alleles and erm –implicated in gonococcal AMR. The population dynamics of the N. gonorrhoeae isolates in SK was investigated by DNA based molecular methods to determine strain distribution, evolution of AMR phenotypes, and association between strain types (STs) and AMR genotypes and phenotypes. N. gonorrhoeae isolates (n=427) from Saskatchewan (2003-2011) were susceptible to antibiotics now recommended for treatment - cefixime, ceftriaxone and spectinomycin. Over 95% of the isolates tested were also susceptible to penicillin (96%) and ciprofloxacin (95.5%), antibiotics no longer recommended for treatment, and azithromycin (99.4%). Tetracycline resistance was also high (50.1%). N. gonorrhoeae isolates that were resistant to the antibiotics tested and also those isolates with MICs ≥0.003 mg/L to cefixime and ceftriaxone were analyzed (n=146) to determine their resistance mechanisms. This analysis revealed that reduced susceptibility to ceftriaxone and cefixime and resistance to penicillin is mediated by specific mutations in penicillin binding protein 2 (PBP2), in the promoter and dimerization domains of MtrR and porin protein (PorB). Novel mutations and combinations of mutations were noted. Ciprofloxacin resistant N. gonorrhoeae isolates carried double mutations in GyrA (S91F and D95G/N) and a S87R or S88P substitution in ParC. Isolates resistant to azithromycin had specific mutations in all the four alleles of 23S rRNA as well as in the DNA binding domain of MtrR. Most resistance was chromosomally mediated while plasmid-mediated resistance to penicillin (0.93% of penicillin resistant isolates) and tetracycline (3.3%) was low. DNA based strain typing methods such as porB-DNA sequencing, N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) showed that the gonococcal population in SK differs appreciably from both other Canadian provinces and from strains reported internationally. MLST analysis, which ascertains the evolution of isolates over time, demonstrated that penicillin and tetracycline resistant isolates in SK evolved through spontaneous mutations in established lineages. Ciprofloxacin and azithromycin resistant N. gonorrhoeae isolates, on the other hand, were introduced into SK from outside the province. Significant associations between particular mutation pattern combinations in resistance determining genes and specific NG-MAST STs were identified e.g. NG-MAST ST 25 was associated with specific combined mutation patterns in PBP2, MtrR and PorB and antibiotic susceptibility; and, NG-MAST ST 3654 was associated with another PBP2/MtrR/PorB mutation pattern, chromosomal resistance to penicillin and tetracycline and elevated MICs to cefixime. This research shows the importance of regional antimicrobial susceptibility monitoring. In the context of SK, this means that local surveillance of gonococcal AMR may be used to develop policies for regional treatment guidelines which promote the prudent use of antimicrobials for treatment, including those antibiotics which may no longer be used in other regions due to higher AMR rates. Further, the significant association between particular AMR mutation pattern combinations and specific STs indicates that AMR might be predicted. These results should assist in the development of non-culture-based tests for the diagnosis of gonococcal AMR similar to nucleic acid amplification tests used to diagnose N. gonorrhoeae infections.

With over 35 million people currently infected, the World Health Organization considers HIV a global pandemic. HIV is characterized by a high mutation rate, which allows it to evade the host immune system and develop resistance to drugs. However, this extraordinary adaptive ability may also be the key to HIV’s demise. Through the field of phylodynamics, the evolutionary behavior of the virus is being studied in an attempt to control the epidemic. In this thesis, three papers are presented in which we analyze sequences generated through the Canadian HIV Strain and Drug Resistance Surveillance program. In chapter 2 we validate a classifier which distinguishes between recent and established infections based on the proportion of mixed bases observed in population-based pol sequences. Our results will help identify recent infections and improve incidence calculations. In chapter 3, we investigate immune-induced patterns in HIV that are shared by patients of the same ethnicity. An understanding of the forces shaping HIV evolution is instrumental to the development of a vaccine relevant to the Canadian epidemic. In chapter 4, we present preliminary results of a historical reconstruction of HIV across the provinces of Canada. This analysis will highlight strategies that have succeeded or failed in controlling the epidemic. Furthermore, our work will establish whether non-B subtypes of HIV are an increasing threat to Canadian public health. Overall, this thesis provides the first country-wide evolutionary and phylogenetic analysis of the HIV epidemic.

Physicians frequently face challenges in predicting which bacterial subpopulations are likely to cause severe infections. A more accurate prediction of virulence would improve diagnostics and limit the extent of antibiotic resistance. Nowadays, bacterial pathogens can be typed with high accuracy with advanced genotyping technologies. However, effective translation of bacterial genotyping data into assessments of clinical risk remains largely unexplored. The discovery of unknown virulence genes is another key determinant of successful prediction of infectious disease outcomes. The trial-and-error method for virulence gene discovery is time-consuming and resource-intensive. Selecting candidate genes with higher precision can thus reduce the number of futile trials. Several in silico candidate gene prioritisation (CGP) methods have been proposed to aid the search for genes responsible for inherited diseases in human. It remains uninvestigated as to how the CGP concept can assist with virulence gene discovery in bacterial pathogens. The main contribution of this thesis is to demonstrate the value of translational bioinformatics methods to address challenges in virulence prediction and virulence gene discovery. This thesis studied an important perinatal bacterial pathogen, group B streptococcus (GBS), the leading cause of neonatal sepsis and meningitis in developed countries. While several antibiotic prophylactic programs have successfully reduced the number of early-onset neonatal diseases (infections that occur within 7 days of life), the prevalence of late-onset infections (infections that occur between 7??30 days of life) remained constant. In addition, the widespread use of intrapartum prophylactic antibiotics may introduce undue risk of penicillin allergy and may trigger the development of antibiotic-resistant microorganisms. To minimising such potential harm, a more targeted approach of antibiotic use is required. Distinguish virulent GBS strains from colonising counterparts thus lays the cornerstone of achieving the goal of tailored therapy. There are three aims of this thesis: 1. Prediction of virulence by analysis of bacterial genotype data: To identify markers that may be associated with GBS virulence, statistical analysis was performed on GBS genotype data consisting of 780 invasive and 132 colonising S. agalactiae isolates. From a panel of 18 molecular markers studied, only alp3 gene (which encodes a surface protein antigen commonly associated with serotype V) showed an increased association with invasive diseases (OR=2.93, p=0.0003, Fisher??s exact test). Molecular serotype II (OR=10.0, p=0.0007) was found to have a significant association with early-onset neonatal disease when compared with late-onset diseases. To investigate whether clinical outcomes can be predicted by the panel of genotype markers, logistic regression and machine learning algorithms were applied to distinguish invasive isolates from colonising isolates. Nevertheless, the predictive analysis only yielded weak predictive power (area under ROC curve, AUC: 0.56??0.71, stratified 10-fold cross-validation). It was concluded that a definitive predictive relationship between the molecular markers and clinical outcomes may be lacking, and more discriminative markers of GBS virulence are needed to be investigated. 2. Development of two computational CGP methods to assist with functional discovery of prokaryotic genes: Two in silico CGP methods were developed based on comparative genomics: statistical CGP exploits the differences in gene frequency against phenotypic groups, while inductive CGP applies supervised machine learning to identify genes with similar occurrence patterns across a range of bacterial genomes. Three rediscovery experiments were carried out to evaluate the CGP methods: a) Rediscovery of peptidoglycan genes was attempted with 417 published bacterial genome sequences. Both CGP methods achieved their best AUC >0.911 in Escherichia coli K-12 and >0.978 Streptococcus agalactiae 2603 (SA-2603) genomes, with an average improvement in precision of >3.2-fold and a maximum of >27-fold using statistical CGP. A median AUC of >0.95 could still be achieved with as few as 10 genome examples in each group in the rediscovery of the peptidoglycan metabolism genes. b) A maximum of 109-fold improvement in precision was achieved in the rediscovery of anaerobic fermentation genes. c) In the rediscovery experiment with genes of 31 metabolic pathways in SA-2603, 14 pathways achieved an AUC >0.9 and 28 pathways achieved AUC >0.8 with the best inductive CGP algorithms. The results from the rediscovery experiments demonstrated that the two CGP methods can assist with the study of functionally uncategorised genomic regions and the discovery of bacterial gene-function relationships. 3. Application of the CGP methods to discover GBS virulence genes: Both statistical and inductive CGP were applied to assist with the discovery of unknown GBS virulence factors. Among a list of hypothetical protein genes, several highly-ranked genes were plausibly involved in molecular mechanisms in GBS pathogenesis, including several genes encoding family 8 glycosyltransferase, family 1 and family 2 glycosyltransferase, multiple adhesins, streptococcal neuraminidase, staphylokinase, and other factors that may have roles in contributing to GBS virulence. Such genes may be candidates for further biological validation. In addition, the co-occurrence of these genes with currently known virulence factors suggested that the virulence mechanisms of GBS in causing perinatal diseases are multifactorial. The procedure demonstrated in this prioritisation task should assist with the discovery of virulence genes in other pathogenic bacteria.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 83-101) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 83-101) Also available online.

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Obrigada Luciana Pizzani on 2018-04-03T18:47:46Z (GMT) / Submitted by MÔNICA DA SILVEIRA null (monysilveira@hotmail.com) on 2018-04-03T19:30:34Z No. of bitstreams: 1 TESE MONICA Envio corrigida final word modificada ok.pdf: 3526672 bytes, checksum: aa12e249981a8458333be778d0062faf (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-04-03T19:40:57Z (GMT) No. of bitstreams: 1 silveira_m_dr_bot.pdf: 3526672 bytes, checksum: aa12e249981a8458333be778d0062faf (MD5) / Made available in DSpace on 2018-04-03T19:40:57Z (GMT). No. of bitstreams: 1 silveira_m_dr_bot.pdf: 3526672 bytes, checksum: aa12e249981a8458333be778d0062faf (MD5) Previous issue date: 2018-03-02 / Estima-se que no Brasil ocorram centenas de milhares de Infecções Relacionadas à Assistência (IRAS) à Saúde todos os anos, e uma proporção significativa desses quadros é causada por Acinetobacter baumannii. Essa bactéria, hiperendêmica em hospitais brasileiros, é geralmente resistente a múltiplas drogas, restando um escasso arsenal terapêutico. Para além dos fatores de risco tradicionais (comorbidades, exposição ao ambiente hospitalar, procedimentos invasivos, uso de antimicrobianos), estudos recentes demonstraram sazonalidade e associação de sua incidência com altas temperaturas ambientais. No entanto, as razões para esses achados permanecem obscuras. Nós conduzimos estudos em três hospitais públicos conveniados do município de Bauru-SP, com o objetivo de colaborar para a compreensão da epidemiologia do A. baumannii, com ênfase nos isolados resistentes a carbapenêmicos (Carbapenem-resistant A. baumannii, CRAB). O primeiro deles, com delineamento ecológico, analisou o comportamento de taxas mensais de incidência de A. baumannii como um todo e em subgrupos (enfermarias versus Unidades de Terapia Intensiva [UTI], amostras clínicas totais versus hemoculturas) no período de 2006 a 2017. Estudamos o impacto de temperatura, umidade e pluviosidade sobre essa incidência. Como resultados, identificamos que temperaturas médias mensais acima do percentil 75 (24,7°C) estavam associadas a maior incidência de A. baumannii como um todo e de CRAB. Curiosamente, esses achados foram mais consistentes nas UTI e para isolados de hemoculturas. No segundo estudo, tipo “caso-controle” analisamos a clonalidade e fatores de risco para infecções da corrente sanguínea causadas por CRAB (CRABBloodstream infections, CRAB-BSI). Foram estudados 38 pacientes com CRABBSI e 76 controles. Fatores de risco significantes (p<0,05) em análise multivariada para aquisição de CRAB-BSI foram a inserção de cateter venoso central e o uso de cefepima ou meropenem. Na genotipagem por Pulsed-Field Gel Electrophoresis (PFGE), identificamos 12 perfis, sendo que 7 deles agrupavam mais de um isolado. Além disso, identificamos a presença de genes codificadores das enzimas OXA-23 e OXA-51, associadas à resistência aos carbapenêmicos. O terceiro estudo comparou os mesmos 38 casos de CRAB e 76 controles, em delineamento tipo “coorte pareada”, para o desfecho “óbito durante a internação”. Como esperado, os casos foram significantemente associados a maior risco de óbito em modelos de análise de sobrevida. Além disso, quando estudados somente os casos, o uso de Aminoglicosídeos e Polimixina após o diagnóstico se associou a melhor prognóstico. Concluímos que o A. baumannii é um agente potencialmente letal, que se transmite no interior de hospitais e entre diferentes serviços, com incidência aumentada em períodos quentes. / It is estimated that in Brazil hundreds of thousands of Healthcare Associated Infections (HCAI) occur every year, and a significant proportion of these are caused by Acinetobacter baumannii. This bacterium, hyperendemic in Brazilian hospitals, is generally resistant to multiple drugs, leaving behind a scarce therapeutic arsenal. In addition to the traditional risk factors (comorbidities, exposure to the hospital environment, invasive procedures, antimicrobial use), recent studies have demonstrated seasonality and association of A. baumannii incidence with high environmental temperatures. However, the reasons underlying these findings remain obscure. We conducted studies in three public hospitals in the city of Bauru-SP, Brazil, in order to contribute to understanding the epidemiology of A. baumannii, with emphasis on carbapenem resistant isolates (CRAB). The first one, with ecological design, analyzed the behavior of monthly rates of incidence of overall A. baumannii and several subgroups (non-critical wards vs. Intensive Care Units (ICU), overall clinical samples vs. blood cultures) from 2006 to 2017. We studied the impact of temperature, humidity and rainfall on this incidence. As a result, we identified that average monthly temperatures above the 75th percentile (24.7°C) were associated with a higher incidence of overall A. baumannii and CRAB. Interestingly, these findings were more consistent in ICUs and for isolates of blood cultures. In the second study (casecontrol), we analyzed the clonality and risk factors for CRAB bloodstream infections (CRAB-BSI). We studied 38 patients with CRAB-BSI and 76 controls. Significant risk factors (p <0.05) in multivariable analysis for CRAB-BSI acquisition were central venous catheter insertion and use of cefepime or meropenem. In the Pulsed-Field Gel Electrophoresis (PFGE) genotyping, we identified 12 patterns, 7 of which grouped more than one isolate. In addition, we identified the presence of genes encoding the enzymes OXA-23 and OXA-51, associated with resistance to carbapenems. The third study compared the same 38 cases of CRAB and 76 controls, in a "matched cohort" design, for the outcome "in-hospital death”. As expected, the cases were significantly associated with a higher risk of death in survival analysis models. In addition, when only cases were studied, the use of Aminoglycosides and Polimyxyns after diagnosis was associated with a better prognosis. We conclude that A. baumannii is a potentially lethal agent, which is transmitted within hospitals and between different services, with an increased incidence during warm periods.