1 A INTRODUCTION RATIONALE Streptococcus suis is a gram positive cocci that could transmit from animals (mainly pigs) to human via ingestion behaviors of uncooked foods and non-intact skin exposure during pig butchering and pork meat processing It could cause an endemic with a high mortality rate raging from 12,8% to 27,9% Patients with S suis infection after discharge could have severe complications sush as hearing loss (from 50% to 66,4%), vestibular disorders (22,7%), limb gangrene and amputation These complications have a low recovery rate, leading to a great burden in healthcare system and economic system In Vietnam, disease due to S.suis is a topical issue in the field of infectious diseases because of specific epidemiological factors and severe clinical consequences, especially in case of inappropriate diagnosis and late treatment This disease ranked on the 6th position among 10 infectious diseases with the highest incidence and mortality rate Currently, the resistance of S.suis to almost antibiotics used in farming results in a high risk of antimicrobial resistance in human Resistance genes and virulence related genes have been studied in the world In Vietnam, major studies are mainly from the South Until now, despite a widespread circulation, there has been no study on comprehensive evaluation of clinical and laboratory characteristics, prognostic and virulence factors, distribution of these features by clinical syndromes of S.suis in Vietnam Therefore, we conduct this research for three following objectives: Describe clinical and laboratory characteristics, clinical syndromes in patients infected with Streptococcus suis Determine prognostic factors of mortality in patients infected with Streptococcus suis Evaluate the antibiotic susceptibility and identify some resistance genes, virulence genes of Streptococcus suis URGENCY OF THESIS Due to a different distribution of clinical syndromes among regions in Vietnam, there should have been an overall picture of clinical and laboratory characteristics, mortality prognostic factors in patients with S.suis infection in order to help physicians avoid a pitfall in practice, access to proper diagnosis and treatment, reduce the mortality risk In addition, the uncontrolled use of antibiotics in farming could lead to an increase in antibiotic resistance associated with the emergence of high virulent strains in pigs, which makes treatment becomes difficulty Therefore, it is necessary to understand genotypes, resistance genes and virulence related genes of S.suis in human THESIS STRUCTURE The length of this thesis was 124 pages, divided into chapters (Background: 30 pages; Material and Method: 23 pages; Results: 32 pages; Discussion: 34 pages There were 40 tables; charts; 10 figures; 115 references (both in English and Vietnamese, with 35% documents from 2014) B CONTENT Chapter BACKGROUND Microbiology features and pathogenesis of Streptococcus suis In 1950s, Streptococcus suis which caused infection in pig was initially reported in England and Netherlands It is facultative gram positive anaerobes with a spherical shape It is transmitted via uncooked ingestion and exposure through wounds in the skin while contact with infected pigs in butchering and uncooked food processing S.suis breach the epithelial barriers, exist in blood then invade multiple organs and cause inflammatory reactions in these organs Moreover, S.suis is able to cross the blood-brain barrier to invade the central nervous system, leading to meningitis Epidemiology of Streptococcus suis infection in human The first case of S.suis infection in human was described in 1968 in Denmark Since then, significantly increasing numbers of human cases have been reported in many countries including Netherlands, Belgium, Denmark, England, France, Germany, Sweden, Canada Asia regions such as Hong Kong, Taiwan, China, Thailand and Vietnam notably have a great proportion of 90.2% S.suis infection could cause an endemic, typically in China where observed two outbreaks in 1999 and 2005 with huge numbers of infected cases and a high mortality rate In Vietnam, a case of S.suis infection was first reported in November 1996 with meningitis manifestation Since then, human cases infected by S.suis have been reported in all three regions in Vietnam According to General Department of Preventive Medicine – Ministry of Health, until 2016, the rate of S.suis infection in human was 0.007 cases per 100,000 persons and this disease was at the th rank among 10 infectious diseases with the highest incidence and mortality rate Clinical syndromes of S suis infection and prognostic factors Streptococcus suis causes a variety of clinical manifestations in human such as meningitis, sepsis, septic shock, endocarditis…Around the world, 68% of observed clinical manifestations are meningitis Hearing loss is the most common complication in patients with S.suis meningitis with approximately 50% and a very low recovery rate Septic shock usually has a rapid progression with a high mortality rate ranging from 60% to 80% depending on the research Factors like coagulation disorders, multiorgan failure, comorbidities and short incubation period are prognostic factors in patients with S Suis infection Diagnosis and treatment of patients with Streptococcus suis infection Diagnosis of S.suis infection is relied on clinical manifestations of meningitis (fever, headache, altered mental status, neck stiffness, nausea, vomiting…), sepsis (fever, myalgia, hemorrhagic necrotic rash, tachycardia…) in association with definitive tests for pathogen identification sush as positive culture or PCR Specific treatments are betalactam antibiotics with supportive care like mechanical ventilation, renal replacement therapy… Antibiotic resistance and virulence factors of S suis S suis is almost resistant to all antibiotics used in the farming like tetracycline, erythromycin; some strains are resistant to ceftriaxon and fluoroquinolone Resistance genes were also identified such as erm(B) (erythromycin), cat (chloramphenicol), tet(M), tet(O), tet(L) (tetracyclin) Virulence factors are predominant existence in type S suis which transmits directly from animals to human Many studies in the world indicated three major virulence factors including: protein muramidase-released protein (MRP) encoded by mrp gene, suilysin encoded by sly, extracellular factor (EF) encoded by epf gene Technique of molecular biology and the applications in S Suis research The discovery of novel gene sequencing technology makes a significant breakthrough compared to Sanger technology in time, quality and cost of sequencing process Whole genome sequencing of S.suis provides aid for determining serotypes, MLST (Multilocus sequence typing), resistance genes and virulence related genes Chapter MATERIAL AND METHOD Study population 1 2 4 – • • Inclusion criteria: During the study period, we recruited 221 patients with S suis infection who met the following criteria: Age > 16, without gender discrimination Signs and symptoms of sepsis and/or meningitis Blood/cerebrospinal fluid (CSF) culture and/or PCR blood/CSF is positive with S suis Agreement on study participation Exclusion criteria Patients had additional microbiology or molecular results of other bacteria beside S suis Patients had comorbidities including liver and kidney failure before S suis infection Patients had co-infection with HIV, HCV, HBV Patients refused to participate in the study at any time regardless of study period Study design: Prospective study Sample size: Convenience sampling, the study will recruited all patients who satisfied inclusion criteria meet in 2.1.1, and who were treated at National hospital of Tropical diseases from May 2015 to May 2018 Method Patients with definitive diagnosis of S suis infection will be classified into groups: (1) Meningitis, (2) Sepsis with meningitis and (3) Septic shock We will not only evaluate and analyse general characteristics but we also compare features of clinical findings, laboratory tests and prognostic factors, resistance genes, virulence genes and serotype 2.4.1 Study process a) Objective 1: Evaluate general clinical characteristics, laboratory results and clinical syndromes in patients infected with Streptococcus suis Clinical syndromes caused by human infection of S suis: Based on clinical manifestations and sample culture or PCR results, clinical syndromes were divided into three following groups: Meningitis: patients with signs and symptoms of meningitis + CSF culture and/or PCR is positive with S suis Sepsis with meningitis: patients with clinical manifestations of sepsis and meningitis had positive result of S suis in both blood and CSF samples Septic shock: patients with clinical presentations of septic shock + at least positive laboratory result of S suis (culture or PCR in blood and/or CSF) ⇒ Record clinical syndromes will be statistically represented in appropriate quantity, percentage and proper tables or charts – Clinical characteristics: research was carried out to investigate not only general clinical characteristics in patient population but also individual features in each group of clinical syndrome A comparison of individual characteristics among clinical syndromes were made based on exposure history (ingestion, contact ), symptoms (fever, headache, myalgia, nausea, vomiting, diarrhea ), signs (altered mental status, neck stiffness, kernig ) – Laboratory characteristics: we recorded laboratory results in clinical syndromes and noticed a difference by using mean or median comparison (chi-squared test and Kruskal-wallis test ) Laboratory tests included routine tests, tests for pathogen identification and tests for follow-up treatment b) Objective 2: Analyze prognostic factors of mortality in patients infected with Streptococcus suis Patients in the study were divided into 02 groups based on treatment outcome Survival group: patients were cured or made a good recovery and were transferred to provincial/district hospital in order to satisfy expectations of patient or family members Death group: patients were died at the hospital or patients went home for death because of treatment unresponsiveness or the severity of disease progression We utilized univariate and multivariate regression analysis of clinical characteristics, laboratory results, exposure history and comorbidities between two groups to identify prognostic factors of mortality Treatment outcome: Mortality rate, average duration of treatment, sequelae (hearing loss, paralysis, mental issues, limb amputation due to gangrene after discharge) c) Objective 3: Evaluate antibiotic susceptibility patterns and identify some resistance genes, virulence genes of Streptococcus suis • We identified antibiotic sensitivity of S suis by susceptibility testing, MIC calculation with E-test technique (E-test strips manufactured by Biomerieux-France), which is based on the guidance of CLSI M100 Identification of resistance genes, virulence genes, serotypes and genotypes via MLST were based on analysis of S suis genome after performing novel sequencing method by Miseq machine, using primer from standard kit of NextaraXT library manufactured by Illumia – American Results were checked, analysed with experts from Hanoi Oxford University Clinical Research Unit (OUCRU) – Evaluate antibiotic sensitivity of S suis: Determine the sensitivity of some common antibiotics in clinical practice (ampicillin, penicillin, ceftriaxon, levofloxacin, linezolid, vancomycin) and in cattle breeding (erythromycin, clindamycin, tetracycllin) Calculate the percentage of sensitivity and resistance – Determine some resistance genes, plasmids and virulence genes: We identify resistance genes, virulence genes based on a search of following genbank: • For resistance genes: from ARG-ANNOT (Antibiotic Resistance GeneANNOTation), CARD (Comprehensive Antibiotic Resistance Database, Resfinder); • For virulence genes: from VFDB (Virulence Factor Database); – Determine serotype, MLST (Multilocus sequencing type) and phylogenetic tree: Identification of S suis serotype was relied on capsular polysaccharide region where encoded genes in locus cps for the synthesis of bio-functional proteins We downloaded database of cps gene from the system via the following link: https:// github.com/streplab/SsuisSerotyping_pipeline MLST classification of isolated S suis strains were determined owing to basic genes (house keeping gene) We compared this data with database in the website: http://www.mlst.net ⇒ Analyze the distribution of serotypes and genotypes in each clinical syndrome and general distribution of overall population ⇒ Compare genome sequences among MSLT strains and with standard strain Criteria, technique in our study – Criteria for diagnosis of sepsis and septic shock: Surviving Sepsis Campaign (2012) – Criteria for clinical diagnosis of meningitis: Based on Principles and Practice of Infectious Diseases (2013) – Criteria for alcoholism: Based on AUDIT-C score of WHO (2001) and developed by Frank (2008) Patients were diagnosed with alcoholism when they had total score ≥ in men and ≥ in women – Technique for Streptococcus suis identification using Realtime PCR: Use a pair of primers and a probe for cps2 gene to recognize S suis o Primers: • cps2JF (5’- GGTTACTTGCTACTTTTGATGGAAATT-3’) • cps2JR (5’-CGCACCTCTTTTATCTCTTCCAA-3’) o Probe: (FAM-TCAAGAATCTGAGCTGCAAAAGTGTCAAATTGATAMRA) Bioresearch (American) + Machine: 7500 Fast Real-time PCR (Applied Biosystem – American) + A process of real-time PCR for cps2 gene (based on the process performed at Laboratory Department in National Hospital of Tropical Diseases and OUCRU in Hanoi – Culture and identification technique of S suis: Equipment: Blood culture system Bactec 9050/9120: Bactec Dickinson, American and some machines from England, Germany, France; Sheep blood agar, chocolate agar (Oxoid, England) Bacteria identification was based on Vitek compact system from Biomeriux, France – Susceptibility testing and MIC identification with E-test: Use equipment and antibiotic strips (E-test) manufactured by Bio merieux, France Interpretation and Report based on CLSI M100 – Novel sequencing technology and data analysis using bioinformatics sofware: Follow current procedures in National Hospital of Tropical Diseases and Oxford University Clinical Research Unit Data collection: Data collection was carried out via designed case report form (CRF) Data analysis: Software SPSS 16.0, STAT with appropiate statistical methods were performed for data analysis Study location: National Hospital of Tropical Diseases Study duration: years from May 2015 to May 2018 10 Medical Ethics: The study was approval by Institutional Review Board of Hanoi Medical University and National Hospital of Tropical Diseases Chapter RESULTS There were 221 patients who fulfilled the inclusion criteria during study period A higher proportion of male patients was observed in the study (92%) Mean age of patient population was 53.5 with the highest proportion (69.23%) belonged to age group of 40 – 60 Disease due to S.suis occurs predominantly from April to October Clinical and laboratory characteristics of patient population General characteristics – Pattern of exposure conditions Table 3.1 Exposure conditions Frequency Proportion History of exposure (n=221) % Contact exposure 59 26.70 • Pig butchering 41 18.55 • Uncooked food processing 11 4.98 • Pig breeding 3.17 Ingestion exposure 46 20.81 • Eat “tiet canh” 41 18.55 • Eat other raw foods 2.26 Ingestion + Contact 12 5.43 Unknown 104 47.06 There were 117 patients (52.94%) with medical history of exposure to source of infection Eating “tiet canh” made the largest contribution of 23.08% There were 47.06% patients with unknown history of exposure Clinical characteristics – Clinical syndromes Chart 3.1 Clinical syndromes Meningitis was the most common clinical syndrome, followed by sepsis with meningitis (35.8%) and septic shock (18.1%) Table 3.2 General symptoms Frequency Proportion % (n=221) Fever 221 100 Headache 197 89.14 Shivering 151 68.33 Nausea 139 62.90 Vomiting 132 59.73 Myalgia 121 55.00 The most common symptom in S.suis patients were fever, headache and shivering Table 3.3 Symptoms by category of clinical syndrome Meningiti Meningitis with Septic p s sepsis shock Symptoms n=102 n=79 n=40 (%) (%) (%) 102 79 40 Fever (100) (100) (100) 63 53 35 Shivering 0.01 (61.76) (67.09) (87.50) 52 43 26 Myalgia 0.35 (51.49) (54.43) (65.00) 96 72 29 Headache 0.001 (94.12) (91.14) (72.5) 76 49 14