Until now, despite a widespread circulation,there has been no study on comprehensive evaluation of clinical andlaboratory characteristics, prognostic and virulence factors, distribution
Trang 1A INTRODUCTION RATIONALE
Streptococcus suis is a gram positive cocci that could transmit from
animals (mainly pigs) to human via ingestion behaviors of uncookedfoods and non-intact skin exposure during pig butchering and pork meatprocessing 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% to66,4%), 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 Thisdisease ranked on the 6th position among 10 infectious diseases with thehighest incidence and mortality rate Currently, the resistance of S.suis
to almost antibiotics used in farming results in a high risk ofantimicrobial resistance in human Resistance genes and virulencerelated genes have been studied in the world In Vietnam, major studiesare mainly from the South Until now, despite a widespread circulation,there has been no study on comprehensive evaluation of clinical andlaboratory 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:
1 Describe clinical and laboratory characteristics, clinical syndromes in patients infected with Streptococcus suis.
2 Determine prognostic factors of mortality in patients infected with Streptococcus suis.
3 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 andlaboratory 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 Inaddition, the uncontrolled use of antibiotics in farming could lead to anincrease in antibiotic resistance associated with the emergence of high
Trang 2virulent strains in pigs, which makes treatment becomes difficulty.Therefore, it is necessary to understand genotypes, resistance genes andvirulence related genes of S.suis in human.
THESIS STRUCTURE
The length of this thesis was 124 pages, divided into 4 chapters(Background: 30 pages; Material and Method: 23 pages; Results: 32pages; Discussion: 34 pages There were 40 tables; 8 charts; 10 figures;
115 references (both in English and Vietnamese, with 35% documentsfrom 2014)
B CONTENT Chapter 1 BACKGROUND
1.1 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 grampositive anaerobes with a spherical shape It is transmitted via uncookedingestion 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
1.2 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 caseshave been reported in many countries including Netherlands, Belgium,Denmark, England, France, Germany, Sweden, Canada Asia regionssuch 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 withhuge 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 GeneralDepartment 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 6th rank among 10 infectious diseases with thehighest incidence and mortality rate
Trang 31.3 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…Aroundthe 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 Septicshock usually has a rapid progression with a high mortality rate rangingfrom 60% to 80% depending on the research Factors like coagulationdisorders, multiorgan failure, comorbidities and short incubation period
are prognostic factors in patients with S Suis infection.
1.4 Diagnosis and treatment of patients with Streptococcus suis
infection
Diagnosis of S.suis infection is relied on clinical manifestations ofmeningitis (fever, headache, altered mental status, neck stiffness, nausea,vomiting…), sepsis (fever, myalgia, hemorrhagic necrotic rash,tachycardia…) in association with definitive tests for pathogenidentification sush as positive culture or PCR Specific treatments arebetalactam antibiotics with supportive care like mechanical ventilation,renal replacement therapy…
1.5 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 2 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.
1.6 Technique of molecular biology and the applications in S Suis
research
The discovery of novel gene sequencing technology makes asignificant 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 sequencetyping), resistance genes and virulence related genes
Chapter 2 MATERIAL AND METHOD
Trang 42.1 Study population
2.1.1 Inclusion criteria: During the study period, we recruited 221
patients with S suis infection who met the following criteria:
1 Age > 16, without gender discrimination
2 Signs and symptoms of sepsis and/or meningitis
3 Blood/cerebrospinal fluid (CSF) culture and/or PCR blood/CSF
is positive with S suis.
4 Agreement on study participation
2.1.2 Exclusion criteria
1 Patients had additional microbiology or molecular results of
other bacteria beside S suis
2 Patients had comorbidities including liver and kidney failure
before S suis infection
3 Patients had co-infection with HIV, HCV, HBV
4 Patients refused to participate in the study at any time regardless
of study period
2.2 Study design: Prospective study.
2.3 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
2.4 Method
Patients with definitive diagnosis of S suis infection will be
classified into 3 groups: (1) Meningitis, (2) Sepsis with meningitis and(3) Septic shock We will not only evaluate and analyse generalcharacteristics but we also compare features of clinical findings,laboratory tests and prognostic factors, resistance genes, virulence genesand 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, clinicalsyndromes 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
Trang 5 Septic shock: patients with clinical presentations of septic shock
+ at least 1 positive laboratory result of S suis (culture or PCR in blood
and/or CSF)
Record clinical syndromes will be statistically represented inappropriate quantity, percentage and proper tables or charts
– Clinical characteristics: research was carried out to investigate
not only general clinical characteristics in patient population but alsoindividual features in each group of clinical syndrome A comparison ofindividual characteristics among 3 clinical syndromes were madebased on exposure history (ingestion, contact ), symptoms (fever,headache, myalgia, nausea, vomiting, diarrhea ), signs (alteredmental status, neck stiffness, kernig )
– Laboratory characteristics: we recorded laboratory results in 3
clinical syndromes and noticed a difference by using mean or mediancomparison (chi-squared test and Kruskal-wallis test ) Laboratory testsincluded routine tests, tests for pathogen identification and tests forfollow-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 ontreatment outcome
1 Survival group: patients were cured or made a good recoveryand were transferred to provincial/district hospital in order to satisfyexpectations of patient or family members
2 Death group: patients were died at the hospital or patients wenthome for death because of treatment unresponsiveness or the severity ofdisease progression
We utilized univariate and multivariate regression analysis ofclinical characteristics, laboratory results, exposure history andcomorbidities between two groups to identify prognostic factors ofmortality
Treatment outcome: Mortality rate, average duration of treatment,
sequelae (hearing loss, paralysis, mental issues, limb amputation due togangrene 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
Trang 6manufactured by Biomerieux-France), which is based on the guidance ofCLSI 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 primerfrom 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 cattlebreeding (erythromycin, clindamycin, tetracycllin) Calculate thepercentage 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 ResistanceGene-ANNOTation), CARD (Comprehensive Antibiotic ResistanceDatabase, 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 thesynthesis of bio-functional proteins We downloaded database of cpsgene from the system via the following link: https://github.com/streplab/SsuisSerotyping_pipeline MLST classification of
isolated S suis strains were determined owing to 7 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 eachclinical syndrome and general distribution of overall population
Compare genome sequences among MSLT strains and withstandard strain
2.5 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)
Trang 7– Criteria for alcoholism: Based on AUDIT-C score of WHO
(2001) and developed by Frank (2008) Patients were diagnosed withalcoholism when they had total score ≥ 4 in men and ≥ 3 in women
– Technique for Streptococcus suis identification using Realtime PCR: Use a pair of primers and a probe for cps2 gene to
+ 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 TropicalDiseases and OUCRU in Hanoi
– Culture and identification technique of S suis: Equipment:
Blood culture system Bactec 9050/9120: Bactec Dickinson, Americanand some machines from England, Germany, France; Sheep blood agar,chocolate agar (Oxoid, England) Bacteria identification was based onVitek 2 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
2.6 Data collection: Data collection was carried out via designed
case report form (CRF)
2.7 Data analysis: Software SPSS 16.0, STAT with appropiate
statistical methods were performed for data analysis
2.8 Study location: National Hospital of Tropical Diseases 2.9 Study duration: 3 years from May 2015 to May 2018 2.10 Medical Ethics: The study was approval by Institutional Review Board of Hanoi Medical University and National Hospital of
Tropical Diseases
Trang 8Chapter 3 RESULTS
There were 221 patients who fulfilled the inclusion criteria duringstudy period A higher proportion of male patients was observed in thestudy (92%) Mean age of patient population was 53.5 with the highestproportion (69.23%) belonged to age group of 40 – 60 Disease due to
S.suis occurs predominantly from April to October.
3.1 Clinical and laboratory characteristics of patient population
3.1.1 General characteristics
– Pattern of exposure conditions
Table 3.1 Exposure conditions History of exposure Frequency (n=221) Proportion %
There were 117 patients (52.94%) with medical history ofexposure to source of infection Eating “tiet canh” made the largestcontribution of 23.08% There were 47.06% patients with unknownhistory of exposure
3.1.2 Clinical characteristics
– Clinical syndromes.
46.1
35.818.1
Meningitis Sepsis with meningitis Septic shock
Chart 3.1 Clinical syndromes
Trang 9Meningitis was the most common clinical syndrome, followed
by sepsis with meningitis (35.8%) and septic shock (18.1%)
Table 3.2 General symptoms Symptoms Frequency (n=221) Proportion %
Meningitis with sepsis
Septic
n=102 (%)
n=79 (%)
n=40 (%)
(100)
79(100)
40(100)
(61.76)
53 (67.09)
35 (87.50) 0.01
(51.49)
43 (54.43)
26 (65.00) 0.35
(94.12)
72 (91.14)
29(72.5) 0.001
(74.51)
49 (62.03)
14(35.00)
<0.001
(64.71)
46 (58.23)
20(50.00) 0.26
(8.82)
14 (17.72)
25 (62.50)
<0.001
Trang 10Specific symptoms in meningitis group were headache, nausea andvomiting Patients with septic shock often presented with shivering,myalgia and diarrhea.
Kernig and altered mental status were the most common signs in
patients with S suis infection.
Table 3.5 Signs by category of clinical syndrome
n=40 (%)
(95.1)
60 (75.95)
14(35.00)
<0.001Altered mental status
(G<15)
68 (66.67)
48 (60.76)
17(42.05)
<0.001
(0.98)
5(6.33)
24 (60.00)
<0.001Hemorrhagic necrotic
rash
6(5.88)
16 (20.25)
33 (82.5)
<0.001
(25.49)
29 (36.71)
23 (63.89) 0.002
(Chi-squared test)
Trang 11Major patients with meningitis had Glasgow coma score below
15 and positive kernig sign In group of septic shock, common signswere hemorrhagic necrotic rash, refill > 2 seconds and cold extremities
bilirubin > 17
(mmol/l)
21(20.59) (45.57)36 (95.00)38 (42.99)95 <0.001AST ≥ 40
(UI/l) (50.98)52 (69.62)55 (97.50)39 (66.06)146 <0.001ALT ≥ 37
(UI/l)
60(58.82)
55(69.62)
37(92.50)
152(68.78) <0.001
(Chi-squared test, * Fisher’s exact test)
Patients with septic shock had more significant decrease in liverand kidney function than that in patients with meningitis
Frequency (%)
Frequency (%)
PT< 70 (%) (29.41)30 (41.77)33 (92.5)37 (45.25)100 <0.001
Fibrinogen <
2 (g/l)
0(0)
7(8.86)
31(77.5)
38(17.19)
<0.001D-Dimer
>500 (ng/l) (43.14)44 (59.49)47 (92.5)37 (57.92)128 <0.001
(Chi-squared test)
There were a marked decline in prothrombin time, fibrinogen andnoticeable increase in D-dimer, APTTs in the group of septic shockcompared to remaining groups
3.1.4 Treatment outcome
Trang 12– Mortality rate: The overall mortality rate was 14.03% The mortality
rate in patients of septic shock was 60% 35.26% patients were deathwithin the first 24 hours of admission
– Sequelae when discharged: Proportion of hearing loss in patient
population was 44.8%
3.2 Prognostic factors of mortality
Table 3.8 Regression analysis of epidemiological factors related to
mortality Characteristics OR 95% CI value aOR 95% CI p value p
function OR Univariate 95 CI p aOR Multivariate 95 CI p
Ure (mmol/l)