Tran Thi Thu Nga

A changing picture of shigellosis in southern Vietnam: shifting species dominance, antimicrobial susceptibility and clinical presentation

BackgroundShigellosis remains considerable public health problem in some developing countries. The nature of Shigellae suggests that they are highly adaptable when placed under selective pressure in a human population. This is demonstrated by variation and fluctuations in serotypes and antimicrobial resistance profile of organisms circulating in differing setting in endemic locations. Antimicrobial resistance in the genus Shigella is a constant threat, with reports of organisms in Asia being resistant to multiple antimicrobials and new generation therapies.MethodsHere we compare microbiological, clinical and epidemiological data from patients with shigellosis over three different periods in southern Vietnam spanning14 years.ResultsOur data demonstrates a shift in dominant infecting species (S. flexneri to S. sonnei) and resistance profile of the organisms circulating in southern Vietnam. We find that there was no significant variation in the syndromes associated with either S. sonnei or S. flexneri, yet the clinical features of the disease are more severe in later observations.ConclusionsOur findings show a change in clinical presentation of shigellosis in this setting, as the disease may be now more pronounced, this is concurrent with a change in antimicrobial resistance profile. These data highlight the socio-economic development of southern Vietnam and should guide future vaccine development and deployment strategies.Trial RegistrationCurrent Controlled Trials ISRCTN55945881

Tracking the establishment of local endemic populations of an emergent enteric pathogen

Significance Shigella sonnei is a globally emerging agent of bacterial dysentery. Here, we use genomics to examine the microevolution of S. sonnei in Vietnam. We show that S. sonnei was introduced into Vietnam in the early 1980s, where it continued to evolve, spreading geographically to establish localized founder populations. The population in Ho Chi Minh City has undergone several localized clonal replacement events, during which a small number of microevolutionary changes have risen to dominance. These changes, induced by horizontal gene transfer and substitution mutations, confer high-level antimicrobial resistance and the ability to kill other gut bacteria. This work provides a unique, high-resolution insight into the microevolution of a pioneering human pathogen during its establishment in a new host population. Shigella sonnei is a human-adapted pathogen that is emerging globally as the dominant agent of bacterial dysentery. To investigate local establishment, we sequenced the genomes of 263 Vietnamese S. sonnei isolated over 15 y. Our data show that S. sonnei was introduced into Vietnam in the 1980s and has undergone localized clonal expansion, punctuated by genomic fixation events through periodic selective sweeps. We uncover geographical spread, spatially restricted frontier populations, and convergent evolution through local gene pool sampling. This work provides a unique, high-resolution insight into the microevolution of a pioneering human pathogen during its establishment in a new host population.

High prevalence of plasmid-mediated quinolone resistance determinants in commensal members of the Enterobacteriaceae in Ho Chi Minh City, Vietnam

Antimicrobial-resistant pathogenic members of the Enterobacteriaceae are a well-defined global problem. We hypothesized that one of the main reservoirs of dissemination of antimicrobial resistance genes in Vietnam is non-pathogenic intestinal flora, and sought to isolate antimicrobial-resistant organisms from hospitalized patients and non-hospitalized healthy individuals in Ho Chi Minh City. The results identified substantial faecal carriage of gentamicin-, ceftazidime- and nalidixic acid-resistant members of the Enterobacteriaceae in both hospitalized patients and non-hospitalized healthy individuals. A high prevalence of quinolone resistance determinants was identified, particularly the qnrS gene, in both community- and hospital-associated strains. Furthermore, the results demonstrated that a combination of quinolone resistance determinants can confer resistance to nalidixic acid and ciprofloxacin, even in the apparent absence of additional chromosomal resistance mutations in wild-type strains and laboratory strains with transferred plasmids. These data suggest that intestinal commensal organisms are a significant reservoir for the dissemination of plasmid-mediated quinolone resistance in Ho Chi Minh City.

Slaughterhouse Pigs Are a Major Reservoir of Streptococcus suis Serotype 2 Capable of Causing Human Infection in Southern Vietnam

Streptococcus suis is a pathogen of major economic significance to the swine industry and is increasingly recognized as an emerging zoonotic agent in Asia. In Vietnam, S. suis is the leading cause of bacterial meningitis in adult humans. Zoonotic transmission is most frequently associated with serotype 2 strains and occupational exposure to pigs or consumption of infected pork. To gain insight into the role of pigs for human consumption as a reservoir for zoonotic infection in southern Vietnam, we determined the prevalence and diversity of S. suis carriage in healthy slaughterhouse pigs. Nasopharyngeal tonsils were sampled from pigs at slaughterhouses serving six provinces in southern Vietnam and Ho Chi Minh City area from September 2006 to November 2007. Samples were screened by bacterial culture. Isolates of S. suis were serotyped and characterized by multi locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). Antibiotic susceptibility profiles and associated genetic resistance determinants, and the presence of putative virulence factors were determined. 41% (222/542) of pigs carried S. suis of one or multiple serotypes. 8% (45/542) carried S. suis serotype 2 which was the most common serotype found (45/317 strains, 14%). 80% of serotype 2 strains belonged to the MLST clonal complex 1,which was previously associated with meningitis cases in Vietnam and outbreaks of severe disease in China in 1998 and 2005. These strains clustered with representative strains isolated from patients with meningitis in PFGE analysis, and showed similar antimicrobial resistance and virulence factor profiles. Slaughterhouse pigs are a major reservoir of S. suis serotype 2 capable of causing human infection in southern Vietnam. Strict hygiene at processing facilities, and health education programs addressing food safety and proper handling of pork should be encouraged.

The Sudden Dominance of blaCTX–M Harbouring Plasmids in Shigella spp. Circulating in Southern Vietnam

Background Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs) has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges. Methodology We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla CTX–M encoding plasmid. Principal Findings We show that two different bla CTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla CTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356) carried the bla CTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids. Significance The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.

A Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children

Background The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC − ssaV −) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile. Objectives We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years. Methods Subjects were randomly assigned to receive either a nominal dose of 5×109 CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA. Principal Findings One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18–5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12–36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI]  = 1.23 [0.550–2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC − ssaV −) ZH9 was detected in 51 (51%; 95% CI, 41–61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92–99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7–29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88–100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested. Conclusions This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children. Trial Registration Controlled-trials.comISRCTN91111837

The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus

Highlights ► A multiplex real-time PCR was developed for the detection and quantitation of Rotavirus A and Norovirus genogroup II. ► An internal extraction and amplification control was incorporated. ► Real-time PCR was compared to the current gold standard, enzyme immunoassay. ► Real-time PCR was significantly more sensitive than enzyme immunoassay. ► Quantitation demonstrated that the viral loads of both pathogens were ten times greater in stools children with diarrhea than in children without diarrhea.

Real-time PCR for detection of Streptococcus suis serotype 2 in cerebrospinal fluid of human patients with meningitis

Streptococcus suis serotype 2 is an emerging zoonotic pathogen and is the main cause of acute bacterial meningitis in adult patients in Vietnam. We developed an internally controlled real-time PCR for detection of S. suis serotype 2 in cerebrospinal fluid (CSF) samples targeted at the cps2J gene. Sensitivity and specificity in culture-confirmed clinical samples were 100%. The PCR detected S. suis serotype 2 infection in 101 of 238 (42.4%) prospectively collected CSF samples, of which 55 (23%) were culture positive. Culture-negative but PCR-positive CSF samples were significantly associated with the use of antimicrobial agents before admission. S. suis serotype 2 infection was more common than infections with Streptococcus pneumoniae and Neisseria meningitidis combined. Our results strikingly illustrate the additional diagnostic value of PCR in patients who are pretreated with antimicrobial agents and demonstrate the extremely high prevalence of S. suis infections among Vietnamese adult patients with bacterial meningitis.

Decreased parasite load and improved cognitive outcomes caused by deworming and consumption of multi-micronutrient fortified biscuits in rural Vietnamese schoolchildren.

Micronutrient deficiencies are associated with impaired growth and cognitive function. A school-based fortification program might benefit schoolchildren but a high prevalence of parasite infestation might affect effectiveness. A randomized, double-blind, placebo-controlled 2 × 2 factorial trial was conducted to assess the efficacy of multi-micronutrient fortified biscuits with or without de-worming on growth, cognitive function, and parasite load in Vietnamese schoolchildren. Schoolchildren (n = 510), 6-8 years of age were randomly allocated to receive albendazole or placebo at baseline and four months of multi-micronutrient fortified biscuits (FB) or non-fortified biscuits. Children receiving FB for four months scored higher on two cognitive tests: Ravens Colored Progressive Matrices and the Digit Span Forward test. Children receiving albendazole plus FB had the lowest parasite load after four months. In children receiving FB, mid-upper arm circumference was slightly improved (+0.082 cm) but there were no differences in other indexes of anthropometry. Combining multi-micronutrient fortified biscuits with de-worming is an effective strategy.

A Prospective Multi-Center Observational Study of Children Hospitalized with Diarrhea in Ho Chi Minh City, Vietnam

We performed a prospective multicenter study to address the lack of data on the etiology, clinical and demographic features of hospitalized pediatric diarrhea in Ho Chi Minh City (HCMC), Vietnam. Over 2,000 (1,419 symptomatic and 609 non-diarrheal control) children were enrolled in three hospitals over a 1-year period in 2009–2010. Aiming to detect a panel of pathogens, we identified a known diarrheal pathogen in stool samples from 1,067/1,419 (75.2%) children with diarrhea and from 81/609 (13.3%) children without diarrhea. Rotavirus predominated in the symptomatic children (664/1,419; 46.8%), followed by norovirus (293/1,419; 20.6%). The bacterial pathogens Salmonella, Campylobacter, and Shigella were cumulatively isolated from 204/1,419 (14.4%) diarrheal children and exhibited extensive antimicrobial resistance, most notably to fluoroquinolones and third-generation cephalosporins. We suggest renewed efforts in generation and implementation of policies to control the sale and prescription of antimicrobials to curb bacterial resistance and advise consideration of a subsidized rotavirus vaccination policy to limit the morbidity due to diarrheal disease in Vietnam.