Genomic Serotyping, Clinical Manifestations, and Antimicrobial Resistance of Nontyphoidal Salmonella Gastroenteritis in Hospitalized Children in Ho Chi Minh City, Vietnam.

Nontyphoidal Salmonella (NTS) are among the most common etiological agents of diarrheal diseases worldwide and have become the most commonly detected bacterial pathogen in children hospitalized with diarrhea in Vietnam. Aiming to better understand the epidemiology, serovar distribution, antimicrobial resistance (AMR), and clinical manifestation of NTS gastroenteritis in Vietnam, we conducted a clinical genomics investigation of NTS isolated from diarrheal children admitted to one of three tertiary hospitals in Ho Chi Minh City. Between May 2014 and April 2016, 3,166 children hospitalized with dysentery were recruited into the study; 478 (∼15%) children were found to be infected with NTS by stool culture. Molecular serotyping of the 450 generated genomes identified a diverse collection of serogroups (B, C1, C2 to C3, D1, E1, G, I, K, N, O, and Q); however, Salmonella enterica serovar Typhimurium was the most predominant serovar, accounting for 41.8% (188/450) of NTS isolates. We observed a high prevalence of AMR to first-line treatments recommended by WHO, and more than half (53.8%; 242/450) of NTS isolates were multidrug resistant (MDR; resistant to ≥3 antimicrobial classes). AMR gene detection positively correlated with phenotypic AMR testing, and resistance to empirical antimicrobials was associated with a significantly longer hospitalization (0.91 days; P = 0.04). Our work shows that genome sequencing is a powerful epidemiological tool to characterize the serovar diversity and AMR profiles in NTS. We propose a revaluation of empirical antimicrobials for dysenteric diarrhea and endorse the use of whole-genome sequencing for sustained surveillance of NTS internationally.

Novel multiplex real-time PCR assays reveal a high prevalence of diarrhoeagenic Escherichia coli pathotypes in healthy and diarrhoeal children in the south of Vietnam.

BACKGROUND: Diarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. RESULTS: The multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p < 0.001), was significantly associated with diarrhoea. Multiplex real-time PCR when applied to clinical samples is an efficient and high-throughput approach to DEC pathotypes. CONCLUSIONS: This approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

No Clinical Benefit of Empirical Antimicrobial Therapy for Pediatric Diarrhea in a High-Usage, High-Resistance Setting.

Background: Pediatric diarrheal disease presents a major public health burden in low- to middle-income countries. The clinical benefits of empirical antimicrobial treatment for diarrhea are unclear in settings that lack reliable diagnostics and have high antimicrobial resistance (AMR). Methods: We conducted a prospective multicenter cross-sectional study of pediatric patients hospitalized with diarrhea containing blood and/or mucus in Ho Chi Minh City, Vietnam. Clinical parameters, including disease outcome and treatment, were measured. Shigella, nontyphoidal Salmonella (NTS), and Campylobacter were isolated from fecal samples, and their antimicrobial susceptibility profiles were determined. Statistical analyses, comprising log-rank tests and accelerated failure time models, were performed to assess the effect of antimicrobials on disease outcome. Results: Among 3166 recruited participants (median age 10 months; interquartile range, 6.5-16.7 months), one-third (1096 of 3166) had bloody diarrhea, and 25% (793 of 3166) were culture positive for Shigella, NTS, or Campylobacter. More than 85% of patients (2697 of 3166) were treated with antimicrobials; fluoroquinolones were the most commonly administered antimicrobials. AMR was highly prevalent among the isolated bacteria, including resistance against fluoroquinolones and third-generation cephalosporins. Antimicrobial treatment and multidrug resistance status of the infecting pathogens were found to have no significant effect on outcome. Antimicrobial treatment was significantly associated with an increase in the duration of hospitalization with particular groups of diarrheal diseases. Conclusions: In a setting with high antimicrobial usage and high AMR, our results imply a lack of clinical benefit for treating diarrhea with antimicrobials; adequately powered randomized controlled trials are required to assess the role of antimicrobials for diarrhea.

Evaluation of Luminex xTAG Gastrointestinal Pathogen Panel Assay for Detection of Multiple Diarrheal Pathogens in Fecal Samples in Vietnam.

Diarrheal disease is a complex syndrome that remains a leading cause of global childhood morbidity and mortality. The diagnosis of enteric pathogens in a timely and precise manner is important for making treatment decisions and informing public health policy, but accurate diagnosis is a major challenge in industrializing countries. Multiplex molecular diagnostic techniques may represent a significant improvement over classical approaches. We evaluated the Luminex xTAG gastrointestinal pathogen panel (GPP) assay for the detection of common enteric bacterial and viral pathogens in Vietnam. Microbiological culture and real-time PCR were used as gold standards. The tests were performed on 479 stool samples collected from people admitted to the hospital for diarrheal disease throughout Vietnam. Sensitivity and specificity were calculated for the xTAG GPP for the seven principal diarrheal etiologies. The sensitivity and specificity for the xTAG GPP were >88% for Shigellaspp.,Campylobacterspp., rotavirus, norovirus genotype 1/2 (GI/GII), and adenovirus compared to those of microbiological culture and/or real-time PCR. However, the specificity was low (∼60%) for Salmonella species. Additionally, a number of important pathogens that are not identified in routine hospital procedures in this setting, such as Cryptosporidiumspp. and Clostridium difficile, were detected with the GPP. The use of the Luminex xTAG GPP for the detection of enteric pathogens in settings, like Vietnam, would dramatically improve the diagnostic accuracy and capacity of hospital laboratories, allowing for timely and appropriate therapy decisions and a wider understanding of the epidemiology of pathogens associated with severe diarrheal disease in low-resource settings.

Availability of different phosphorus forms in agricultural soil to Microcystis aeruginosa.

We investigated the availability of different forms of particulate soil phosphorus (P) to Microcystis aeruginosa by sequential extraction and bioassay. We cultured M. aeruginosa in media containing, as the sole source of P, soils sequentially extracted with 1 M NH4Cl, 0.11 M bicarbonate dithionite, 1 M NaOH, and 0.5 M HCl. Analyses of chlorophyll-a, particulate organic carbon, and particulate organic nitrogen showed that M. aeruginosa could utilize some of the P remaining in the soil after each extraction. Alkaline phosphatase (AP) assays of sequentially extracted soils showed distinct patterns that depended on the type of co-cultured soil. A direct relationship between cellular P concentrations and the level of alkaline phosphatase activity was observed in only some media, an indication that not all forms of P were equally suitable substrates for AP hydrolysis. These results imply that cyanobacterial-available P included not only HCl-extractable P, which is assumed to consist of carbonate or apatite bound-P and organic P, but also refractory P, which has been considered to be unavailable to algae. Both HCl-extracted P and refractory P enhance the production of chlorophyll a, but did not lead to the storage of P by M. aeruginosa.

Developing a method to measure bioavailable phosphorus in river water via simultaneous multisample ultrasonic extraction.

To reduce aquatic eutrophication, measurements of bioavailable phosphorus (BAP) rather than total phosphorus (TP) are deemed critical. However, current methods require much time to separate sediments from river water, which limits the routine measurement of BAP in rivers. Therefore, in this study, a simultaneous multisample ultrasonic extraction method is proposed to directly measure total BAP (TBAP) in river water without the separation of sediment and water. Spike-and-recovery assessments showed that at least three extractions are required to maintain efficiency. A process including 2-min extraction time and three extractions was suggested. The concentrations of TBAP extracted by this process showed no significant differences with the spike calculations. Furthermore, river water TBAP was quantified using the conventional and proposed method to examine the practicality of using the proposed method for simultaneous multisample ultrasonic extraction and to evaluate its adaptability to actual river water analysis. The extracted concentrations matched those obtained using the conventional method, in which total BAP is calculated as the sum of dissolved BAP and particulate BAP; no significant difference was observed between the concentrations. Ultrasonic extraction was considerably less time-consuming than the conventional method because more samples could be analyzed during a single run. Therefore, the simultaneous multisample ultrasonic extraction method proposed in this study can be used to directly quantify total BAP in river water.

Multidrug resistance plasmids underlie clonal expansions and international spread of Salmonella enterica serotype 1,4,[5],12:i:- ST34 in Southeast Asia.

Salmonella enterica serotype 1,4,[5],12:i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades. Despite increasing reports of resistance to antimicrobials in Southeast Asia, Salmonella ST34 population structure and evolution remained understudied in the region. Here we performed detailed genomic investigations on 454 ST34 genomes collected from Vietnam and diverse geographical sources to elucidate the pathogen's epidemiology, evolution and antimicrobial resistance. We showed that ST34 has been introduced into Vietnam in at least nine occasions since 2000, forming five co-circulating major clones responsible for paediatric diarrhoea and bloodstream infection. Most expansion events were associated with acquisitions of large multidrug resistance plasmids of IncHI2 or IncA/C2. Particularly, the self-conjugative IncA/C2 pST34VN2 (co-transferring blaCTX-M-55, mcr-3.1, and qnrS1) underlies local expansion and intercontinental spread in two separate ST34 clones. At the global scale, Southeast Asia was identified as a potential hub for the emergence and dissemination of multidrug resistant Salmonella ST34, and mutation analysis suggests of selection in antimicrobial responses and key virulence factors.

Mobility of antimicrobial resistance across serovars and disease presentations in non-typhoidal Salmonella from animals and humans in Vietnam.

Non-typhoidal Salmonella (NTS) is a major cause of bacterial enterocolitis globally but also causes invasive bloodstream infections. Antimicrobial resistance (AMR) hampers the treatment of these infections and understanding how AMR spreads between NTS may help in developing effective strategies. We investigated NTS isolates associated with invasive disease, diarrhoeal disease and asymptomatic carriage in animals and humans from Vietnam. Isolates included multiple serovars and both common and rare phenotypic AMR profiles; long- and short-read sequencing was used to investigate the genetic mechanisms and genomic backgrounds associated with phenotypic AMR profiles. We demonstrate concordance between most AMR genotypes and phenotypes but identified large genotypic diversity in clinically relevant phenotypes and the high mobility potential of AMR genes (ARGs) in this setting. We found that 84 % of ARGs identified were located on plasmids, most commonly those containing IncHI1A_1 and IncHI1B(R27)_1_R27 replicons (33%), and those containing IncHI2_1 and IncHI2A_1 replicons (31%). The vast majority (95%) of ARGS were found within 10 kbp of IS6/IS26 elements, which provide plasmids with a mechanism to exchange ARGs between plasmids and other parts of the genome. Whole genome sequencing with targeted long-read sequencing applied in a One Health context identified a comparatively limited number of insertion sequences and plasmid replicons associated with AMR. Therefore, in the context of NTS from Vietnam and likely for other settings as well, the mechanisms by which ARGs move contribute to a more successful AMR profile than the specific ARGs, facilitating the adaptation of bacteria to different environments or selection pressures.

Medication Adherence in Vietnamese Patients with Cardiovascular and Endocrine-Metabolic Diseases.

(1) Background: COVID-19 has significantly affected the quality of life and the medication adherence of patients with chronic diseases. Attitudes towards the disease and preventive measures are the things that need to be considered for patient adherence to medication during the COVID-19 pandemic. We aimed to evaluate the rate and compare the medication adherence and the impact of the COVID-19 pandemic on medication adherence in Vietnamese patients with cardiovascular and endocrine−metabolic diseases. (2) Methods: A cross-sectional study was conducted on outpatients having chronic diseases such as cardiovascular or/and endocrine−metabolic diseases in some southern provinces in Vietnam. In each group of patients, medication adherence was measured and assessed with the General Medication Adherence Scale (GMAS), adjusted and validated in Vietnam. In addition, the study also investigated attitudes and practices to prevent COVID-19. (3) Results: Out of 1444 patients in our study, the level of adherence was recorded in 867 cases, accounting for 61.1%. The group of patients with only cardiovascular disease and patients with only endocrine−metabolic disease had relatively similar compliance rates of 62 and 61.1%, respectively. The leading cause of non-adherence to treatment in all three groups of patients in the study, as assessed by the GMAS, was non-adherence due to financial constraints. Our study showed that 71.6% of patients felt anxious when going to the hospital for a medical examination. However, only 53.7% identified the COVID-19 pandemic as obstructing treatment follow-up visits. The research results showed that the COVID-19 epidemic influences the patient's psychology with regard to re-examination and treatment adherence, with p coefficients of 0.003 and <0.001, respectively. (4) Conclusion: Medication adherence rates in two disease groups are close, and financial constraint is the fundamental reason for medication non-adherence. Regulatory agencies must take care of people's welfare to improve adherence in the epidemic context.

Development of a novel peptide aptamer-based immunoassay to detect Zika virus in serum and urine.

Zika virus (ZIKV) has been identified as a cause of adverse outcomes of pregnancy, including microcephaly and other congenital diseases. Most people infected with ZIKV do not show any symptoms. Development of a method to discriminate dengue virus (DENV) and ZIKV infections has been challenging, and efficient assays for patient management are limited, attributable to high levels of cross-reactivity among co-circulating Flaviviruses. Thus, there is an urgent need for a specific high-throughput diagnostic assay to discriminate ZIKV infections from other Flavivirus infections. Methods: A novel epitope peptide of the ZIKV envelope protein was predicted using three immune epitope database analysis tools and then further modified. A molecular docking study was conducted using three-dimensional structures of the ZIKV envelope and peptide. Experimentally, interactions between the selected peptides and virus were assessed via a fluorescence-linked sandwich immunosorbent assay (FLISA), and performance of peptide-linked sandwich FLISA was evaluated in virus-spiked human serum and urine. Results: The Z_10.8 peptide (KRAVVSCAEA) was predicted to be a suitable detector, with a higher binding affinity than other candidates based on four criteria (binding affinity, root mean square deviation, position of amine residue of lysine at the N-terminus, and interactive site) in a docking study. Z_10.8 was significantly more efficient at detecting ZIKV than the other two peptides, as shown in the direct FLISA (P < 0.001). Further, the equilibrium dissociation constant (Kd) for the Z_10.8 peptide was 706.0 ± 177.9 (mean ± SD, nM), with specificity to discriminate ZIKV from DENV. The limit of detection for the sandwich FLISA was calculated as 1×104 tissue culture infective dose (TCID)50/mL. The presence of serum or urine did not interfere with the performance of the Z_10.8-linked sandwich FLISA. Conclusion: Four criteria are suggested for the development of an in silico modeled peptide aptamer; this computerized peptide aptamer discriminated ZIKV from DENV via immunoassay.

A Double-blind, Randomized, Placebo-controlled Trial of Lactobacillus acidophilus for the Treatment of Acute Watery Diarrhea in Vietnamese Children.

BACKGROUND: Probiotics are the most frequently prescribed treatment for children hospitalized with diarrhea in Vietnam. We were uncertain of the benefits of probiotics for the treatment of acute watery diarrhea in Vietnamese children. METHODS: We conducted a double-blind, placebo-controlled, randomized trial of children hospitalized with acute watery diarrhea in Vietnam. Children meeting the inclusion criteria (acute watery diarrhea) were randomized to receive either 2 daily oral doses of 2 × 10 CFUs of a local probiotic containing Lactobacillus acidophilus or placebo for 5 days as an adjunct to standard of care. The primary end point was time from the first dose of study medication to the start of the first 24-hour period without diarrhea. Secondary outcomes included the total duration of diarrhea and hospitalization, daily stool frequency, treatment failure, daily fecal concentrations of rotavirus and norovirus, and Lactobacillus colonization. RESULTS: One hundred and fifty children were randomized into each study group. The median time from the first dose of study medication to the start of the first 24-hour diarrhea-free period was 43 hours (interquartile range, 15-66 hours) in the placebo group and 35 hours (interquartile range, 20-68 hours) in the probiotic group (acceleration factor 1.09 [95% confidence interval, 0.78-1.51]; P = 0.62). There was also no evidence that probiotic treatment was efficacious in any of the predefined subgroups nor significantly associated with any secondary end point. CONCLUSIONS: This was a large double-blind, placebo-controlled trial in which the probiotic underwent longitudinal quality control. We found under these conditions that L. acidophilus was not beneficial in treating children with acute watery diarrhea.

Rapid Detection of Avian Influenza Virus by Fluorescent Diagnostic Assay using an Epitope-Derived Peptide.

Currently, the point of care testing (POCT) is not fully developed for subtype-specific avian influenza virus detection. In this study, an H5N1 hemaglutinin 1 (HA1) epitope (P0: KPNDAINF) and three modified peptides (P1: KPNTAINF, P2: KPNGAINF, P3: KPNDAINDAINF) were evaluated as POCT elements for rapid detection of avian influenza virus. Based on modeling predictions by Autodock Vina, binding affinity varied depending on alteration of one amino acid in these peptides. The binding energy of P2 indicated its potential for a strong interaction with HA. Fluorescence-linked immunosorbent assay experimentally demonstrated the interaction between these peptides and virus. The four peptides interacted with HA1 of H5N3 with different binding affinities with P2 showing the strongest binding affinity. When P0 and P2 peptides were used in rapid fluorescent immunochromatographic test (FICT) as detection elements, the inter-assay coefficients of variation (CV) indicated that P2-linked FICT was more acceptable than the P0-linked FICT in the presence of human specimens. Antibody pair-linked FICT was influenced by clinical samples more than the P2-linked FICT assay, which showed a 4-fold improvement in the detection limit of H5N3 and maintained H5 subtype-specificity. Compared to the rapid diagnostic test (RDT) which is not specific for influenza subtypes, P2-linked FICT could increase virus detection. In conclusion, results of this study suggest that HA epitope-derived peptides can be used as alternatives to antibodies for a rapid fluorescent diagnostic assay to detect avian influenza virus.

Smartphone-Based Fluorescent Diagnostic System for Highly Pathogenic H5N1 Viruses.

Field diagnostic tools for avian influenza (AI) are indispensable for the prevention and controlled management of highly pathogenic AI-related diseases. More accurate, faster and networked on-site monitoring is demanded to detect such AI viruses with high sensitivity as well as to maintain up-to-date information about their geographical transmission. In this work, we assessed the clinical and field-level performance of a smartphone-based fluorescent diagnostic device with an efficient reflective light collection module using a coumarin-derived dendrimer-based fluorescent lateral flow immunoassay. By application of an optimized bioconjugate, a smartphone-based diagnostic device had a two-fold higher detectability as compared to that of the table-top fluorescence strip reader for three different AI subtypes (H5N3, H7N1, and H9N2). Additionally, in a clinical study of H5N1-confirmed patients, the smartphone-based diagnostic device showed a sensitivity of 96.55% (28/29) [95% confidence interval (CI): 82.24 to 99.91] and a specificity of 98.55% (68/69) (95% CI: 92.19 to 99.96). The measurement results from the distributed individual smartphones were wirelessly transmitted via short messaging service and collected by a centralized database system for further information processing and data mining. Smartphone-based diagnosis provided highly sensitive measurement results for H5N1 detection within 15 minutes. Because of its high sensitivity, portability and automatic reporting feature, the proposed device will enable agile identification of patients and efficient control of AI dissemination.

Knowledge of primary health care and career choice at primary health care settings among final year medical students - challenges to human resources for health in Vietnam.

There is a shortage of medical doctors in primary health care (PHC) settings in Vietnam. Evidence about the knowledge medical students have about PHC and their career decision-making is important for making policy in human resources for health. The objective of this study was to analyse knowledge and attitudes about PHC among medical students in their final year and their choice to work in PHC after graduation. A cross-sectional study was conducted among 400 final year general medical students from Hanoi Medical University. Self-administered interviews were conducted. Key variables were knowledge, awareness of the importance of PHC and PHC career choices. Descriptive and analytic statistics were performed. Students had essential knowledge of the concept and elements of PHC and were well aware of its importance. However, only one-third to one half of them valued PHC with regard to their professional development or management opportunities. Less than 1% of students would work at commune or district health facilities after graduation. This study evidences challenges related to increasing the number of medical doctors working in PHC settings. Immediate and effective interventions are needed to make PHC settings more attractive and to encourage medical graduates to start and continue a career in PHC.

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.