Isolation of New Delhi metallo‐β‐lactamase 1‐producing Vibrio cholerae non‐O1, non‐O139 strain carrying ctxA,st and hly genes in southern Vietnam

Vibrio cholerae non‐O1, non‐O139 (VC_NAG) organisms are universally present in the aquatic environment and regarded as non‐pathogenic bacteria. However, considering that they do occasionally induce gastroenteritis, a study of their virulence and antibiotic resistance genes is important. The presence of enteropathogenic genes, including ctxA, VC_NAG‐specific heat‐stable toxin gene (st), hemolysin (hly), and zona occludens toxin (zot) was determined by PCR in 100 VC_NAG strains isolated in southern Vietnam in 2010–2013 from 94 environmental and six human origins. These 100 VC_NAG strains were also tested phenotypically and genotypically for the presence of the New Delhi metallo‐β‐lactamase (NDM‐1). Of the 100 VC_NAG strains tested, six were positive for ctxA; five from the environment and one of human origin. The st gene was detected in 17 isolates, 15 and two of which were of environmental and human origins, respectively. Gene hly was detected in 19 VC_NAG strains examined, two of which were isolated from humans and 17 from environments. The zot gene was not detected in any of the strains tested. Three VC_NAG strains of environmental origin were confirmed to produce NDM‐1 and the bla_NDM‐1 gene was detected in those strains by PCR. Of note, one of the three NDM‐1‐producing VC_NAG strains was confirmed to carry ctxA, st and hly genes concurrently. This is the first report of isolation of NDM‐1‐producing VC_NAG strains in Vietnam.     

Larval Gnathostoma spinigerum Detected in Asian Swamp Eels,Monopterus albus,Purchased from a Local Market in Yangon,Myanmar

The present study was performed to determine the infection status of swamp eels with Gnathostoma sp. larvae in Myanmar. We purchased total 37 Asian swamp eels, Monopterus albus, from a local market in Yangon in June and December 2013 and 2014. All collected eels were transferred with ice to our laboratory and each of them was examined by the artificial digestion technique. A total of 401 larval gnathostomes (1-96 larvae/eel) were detected in 33 (89.2%) swamp eels. Most of the larvae (n=383; 95.5%) were found in the muscle. The remaining 18 larvae were detected in the viscera. The advanced third-stage larvae (AdL₃) were 2.3-4.4 mm long and 0.25-0.425 mm wide. The characteristic head bulb (0.093 × 0.221 mm in average size) with 4 rows of hooklets, muscular long esophagus (1.025 mm), and 2 pairs of cervical sacs (0.574 mm) were observed by light microscopy. The average number of hooklets in the 1st, 2nd, 3rd, and 4th rows was 41, 45, 48, and 51, respectively. As scanning electron microscopic findings, the characteristic 4-5 rows of hooklets on the head bulb, a cervical papilla, tegumental spines regularly arranged in the transverse striations, and an anus were well observed. Based on these morphological characters, they were identified as the AdL₃ of Gnathostoma spinigerum. By the present study, it has been confirmed for the first time that Asian swamp eels, M. albus, from Yangon, Myanmar are heavily infected with G. spinigerum larvae.     

Highly expressed cholera toxin B subunit in the fruit of a transgenic tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> L.)

The cholera toxin B subunit (CTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. A gene encoding CTB, which was modified based on the optimized codon usage in the plant, was synthesized and fused to the endoplasmic reticulum retention signal KDEL to enhance its expression level in plants. The synthetic CTB (sCTB) gene was introduced into a plant expression vector adjacent to the CaMV 35S promoter, and was transformed into tomato using an Agrobacterium-mediated transformation method. The integration of the sCTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The synthesis and assembly of CTB protein in transgenic plants was demonstrated through immunoblot analysis and G_M1-ELISA. The highest amount of CTB protein produced in transgenic tomatoes was approximately 0.9% of total soluble fruit protein which was 10-fold greater than the previously 0.081%. G_M1-ELISA indicated that plant-synthesized CTB protein bound specifically to G_M1-gangliosides, suggesting that the CTB subunits formed active pentamers.     

Quantifying the emergence of dengue in Hanoi, Vietnam: 1998-2009

Background

An estimated 2.4 billion people live in areas at risk of dengue transmission, therefore the factors determining the establishment of endemic dengue in areas where transmission suitability is marginal is of considerable importance. Hanoi, Vietnam is such an area, and following a large dengue outbreak in 2009, we set out to determine if dengue is emerging in Hanoi.

Methods and Principal Findings

We undertook a temporal and spatial analysis of 25,983 dengue cases notified in Hanoi between 1998 and 2009. Age standardized incidence rates, standardized age of infection, and Standardized Morbidity Ratios (SMR) were calculated. A quasi-Poisson regression model was used to determine if dengue incidence was increasing over time. Wavelet analysis was used to explore the periodicity of dengue transmission and the association with climate variables. After excluding the two major outbreak years of 1998 and 2009 and correcting for changes in population age structure, we identified a significant annual increase in the incidence of dengue cases over the period 1999–2008 (incidence rate ratio  = 1.38, 95% confidence interval  = 1.20–1.58, p value  = 0.002). The age of notified dengue cases in Hanoi is high, with a median age of 23 years (mean 26.3 years). After adjusting for changes in population age structure, there was no statistically significant change in the median or mean age of dengue cases over the period studied. Districts in the central, highly urban, area of Hanoi have the highest incidence of dengue (SMR>3).

Conclusions

Hanoi is a low dengue transmission setting where dengue incidence has been increasing year on year since 1999. This trend needs to be confirmed with serological surveys, followed by studies to determine the underlying drivers of this emergence. Such studies can provide insights into the biological, demographic, and environmental changes associated with vulnerability to the establishment of endemic dengue.     

A retrospective analysis of the haemodynamic and metabolic effects of fluid resuscitation in Vietnamese adults with severe falciparum malaria

Background

Optimising the fluid resuscitation of patients with severe malaria is a simple and potentially cost-effective intervention. Current WHO guidelines recommend central venous pressure (CVP) guided, crystalloid based, resuscitation in adults.

Methods

Prospectively collected haemodynamic data from intervention trials in Vietnamese adults with severe malaria were analysed retrospectively to assess the responses to fluid resuscitation.

Results

43 patients were studied of whom 24 received a fluid load. The fluid load resulted in an increase in cardiac index (mean increase: 0.75 L/min/m² (95% Confidence interval (CI): 0.41 to 1.1)), but no significant change in acid-base status post resuscitation (mean increase base deficit 0.6 mmol/L (95% CI: −0.1 to 1.3). The CVP and PAoP (pulmonary artery occlusion pressure) were highly inter-correlated (r_s = 0.7, p<0.0001), but neither were correlated with acid-base status (arterial pH, serum bicarbonate, base deficit) or respiratory status (PaO₂/FiO₂ ratio). There was no correlation between the oxygen delivery (DO₂) and base deficit at the 63 time-points where they were assessed simultaneously (r_s = −0.09, p = 0.46).

Conclusions

In adults with severe falciparum malaria there was no observed improvement in patient outcomes or acid-base status with fluid loading. Neither CVP nor PAoP correlated with markers of end-organ perfusion or respiratory status, suggesting these measures are poor predictors of their fluid resuscitation needs.     

Molecular characterization of HIV-1 CRF01_AE in Mekong Delta, Vietnam, and impact of T-cell epitope mutations on HLA recognition (ANRS 12159)

Background

To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding.

Methods

We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores) and HLA binding (MHC score).

Results

All sequences clustered with CRF01_AE. HLA class I genotyping showed the predominance of Asian alleles as A*11:01 and B*46:01 with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9) versus 2 (±0.7) in non-divergent ones (p<0.0001).

Conclusions

Our study confirms the wide predominance of CRF01_AE in the Mekong Delta where patients harbor a specific HLA pattern. Moreover, it demonstrates the lower MHC binding affinity among divergent epitopes. This weak immune pressure combined with a narrow genetic diversity favors immune escape and could explain why CRF01_AE is still predominant in Vietnam, particularly in the Mekong area.     

Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.