High prevalence of colonisation with carbapenem-resistant Enterobacteriaceae among patients admitted to Vietnamese hospitals: Risk factors and burden of disease.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) is an increasing problem worldwide, but particularly problematic in low- and middle-income countries (LMIC) due to limitations of resources for surveillance of CRE and infection prevention and control (IPC). METHODS: A point prevalence survey (PPS) with screening for colonisation with CRE was conducted on 2233 patients admitted to neonatal, paediatric and adult care at 12 Vietnamese hospitals located in northern, central and southern Vietnam during 2017 and 2018. CRE colonisation was determined by culturing of faecal specimens on selective agar for CRE. Risk factors for CRE colonisation were evaluated. A CRE admission and discharge screening sub-study was conducted among one of the most vulnerable patient groups; infants treated at an 80-bed Neonatal ICU from March throughout June 2017 to assess CRE acquisition, hospital-acquired infection (HAI) and treatment outcome. RESULTS: A total of 1165 (52%) patients were colonised with CRE, most commonly Klebsiella pneumoniae (n = 805), Escherichia coli (n = 682) and Enterobacter spp. (n = 61). Duration of hospital stay, HAI and treatment with a carbapenem were independent risk factors for CRE colonisation. The PPS showed that the prevalence of CRE colonisation increased on average 4.2% per day and mean CRE colonisation rates increased from 13% on the day of admission to 89% at day 15 of hospital stay. At the NICU, CRE colonisation increased from 32% at admission to 87% at discharge, mortality was significantly associated (OR 5·5, P < 0·01) with CRE colonisation and HAI on admission. CONCLUSION: These data indicate that there is an epidemic spread of CRE in Vietnamese hospitals with rapid transmission to hospitalised patients.

Co-infection of human parvovirus B19 with Plasmodium falciparum contributes to malaria disease severity in Gabonese patients.

BACKGROUND: High seroprevalence of parvovirus B19 (B19V) coinfection with Plasmodium falciparum has been previously reported. However, the impact of B19V-infection on the clinical course of malaria is still elusive. In this study, we investigated the prevalence and clinical significance of B19V co-infection in Gabonese children with malaria. METHODS: B19V prevalence was analyzed in serum samples of 197 Gabonese children with P. falciparum malaria and 85 healthy controls using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and direct DNA-sequencing. RESULTS: B19V was detected in 29/282 (10.28%) of Gabonese children. B19V was observed more frequently in P. falciparum malaria patients (14.21%) in comparison to healthy individuals (1.17%) (P<0.001). Notably, the mild-malaria group revealed significantly lower hematocrit levels in B19V/P. falciparum co-infection than in P. falciparum mono-infection (P<0.05). Genetic analysis revealed a predominance of B19V genotype-1 (71.43%) in the studied population. However, B19V-genotype 2 was observed significantly more often in children with severe-malaria than in mild-malaria (P=0.04). CONCLUSION: Our findings reveal that B19V-infection is frequent in Gabonese children with P. falciparum malaria and signifies a possible contribution of B19V on the clinical course of malaria in a genotype-dependent manner. B19V co-infection should be considered as a additional diagnostic measure in malaria patients with life threatening anemia.

A trivial role of STAT4 variant in chronic hepatitis B induced hepatocellular carcinoma.

Two polymorphisms in the STAT4 and HLA-DQ loci were more recently reported to associate with chronic hepatitis B (CHB) induced hepatocellular carcinoma (HCC). We utilised an independent Vietnamese cohort of clinically classified HBV patients of chronic hepatitis B carriers (n=206), liver cirrhosis (n=222) and hepatocellular carcinoma (n=239) and assessed the influence of the reported variants. The STAT4 variant (rs7574865) was marginally associated with HCC susceptibility in CHB carriers in allelic and recessive genetic models (OR=0.84, 95%CI=0.7-0.99, P=0.048 and OR=0.7, 95%CI=0.5-0.99, P=0.047). No significant association between the studied variant with several clinical parameters such as liver enzymes (ALT, AST), total and direct bilirubin, AFP, HBV genotype and viral loads were observed. Our study highlights the reported variant to be a trivial factor and possibly other confounding factors may regulate STAT4 expression during HCC development.

IL-4 haplotype -590T, -34T and intron-3 VNTR R2 is associated with reduced malaria risk among ancestral indian tribal populations.

BACKGROUND: Interleukin 4 (IL-4) is an anti-inflammatory cytokine, which regulates balance between T(H)1 and T(H)2 immune response, immunoglobulin class switching and humoral immunity. Polymorphisms in this gene have been reported to affect the risk of infectious and autoimmune diseases. METHODS: We have analyzed three regulatory IL-4 polymorphisms; -590C>T, -34C>T and 70 bp intron-3 VNTR, in 4216 individuals; including: (1) 430 ethnically matched case-control groups (173 severe malaria, 101 mild malaria and 156 asymptomatic); (2) 3452 individuals from 76 linguistically and geographically distinct endogamous populations of India, and (3) 334 individuals with different ancestry from outside India (84 Brazilian, 104 Syrian, and 146 Vietnamese). RESULTS: The -590T, -34T and intron-3 VNTR R2 alleles were found to be associated with reduced malaria risk (P<0.001 for -590C>T and -34C>T, and P = 0.003 for VNTR). These three alleles were in strong LD (r²>0.75) and the TTR2 (-590T, -34T and intron-3 VNTR R2) haplotype appeared to be a susceptibility factor for malaria (P = 0.009, OR = 0.552, 95% CI = 0.356 -0.854). Allele and genotype frequencies differ significantly between caste, nomadic, tribe and ancestral tribal populations (ATP). The distribution of protective haplotype TTR2 was found to be significant (χ²3 =  182.95, p-value <0.001), which is highest in ATP (40.5%); intermediate in tribes (33%); and lowest in caste (17.8%) and nomadic (21.6%). CONCLUSIONS: Our study suggests that the IL-4 polymorphisms regulate host susceptibility to malaria and disease progression. TTR2 haplotype, which gives protection against malaria, is high among ATPs. Since they inhabited in isolation and mainly practice hunter-gatherer lifestyles and exposed to various parasites, IL-4 TTR2 haplotype might be under positive selection.

Reliable and rapid characterization of functional FCN2 gene variants reveals diverse geographical patterns.

BACKGROUND: Ficolin-2 coded by FCN2 gene is a soluble serum protein and an innate immune recognition element of the complement system. FCN2 gene polymorphisms reveal distinct geographical patterns and are documented to alter serum ficolin levels and modulate disease susceptibility. METHODS: We employed a real-time PCR based on Fluorescence Resonance Energy Transfer (FRET) method to genotype four functional SNPs including -986 G > A (#rs3124952), -602 G > A (#rs3124953), -4A > G (#rs17514136) and +6424 G > T (#rs7851696) in the ficolin-2 (FCN2) gene. We characterized the FCN2 variants in individuals representing Brazilian (n = 176), Nigerian (n = 180), Vietnamese (n = 172) and European Caucasian ethnicity (n = 165). RESULTS: We observed that the genotype distribution of three functional SNP variants (-986 G > A, -602 G > A and -4A > G) differ significantly between the populations investigated (p < 0.0001). The SNP variants were highly linked to each other and revealed significant population patterns. Also the distribution of haplotypes revealed distinct geographical patterns (p < 0.0001). CONCLUSIONS: The observed distribution of the FCN2 functional SNP variants may likely contribute to altered serum ficolin levels and this may depend on the different disease settings in world populations. To conclude, the use of FRET based real-time PCR especially for FCN2 gene will benefit a larger scientific community who extensively depend on rapid, reliable method for FCN2 genotyping.

Association of CISH -292A/T genetic variant with hepatitis B virus infection.

Cytokine-inducible SRC homology 2 domain protein (CISH) is a suppressor of cytokine signaling that controls interleukin-2 signaling pathway. We investigated the single nucleotide polymorphism (SNP) -292A>T in 473 Vietnamese hepatitis B virus (HBV) carriers and 416 healthy controls. CISH variants at -292A>T were associated to HBV infection (Allelic: OR, 1.22 95% CI, 1-1.49; P = 0.04; Recessive: OR, 1.69 95% CI 1.23-2.54; P = 0.007). A gene dose effect for the risk allele -292T was observed (P = 0.04). The level of interleukin 2 and liver enzymes such as alanine transaminase, aspartate transaminase, total bilirubin, and direct bilirubin were not associated to CISH polymorphism at position -292A>T This study associated the vital role of CISH SNP -292A>T variant to hepatitis B virus infection in a Vietnamese population.

Ficolin-2 levels and FCN2 haplotypes influence hepatitis B infection outcome in Vietnamese patients.

Human Ficolin-2 (L-ficolins) encoded by FCN2 gene is a soluble serum protein that plays an important role in innate immunity and is mainly expressed in the liver. Ficolin-2 serum levels and FCN2 single nucleotide polymorphisms were associated to several infectious diseases. We initially screened the complete FCN2 gene in 48 healthy individuals of Vietnamese ethnicity. We genotyped a Vietnamese cohort comprising of 423 clinically classified hepatitis B virus patients and 303 controls for functional single nucleotide polymorphisms in the promoter region (-986G>A, -602G>A, -4A>G) and in exon 8 (+6424G>T) by real-time PCR and investigated the contribution of FCN2 genotypes and haplotypes to serum Ficolin-2 levels, viral load and liver enzyme levels. Haplotypes differed significantly between patients and controls (P = 0.002) and the haplotype AGGG was found frequently in controls in comparison to patients with hepatitis B virus and hepatocellular carcinoma (P = 0.0002 and P<0.0001) conferring a protective effect. Ficolin-2 levels differed significantly between patients and controls (p<0.0001). Patients with acute hepatitis B had higher serum Ficolin-2 levels compared to other patient groups and controls.The viral load was observed to be significantly distributed among the haplotypes (P = 0.04) and the AAAG haplotype contributed to higher Ficolin-2 levels and to viral load. Four novel single nucleotide polymorphisms in introns (-941G>T, -310G>A, +2363G>A, +4882G>A) and one synonymous mutation in exon 8 (+6485G>T) was observed. Strong linkage was found between the variant -986G>A and -4A>G. The very first study on Vietnamese cohort associates both Ficolin-2 serum levels and FCN2 haplotypes to hepatitis B virus infection and subsequent disease progression.

Association of two variants of the interferon-alpha receptor-1 gene with the presentation of hepatitis B virus infection.

Interferon-alpha (IFNalpha) is a critical mediator of immunity to hepatitis B virus (HBV) infection. Although IFN has been used in the treatment of viral hepatitis for more than a decade, the role of IFN-alpha-receptor in HBV infection has not been intensively studied. We have evaluated the impact of two variants of the IFNAR1 gene on the outcome of HBV infection. Four hundred and fifty eight HBV-infected Vietnamese patients, with well-characterised clinical profiles including all forms of hepatic disease, and 160 non-infected, healthy Vietnamese individuals were enrolled in the study. Of these patients, 54 had acute hepatitis B, 88 had chronic hepatitis B, 118 had liver cirrhosis, 146 had a hepatocellular carcinoma and 52 were asymptomatic carriers of HBV. We analysed two SNPs for unequal distribution between these groups. The first SNP, rs1012335 is situated in intron 3 of the interferon alpha receptor 1 (IFNAR1). A C at position 17470 in the IFNAR1 on both chromosomes was detected more frequently in HBV-infected patients compared to healthy controls (OR: 2.6; 95% CI: 1.46-4.72, p < 0.001). The same homozygosity is also associated with higher concentrations of AST and ALT (aspartate and alanine amino-transferase) in the plasma of the patients. The second SNP (rs2257167) is situated in exon 4, causing a change of amino acids from Val (GTT) to Leu (CTT). Subjects having GTT on both chromosomes were more frequent in the healthy control group (OR: 0.54, 95% CI: 0.35-0.84, p = 0.004) and had lower plasma ALT concentrations. The findings indicate that two variants of the IFNAR1 gene are associated with the clinical presentation of HBV infection.

A globally occurring indel polymorphism in the promoter of the IFNA2 gene is not associated with severity of malaria but with the positivity rate of HCV.

BACKGROUND: Type I Interferons (IFNs) are well known cytokines which exert antiviral activity, antitumor activity and immunomodulatory effects. Single-nucleotide polymorphisms (SNP) and deletions in the gene coding for IFNA2 have been shown to influence the level of expression in vitro. The indel polymorphism -305_-300delAACTTT showed the strongest effect in vitro. To analyse the worldwide distribution of this polymorphism we analyzed five different populations (586 Vietnamese, 199 Central Africans, 265 Brazilians, 108 Kaingang and 98 Guarani). To investigate a possible association with susceptibility to infectious diseases we determined the polymorphism in malaria patients suffering either mild or severe malaria and in a cohort of hepatitis C virus infected individuals. RESULTS: We could detect the indel polymorphism in all populations analysed. There was no association with this polymorphism and the outcome of malaria but we found an increase of this indel polymorphism in hepatitis C virus positive individuals compared to healthy controls (p = 0.014). CONCLUSION: Polymorphisms in genes involved in the interferon pathway have been implicated in the resistance or susceptibility against cerebral malaria and HBV. Here we show that an indel polymorphism, which mediates a disadvantageous effect in HBV patients, may also play a disadvantageous role in HCV infections stressing the importance of a fully functional interferon pathway.

Subcellular mislocalization of mutant hepatitis B X proteins contributes to modulation of STAT/SOCS signaling in hepatocellular carcinoma.

OBJECTIVE: The hepatitis B virus X (HBx) protein plays an important role in the pathogenesis of hepatocellular carcinoma (HCC). One potential mechanism by which HBx can cause liver cancer may involve intracellular distribution and consecutively modulation of the proliferative important STAT/SOCS signaling with upregulation of STAT3. METHODS: 153 Vietnamese HBV-infected patients, including 48 patients with HCC, were analyzed. HBx sequences were determined by sequencing and subcloned for functional experiments. Intracellular localization of HBx mutants was determined by immunofluorescence assays. The impact of HBx mutants on JAK/STAT/SOCS signaling was investigated using Western blot and PCR analyses. RESULTS: In 4/48 HCC patients, truncated HBx together with full-length mutated HBx proteins were observed. Expression of HBx mutant proteins demonstrated an atypical nuclear and perinuclear localization. Functional experiments to determine the effect of HBx mutants on STAT/SOCS signaling demonstrated a significantly increased upregulation of STAT3 activation (p > 0.001) in comparison to wild-type (wt)-HBx. STAT1 was not activated either by wt-HBx or HBx mutants. Interestingly, SOCS1 and SOCS3 expression was not activated by wt-HBx and HBx mutants. CONCLUSIONS: Our results suggest that atypical nuclear/perinuclear localization of HBx mutants might be responsible for an enhanced activation of STAT3, inhibition of STAT1 and silencing of SOCS1/SOCS3 expression. This observation points to an active role of HBx mutants in hepatocarcinogenesis that involves dysregulation of STAT/SOCS signaling.

Phylogenetic analysis of human parvovirus B19, indicating two subgroups of genotype 1 in Vietnamese patients.

Recently, three distinct genotypes (1, 2 and 3) of human parvovirus B19 (B19) have been identified. However, the characteristics and distribution of B19 genotypes in Vietnam have not been investigated. Phylogenetic analysis using 49 subgenomic NS1/VP1u regions and two coding NS1-VP1/VP2 regions has been applied to investigate the prevalence of B19 genotypes in Vietnamese patients co-infected with Hepatitis B virus. Genetic analysis of the subgenomic NS1/VP1u region of B19 revealed that two genotypes of B19 were identified in these populations, with predominance of genotype 1 (47/49, 96 %) followed by genotype 2 (2/49, 4 %), but not genotype 3. Further, phylogenetic analysis of subgenomic B19 genomes revealed two major subgroups within genotype 1 (B19-1A and B19-1B) with an estimated nucleotide difference of >5 % between each subgroup, forming different branches. The mean percentage of amino acid variation between subgroup B19-1A and B19-1B was >2 % of the NS1, VP1 and VP2 proteins. Our results indicated that two of the three known genotypes of B19 were present in Vietnamese patients, with genotype 1 predominating, and that this genotype can be classified into at least two subgroups, B19-1A and B19-1B.

A promoter polymorphism in the interferon alpha-2 gene is associated with the clinical presentation of hepatitis B.

Cytokine gene polymorphisms influence the severity of infectious diseases of viral and parasitic origin. Interferon alpha (IFN-alpha) is known to be involved in the defence against hepatitis B. The promoter of the IFN-alpha-2 gene was investigated for mutations in 344 hepatitis B virus (HBV)-infected Vietnamese patients and 293 uninfected Vietnamese. We found a deletion in the promoter, which was present significantly more frequently in HBV-infected patients than in control individuals; 20% of the healthy, whereas 35% of the HBV-infected cohort carries this deletion (P<0.001). Reporter gene assays showed that a construct with the deletion had a lower level of transcription in comparison to the wild type (P=0.011). These findings indicate that the deletion in the promoter of the IFN-alpha-2 gene reduces the transcription of this gene in vitro. This reduction could explain the individually different interferon levels in humans and could also be one cause of susceptibility to hepatitis B.

Impact of the hepatitis B virus genotype and genotype mixtures on the course of liver disease in Vietnam.

Eight genotypes (A-H) of hepatitis B virus (HBV) have been identified. However, the impact of different genotypes on the clinical course of hepatitis B infection remains controversial. We investigated the frequency and clinical outcome of HBV genotypes and genotype mixtures in HBV-infected patients from Vietnam, Europe, and Africa. In addition, we analyzed the effects of genotype mixtures on alterations in in vitro viral replication. In Asian patients, seven genotypes (A-G) were detected, with A, C, and D predominating. In European and African patients, only genotypes A, C, D, and G were identified. Genotype mixtures were more frequently encountered in African than in Asian (P = .01) and European patients (P = .06). In Asian patients, the predominant genotype mixtures included A/C and C/D, compared to C/D in European and A/D in African patients. Genotype A was more frequent in asymptomatic compared with symptomatic patients (P < .0001). Genotype C was more frequent in patients with hepatocellular carcinoma (HCC; P = .02). Genotype mixtures were more frequently encountered in patients with chronic hepatitis in comparison to patients with acute hepatitis B (P = .015), liver cirrhosis (P = .013), and HCC (P = .002). Viral loads in patients infected with genotype mixtures were significantly higher in comparison to patients with a single genotype (P = .019). Genotype mixtures were also associated with increased in vitro HBV replication. In conclusion, infection with mixtures of HBV genotypes is frequent in Asia, Africa, and Europe. Differences in the replication-phenotype of single genotypes compared to genotype-mixtures suggest that co-infection with different HBV-genotypes is associated with altered pathogenesis and clinical outcome.

Co-infection of human parvovirus B19 in Vietnamese patients with hepatitis B virus infection.

BACKGROUND/AIMS: Human parvovirus B19 (B19) has been identified in the serum of hepatitis B virus (HBV) infected patients. However, the effect of B19-infection on the course of HBV-associated liver disease has not previously been investigated. We examined the prevalence of B19-DNA in HBV-infected Vietnamese patients and analysed the association between co-infection and the clinical outcome of HBV-infection. METHODS: Serum samples from 399 HBV-infected patients and 64 healthy individuals were analysed for the presence of B19-DNA by PCR and DNA-sequencing. RESULTS: B19-DNA was detected in 99/463 (21.4%) individuals. The proportion of HBV-infected patients who were also co-infected with B19 was higher than the healthy controls (P<0.001). B19-DNA was detected more frequently in patients with HBV-associated hepatocellular carcinoma compared to patients with acute and chronic HBV, HBV-associated liver cirrhosis and healthy subjects (P<0.006). A positive correlation was also found between B19-DNA loads and both serum HBV-DNA loads and alanine aminotransferase (rho>0.250 and P<0.05). CONCLUSIONS: Our findings demonstrate that B19-infection is frequent in HBV-infected Vietnamese patients. Also, a significant correlation exists between HBV/B19 co-infection and a greater likelihood of progression to more severe hepatitis B-associated liver disease. Further studies are required to determine the role of B19-infection on HBV-associated pathogenesis.

Variations in the serum concentrations of soluble Fas and soluble Fas ligand in Vietnamese patients infected with hepatitis B virus.

Earlier studies of both chronic hepatitis B and C virus (HBV and HCV) patients have shown a strong correlation between the soluble membrane Fas (sFas) and Fas protein expression on hepatocytes. The serum concentrations of sFas and soluble Fas ligand (sFasL) was examined in both healthy and HBV-infected Vietnamese patients to determine their relationship with the outcome of HBV infection. Patients with chronic rather than acute HBV had significantly higher amounts of sFas and sFasL, whilst the highest concentrations of both molecules were detected in those with malignant forms of HBV infection. sFas and sFasL concentrations tended to increase with a profile that paralleled the progression from asymptomatic to acute through chronic to malignant states, most markedly in the case of sFas. The sFas:sFasL ratio highlighted the relative predominance of sFas in those with acute and chronic HBV compared with asymptomatic or severe forms. In patients with hepatocellular carcinoma (HCC) a significant correlation was also observed between sFasL and alpha-feto protein (AFP) levels. The results indicate that sFas and to a lesser extent sFasL levels are to some degree associated with clinical progression in HBV infection.

Serum cytokine profiles associated with clinical presentation in Vietnamese infected with hepatitis B virus.

BACKGROUND/OBJECTIVES: An ineffective cytokine response is thought to be one of the reasons for the failure to suppress hepatitis B virus (HBV) replication and to eliminate the virus. We investigated the serum levels of interleukin (IL)-6, IL-10, IL-12, and interferon (IFN)-gamma in HBV-infected Vietnamese patients to determine whether they were related to the outcome of HBV infection. STUDY DESIGN: Samples from a total of 154 HBV-infected patients with well-characterised clinical profiles and 56 healthy controls were assessed. RESULTS: Serum IL-6 levels, which were inversely correlated with transaminase levels, were highest in patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) and lowest in those with either asymptomatic (ASYM), acute or chronic HBV, and thus, represented the best marker of HBV-related clinical progression. Compared with the healthy control group, serum IL-12 was uniformly elevated in all HBV-infected patients apart from those with ASYM infections, implying no impairment of production of this cytokine in HBV-infected individuals. Serum IL-10 and IFN-gamma levels, however, were uniformly low and showed no association with clinical presentation. Cytokine profiles were not influenced by the presence of hepatitis B e antigen (HbeAg). CONCLUSIONS: Serum IL-6 and IL-12 but not IL-10 and IFN-gamma are associated with the clinical presentation in HBV-infected Vietnamese patients.

Mannose-binding lectin gene polymorphisms and hepatitis B virus infection in Vietnamese patients.

Mannose-binding lectin (MBL) is a constituent of the human innate immune system which may play an important role in combating a variety of infectious diseases. We investigated the distribution of MBL gene mutations in a Vietnamese population, using polymerase chain reaction and DNA sequence analysis, and sought associations with the outcome of hepatitis B virus (HBV) infection. For this purpose we used samples from a total of 123 patients with confirmed, well-defined HBV infections, representing a full spectrum of clinical presentation from acute to chronic to malignant states, as well as from 112 healthy controls. The only MBL gene mutation found in this population, that at codon 54 of exon 1, was present at an overall frequency of 0.12, with a trend towards a higher frequency in the HBV-infected group compared with controls (0.15 versus 0.08, P = 0.079). Within the HBV-infected group there was a non-significant trend towards higher viral loads in those with this mutation, accompanied by significantly higher serum transaminase levels in the same individuals. Segregation according to clinical presentation showed that the mutation was present at a significantly higher frequency in the group with acute hepatitis B (AHB) compared with the healthy control group (0.25 versus 0.08, P = 0.01), and was associated with higher serum transaminase levels. Our results indicate that a mutation of the MBL gene might influence the clinical outcome of HBV infection in Vietnamese patients.