Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling.

Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to -80°C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling. IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.

Diversity of macaque microbiota compared to the human counterparts.

Studies on the microbial communities in non-human primate hosts provide unique insights in both evolution and function of microbes related to human health and diseases. Using 16S rRNA gene amplicon profiling, we examined the oral, anal and vaginal microbiota in a group of non-captive rhesus macaques (N = 116) and compared the compositions with the healthy communities from Human Microbiome Project. The macaque microbiota was dominated by Bacteroidetes, Firmicutes and Proteobacteria; however, there were marked differences in phylotypes enriched across body sites indicative of strong niche specialization. Compared to human gut microbiota where Bacteroides predominately enriched, the surveyed macaque anal community exhibited increased abundance of Prevotella. In contrast to the conserved human vaginal microbiota extremely dominated by Lactobacillus, the macaque vaginal microbial composition was highly diverse while lactobacilli were rare. A constant decrease of the vaginal microbiota diversity was observed among macaque samples from juvenile, adult without tubectomy, and adult with tubectomy, with the most notable distinction being the enrichment of Halomonas in juvenile and Saccharofermentans in contracepted adults. Both macaque and human oral microbiota were colonized with three most common oral bacterial genera: Streptococcus, Haemophilus and Veillonella, and shared relatively conserved communities to each other. A number of bacteria related to human pathogens were consistently detected in macaques. The findings delineate the range of structure and diversity of microbial communities in a wild macaque population, and enable the application of macaque as an animal model for future characterization of microbes in transmission, genomics and function.

Frequent Genetic Mismatch between Vaccine Strains and Circulating Seasonal Influenza Viruses, Hong Kong, China, 1996-2012.

The World Health Organization selects influenza vaccine compositions biannually to cater to peaks in temperate regions. In tropical and subtropical regions, where influenza seasonality varies and epidemics can occur year-round, the choice of vaccine remains uncertain. Our 17-year molecular epidemiologic survey showed that most influenza A(H3N2) (9/11) and B (6/7) vaccine strains had circulated in East Asia >1 year before inclusion into vaccines. Northern Hemisphere vaccine strains and circulating strains in East Asia were closely matched in 7 (20.6%) of 34 seasons for H3N2 and 5 (14.7%) of 34 seasons for B. Southern Hemisphere vaccines also had a low probability of matching (H3N2, 14.7%; B, 11.1%). Strain drift among seasons was common (H3N2, 41.2%; B, 35.3%), and biannual vaccination strategy (Northern Hemisphere vaccines in November followed by Southern Hemisphere vaccines in May) did not improve matching. East Asia is an important contributor to influenza surveillance but often has mismatch between vaccine and contemporarily circulating strains.

Age-specific seroprevalence of dengue infection in Hong Kong.

A newly developed dengue virus vaccine (chimeric yellow fever virus-tetravalent dengue vaccine [CYD-TDV]) has recently been licensed for clinical use. The World Health Organization recommends vaccination for populations with seroprevalence of at least 70% to maximize public health impact. This study aimed to delineate the seroprevalence of dengue infection in Hong Kong. A total of 105 972 serum samples submitted for clinical testing during the period 2013-2015 were age-stratified and sex-stratified. For each year of collection, 25 samples were randomly selected from each age-sex group. Altogether, 2100 samples were tested for the dengue immunoglobulin G (IgG) antibody using a non-type-specific ELISA kit. The overall dengue IgG-positive rate was 4.6% and showed no significant change over the 3 years. The positive rate was not associated with sex, but a steep rise in seroprevalence for persons above 65 years (32.7%) was observed. The low dengue seroprevalence in Hong Kong does not support implementation of a national immunization program. Majority of the population in Hong Kong are susceptible to dengue infection, and a substantial proportion of persons older than 65 years could acquire secondary infection and are prone to develop severe dengue.

Bacteriophage transfer during faecal microbiota transplantation in Clostridium difficile infection is associated with treatment outcome.

OBJECTIVE: Faecal microbiota transplantation (FMT) is effective for the treatment of recurrent Clostridium difficile infection (CDI). Studies have shown bacterial colonisation after FMT, but data on viral alterations in CDI are scarce. We investigated enteric virome alterations in CDI and the association between viral transfer and clinical outcome in patients with CDI. DESIGN: Ultra-deep metagenomic sequencing of virus-like particle preparations and bacterial 16S rRNA sequencing were performed on stool samples from 24 subjects with CDI and 20 healthy controls. We longitudinally assessed the virome and bacterial microbiome changes in nine CDI subjects treated with FMT and five treated with vancomycin. Enteric virome alterations were assessed in association with treatment response. RESULTS: Subjects with CDI demonstrated a significantly higher abundance of bacteriophage Caudovirales and a lower Caudovirales diversity, richness and evenness compared with healthy household controls. Significant correlations were observed between bacterial families Proteobacteria, Actinobacteria and Caudovirales taxa in CDI. FMT treatment resulted in a significant decrease in the abundance of Caudovirales in CDI. Cure after FMT was observed when donor-derived Caudovirales contigs occupied a larger fraction of the enteric virome in the recipients (p=0.024). In treatment responders, FMT was associated with alterations in the virome and the bacterial microbiome, while vancomycin treatment led to alterations in the bacterial community alone. CONCLUSIONS: In a preliminary study, CDI is characterised by enteric virome dysbiosis. Treatment response in FMT was associated with a high colonisation level of donor-derived Caudovirales taxa in the recipient. Caudovirales bacteriophages may play a role in the efficacy of FMT in CDI. TRIAL REGISTRATION NUMBER: NCT02570477.

Increased Detection of Emergent Recombinant Norovirus GII.P16-GII.2 Strains in Young Adults, Hong Kong, China, 2016-2017.

A new recombinant norovirus GII.P16-GII.2 outnumbered pandemic GII.4 as the predominant GII genotype in the winter of 2016-2017 in Hong Kong, China. Half of hospitalized case-patients were older children and adults, including 13 young adults. This emergent norovirus targets a wider age population compared with circulating pandemic GII.4 strains.

Clinical features and seasonality of parechovirus infection in an Asian subtropical city, Hong Kong.

BACKGROUND: The epidemiology of human parechovirus (HPeV) in Asia remains obscure. We elucidated the prevalence, seasonality, type distribution and clinical presentation of HPeV among children in Hong Kong. METHODS: A 24-month prospective study to detect HPeV in children ≤36 months hospitalized for acute viral illnesses. RESULTS: 2.3% of the 3911 children examined had HPeV infection, with most (87.5%) concentrated in September-January (autumn-winter). 81.3% were HPeV1 and 12.5% were HPeV4, while HPeV3 was rare (2.5%). HPeV was a probable cause of the disease in 47.7% (42/88), mostly self-limiting including acute gastroenteritis, upper respiratory tract infection and maculopapular rash. A neonate developed severe sepsis-like illness with HPeV3 as the only pathogen detected. A high proportion (60.0%) of children coinfected with HPeV and other respiratory virus(es) had acute bronchiolitis or pneumonia. Six children with HPeV coinfections developed convulsion / pallid attack. Most rash illnesses exhibited a generalized maculopapular pattern involving the trunk and limbs, and were more likely associated with HPeV4 compared to other syndrome groups (36.4% vs. 3.1%, p = 0.011). CONCLUSIONS: In Hong Kong, HPeV exhibits a clear seasonality (autumn-winter) and was found in a small proportion (2.3%) of young children (≤36 months) admitted with features of acute viral illnesses. The clinical presentation ranged from mild gastroenteritis, upper respiratory tract infection and febrile rash to convulsion and severe sepsis-like illness. HPeV3, which is reported to associate with more severe disease in neonates, is rare in Hong Kong. HPeV coinfection might associate with convulsion and aggravate other respiratory tract infections.

Ancient Evolution and Dispersion of Human Papillomavirus 58 Variants.

Human papillomavirus 58 (HPV58) is found in 10 to 18% of cervical cancers in East Asia but is rather uncommon elsewhere. The distribution and oncogenic potential of HPV58 variants appear to be heterogeneous, since the E7 T20I/G63S variant is more prevalent in East Asia and confers a 7- to 9-fold-higher risk of cervical precancer and cancer. However, the underlying genomic mechanisms that explain the geographic and carcinogenic diversity of HPV58 variants are still poorly understood. In this study, we used a combination of phylogenetic analyses and bioinformatics to investigate the deep evolutionary history of HPV58 complete genome variants. The initial splitting of HPV58 variants was estimated to occur 478,600 years ago (95% highest posterior density [HPD], 391,000 to 569,600 years ago). This divergence time is well within the era of speciation between Homo sapiens and Neanderthals/Denisovans and around three times longer than the modern Homo sapiens divergence times. The expansion of present-day variants in Eurasia could be the consequence of viral transmission from Neanderthals/Denisovans to non-African modern human populations through gene flow. A whole-genome sequence signature analysis identified 3 amino acid changes, 16 synonymous nucleotide changes, and a 12-bp insertion strongly associated with the E7 T20I/G63S variant that represents the A3 sublineage and carries higher carcinogenetic potential. Compared with the capsid proteins, the oncogenes E7 and E6 had increased substitution rates indicative of higher selection pressure. These data provide a comprehensive evolutionary history and genomic basis of HPV58 variants to assist further investigation of carcinogenic association and the development of diagnostic and therapeutic strategies.IMPORTANCE Papillomaviruses (PVs) are an ancient and heterogeneous group of double-stranded DNA viruses that preferentially infect the cutaneous and mucocutaneous epithelia of vertebrates. Persistent infection by specific oncogenic human papillomaviruses (HPVs), including HPV58, has been established as the primary cause of cervical cancer. In this work, we reveal the complex evolutionary history of HPV58 variants that explains the heterogeneity of oncogenic potential and geographic distribution. Our data suggest that HPV58 variants may have coevolved with archaic hominins and dispersed across the planet through host interbreeding and gene flow. Certain genes and codons of HPV58 variants representing higher carcinogenic potential and/or that are under positive selection may have important implications for viral host specificity, pathogenesis, and disease prevention.

Global Spread of Norovirus GII.17 Kawasaki 308, 2014-2016.

Analysis of complete capsid sequences of the emerging norovirus GII.17 Kawasaki 308 from 13 countries demonstrated that they originated from a single haplotype since the initial emergence in China in late 2014. Global spread of a sublineage SL2 was identified. A new sublineage SL3 emerged in China in 2016.

Hepatitis B infection acquired after haematopioetic stem cell transplant through horizontal mode.

A 22 month old child with thalassaemia major received unrelated umbilical cord blood transplantation. She was born to mother of HBsAg carrier and received hepatitis B immunoglobulin at birth and hepatitis B vaccination. She was HBsAg negative and anti-HBs positive before transplantation. After transplant, she was taken care by her mother and found to be HBsAg positive at 2 year post-transplant. Genotyping of the mother's and child's HBV status confirmed to be of same genotype and demonstrated horizontal transmission in post-transplant setting. Passive immunization of HBV may be considered in early post-transplant phase to prevent horizontal transmission of HBV, and antiviral treatment of the carer should be offered to prevent transmission of infection to immunocompromised child.

Complete Genome Sequence of an Emergent Recombinant GII.P16-GII.2 Norovirus Strain Associated with an Epidemic Spread in the Winter of 2016-2017 in Hong Kong, China.

A new recombinant norovirus, GII.P16-GII.2, emerged in the winter of 2016-2017. Here, we report the complete genome of this strain (Hu/GII/HK/2016/GII.P16-GII.2/CUHK-NS-1082), which was collected from a patient hospitalized with gastroenteritis in September 2016 in Hong Kong, China, and sequenced using next-generation sequencing. This genome had a 95.2% nucleotide identity to the closest sequence in GenBank.

Anti-inflammatory effects of adjunctive macrolide treatment in adults hospitalized with influenza: A randomized controlled trial.

INTRODUCTION: - Macrolides can ameliorate inflammation in respiratory diseases, providing clinical benefits. Data in influenza is lacking. METHOD: - A randomized, open-label, multicenter trial among adults hospitalized for laboratory-confirmed influenza was conducted. Study treatments of oseltamivir and azithromycin (500 mg/day), or oseltamivir alone, both for 5 days, were allocated at 1:1 ratio. The primary outcome was plasma cytokine/chemokine concentration change over time (Day 0-10); secondary outcomes were viral load and symptom score changes. Generalized Estimating Equation (GEE) models were used to analyze longitudinal data. RESULTS: - Fifty patients were randomized to the oseltamivir-azithromycin or oseltamivir groups, with comparable baseline characteristics (age, 57 ± 18 years; A/H3N2, 70%), complications (72%), and viral load. Pro-inflammatory cytokines IL-6 (GEE: ß -0.037, 95%CI-0.067,-0.007, P = 0.016; reduction from baseline -83.4% vs -59.5%), CXCL8/IL-8 (ß -0.018, 95%CI-0.037,0.000, P = 0.056; -80.5% vs -58.0%), IL-17 (ß -0.064, 95%CI-0.117,-0.012, P = 0.015; -74.0% vs -34.3%), CXCL9/MIG (ß -0.010, 95%CI-0.020,0.000, P = 0.043; -71.3% vs -56.0%), sTNFR-1, IL-18, and CRP declined faster in the oseltamivir-azithromycin group. There was a trend toward faster symptom resolution (ß -0.463, 95%CI-1.297,0.371). Viral RNA decline (P = 0.777) and culture-negativity rates were unaffected. Additional ex vivo studies confirmed reduced induction of IL-6 (P = 0.017) and CXCL8/IL-8 (P = 0.005) with azithromycin. CONCLUSION: - We found significant anti-inflammatory effects with adjunctive macrolide treatment in adults with severe influenza infections. Virus control was unimpaired. Clinical benefits of a macrolide-containing regimen deserve further study. [ClinicalTrials.gov NCT01779570].

Molecular Epidemiology and Strain Comparison between Hepatitis E Viruses in Human Sera and Pig Livers during 2014 to 2016 in Hong Kong.

Hepatitis E virus (HEV) causes substantial morbidity and mortality in developing countries and is considered an emerging foodborne pathogen in developed countries in which it was previously not endemic. To investigate genetic association between human HEV infection and HEV-contaminated high-risk food in Hong Kong, we compared local virus strains obtained from hepatitis E patient sera with those surveyed from high-risk food items during 2014 to 2016. Twenty-four cases of laboratory-confirmed human HEV infections were identified from January 2014 to March 2016 in our hospitals. Five types of food items at risk of HEV contamination were purchased on a biweekly basis from April 2014 to March 2016 in two local market settings: supermarkets (lamb, oyster, and pig liver) and wet markets (oyster, pig blood curd, pig large intestine, and pig liver). HEV RNA detection was performed by a real-time reverse transcription-PCR assay. HEV RNA was detected in pig liver, pig intestine, and oyster samples with prevalences of 1.5%, 0.4%, and 0.2%, respectively. Neighbor-joining phylogenetic inference showed that all human and swine HEV strains belonged to genotype 4. HEV subtype distributions in humans and swine were highly comparable: subtype 4b predominated, while subtype 4d was the minority. Local human and swine HEV genotype 4 strains shared over 95% nucleotide identity and were genetically very similar, implicating swine as an important foodborne source of autochthonous human HEV infections in Hong Kong. Action should be taken to raise the awareness among public and health care professionals of hepatitis E as an emerging foodborne disease.

Rotavirus vaccine effectiveness in Hong Kong children.

BACKGROUND: Rotavirus is a common infectious cause of childhood hospitalisation in Hong Kong. Rotavirus vaccines have been used in the private sector since licensure in 2006 but have not been incorporated in the government's universal Childhood Immunisation Programme. This study aimed to evaluate rotavirus vaccine effectiveness against hospitalisation. METHODS: This case-control study was conducted in the 2014/2015 rotavirus season in six public hospitals. Hospitalised acute gastroenteritis patients meeting inclusion criteria were recruited and copies of their immunisation records were collected. Case-patients were defined as enrolled subjects with stool specimens obtained in the first 48h of hospitalisation that tested positive for rotavirus, whereas control-patients were those with stool specimens obtained in the first 48h of hospitalisation testing negative for rotavirus. Vaccine effectiveness for administration of at least one dose of either Rotarix(®) (GlaxoSmithKline Biologicals) or RotaTeq(®) (Merck Research Laboratories) was calculated as 1 minus the odds ratio for rotavirus vaccination history for case-patients versus control-patients. RESULTS: Among the 525 eligible subjects recruited, immunisation records were seen in 404 (77%) subjects. 31% (162/525 and 126/404) tested positive for rotavirus. In the 404 subjects assessed for vaccine effectiveness, 2.4% and 24% received at least 1 dose of either rotavirus vaccine in case- and control-patients respectively. The unmatched vaccine effectiveness against hospitalisation for administration of at least one dose of either rotavirus vaccines was 92% (95% confidence interval [CI]: 75%, 98%). The matched analyses by age only and both age and admission date showed 96% (95% CI: 72%, 100%) and 89% (95% CI: 51%, 97%) protection against rotavirus hospitalisation respectively. CONCLUSIONS: Rotavirus vaccine is highly effective in preventing hospitalisation from rotavirus disease in young Hong Kong children.

Ad Hoc Influenza Vaccination During Years of Significant Antigenic Drift in a Tropical City With 2 Seasonal Peaks: A Cross-Sectional Survey Among Health Care Practitioners.

We evaluated the acceptability of an additional ad hoc influenza vaccination among the health care professionals following seasons with significant antigenic drift.Self-administered, anonymous surveys were performed by hard copy questionnaires in public hospitals, and by an on-line platform available to all healthcare professionals, from April 1st to May 31st, 2015. A total of 1290 healthcare professionals completed the questionnaires, including doctors, nurses, and allied health professionals working in both the public and private systems.Only 31.8% of participating respondents expressed an intention to receive the additional vaccine, despite that the majority of them agreed or strongly agreed that it would bring benefit to the community (88.9%), save lives (86.7%), reduce medical expenses (76.3%), satisfy public expectation (82.8%), and increase awareness of vaccination (86.1%). However, a significant proportion expressed concern that the vaccine could disturb the normal immunization schedule (45.5%); felt uncertain what to do in the next vaccination round (66.0%); perceived that the summer peak might not occur (48.2%); and believed that the summer peak might not be of the same virus (83.5%). Furthermore, 27.8% of all respondents expected that the additional vaccination could weaken the efficacy of previous vaccinations; 51.3% was concerned about side effects; and 61.3% estimated that there would be a low uptake rate. If the supply of vaccine was limited, higher priority groups were considered to include the elderly aged ≥65 years with chronic medical conditions (89.2%), the elderly living in residential care homes (87.4%), and long-stay residents of institutions for the disabled (80.7%). The strongest factors associated with accepting the additional vaccine included immunization with influenza vaccines in the past 3 years, higher perceived risk of contracting influenza, and higher perceived severity of the disease impact.The acceptability to an additional ad hoc influenza vaccination was low among healthcare professionals. This could have a negative impact on such additional vaccination campaigns since healthcare professionals are a key driver for vaccine acceptance. The discordance in perceived risk and acceptance of vaccination regarding self versus public deserves further evaluation.

Reduced Diagnostic Performance of Two Norovirus Antigen Enzyme Immunoassays for the Emergent Genogroup II Genotype 17 Kawasaki 2014 Variant.

Two commonly used norovirus enzyme immunoassays have reduced diagnostic performance, with clinical sensitivities ranging from 11% to 35% for the detection of the recently emerging genogroup II genotype 17 (GII.17) Kawasaki 2014 variant that caused the majority of infections in Asia during the winter of 2014 to 2015. False-negative results can compromise infection control and patient management.

Rapid emergence and predominance of a broadly recognizing and fast-evolving norovirus GII.17 variant in late 2014.

Norovirus genogroup II genotype 4 (GII.4) has been the predominant cause of viral gastroenteritis since 1996. Here we show that during the winter of 2014-2015, an emergent variant of a previously rare norovirus GII.17 genotype, Kawasaki 2014, predominated in Hong Kong and outcompeted contemporary GII.4 Sydney 2012 in hospitalized cases. GII.17 cases were significantly older than GII.4 cases. Root-to-tip and Bayesian BEAST analyses estimate GII.17 viral protein 1 (VP1) evolves one order of magnitude faster than GII.4 VP1. Residue substitutions and insertion occur in four of five inferred antigenic epitopes, suggesting immune evasion. Sequential GII.4-GII.17 infections are noted, implicating a lack of cross-protection. Virus bound to saliva of secretor histo-blood groups A, B and O, indicating broad susceptibility. This fast-evolving, broadly recognizing and probably immune-escaped emergent GII.17 variant causes severe gastroenteritis and hospitalization across all age groups, including populations who were previously less vulnerable to GII.4 variants; therefore, the global spread of GII.17 Kawasaki 2014 needs to be monitored.

Hospitalization Incidence, Mortality, and Seasonality of Common Respiratory Viruses Over a Period of 15 Years in a Developed Subtropical City.

Information on respiratory viruses in subtropical region is limited.Incidence, mortality, and seasonality of influenza (Flu) A/B, respiratory syncytial virus (RSV), adenovirus (ADV), and parainfluenza viruses (PIV) 1/2/3 in hospitalized patients were assessed over a 15-year period (1998-2012) in Hong Kong.Male predominance and laterally transversed J-shaped distribution in age-specific incidence was observed. Incidence of Flu A, RSV, and PIV decreased sharply from infants to toddlers; whereas Flu B and ADV increased slowly. RSV conferred higher fatality than Flu, and was the second killer among hospitalized elderly. ADV and PIV were uncommon, but had the highest fatality. RSV, PIV 2/3 admissions increased over the 15 years, whereas ADV had decreased significantly. A "high season," mainly contributed by Flu, was observed in late-winter/early-spring (February-March). The "medium season" in spring/summer (April-August) was due to Flu and RSV. The "low season" in late autumn/winter (October-December) was due to PIV and ADV. Seasonality varied between viruses, but predictable distinctive pattern for each virus existed, and temperature was the most important associating meteorological variable.Respiratory viruses exhibit strong sex- and age-predilection, and with predictable seasonality allowing strategic preparedness planning. Hospital-based surveillance is crucial for real-time assessment on severity of new variants.

Complete Genome Sequence of a Novel Recombinant GII.Pe_GII.17 Norovirus Strain from Hong Kong in 2015.

The complete genome sequence of a novel recombinant GII.Pe_GII.17 norovirus strain, tentatively named GII.17 Hong Kong 2015, was determined. RNA-dependent RNA polymerase has 95.6% and 98.4% and viral protein 1 has 90.6% and 95.9% identity at the nucleotide and amino acid levels, respectively, to the closest sequences in GenBank.