Hepatitis C virus antigen detection is an appropriate test for screening and early diagnosis of hepatitis C virus infection in at-risk populations and immunocompromised hosts.

Early diagnosis remains key for effective prevention and treatment. Unfortunately, current screening with anti-hepatitis C virus antibody (anti-HCV Ab) test may have limited utility in the diagnosis of HCV infection and reinfection. This is of special concern to at-risk population, such as immunocompromised hosts and end-stage renal failure patients on hemodialysis. HCV antigen (Ag) could be useful in identifying the ongoing infection in such clinical scenarios. Hence, we aimed to study the utility of HCV Ag testing for the diagnosis of acute and chronic hepatitis C. Of 89 samples studied, 19 were from acute hepatitis C patients who were immunocompromised or were on hemodialysis, 43 were from active chronic hepatitis C patients and 27 were from patients treated for chronic hepatitis C. All samples were tested for HCV Ag using the Abbott ARCHITECT HCV Ag assay. HCV Ag was reactive in 19/19 samples from acute hepatitis C patients and 42/43 samples from active chronic hepatitis C patients. It was nonreactive in all samples from treated patients. The test showed a sensitivity and specificity of 98.4% and 100.0%, respectively. The positive and negative predictive values were 100.0% and 96.4%, respectively. The HCV antigen test has high clinical sensitivity and specificity and is useful for the diagnosis of acute and chronic hepatitis C infection in at-risk and immunocompromised patients. Its short turnaround time and relatively low cost are advantageous for use in patients on hemodialysis and other at-risk patients who require monitoring of HCV infection and reinfection.

Adenoviral Infections in Singapore: Should New Antiviral Therapies and Vaccines Be Adopted?

BACKGROUND: A number of serious human adenovirus (HAdV) outbreaks have been recently reported: HAdV-B7 (Israel, Singapore, and USA), HAdV-B7d (USA and China), HAdV-D8, -D54, and -C2 (Japan), HAdV-B14p1 (USA, Europe, and China), and HAdV-B55 (China, Singapore, and France). METHODS: To understand the epidemiology of HAdV infections in Singapore, we studied 533 HAdV-positive clinical samples collected from 396 pediatric and 137 adult patients in Singapore from 2012 to 2018. Genome sequencing and phylogenetic analyses were performed to identify HAdV genotypes, clonal clusters, and recombinant or novel HAdVs. RESULTS: The most prevalent genotypes identified were HAdV-B3 (35.6%), HAdV-B7 (15.4%), and HAdV-E4 (15.2%). We detected 4 new HAdV-C strains and detected incursions with HAdV-B7 (odds ratio [OR], 14.6; 95% confidence interval [CI], 4.1-52.0) and HAdV-E4 (OR, 13.6; 95% CI, 3.9-46.7) among pediatric patients over time. In addition, immunocompromised patients (adjusted OR [aOR], 11.4; 95% CI, 3.8-34.8) and patients infected with HAdV-C2 (aOR, 8.5; 95% CI, 1.5-48.0), HAdV-B7 (aOR, 3.7; 95% CI, 1.2-10.9), or HAdV-E4 (aOR, 3.2; 95% CI, 1.1-8.9) were at increased risk for severe disease. CONCLUSIONS: Singapore would benefit from more frequent studies of clinical HAdV genotypes to identify patients at risk for severe disease and help guide the use of new antiviral therapies, such as brincidofovir, and potential administration of HAdV 4 and 7 vaccine.

Epidemiology and risk factors for cytomegalovirus infection in glomerular diseases treated with immunosuppressive therapy.

AIM: Cytomegalovirus (CMV) infections are associated with morbidity and mortality. We aimed to describe the epidemiology, risk factors and outcomes of CMV infection among patients with glomerulonephritis (GN) who received potent immunosuppressants (IS). METHODS: Single-centre retrospective study of adults with biopsy-proven GN prescribed methylprednisolone (MP), cyclophosphamide (CYC) or rituximab (RTX). Primary endpoint was CMV infection defined by significant CMV antigenaemia (>10 positive cells in 106 cells) or viraemia (>2000 copies/mL). Death was related to CMV if CMV infection occurred within the same hospitalization as death. RESULTS: Ninety-four patients were studied. CYC was prescribed in 65% and MP in 71% of the cohort. Only two patients received RTX and 15 patients received plasma exchanges (PEX). Median follow up was 31.9 (IQR: 13.7, 53.6) months. CMV infection occurred in 13 patients (13.8%) at 1.3 (0.6, 3.0) months from biopsy. Patients with CMV infection had higher serum creatinine [404 (272, 619) vs. 159 (93, 317) µmol/L, P < 0.001] and greater proteinuria [UPCR 7.5, (4.8, 11.8) vs. 4.2 (2.3, 8.4) g/g, P = 0.02] than those who did not have CMV infection. Also, more patients received CYC (92% vs. 60%, P = 0.03), RTX (15% vs. 0, P = 0.02) and PEX (38% vs. 12%, P = 0.01) than those who did not have CMV infection. Two patients had CMV-related deaths. CONCLUSION: Cytomegalovirus infection is common in GN patients receiving potent IS. Surveillance and possibly anti-viral prophylaxis should be considered for high-risk patients.

A highly divergent Encephalomyocarditis virus isolated from nonhuman primates in Singapore.

BACKGROUND: In 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens. METHODS: Of the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy. Reverse transcription and polymerase chain reaction with Encephalomyocarditis virus (EMCV)-specific primers targeting the gene regions of VP3/VP1 and 3D polymerase (3Dpol) confirmed the virus genus and species. The two EMCV isolates were sequenced and phylogenetic analyses of the virus genes performed. Serological testing on other animal species in the Singapore Zoological Gardens was also conducted. RESULTS: Electron microscopy of the two EMCV isolates, designated Sing-M100-02 and Sing-M105-02, revealed spherical viral particles of about 20 to 30 nm, consistent with the size and morphology of members belonging to the family Picornaviridae. In addition, infected-Vero cells showed positive immunoflorescence staining with antiserum to EMCV. Sequencing of the viral genome showed that the two EMCV isolates were 99.9% identical at the nucleotide level, indicating a similar source of origin. When compared with existing EMCV sequences in the VP1 and 3Dpol gene regions, the nucleotide divergence were at a maximum of 38.8% and 23.6% respectively, while the amino acid divergence were at a maximum of 33.9% and 11.3% respectively. Phylogenetic analyses of VP1 and 3Dpol genes further grouped the Sing-M100-02 and Sing-M105-02 isolates to themselves, away from existing EMCV lineages. This strongly suggested that Sing-M100-02 and Sing-M105-02 isolates are highly divergent variants of EMCV. Apart from the two deceased orang utans, a serological survey conducted among other zoo animals showed that a number of other animal species had neutralizing antibodies to Sing-M105-02 isolate, indicating that the EMCV variant has a relatively wide host range. CONCLUSIONS: The etiological agent responsible for the fatal myocarditis cases among two of the four orang utans in the Singapore Zoological Gardens was a highly divergent variant of EMCV. This is the first report of an EMCV infection in Singapore and South East Asia.

Vaccine-associated Rubella - a report of two cases and a review of the literature.

We report the clinical characteristics of two adult patients, presenting with a typical erythematous rash consistent with rubella disease after MMR vaccination. Both patients had an uncomplicated clinical course and recovered uneventfully. One patient was confirmed to have vaccine-associated rubella via sequencing of virus isolated in viral culture. The other patient had a pharyngeal swab positive for rubella virus PCR, with sequencing matching the vaccine strain. There are few reports of clinical disease from rubella vaccine-strains in the literature. Previous authors have reported severe disseminated vaccine-associated rubella in both immunodeficient and immunocompetent patients. Further study is required to ascertain the incidence, risk factors, and clinical characteristics of this condition; as well as investigate the extent of horizontal transmission to guide infection control recommendations.

Emergence of oseltamivir-resistant pandemic (H1N1) 2009 virus within 48 hours.

An oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus evolved and emerged from zero to 52% of detectable virus within 48 hours of a patient's exposure to oseltamivir. Phylogenetic analysis and data gathered by pyrosequencing and cloning directly on clinical samples suggest that the mutant emerged de novo.

Molecular insights into evolution, mutations and receptor-binding specificity of influenza A and B viruses from outpatients and hospitalized patients in Singapore.

BACKGROUND: This study compared the genomes of influenza viruses that caused mild infections among outpatients and severe infections among hospitalized patients in Singapore, and characterized their molecular evolution and receptor-binding specificity. METHODS: The complete genomes of influenza A/H1N1, A/H3N2 and B viruses that caused mild infections among outpatients and severe infections among inpatients in Singapore during 2012-2015 were sequenced and characterized. Using various bioinformatics approaches, we elucidated their evolutionary, mutational and structural patterns against the background of global and vaccine strains. RESULTS: The phylogenetic trees of the 8 gene segments revealed that the outpatient and inpatient strains overlapped with representative global and vaccine strains. We observed a cluster of inpatients with A/H3N2 strains that were closely related to vaccine strain A/Texas/50/2012(H3N2). Several protein sites could accurately discriminate between outpatient versus inpatient strains, with site 221 in neuraminidase (NA) achieving the highest accuracy for A/H3N2. Interestingly, amino acid residues of inpatient but not outpatient isolates at those sites generally matched the corresponding residues in vaccine strains, except at site 145 of hemagglutinin (HA). This would be especially relevant for future surveillance of A/H3N2 strains in relation to their antigenicity and virulence. Furthermore, we observed a trend in which the HA proteins of influenza A/H3N2 and A/H1N1 exhibited enhanced ability to bind both avian and human host cell receptors. In contrast, the binding ability to each receptor was relatively stable for the HA of influenza B. CONCLUSIONS: Overall, our findings extend our understanding of the molecular and structural evolution of influenza virus strains in Singapore within the global context of these dynamic viruses.

Prevalence of hepatitis C virus infection and the IL28B genotype polymorphism among blood donors and high-risk populations.

INTRODUCTION: To study the prevalence of hepatitis C virus (HCV) infection in blood donor (BD), haemodialysis (HD) and intravenous drug user (IVDU) populations in Singapore and assess the IL28B polymorphism if HCV positive. METHODS: The BD population were healthy volunteers, the HD population were patients who were on haemodialysis for at least six months of follow-up between January 2009 and December 2014. IVDU population was from inmates at halfway houses who consented. RESULTS: Between 2011 and 2014, of 161,658 individuals who underwent screening prior to blood donation, 95 (0.059%) were positive for HCV. Of the 42 sera available, common genotypes (GTs) were GT-3 (47.6%) and GT-1 (31.0%). Of 1,575 HD patients, 2.2% were anti-HCV positive. The HCV GT distribution was HCV GT-1 (32.4%), HCV GT-3 (20.5%) and GT-6 (8.8%). 83 halfway house inmates were screened. Of the 47 IVDUs, 36.2% were anti-HCV positive with predominant GT-3 (%). IL28B polymorphism was noted to be CC predominantly 85.3%. CONCLUSION: Prevalence of HCV infection has decreased in both the BD and HD populations. However, it remains high in the IVDU population. GT-1 remains the most common in the HD population; however, GT-3 infection is now more common among the BD population in Singapore. IL28B - CC is the predominant variant among the HCV-infected individuals in Singapore.

Seroepidemiology and genotyping of hepatitis E virus in Singapore reveal rise in number of cases and similarity of human strains to those detected in pig livers.

Hepatitis E virus (HEV) causes 20 million infections worldwide yearly, of which only about 3.3 million are symptomatic. In developed Asian countries, HEV strains detected in human sera and in food sources were genetically similar, suggesting that indigenous HEV infections may be largely food-borne. To assess the burden of hepatitis E in Singapore, we performed a seroepidemiologic study of the infection. Additionally, we carried out HEV genotyping on archived, residual HEV IgM-positive serum samples collected between 2014 and 2016 (n = 449), and on pig liver samples (n = 36) purchased from wet markets and supermarkets. Our study shows a rise in hepatitis E incidence (IgM) from 1.7 to 4.1 cases per 100,000 resident population from 2012 to 2016 and an increase in hepatitis E IgG positivity rate among residents from 14% in 2007 to 35% in 2016. Other findings also suggest the epidemiology of hepatitis E in Singapore has shifted, from it being mainly a disease imported from the Indian subcontinent, to one that is now increasingly prevalent in our resident population. Genotypes obtained from 143 human samples identified the majority to be genotype 3 (n = 121), 21 to be genotype 1 and one to be genotype 4. Further phylogenetic analyses suggest genotype 3a to be the cause of indigenous infections in residents, which showed genetic similarity to the genotype 3a strains detected in pig livers. This link between the strains in the majority of human samples and those in pig livers consumed by the public suggests a possible food-borne route of HEV infection in Singapore.

Performance of the HIV Blot 2.2, INNO-LIA HIV I/II Score, and Geenius HIV 1/2 Confirmatory Assay for use in HIV confirmation.

In view of recent revised recommendations for human immunodeficiency virus (HIV) confirmatory testing, the performance of 3 HIV confirmatory assays was compared. Using the HIV Blot 2.2 (MP-WB), the INNO-LIA HIV I/II Score (INNO), and the Geenius HIV 1/2 Confirmatory Assay (Geenius), we tested 199 HIV-1 positive, 161 HIV negative, 65 HIV western blot indeterminate, 26 HIV seroconversion, 34 early HIV infection and 4 HIV-2 positive archived specimens. We show that all 3 assays had comparable test sensitivity in the detection of HIV-1 positive cases. However, less non-specific reactivity was observed with the INNO and Geenius assays, where both of them were able to resolve MP-WB indeterminate cases. When early HIV cases were considered, INNO and Geenius were more likely to confirm an early-stage infection as positive. Nevertheless, overall poor sensitivity (25.5% - 44.7%) of these assays for the detection of early cases was observed, likely because these cases had very low or non-detectable levels of HIV antibodies. Hence, further testing by a nucleic acid test or a p24 antigen test of specimens reactive on screening with a fourth generation Ag/Ab assay that are negative on confirmatory testing for HIV-specific antibody, may be useful. In conclusion, INNO and Geenius had comparable test performance, although the ease of use and shorter assay time for Geenius may make it the preferred choice for laboratories. In that regard, of note is our observation of non-specific reactivity of lipaemic specimens to the HIV-2 gp140 band in the Geenius assay, which should prompt caution when interpreting results of such specimens.

Clinical and laboratory findings of SARS in Singapore.

INTRODUCTION: Singapore was one of 29 countries worldwide affected by severe acute respiratory syndrome (SARS) in 2003. MATERIALS AND METHODS: There were 238 cases identified during the outbreak. We performed a retrospective analysis of the clinical and laboratory data of 234 patients admitted to Tan Tock Seng Hospital and Singapore General Hospital. RESULTS: The mean age of patients was 21 years, 31.6% of patients were males and 41.8% were healthcare workers. At presentation, the common symptoms were fever, myalgia, cough and headache; rhinorrhoea was uncommon. On admission, 21% had leukopenia, 18% had thrombocytopaenia, 29% had hyponatraemia, 31% had hypokalaemia, 21% had transaminitis. Polymerase chain reaction (PCR) testing of respiratory and stool samples provided the best yield at the end of the first week of illness. Thirty-two patients were initially not recognised as probable SARS and were reclassified when the serology test results were available. The chief reasons for not identifying these patients early were persistently normal chest X-rays (68.8%), very mild presentation (43.8%) and the presence of a concomitant illness (12.5%). Overall, 12% of the patients were probable SARS with atypical presentations. Overall mortality was 11.8%. CONCLUSION: Patients infected with the SARS coronavirus had a wide clinical presentation with non-specific symptoms.

Impedimetric cell-based biosensor for real-time monitoring of cytopathic effects induced by dengue viruses.

We describe an impedimetric cell-based biosensor constructed from poly-l-lysine (PLL)-modified screen-printed carbon electrode for real-time monitoring of dengue virus (DENV) infection of surface-immobilized baby hamster kidney (BHK-21) fibroblast cells. Cytopathic effects (CPE) induced by DENV-2 New Guinea C strain (including degenerative morphological changes, detachment, membrane degradation and death of host cells), were reflected by drastic decrease in impedance signal response detected as early as ~30 hours post-infection (hpi). In contrast, distinct CPE by conventional microscopy was evident only at ~72 hpi at the corresponding multiplicity of infection (MOI) of 10. A parameter that describes the kinetics of cytopathogenesis, CIT50, which refers to the time taken for 50% reduction in impedance signal response, revealed an inverse linear relationship with virus titer and MOI. CIT50 values were also delayed by 31.5h for each order of magnitude decrease in MOI. Therefore, based on the analysis of CIT50, the virus titer of a given sample can be determined from the measured impedance signal response. Furthermore, consistent impedance results were also obtained with clinical isolates of the four DENV serotypes verified by RT-PCR and cycle sequencing. This impedimetric cell-based biosensor represents a label-free and continuous approach for the dynamic measurement of cellular responses toward DENV infection, and for detecting the presence of infectious viral particles.

Comparison of three methods for respiratory virus detection between induced sputum and nasopharyngeal aspirate specimens in acute asthma.

Viral respiratory tract infections are associated frequently with acute exacerbations of asthma. Nasopharyngeal aspirates and bronchoalveolar lavage specimens are used extensively for detecting viral respiratory tract infections, but not sputum. The aim of the study was to determine the efficiency of viral detection in induced sputum versus nasopharyngeal aspirate obtained during acute exacerbations of asthma, comparing three laboratory methods of viral diagnosis. Paired samples of induced sputum and nasopharyngeal aspirate obtained from 32 adults admitted to hospital with acute asthma were subjected to reverse transcription-polymerase chain reaction (RT-PCR), viral culture, and immunofluorescence assay. The results show that RT-PCR was associated with significantly higher rates of viral detection than culture (P=0.005) or immunofluorescence (P=0.001), without significant differences in the rates of viral detection between induced sputum and nasopharyngeal aspirate. It is concluded that induced sputum specimens are feasible for detection of viral respiratory tract infections by RT-PCR during acute exacerbations of asthma.

Progress towards measles elimination in Singapore.

OBJECTIVE: We describe the epidemiological trends of measles in Singapore in relation to its progress towards measles elimination and identify gaps in fulfilling the World Health Organization Western Pacific Regional Office regional measles elimination criteria. METHODS: Epidemiological data on measles maintained by the Communicable Diseases Division, Ministry of Health from 1981 to 2012 were collated and analysed. Data on measles vaccination coverage were obtained from the National Immunization Registry and School Health Services, Health Promotion Board. To assess the seroprevalence of the population, the findings of periodic seroepidemiological surveys on measles were traced and reviewed. FINDINGS: With the successful implementation of the National Childhood Immunization Programme using the monovalent measles vaccine, measles incidence declined from 88.5 cases per 100,000 in 1984 to 6.9 per 100,000 in 1991. Resurgences were observed in 1992, 1993 and 1997. A 'catch-up' vaccination programme using the trivalent measles, mumps and rubella (MMR) vaccine was conducted in 1997, followed by introduction of the two-dose vaccination schedule in January 1998. Measles incidence subsequently declined sharply to 2.9 per 100,000 in 1998. Vaccination coverage was maintained at 95% for the first dose and 92-94% for the second dose. Seroprevalence surveys showed seropositivity for measles IgG antibodies in over 95% of adults in 2004, and in 83.1% of children aged 1-17 years in 2008-2010. Sporadic cases with occasional clusters of two or more cases continued to occur among the unvaccinated population, especially children aged below 4 years. The predominant measles virus genotype has shifted from D9 to the B3 and G3 genotypes, which are endemic in neighbouring countries. CONCLUSION: Singapore has made good progress towards the elimination of endemic measles. To further eliminate sporadic cases of measles, the national immunisation schedule has recently been amended to vaccinate children with 2 doses of MMR vaccine before 2 years of age.

Epidemiology of acute hepatitis E in Singapore.

OBJECTIVES: We undertook an epidemiological review to determine the trend and characteristics of acute hepatitis E in Singapore over the last 12 years. METHODS: We analysed the epidemiological records of all laboratory-confirmed cases of acute hepatitis E maintained at the Communicable Diseases Division, Ministry of Health, from 2000 to 2011. RESULTS: A total of 540 laboratory-confirmed cases of acute hepatitis E was reported with more than half imported, mainly from India and Bangladesh. Among the indigenous cases, the mean annual incidence per 100,000 population increased from 0.05 in 2000-2002 to 0.92 in 2009-2011. There was a male predominance and the median age was 46 years. Among the 3 major ethnic groups of Singapore residents, Chinese and Indians had higher mean annual incidence rate compared to Malays. All the indigenous cases occurred singly and sporadically and could not be epidemiologically linked to one another by person, place or time. No common food item was implicated. CONCLUSIONS: Indigenous acute hepatitis E has emerged as a major cause of acute viral hepatitis in Singapore. While epidemiological investigations are ongoing to elucidate the risk factors and modes of transmission, travellers should be reminded to practise a high standard of personal and food hygiene when visiting endemic countries.

Certification of poliomyelitis eradication in Singapore and the challenges ahead.

INTRODUCTION: This study reviewed the epidemiological trends of poliomyelitis from 1946 to 2010, and the impact of the national immunisation programme in raising the population herd immunity against poliovirus. We also traced the efforts Singapore has made to achieve certification of poliomyelitis eradication by the World Health Organisation. MATERIALS AND METHODS: Epidemiological data on all reported cases of poliomyelitis were obtained from the Communicable Diseases Division of the Ministry of Health as well as historical records. Coverage of the childhood immunisation programme against poliomyelitis was based on the immunisation data maintained by the National Immunisation Registry, Health Promotion Board. To assess the herd immunity of the population against poliovirus, 6 serological surveys were conducted in 1962, 1978, 1982 to 1984, 1989, 1993 and from 2008 to 2010. RESULTS: Singapore was among the fi rst countries in the world to introduce live oral poliovirus vaccine (OPV) on a mass scale in 1958. With the comprehensive coverage of the national childhood immunisation programme, the incidence of paralytic poliomyelitis declined from 74 cases in 1963 to 5 cases from 1971 to 1973. The immunisation coverage for infants, preschool and primary school children has been maintained at 92% to 97% over the past decade. No indigenous poliomyelitis case had been reported since 1978 and all cases reported subsequently were imported. CONCLUSION: Singapore was certified poliomyelitis free along with the rest of the Western Pacific Region in 2000 after fulfilling all criteria for poliomyelitis eradication, including the establishment of a robust acute flaccid paralysis surveillance system. However, post-certification challenges remain, with the risk of wild poliovirus importation. Furthermore, it is timely to consider the replacement of OPV with the inactivated poliovirus vaccine in Singapore's national immunisation programme given the risk of vaccine-associated paralytic poliomyelitis and circulating vaccine-derived polioviruses.

Pandemic (H1N1) 2009 infection in adult solid organ transplant recipients in Singapore.

BACKGROUND: Influenza can produce significant complications in immunocompromised persons. METHODS: We studied the effects of the pandemic (H1N1) 2009 (pH1N1) infection on solid organ transplant recipients in our hospital, with emphasis on clinical information, duration of viral culture positivity, polymerase chain reaction positivity, effects of oseltamivir therapy, and graft status at 6 months of follow-up. RESULTS: Twenty-two cases of pH1N1 infection involving 18 renal, two lung, one heart, and one liver transplant recipients were seen from July 14 to September 8, 2009. Their median age was 50.5 years (range 20-70 years); 64% were women, and median time posttransplant was 40 months (range 6-204 months). Common symptoms were fever (86%), cough (77%), sore throat (55%), phlegm (32%), and myalgia (27%). The median duration of symptoms (n=21) and duration of polymerase chain reaction positivity (n=15) were 7 (range 4-13 days) and 8 days (range 4-16 days), respectively. Mean (± SD) duration of symptom resolution (7.4 ± 3.0 vs. 7.8 ± 3.0 days, P=0.76) and viral culture positivity (5.3 ± 2.8 vs. 4.3 ± 3.2 days, P=0.65) did not differ between those who received a 5-day (n=9) or 10-day (n=12) course of oseltamivir. Five patients (22.7%) developed pneumonia with three needing intensive care. Mortality rate was 4.5% (1/22). At 6 months, three graft rejections involving two renal and one lung developed. CONCLUSIONS: Our findings indicate that the pH1N1 infection in solid organ transplant recipients is associated with some degree of morbidity and may affect the function of the transplanted organ. In this nonrandomized comparison, patients treated with 5 days of oseltamivir did not fare worse compared with those who received 10 days.