Exploring the potential of next-generation sequencing in detection of respiratory viruses.

Efficient detection of human respiratory viral pathogens is crucial in the management of patients with acute respiratory tract infection. Sequence-independent amplification of nucleic acids combined with next-generation sequencing technology and bioinformatics analyses is a promising strategy for identifying pathogens in clinical and public health settings. It allows the characterization of hundreds of different known pathogens simultaneously and of novel pathogens that elude conventional testing. However, major hurdles for its routine use exist, including cost, turnaround time, and especially sensitivity of the assay, as the detection limit is dependent on viral load, host genetic material, and sequencing depth. To obtain insights into these aspects, we analyzed nasopharyngeal aspirates from a cohort of 81 Thai children with respiratory disease for the presence of respiratory viruses using a sequence-independent next-generation sequencing approach and routinely used diagnostic real-time reverse transcriptase PCR (real-time RT-PCR) assays. With respect to the detection of rhinovirus and human metapneumovirus, the next-generation sequencing approach was at least as sensitive as diagnostic real-time RT-PCR in this small cohort, whereas for bocavirus and enterovirus, next-generation sequencing was less sensitive than real-time RT-PCR. The advantage of the sequencing approach over real-time RT-PCR was the immediate availability of virus-typing information. Considering the development of platforms capable of generating more output data at declining costs, next-generation sequencing remains of interest for future virus diagnosis in clinical and public health settings and certainly as an additional tool when screening results from real-time RT-PCR are negative.

Evaluation of rapid influenza virus tests in patients with influenza-like illness in Thailand.

BACKGROUND: The influenza virus is responsible for causing major respiratory tract symptoms. A fast, accurate diagnosis is essential for efficient treatment, especially in patients with complications. The Rapid Influenza Diagnostic Tests (RIDTs) for influenza detection have been developed to subtype the influenza virus. The re-evaluation of the rapid test is needed in terms of specificity, sensitivity, and accuracy. METHODS: From August 13, 2010 to September 22, 2011, 1,076 nasal aspirates were obtained from patients, ages ranging from 15 days to 98 years, with symptoms of influenza-like illness (ILI) and evaluated by 2 types of RIDTs, Standard Diagnosis (SD) and QuickVue (QV) Rapid tests followed by real-time RT-PCR. The results from the rapid test diagnoses were compared to those from real-time RT-PCR. RESULTS: During 2010 and 2011, the estimated sensitivity of the SD rapid test for seasonal H3, human pandemic H1N1, and influenza B infection was 49.4%, specificity was 84.1%, positive predictive value was 47.6%, and negative predictive value was 85% while those of the QV rapid test were 63.4%, 96.7%, 94.8%, and 80.3%, respectively. Infant patients (< or = 5 years) yielded less false negatives while adolescents and adults (older than 5 years) showed more false negatives 8.8% and 15.2%, respectively. Using rapid test diagnosis, H3N2 influenza virus was found with more false negative results (11.1%) than the other viruses (1.1 - 3.5%). The SD rapid test appeared to be more sensitive than the QV test during high season activity while the QV test was more sensitive during the period of low influenza virus activity. CONCLUSIONS: Due to persistent genetic drift of the influenza virus, the available RIDTs should be re-evaluated each year. During 2010 - 2011, the QV rapid test showed more reliable results than the SD rapid test. However, the false negative results of H3N2 influenza virus detection during its peak should be cause for concern. Some of the results, e.g. patients with complications, should be compared with real-time RT-PCR as the gold standard method for detecting influenza virus infection.

Influenza surveillance in southern Thailand during 2009-2010.

The aim of this study was to determine the epidemiology of influenza infection among patients with influenza-like illness by real-time RT-PCR in southern Thailand from August 2009 to January 2011. The predominant strain in Thung Song District was influenza A. Sporadic cases of influenza occured year round but the incidence peaked from August to November 2009 and July to November 2010. During August to November 2009, pandemic H1N1 (pH1N1) activity was observed along with a low level of seasonal influenza co-circulation. Subsequently, seasonal influenza (H3) activity increased and became the predominant influenza strain, with co-circulation with pH1N1 and influenza B during the 2010 influenza season. Continual surveillance of influenza activity is useful for public health planning in southern Thailand and plays a major role in future influenza control and prevention measures.

Serological analysis of human pandemic influenza (H1N1) in Thailand.

The study was aimed at determining the prevalence of pandemic influenza (H1N1) 2009 among patients with respiratory tract diseases during July-December 2009 using real-time reverse transcription polymerase chain reaction. Haemagglutination inhibition (HI) assay was performed to detect antibody titres against pandemic influenza in 255 medical personnel, 307 members of the general population during the second week of December 2009 in Khon Kaen province, Thailand, and in 100 stored sera collected from people of different age-groups during 2008. The results showed that the pandemic (H1N1) 2009 had occurred during July-December 2009. The results of the HI test after the wave of this outbreak showed that 123 (48%) of the 255 sera collected from the medical personnel, 109 (36%) of the 307 sera obtained from the general population, and only two of the 100 stored sera from 2008 contained antibodies (HI titres > or = 40) against pandemic influenza. Antibody against the pandemic (H1N1) 2009 was found in at least one-third of the population. In conclusion, the prevalence of virus and serological data obtained from the study can be used as the serological background level of the Thai population after the July-December pandemic. Finally, the serological data might be useful for outbreak-prevention and control strategies and for the management of vaccination for the pandemic (H1N1) 2009 in Thailand.

Determination of antibody response to the human pandemic influenza H1N1 2009 among patients with influenza-like illness and high risk groups.

The global population has been exposed to the novel pandemic H1N1 influenza virus since mid March 2009, causing the expansion of respiratory illness around the world, including Thailand. To evaluate the antibody titers against human pandemic influenza (H1N1) in Thai people with influenza-like illness (ILI), 45 paired serum samples (acute and convalescent) were subjected to hemagglutination inhibition (HI) test and real-time RT-PCR. Most serum samples of ILI patients positive by real-time RT-PCR displayed an at least four-fold antibody increase of HI titers against pandemic influenza (H1N1). In addition, to determine cross-reactivity with human seasonal H1N1 influenza, viral antigen from the seasonal H1N1 was used to detect antibody against seasonal H1N1 influenza and all sera showed negative results. We also studied the single sera samples from the high risk medical personals collected before and after the pandemic influenza (HIN1) outbreaks for antibodies against seasonal H3 influenza virus infection. The results showed lack of cross-reactivity to the human pandemic H1N1 influenza virus. HI antibody testing to pandemic influenza (H1N1) can be used for the diagnosis, preventive and control measures of potential outbreaks.

Influenza activity in Thailand and occurrence in different climates.

This study observed influenza activity between June 2009 and July 2014 in Thailand, a country in the Northern hemisphere with a tropical climate, and compared the results to activity in the United States (US) and Australia, which represent temperate climates in the Northern and Southern hemispheres, respectively. From Thailand, a total of 17,416 specimens were collected from patients exhibiting influenza-like illnesses and subjected to real-time PCR for the detection of influenza viruses. For comparison, laboratory confirmations of influenza originating from the US and Australia were obtained from the US CDC's FluView surveillance reports and the Australian Government's Department of Health and Ageing websites. We found that, generally, the influenza season in Thailand starts with the rainy season. This observation of influenza's annual incidence pattern provides a better understanding of its occurrence, suggesting that vaccination campaigns should be started before the influenza season begins in order to reduce transmission.

Molecular epidemiology and phylogenetic analyses of influenza B virus in Thailand during 2010 to 2014.

Influenza B virus remains a major contributor to the seasonal influenza outbreak and its prevalence has increased worldwide. We investigated the epidemiology and analyzed the full genome sequences of influenza B virus strains in Thailand between 2010 and 2014. Samples from the upper respiratory tract were collected from patients diagnosed with influenza like-illness. All samples were screened for influenza A/B viruses by one-step multiplex real-time RT-PCR. The whole genome of 53 influenza B isolates were amplified, sequenced, and analyzed. From 14,418 respiratory samples collected during 2010 to 2014, a total of 3,050 tested positive for influenza virus. Approximately 3.27% (471/14,418) were influenza B virus samples. Fifty three isolates of influenza B virus were randomly chosen for detailed whole genome analysis. Phylogenetic analysis of the HA gene showed clusters in Victoria clades 1A, 1B, 3, 5 and Yamagata clades 2 and 3. Both B/Victoria and B/Yamagata lineages were found to co-circulate during this time. The NA sequences of all isolates belonged to lineage II and consisted of viruses from both HA Victoria and Yamagata lineages, reflecting possible reassortment of the HA and NA genes. No significant changes were seen in the NA protein. The phylogenetic trees generated through the analysis of the PB1 and PB2 genes closely resembled that of the HA gene, while trees generated from the analysis of the PA, NP, and M genes showed similar topology. The NS gene exhibited the pattern of genetic reassortment distinct from those of the PA, NP or M genes. Thus, antigenic drift and genetic reassortment among the influenza B virus strains were observed in the isolates examined. Our findings indicate that the co-circulation of two distinct lineages of influenza B viruses and the limitation of cross-protection of the current vaccine formulation provide support for quadrivalent influenza vaccine in this region.

Seroprevalence of antibody against diphtheria among the population in Khon Kaen province, Thailand.

To assess diphtheria immunity in the northeastern region of Thailand, a seroepidemiological survey was undertaken in 2011 from 516 healthy individuals (age range 2-87 years) in Khon Kaen province. Diphtheria antitoxin levels were measured by enzyme-linked immunosorbent assay and titers of ≥0.1 IU/mL were considered to be protective antitoxin levels. Among the studied population, 94.8% have fully protective levels. The younger population (age range 2-19 years) has higher diphtheria immunity with seroprotection rates of 96.8% to 97.9%, compared with the adult population. The proportion of protective diphtheria antitoxin levels declines to 88.3% to 91.9% in the middle-aged group (20-50 years), and appeared to be higher again in the older age-group (50-70 years). To avoid epidemic spreading, promoting immunization booster programs will be helpful, especially among the adult population (20-50 years). Finally, this study may serve as a valuable guide in deciding exactly which age-groups should be targeted by such an effort.

Assessing Antigenic Drift of Seasonal Influenza A(H3N2) and A(H1N1)pdm09 Viruses.

Under selective pressure from the host immune system, antigenic epitopes of influenza virus hemagglutinin (HA) have continually evolved to escape antibody recognition, termed antigenic drift. We analyzed the genomes of influenza A(H3N2) and A(H1N1)pdm09 virus strains circulating in Thailand between 2010 and 2014 and assessed how well the yearly vaccine strains recommended for the southern hemisphere matched them. We amplified and sequenced the HA gene of 120 A(H3N2) and 81 A(H1N1)pdm09 influenza virus samples obtained from respiratory specimens and calculated the perfect-match vaccine efficacy using the pepitope model, which quantitated the antigenic drift in the dominant epitope of HA. Phylogenetic analysis of the A(H3N2) HA1 genes classified most strains into genetic clades 1, 3A, 3B, and 3C. The A(H3N2) strains from the 2013 and 2014 seasons showed very low to moderate vaccine efficacy and demonstrated antigenic drift from epitopes C and A to epitope B. Meanwhile, most A(H1N1)pdm09 strains from the 2012-2014 seasons belonged to genetic clades 6A, 6B, and 6C and displayed the dominant epitope mutations at epitopes B and E. Finally, the vaccine efficacy for A(H1N1)pdm09 (79.6-93.4%) was generally higher than that of A(H3N2). These findings further confirmed the accelerating antigenic drift of the circulating influenza A(H3N2) in recent years.

Global alert to avian influenza virus infection: from H5N1 to H7N9.

Outbreak of a novel influenza virus is usually triggered by mutational change due to the process known as 'antigenic shift' or re-assortment process that allows animal-to-human or avian-to-human transmission. Birds are a natural reservoir for the influenza virus, and subtypes H5, H7, and H9 have all caused outbreaks of avian influenza in human populations. An especially notorious strain is the HPAI influenza virus H5N1, which has a mortality rate of approximately 60% and which has resulted in numerous hospitalizations, deaths, and significant economic loss. In March 2013, in Eastern China, there was an outbreak of the novel H7N9 influenza virus, which although less pathogenic in avian species, resulted in 131 confirmed cases and 36 deaths in humans over a two-month span. The rapid outbreak of this virus caused global concern but resulted in international cooperation to control the outbreak. Furthermore, cooperation led to valuable research-sharing including genome sequencing of the virus, the development of rapid and specific diagnosis, specimen sharing for future studies, and vaccine development. Although a H7N9 pandemic in the human population is possible due to its rapid transmissibility and extensive surveillance, the closure of the live-bird market will help mitigate the possibility of another H7N9 outbreak. In addition, further research into the source of the outbreak, pathogenicity of the virus, and the development of specific and sensitive detection assays will be essential for controlling and preparing for future H7N9 outbreaks.

Epidemiology of seasonal influenza in Bangkok between 2009 and 2012.

INTRODUCTION: This study investigated influenza activity in Bangkok, Thailand between June 2009 and July 2012. METHODOLOGY: Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect influenza viruses among patients with influenza-like illnesses. RESULTS: Of the 6417 patients tested, influenza virus infection was detected in 42% (n = 2697) of the specimens. Influenza A pH1N1 viruses comprised the predominant strain between 2009 and 2010, and seasonal influenza (H3) had a high prevalence in 2011. Laboratory data showed a prevalence and seasonal pattern of influenza viruses. In 2009, influenza activity peaked in July, the rainy season. In 2010, influenza activity happened in two phases, with the initial one at the beginning of the year and another peak between June and August 2010, which again corresponded to the rainy period. Influenza activity was low for several consecutive weeks at the beginning of 2011, and high H3N2 activity was recorded during the rainy season between July and September 2011. However, from the beginning of 2012 through July 2012, pH1N1, influenza H3N2, and influenza B viruses continuously circulated at a very low level. CONCLUSION: The seasonal pattern of influenza activity in Thailand tended to peak during rainy season between July and September.

Erythrocyte binding preference of human pandemic influenza virus a and its effect on antibody response detection.

BACKGROUND: Validation of hemagglutination inhibition (HI) assays is important for evaluating antibody responses to influenza virus, and selection of erythrocytes for use in these assays is important. This study aimed to determine the correlation between receptor binding specificity and effectiveness of the HI assay for detecting antibody response to pandemic influenza H1N1 (pH1N1) virus. METHODS: Hemagglutination (HA) tests were performed using erythrocytes from 6 species. Subsequently, 8 hemagglutinating units of pH1N1 from each species were titrated by real-time reverse transcription-PCR. To investigate the effect of erythrocyte binding preference on HI antibody titers, comparisons of HI with microneutralization (MN) assays were performed. RESULTS: Goose erythrocytes showed most specific binding with pH1N1, while HA titers using human erythrocytes were comparable to those using turkey erythrocytes. The erythrocyte binding efficiency was shown to have an impact on antibody detection. Comparing MN titers, HI titers using turkey erythrocytes yielded the most accurate results, while those using goose erythrocytes produced the highest geometric mean titer. Human blood group O erythrocytes lacking a specific antibody yielded results most comparable to those obtained using turkey erythrocytes. Further, pre-existing antibody to pH1N1 and different erythrocyte species can distort HI assay results. CONCLUSIONS: HI assay, using turkey and human erythrocytes, yielded the most comparable and applicable results for pH1N1 than those by MN assay, and using goose erythrocytes may lead to overestimated titers. Selection of appropriate erythrocyte species for HI assay allows construction of a more reliable database, which is essential for further investigations and control of virus epidemics.

Whole genome characterization, phylogenetic and genome signature analysis of human pandemic H1N1 virus in Thailand, 2009-2012.

BACKGROUND: Three waves of human pandemic influenza occurred in Thailand in 2009-2012. The genome signature features and evolution of pH1N1 need to be characterized to elucidate the aspects responsible for the multiple waves of pandemic. METHODOLOGY/FINDINGS: Forty whole genome sequences and 584 partial sequences of pH1N1 circulating in Thailand, divided into 1(st), 2(nd) and 3(rd) wave and post-pandemic were characterized and 77 genome signatures were analyzed. Phylogenetic trees of concatenated whole genome and HA gene sequences were constructed calculating substitution rate and d(N)/d(S) of each gene. Phylogenetic analysis showed a distinct pattern of pH1N1 circulation in Thailand, with the first two isolates from May, 2009 belonging to clade 5 while clades 5, 6 and 7 co-circulated during the first wave of pH1N1 pandemic in Thailand. Clade 8 predominated during the second wave and different proportions of the pH1N1 viruses circulating during the third wave and post pandemic period belonged to clades 8, 11.1 and 11.2. The mutation analysis of pH1N1 revealed many adaptive mutations which have become the signature of each clade and may be responsible for the multiple pandemic waves in Thailand, especially with regard to clades 11.1 and 11.2 as evidenced with V731I, G154D of PB1 gene, PA I330V, HA A214T S160G and S202T. The substitution rate of pH1N1 in Thailand ranged from 2.53×10(-3)±0.02 (M2 genes) to 5.27×10(-3)±0.03 per site per year (NA gene). CONCLUSIONS: All results suggested that this virus is still adaptive, maybe to evade the host's immune response and tends to remain in the human host although the d(N)/d(S) were under purifying selection in all 8 genes. Due to the gradual evolution of pH1N1 in Thailand, continuous monitoring is essential for evaluation and surveillance to be prepared for and able to control future influenza activities.

Pandemic (H1N1) 2009 virus infection: persistent viral shedding after Oseltamivir treatment.

OBJECTIVES: To study pandemic (H1N1) 2009 virological outcomes after Oseltamivir treatment in confirmed cases of pandemic (H1N1) 2009 virus infections. A hospital-based cohort study was conducted in south Thailand, between June and September 2009. METHODS: Throat/swab specimens were tested by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) for pandemic (H1N1) 2009. All 357 confirmed cases (122 inpatients, 235 outpatients), whose received a 5-day Oseltamivir treatment. Post-treatment virological follow-up was performed in 91 eligible cases. The NA gene was screened for the H275Y mutation responsible for Oseltamivir resistance. RESULTS: Thirty-three of 91 patients (36%) had underlying diseases. The duration from the onset of illness to the detection of virus ranged 1-14 days (median 3 days). The rRT-PCR was positive on day 5 of treatment in 24 of 91 patients (26%). Patients with underlying diseases had a higher proportion of post-treatment positive test than those without underlying diseases (15/33 vs 9/58). The rRT-PCR-confirmed viruses detected in all 125 throat swab specimens did not show evidence suggesting Oseltamivir resistance. CONCLUSIONS: Prolonged presence of pandemic (H1N1) 2009 detected by rRT-PCR was found. An extended course of antiviral treatment should be considered in patients with underlying diseases and severe clinical symptoms.

Epidemiological and serological surveillance of human pandemic influenza A virus infections during 2009-2010 in Thailand.

Since April 2009, the outbreak of human pandemic influenza A (H1N1) virus (pH1N1) infection has spread from North America to other parts of the world, and currently, pH1N1 is the predominant circulating strain of influenza viruses. Our objectives were to perform a serological survey of medical personnel at the Chumphae Hospital in Thailand and to investigate the prevalence of pH1N1 in randomly selected patients diagnosed with respiratory tract disease. Prevalence of pH1N1 in the patients was determined by performing real-time reverse transcription-polymerase chain reaction. The study was carried out between July 2009 and November 2010. Seroprevalence of hemaglutination inhibition (HI) titers among medical personnel was established in three cross-sectional studies at the end of each wave of the pandemic by performing HI assay to detect antibodies against pH1N1. Infection by the pH1N1 peaked between July and October 2009; the second wave was from January to March 2010 and the third wave from June to November 2010. The HI titers after the first, second, and third waves were 48.2%, 22.4%, and 25.7%, respectively. After the second and third waves, 52.1% and 45.3% of the medical personnel who had received pH1N1 vaccination had HI titers ≥ 40. These findings show that seasonal influenza strain in Chumphae and the predominant influenza strain from each wave was pH1N1. HI assay results also represent the severity of the attack rate in each wave.