Phylodynamics of Highly Pathogenic Avian Influenza A(H5N1) Virus Circulating in Indonesian Poultry.

After its first detection in 1996, the highly pathogenic avian influenza A(H5Nx) virus has spread extensively worldwide. HPAIv A(H5N1) was first detected in Indonesia in 2003 and has been endemic in poultry in this country ever since. However, Indonesia has limited information related to the phylodynamics of HPAIv A(H5N1) in poultry. The present study aimed to increase the understanding of the evolution and temporal dynamics of HPAIv H5N1 in Indonesian poultry between 2003 and 2016. To this end, HPAIv A(H5N1) hemagglutinin sequences of viruses collected from 2003 to 2016 were analyzed using Bayesian evolutionary analysis sampling trees. Results indicated that the common ancestor of Indonesian poultry HPAIv H5N1 arose approximately five years after the common ancestor worldwide of HPAI A(H5Nx). In addition, this study indicated that only two introductions of HPAIv A(H5N1) occurred, after which these viruses continued to evolve due to extensive spread among poultry. Furthermore, this study revealed the divergence of H5N1 clade 2.3.2.1c from H5N1 clade 2.3.2.1b. Both clades 2.3.2.1c and 2.3.2.1b share a common ancestor, clade 1, suggesting that clade 2.3.2.1 originated and diverged from China and other Asian countries. Since there was limited sequence and surveillance data for the HPAIv A(H5N1) from wild birds in Indonesia, the exact role of wild birds in the spread of HPAIv in Indonesia is currently unknown. The evolutionary dynamics of the Indonesian HPAIv A(H5N1) highlight the importance of continuing and improved genomic surveillance and adequate control measures in the different regions of both the poultry and wild birds. Spatial genomic surveillance is useful to take adequate control measures. Therefore, it will help to prevent the future evolution of HPAI A(H5N1) and pandemic threats.

Improving Linkage to Care of Hepatitis C: Clinical Validation of GeneXpert® HCV Viral Load Point-of-Care Assay in Indonesia.

Hepatitis C virus (HCV) infection large-scale diagnosis and treatment are hampered by lack of a simple, rapid, and reliable point-of-care (POC) test, which poses a challenge for the elimination of hepatitis C as a public health problem. This study aimed to evaluate Cepheid Xpert® HCV Viral Load performance in comparison with the Roche Cobas® TaqMan® HCV Test using serum samples of HCV-infected patients in Indonesia. Viral load quantification was performed on 243 anti-HCV positive patients' samples using both Xpert HCV VL and Roche HCV tests, followed by HCV genotyping by reverse hybridization. Strength of the relationship between the assays was measured by Pearson correlation coefficient, while level of agreement was analyzed by Deming regression and Bland-Altman plot analysis using log10-transformed viral load values. Quantifiable viral load was detected in 180/243 (74.1%), with Xpert HCV VL sensitivity of 100% (95% CI 0.98, 1.00) and specificity of 98.4% (95% CI 0.91, 0.99) based on the Roche HCV test, while HCV genotypes were determined in 172/180 (95.6%) samples. There was a good correlation between both assays (r = 0.97, P < 0.001), overall and per genotype, with good concordance by Deming regression and a mean difference of -0.25 log10 IU/mL (95% CI -0.33, -0.18) by Bland-Altman plot analysis. Xpert HCV VL test was demonstrated as a POC platform with good performance for HCV diagnosis and treatment decision that would be beneficial for decentralized services in resource-limited areas. HCV testing sites, alongside additional GeneXpert modular systems distributed toward the fight against COVID-19, could ensure some continuity, once this pandemic is controlled.

Asian consensus recommendations on optimizing the diagnosis and initiation of treatment of hepatitis B virus infection in resource-limited settings.

Asia has an intermediate-to-high prevalence of and high morbidity and mortality from hepatitis B virus (HBV) infection. Optimization of diagnosis and initiation of treatment is one of the crucial strategies for lowering disease burden in this region. Therefore, a panel of 24 experts from 10 Asian countries convened, and reviewed the literature, to develop consensus guidance on diagnosis and initiation of treatment of HBV infection in resource-limited Asian settings. The panel proposed 11 recommendations related to diagnosis, pre-treatment assessment, and indications of therapy of HBV infection, and management of HBV-infected patients with co-infections. In resource-limited Asian settings, testing for hepatitis B surface antigen may be considered as the primary test for diagnosis of HBV infection. Pre-treatment assessments should include tests for complete blood count, liver and renal function, hepatitis B e-antigen (HBeAg), anti-HBe, HBV DNA, co-infection markers and assessment of severity of liver disease. Noninvasive tests such as AST-to-platelet ratio index, fibrosis score 4 or transient elastography may be used as alternatives to liver biopsy for assessing disease severity. Considering the high burden of HBV infection in Asia, the panel adopted an aggressive approach, and recommended initiation of antiviral therapy in all HBV-infected, compensated or decompensated cirrhotic individuals with detectable HBV DNA levels, regardless of HBeAg status or alanine transaminase levels. The panel also developed a simple algorithm for guiding the initiation of treatment in noncirrhotic, HBV-infected individuals. The recommendations proposed herein, may help guide clinicians, to optimize the diagnosis and improvise the treatment rates for HBV infection in Asia.

Highly Pathogenic Avian Influenza A(H5N1) Outbreaks in West Java Indonesia 2015-2016: Clinical Manifestation and Associated Risk Factors.

Knowledge of outbreaks and associated risk factors is helpful to improve control of the Highly Pathogenic Avian Influenza A(H5N1) virus (HPAI) in Indonesia. This study was conducted to detect outbreaks of HPAI H5N1 in endemically infected regions by enhanced passive surveillance, to describe the clinical manifestation of these outbreaks and identify associated risk factors. From November 2015 to November 2016, HPAI outbreak investigations were conducted in seven districts of West Java. In total 64 outbreaks were confirmed out of 75 reported suspicions and outbreak characteristics were recorded. The highest mortality was reported in backyard chickens (average 59%, CI95%: 49-69%). Dermal apoptosis and lesions (64%, CI95%: 52-76%) and respiratory signs (39%, CI95%: 27-51%) were the clinical signs observed overall most frequently, while neurological signs were most frequently observed in ducks (68%, CI95%: 47-90%). In comparison with 60 non-infected control farms, the rate of visitor contacts onto a farm was associated with the odds of HPAI infection. Moreover, duck farms had higher odds of being infected than backyard farms, and larger farms had lower odds than small farms. Results indicate that better external biosecurity is needed to reduce transmission of HPAI A(H5N1) in Indonesia.

Reassortments among Avian Influenza A(H5N1) Viruses Circulating in Indonesia, 2015-2016.

Highly pathogenic avian influenza (HPAI) A(H5N1) viruses have been circulating since 2003 in Indonesia, with major impacts on poultry health, severe economic losses, and 168 fatal laboratory-confirmed human cases. We performed phylogenetic analysis on 39 full-genome H5N1 virus samples collected during outbreaks among poultry in 2015-2016 in West Java and compared them with recently published sequences from Indonesia. Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to 2 genetic groups in clade 2.3.2.1c. We also observed these groups for the neuraminidase, nucleoprotein, polymerase, and polymerase basic 1 genes. Matrix, nonstructural protein, and polymerase basic 2 genes of some HPAI were most closely related to clade 2.1.3 instead of clade 2.3.2.1c, and a polymerase basic 2 gene was most closely related to Eurasian low pathogenicity avian influenza. Our results detected a total of 13 reassortment types among HPAI in Indonesia, mostly in backyard chickens in Indramayu.

Evolutionary study and phylodynamic pattern of human influenza A/H3N2 virus in Indonesia from 2008 to 2010.

Influenza viruses are by nature unstable with high levels of mutations. The sequential accumulation of mutations in the surface glycoproteins allows the virus to evade the neutralizing antibodies. The consideration of the tropics as the influenza reservoir where viral genetic and antigenic diversity are continually generated and reintroduced into temperate countries makes the study of influenza virus evolution in Indonesia essential. A total of 100 complete coding sequences (CDS) of Hemagglutinin (HA) and Neuraminidase (NA) genes of H3N2 virus were obtained from archived samples of Influenza-Like Illness (ILI) surveillance collected from 2008 to 2010. Our evolutionary and phylogenetic analyses provide insight into the dynamic changes of Indonesian H3N2 virus from 2008 to 2010. Obvious antigenic drift with typical 'ladder-like' phylogeny was observed with multiple lineages found in each year, suggesting co-circulation of H3N2 strains at different time periods. The mutational pattern of the Indonesian H3N2 virus was not geographically related as relatively low levels of mutations with similar pattern of relative genetic diversity were observed in various geographical origins. This study reaffirms that the existence of a particular lineage is most likely the result of adaptation or competitive exclusion among different host populations and combination of stochastic ecological factors, rather than its geographical origin alone.

Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus.

BACKGROUND: Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009. RESULTS: An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested. CONCLUSIONS: The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.

Genogeography and Immune Epitope Characteristics of Hepatitis B Virus Genotype C Reveals Two Distinct Types: Asian and Papua-Pacific.

Distribution of hepatitis B virus (HBV) genotypes/subgenotypes is geographically and ethnologically specific. In the Indonesian archipelago, HBV genotype C (HBV/C) is prevalent with high genome variability, reflected by the presence of 13 of currently existing 16 subgenotypes. We investigated the association between HBV/C molecular characteristics with host ethnicity and geographical distribution by examining various subgenotypes of HBV/C isolates from the Asia and Pacific region, with further analysis on the immune epitope characteristics of the core and surface proteins. Phylogenetic tree was constructed based on complete HBV/C genome sequences from Asia and Pacific region, and genetic distance between isolates was also examined. HBV/C surface and core immune epitopes were analyzed and grouped by comparing the amino acid residue characteristics and geographical origins. Based on phylogenetic tree and geographical origins of isolates, two major groups of HBV/C isolates--East-Southeast Asia and Papua-Pacific--were identified. Analysis of core and surface immune epitopes supported these findings with several amino acid substitutions distinguishing the East-Southeast Asia isolates from the Papua-Pacific isolates. A west-to-east gradient of HBsAg subtype distribution was observed with adrq+ prominent in the East and Southeast Asia and adrq- in the Pacific, with several adrq-indeterminate subtypes observed in Papua and Papua New Guinea (PNG). This study indicates that HBV/C isolates can be classified into two types, the Asian and the Papua-Pacific, based on the virus genome diversity, immune epitope characteristics, and geographical distribution, with Papua and PNG as the molecular evolutionary admixture region in the switching from adrq+ to adrq-.

Seroepidemiology and occult hepatitis B virus infection in young adults in Banjarmasin, Indonesia.

Hepatitis B virus (HBV) infection remains a public health problem in Indonesia. There has been limited data regarding HBV infection in young adult population. This study aimed to evaluate the seroepidemiology of HBV infection and characterize occult HBV variants in healthy young adults in Banjarmasin, Indonesia, who were born before the implementation of the universal infant hepatitis B vaccination. Serum samples of 195 healthy young adults were tested for HBsAg, anti-HBc, and anti-HBs. The prevalence of HBsAg, anti-HBc, and anti-HBs was 9 (4.6%), 62 (31.8%), and 96 (49.2%), respectively. Seventy four (37.9%) samples were seronegative for all three parameters, indicating the susceptibility to HBV infection. Among 66 samples positive for HBsAg and/or anti-HBc, 13 (19.7%) were HBV DNA positive; of these, four were HBsAg positive and nine were HBsAg negative, and categorized as occult HBV infection. Most occult HBV cases had high-level anti-HBs (>100 IU/l), suggesting that blood with positive anti-HBs and anti-HBc could not be regarded as noninfectious. Thirteen amino acid substitutions were identified: T126S, P127S, Q129R, T131N, M133T, and Y161S in the HBsAg-positive group; P120T, T126I, G145S, Y161F, E164V, and V168F in the occult-HBV group; and T143S in both groups. More studies are required to provide data on the prevalence and characteristics of mutants to ensure reliable diagnosis. The occult HBV infection, combined with the HBsAg prevalence, could indicate the high HBV carriage among young adults in this area. The high percentage of individuals susceptible to HBV infection reiterates the need for catch-up immunization strategies targeted at young adults.

HBsAg, HBeAg and HBV DNA level changes and precore/basal core promoter mutations in the natural history of chronic hepatitis B in Indonesian patients.

INTRODUCTION: Chronic hepatitis B (CHB) is a state of complex interactions between the hepatitis B virus (HBV) and host. We studied the changes in hepatitis B surface antigen (HBsAg), hepatitis B 'e' antigen (HBeAg) and HBV DNA levels, considering the implications of HBV genotype, basal core promoter (BCP) A1762T/G1764A and precore G1896A mutations in CHB. METHODS: One hundred fifty-two treatment-naïve CHB patients were classified into immune-tolerant (IT), immune-clearance (IC), low/non-replicative (LR) and 'e'-negative hepatitis B (ENH) phases, based on HBeAg status, HBV DNA and ALT levels. HBV DNA was detected and quantified by polymerase chain reaction, then analyzed by sequencing. HBsAg and HBeAg levels were measured serologically. RESULTS: HBsAg and HBV DNA levels varied between CHB phases, with HBsAg highest in IT and lowest in LR, and HBV DNA high in IT and IC, and lowest in LR. Both markers increased in ENH. Correlation between HBsAg and HBV DNA was significant in IT and IC, modest in ENH, but missing in LR. HBeAg and HBV DNA levels were dissociated in HBeAg-positive patients. Genotypes B and C were similarly distributed, with precore mutations higher in HBeAg-negative patients and BCP mutations comparable in all phases. Temporal association between HBeAg seroconversion and an increase of BCP/precore mutations was observed. CONCLUSION: HBsAg and HBV DNA levels were high and correlated in early CHB phases and dissociated after HBeAg seroconversion, indicating different controls affecting HBV replication and HBsAg production. Selection of BCP/precore mutants may affect disease course and explain the HBeAg-HBV DNA dissociation, a precaution for clinical application of quantitative HBeAg.

Ethnogeographical structure of hepatitis B virus genotype distribution in Indonesia and discovery of a new subgenotype, B9.

The distribution of hepatitis B virus (HBV) in the populations of island Southeast Asia is of medical and anthropological interest and is associated with an unusually high genetic diversity. This study examined the association of this HBV genetic diversity with the ethnogeography of the populations of the Indonesian archipelago. Whole genome analysis of 21 HBV isolates from East Nusa Tenggara and Papua revealed two recently reported HBV/B subgenotypes unique to the former, B7 (7 isolates) and B8 (5 isolates), and uncovered a further novel subgenotype designated B9 (4 isolates). Further isolates were collected from 419 individuals with defined ethnic backgrounds representing 40 populations. HBV/B was predominant in Austronesian-language-speaking populations, whereas HBV/C was the major genotype in Papua and Papua-influenced populations of Moluccas; HBV/B3 was the predominant subgenotype in the western half of the archipelago (speakers of the Western Malayo-Polynesian [WMP] branch of Austronesian languages), whereas B7, B8 and B9 were specific to Nusa Tenggara (Central Malayo-Polynesian (CMP)). The result provides the first direct evidence that the distribution of HBV genotypes/subgenotypes in the Indonesian archipelago is related to the ethnic origin of its populations and suggests that the HBV distribution is associated with the ancient migratory events in the peopling of the archipelago.