Publications by authors named "Wiriya Rutvisuttinunt"

35 Publications

Enhanced dengue vaccine virus replication and neutralizing antibody responses in immune primed rhesus macaques.

NPJ Vaccines 2021 May 21;6(1):77. Epub 2021 May 21.

Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MA, USA.

Antibody-dependent enhancement (ADE) is suspected to influence dengue virus (DENV) infection, but the role ADE plays in vaccination strategies incorporating live attenuated virus components is less clear. Using a heterologous prime-boost strategy in rhesus macaques, we examine the effect of priming with DENV purified inactivated vaccines (PIVs) on a tetravalent live attenuated vaccine (LAV). Sera exhibited low-level neutralizing antibodies (NAb) post PIV priming, yet moderate to high in vitro ADE activity. Following LAV administration, the PIV primed groups exhibited DENV-2 LAV peak viremias up to 1,176-fold higher than the mock primed group, and peak viremia correlated with in vitro ADE. Furthermore, PIV primed groups had more balanced and higher DENV-1-4 NAb seroconversion and titers than the mock primed group following LAV administration. These results have implications for the development of effective DENV vaccine prime-boost strategies and for our understanding of the role played by ADE in modulating DENV replication.
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http://dx.doi.org/10.1038/s41541-021-00339-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140083PMC
May 2021

From e-voucher to genomic data: Preserving archive specimens as demonstrated with medically important mosquitoes (Diptera: Culicidae) and kissing bugs (Hemiptera: Reduviidae).

PLoS One 2021 25;16(2):e0247068. Epub 2021 Feb 25.

Walter Reed Biosystematics Unit, Smithsonian Institution Museum Support Center, Suitland, MD, United States of America.

Scientific collections such as the U.S. National Museum (USNM) are critical to filling knowledge gaps in molecular systematics studies. The global taxonomic impediment has resulted in a reduction of expert taxonomists generating new collections of rare or understudied taxa and these large historic collections may be the only reliable source of material for some taxa. Integrated systematics studies using both morphological examinations and DNA sequencing are often required for resolving many taxonomic issues but as DNA methods often require partial or complete destruction of a sample, there are many factors to consider before implementing destructive sampling of specimens within scientific collections. We present a methodology for the use of archive specimens that includes two crucial phases: 1) thoroughly documenting specimens destined for destructive sampling-a process called electronic vouchering, and 2) the pipeline used for whole genome sequencing of archived specimens, from extraction of genomic DNA to assembly of putative genomes with basic annotation. The process is presented for eleven specimens from two different insect subfamilies of medical importance to humans: Anophelinae (Diptera: Culicidae)-mosquitoes and Triatominae (Hemiptera: Reduviidae)-kissing bugs. Assembly of whole mitochondrial genome sequences of all 11 specimens along with the results of an ortholog search and BLAST against the NCBI nucleotide database are also presented.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247068PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906454PMC
August 2021

Temporally integrated single cell RNA sequencing analysis of PBMC from experimental and natural primary human DENV-1 infections.

PLoS Pathog 2021 01 29;17(1):e1009240. Epub 2021 Jan 29.

Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America.

Dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To determine the kinetic transcriptional signature associated with experimental primary DENV-1 infection and to assess how closely this profile correlates with the transcriptional signature accompanying natural primary DENV-1 infection, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both experimental and natural primary DENV-1 infection resulted in overlapping patterns of inflammatory gene upregulation, natural primary DENV-1 infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by experimental and natural primary DENV infection are similar, but that natural primary DENV-1 infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state.
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http://dx.doi.org/10.1371/journal.ppat.1009240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875406PMC
January 2021

The evolution of dengue-2 viruses in Malindi, Kenya and greater East Africa: Epidemiological and immunological implications.

Infect Genet Evol 2021 06 6;90:104617. Epub 2020 Nov 6.

Basic Science Laboratory, US Army Medical Research Directorate - Africa (USAMRD-A), Kisumu, Kenya.

Kenya experiences a substantial burden of dengue, yet there are very few DENV-2 sequence data available from this country and indeed the entire continent of Africa. We therefore undertook whole genome sequencing and evolutionary analysis of fourteen dengue virus (DENV)-2 strains sampled from Malindi sub-County Hospital during the 2017 DENV-2 outbreak in the Kenyan coast. We further performed an extended East African phylogenetic analysis, which leveraged 26 complete African env genes. Maximum likelihood analysis showed that the 2017 outbreak was due to the Cosmopolitan genotype, indicating that this has been the only confirmed human DENV-2 genotype circulating in Africa to date. Phylogeographic analyses indicated transmission of DENV-2 viruses between East Africa and South/South-West Asia. Time-scaled genealogies show that DENV-2 viruses shows spatial structure at the country level in Kenya, with a time-to-most-common-recent ancestor analysis indicating that these DENV-2 strains were circulating for up to 5.38 years in Kenya before detection in the 2017 Malindi outbreak. Selection pressure analyses indicated sampled Kenyan DENV strains uniquely being under positive selection at 6 sites, predominantly across the non-structural genes, and epitope prediction analyses showed that one of these sites corresponds to a putative predicted MHC-I CD8+ DENV-2 Cosmopolitan virus epitope only evident in a sampled Kenyan virus. Taken together, our findings indicate that the 2017 Malindi DENV-2 outbreak arose from a strain which had circulated for several years in Kenya before recent detection, has experienced diversifying selection pressure, and may contain new putative immunogens relevant to vaccine design. These findings prompt further genomic epidemiology studies in this and other Kenyan locations to further elucidate the transmission dynamics of DENV in this region.
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http://dx.doi.org/10.1016/j.meegid.2020.104617DOI Listing
June 2021

A Department of Defense Laboratory Consortium Approach to Next Generation Sequencing and Bioinformatics Training for Infectious Disease Surveillance in Kenya.

Front Genet 2020 25;11:577563. Epub 2020 Sep 25.

Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD, United States.

Epidemics of emerging and re-emerging infectious diseases are a danger to civilian and military populations worldwide. Health security and mitigation of infectious disease threats is a priority of the United States Government and the Department of Defense (DoD). Next generation sequencing (NGS) and Bioinformatics (BI) enhances traditional biosurveillance by providing additional data to understand transmission, identify resistance and virulence factors, make predictions, and update risk assessments. As more and more laboratories adopt NGS and BI technologies they encounter challenges in building local capacity. In addition to choosing the right sequencing platform and approach, considerations must also be made for the complexity of bioinformatics analyses, data storage, as well as personnel and computational requirements. To address these needs, a comprehensive training program was developed covering wet lab and bioinformatics approaches to NGS. The program is meant to be modular and adaptive to meet both common and individualized needs of medical research and public health laboratories across the DoD. The training program was first deployed internationally to the Basic Science Laboratory of the US Army Medical Research Directorate-Africa in Kisumu, Kenya, which is an overseas Lab of the Walter Reed Army Institute of Research (WRAIR). A week-long workshop with intensive focus on targeted sequencing and the bioinformatics of genome assembly ( = 24 participants) was held. Post-workshop self-assessment (completed by 21 participants) noted significant median gains in knowledge domains related to NGS targeted sequencing, bioinformatics for genome assembly, and sequence quality assessment. The participants also reported that the information on study design, sample preparation, sequencing quality control, data quality assessment, reporting, and basic and advanced bioinformatics analysis were the most useful information presented in the training. While longer-term evaluations are planned, the training resulted in significant short-term improvement of a laboratory's self-reported wet lab and bioinformatics capabilities. This framework can be used for future DoD laboratory development in the area of NGS and BI for infectious disease surveillance, ultimately enhancing this global DoD capability.
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http://dx.doi.org/10.3389/fgene.2020.577563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546821PMC
September 2020

Complete Genome Sequences of 10 Phages Lytic against Multidrug-Resistant Pseudomonas aeruginosa.

Microbiol Resour Announc 2020 Jul 16;9(29). Epub 2020 Jul 16.

Wound Infections Department, Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA

We report the genome sequences of 10 phages studied for their potential for formulation of a therapeutic cocktail; they represent the families , , and Genome sizes ranged from 43,299 to 88,728 nucleotides, with G+C contents of 52.1% to 62.2%. The genomes contained 68 to 168 coding sequences.
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http://dx.doi.org/10.1128/MRA.00503-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365796PMC
July 2020

A Phase 1, Open-Label Assessment of a Dengue Virus-1 Live Virus Human Challenge Strain.

J Infect Dis 2021 02;223(2):258-267

Institute for Global Health and Translational Science, Department of Microbiology and Immunology, and Department of Public Health and Preventive Medicine, State University of New York, Upstate Medical University, Syracuse, New York, USA.

Background: Dengue human infection models (DHIM) have been used as a safe means to test the viability of prophylaxis and therapeutics.

Methods: A phase 1 study of 12 healthy adult volunteers using a challenge virus, DENV-1-LVHC strain 45AZ5, was performed. A dose escalating design was used to determine the safety and performance profile of the challenge virus. Subjects were evaluated extensively until 28 days and then out to 6 months.

Results: Twelve subjects received the challenge virus: 6 with 0.5 mL of 6.5 × 103 plaque-forming units (PFU)/mL (low-dose group) and 6 with 0.5 mL of 6.5 × 104 PFU/mL (mid-dose group). All except 1 in the low-dose group developed detectable viremia. For all subjects the mean incubation period was 5.9 days (range 5-9 days) and mean time of viremia was 6.8 days (range 3-9 days). Mean peak for all subjects was 1.6 × 107 genome equivalents (GE)/mL (range 4.6 × 103 to 5 × 107 GE/mL). There were no serious adverse events or long-term safety signals noted.

Conclusions: We conclude that DENV-1-LVHC was well-tolerated, resulted in an uncomplicated dengue illness, and may be a suitable DHIM for therapeutic and prophylactic product testing.

Clinical Trials Registration: NCT02372175.
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http://dx.doi.org/10.1093/infdis/jiaa351DOI Listing
February 2021

Transcriptional and clonal characterization of B cell plasmablast diversity following primary and secondary natural DENV infection.

EBioMedicine 2020 Apr 18;54:102733. Epub 2020 Apr 18.

Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD, United States.

Antibody-mediated humoral immunity is thought to play a central role in mediating the immunopathogenesis of acute DENV infection, but limited data are available on the diversity, specificity, and functionality of the antibody response at the molecular level elicited by primary or secondary DENV infection. In order to close this functional gap in our understanding of DENV-specific humoral immunity, we utilized high-throughput single cell RNA sequencing to investigate B cells circulating in both primary and secondary natural DENV infections. We captured full-length paired immunoglobulin receptor sequence data from 9,027 B cells from a total of 6 subjects, including 2,717 plasmablasts. In addition to IgG and IgM class-switched cells, we unexpectedly found a high proportion of the DENV-elicited plasmablasts expressing IgA, principally in individuals with primary DENV infections. These IgA class-switched cells were extensively hypermutated even in individuals with a serologically confirmed primary DENV infection. Utilizing a combination of conventional biochemical assays and high-throughput shotgun mutagenesis, we determined that DENV-reactive IgA class-switched antibodies represent a significant fraction of DENV-reactive Igs generated in response to DENV infection, and that they exhibit a comparable epitope specificity to DENV-reactive IgG antibodies. These results provide insight into the molecular-level diversity of DENV-elicited humoral immunity and identify a heretofore unappreciated IgA plasmablast response to DENV infection.
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http://dx.doi.org/10.1016/j.ebiom.2020.102733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170960PMC
April 2020

Route of inoculation and mosquito vector exposure modulate dengue virus replication kinetics and immune responses in rhesus macaques.

PLoS Negl Trop Dis 2020 04 8;14(4):e0008191. Epub 2020 Apr 8.

GSK Vaccines, Rixensart, Belgium.

Dengue virus (DENV) is transmitted by infectious mosquitoes during blood-feeding via saliva containing biologically-active proteins. Here, we examined the effect of varying DENV infection modality in rhesus macaques in order to improve the DENV nonhuman primate (NHP) challenge model. NHPs were exposed to DENV-1 via subcutaneous or intradermal inoculation of virus only, intradermal inoculation of virus and salivary gland extract, or infectious mosquito feeding. The infectious mosquito feeding group exhibited delayed onset of viremia, greater viral loads, and altered clinical and immune responses compared to other groups. After 15 months, NHPs in the subcutaneous and infectious mosquito feeding groups were re-exposed to either DENV-1 or DENV-2. Viral replication and neutralizing antibody following homologous challenge were suggestive of sterilizing immunity, whereas heterologous challenge resulted in productive, yet reduced, DENV-2 replication and boosted neutralizing antibody. These results show that a more transmission-relevant exposure modality resulted in viral replication closer to that observed in humans.
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http://dx.doi.org/10.1371/journal.pntd.0008191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141610PMC
April 2020

The origins of dengue and chikungunya viruses in Ecuador following increased migration from Venezuela and Colombia.

BMC Evol Biol 2020 02 19;20(1):31. Epub 2020 Feb 19.

Viral Diseases Branch, Walter Reed Army institute of Research, Silver Spring, MD, USA.

Background: In recent years, Ecuador and other South American countries have experienced an increase in arboviral diseases. A rise in dengue infections was followed by introductions of chikungunya and Zika, two viruses never before seen in many of these areas. Furthermore, the latest socioeconomic and political instability in Venezuela and the mass migration of its population into the neighboring countries has given rise to concerns of infectious disease spillover and escalation of arboviral spread in the region.

Results: We performed phylogeographic analyses of dengue (DENV) and chikungunya (CHIKV) virus genomes sampled from a surveillance site in Ecuador in 2014-2015, along with genomes from the surrounding countries. Our results revealed at least two introductions of DENV, in 2011 and late 2013, that initially originated from Venezuela and/or Colombia. The introductions were subsequent to increases in the influx of Venezuelan and Colombian citizens into Ecuador, which in 2013 were 343% and 214% higher than in 2009, respectively. However, we show that Venezuela has historically been an important source of DENV dispersal in this region, even before the massive exodus of its population, suggesting already established paths of viral distribution. Like DENV, CHIKV was introduced into Ecuador at multiple time points in 2013-2014, but unlike DENV, these introductions were associated with the Caribbean. Our findings indicated no direct CHIKV connection between Ecuador, Colombia, and Venezuela as of 2015, suggesting that CHIKV was, at this point, not following the paths of DENV spread.

Conclusion: Our results reveal that Ecuador is vulnerable to arbovirus import from many geographic locations, emphasizing the need of continued surveillance and more diversified prevention strategies. Importantly, increase in human movement along established paths of viral dissemination, combined with regional outbreaks and epidemics, may facilitate viral spread and lead to novel virus introductions. Thus, strengthening infectious disease surveillance and control along migration routes and improving access to healthcare for the vulnerable populations is of utmost importance.
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http://dx.doi.org/10.1186/s12862-020-1596-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031975PMC
February 2020

Potent Zika and dengue cross-neutralizing antibodies induced by Zika vaccination in a dengue-experienced donor.

Nat Med 2020 02 3;26(2):228-235. Epub 2020 Feb 3.

Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, Saint Louis, MO, USA.

Zika virus (ZIKV) has caused significant disease, with widespread cases of neurological pathology and congenital neurologic defects. Rapid vaccine development has led to a number of candidates capable of eliciting potent ZIKV-neutralizing antibodies (reviewed in refs. ). Despite advances in vaccine development, it remains unclear how ZIKV vaccination affects immune responses in humans with prior flavivirus immunity. Here we show that a single-dose immunization of ZIKV purified inactivated vaccine (ZPIV) in a dengue virus (DENV)-experienced human elicited potent cross-neutralizing antibodies to both ZIKV and DENV. Using a unique ZIKV virion-based sorting strategy, we isolated and characterized multiple antibodies, including one termed MZ4, which targets a novel site of vulnerability centered on the Envelope (E) domain I/III linker region and protects mice from viremia and viral dissemination following ZIKV or DENV-2 challenge. These data demonstrate that Zika vaccination in a DENV-experienced individual can boost pre-existing flavivirus immunity and elicit protective responses against both ZIKV and DENV. ZPIV vaccination in Puerto Rican individuals with prior flavivirus experience yielded similar cross-neutralizing potency after a single vaccination, highlighting the potential benefit of ZIKV vaccination in flavivirus-endemic areas.
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http://dx.doi.org/10.1038/s41591-019-0746-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018608PMC
February 2020

Next Generation Sequencing and Bioinformatics Methodologies for Infectious Disease Research and Public Health: Approaches, Applications, and Considerations for Development of Laboratory Capacity.

J Infect Dis 2020 03;221(Suppl 3):S292-S307

Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland.

Next generation sequencing (NGS) combined with bioinformatics has successfully been used in a vast array of analyses for infectious disease research of public health relevance. For instance, NGS and bioinformatics approaches have been used to identify outbreak origins, track transmissions, investigate epidemic dynamics, determine etiological agents of a disease, and discover novel human pathogens. However, implementation of high-quality NGS and bioinformatics in research and public health laboratories can be challenging. These challenges mainly include the choice of the sequencing platform and the sequencing approach, the choice of bioinformatics methodologies, access to the appropriate computation and information technology infrastructure, and recruiting and retaining personnel with the specialized skills and experience in this field. In this review, we summarize the most common NGS and bioinformatics workflows in the context of infectious disease genomic surveillance and pathogen discovery, and highlight the main challenges and considerations for setting up an NGS and bioinformatics-focused infectious disease research public health laboratory. We describe the most commonly used sequencing platforms and review their strengths and weaknesses. We review sequencing approaches that have been used for various pathogens and study questions, as well as the most common difficulties associated with these approaches that should be considered when implementing in a public health or research setting. In addition, we provide a review of some common bioinformatics tools and procedures used for pathogen discovery and genome assembly, along with the most common challenges and solutions. Finally, we summarize the bioinformatics of advanced viral, bacterial, and parasite pathogen characterization, including types of study questions that can be answered when utilizing NGS and bioinformatics.
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http://dx.doi.org/10.1093/infdis/jiz286DOI Listing
March 2020

Dissecting the heterogeneity of DENV vaccine-elicited cellular immunity using single-cell RNA sequencing and metabolic profiling.

Nat Commun 2019 08 14;10(1):3666. Epub 2019 Aug 14.

Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD, USA.

Generating effective and durable T cell immunity is a critical prerequisite for vaccination against dengue virus (DENV) and other viral diseases. However, understanding the molecular mechanisms of vaccine-elicited T cell immunity remains a critical knowledge gap in vaccinology. In this study, we utilize single-cell RNA sequencing (scRNAseq) and longitudinal TCR clonotype analysis to identify a unique transcriptional signature present in acutely activated and clonally-expanded T cells that become committed to the memory repertoire. This effector/memory-associated transcriptional signature is dominated by a robust metabolic transcriptional program. Based on this transcriptional signature, we are able to define a set of markers that identify the most durable vaccine-reactive memory-precursor CD8 T cells. This study illustrates the power of scRNAseq as an analytical tool to assess the molecular mechanisms of host control and vaccine modality in determining the magnitude, diversity and persistence of vaccine-elicited cell-mediated immunity.
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http://dx.doi.org/10.1038/s41467-019-11634-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694189PMC
August 2019

Aerosol Transmission of Gull-Origin Iceland Subtype H10N7 Influenza A Virus in Ferrets.

J Virol 2019 07 14;93(13). Epub 2019 Jun 14.

Department of Basic Science, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi, USA

Subtype H10 influenza A viruses (IAVs) have been recovered from domestic poultry and various aquatic bird species, and sporadic transmission of these IAVs from avian species to mammals (i.e., human, seal, and mink) are well documented. In 2015, we isolated four H10N7 viruses from gulls in Iceland. Genomic analyses showed four gene segments in the viruses were genetically associated with H10 IAVs that caused influenza outbreaks and deaths among European seals in 2014. Antigenic characterization suggested minimal antigenic variation among these H10N7 isolates and other archived H10 viruses recovered from human, seal, mink, and various avian species in Asia, Europe, and North America. Glycan binding preference analyses suggested that, similar to other avian-origin H10 IAVs, these gull-origin H10N7 IAVs bound to both avian-like alpha 2,3-linked sialic acids and human-like alpha 2,6-linked sialic acids. However, when the gull-origin viruses were compared with another Eurasian avian-origin H10N8 IAV, which caused human infections, the gull-origin virus showed significantly higher binding affinity to human-like glycan receptors. Results from a ferret experiment demonstrated that a gull-origin H10N7 IAV replicated well in turbinate, trachea, and lung, but replication was most efficient in turbinate and trachea. This gull-origin H10N7 virus can be transmitted between ferrets through the direct contact and aerosol routes, without prior adaptation. Gulls share their habitat with other birds and mammals and have frequent contact with humans; therefore, gull-origin H10N7 IAVs could pose a risk to public health. Surveillance and monitoring of these IAVs at the wild bird-human interface should be continued. Subtype H10 avian influenza A viruses (IAVs) have caused sporadic human infections and enzootic outbreaks among seals. In the fall of 2015, H10N7 viruses were recovered from gulls in Iceland, and genomic analyses showed that the viruses were genetically related with IAVs that caused outbreaks among seals in Europe a year earlier. These gull-origin viruses showed high binding affinity to human-like glycan receptors. Transmission studies in ferrets demonstrated that the gull-origin IAV could infect ferrets, and that the virus could be transmitted between ferrets through direct contact and aerosol droplets. This study demonstrated that avian H10 IAV can infect mammals and be transmitted among them without adaptation. Thus, avian H10 IAV is a candidate for influenza pandemic preparedness and should be monitored in wildlife and at the animal-human interface.
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http://dx.doi.org/10.1128/JVI.00282-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580963PMC
July 2019

Molecular epidemiology of a primarily MSM acute HIV-1 cohort in Bangkok, Thailand and connections within networks of transmission in Asia.

J Int AIDS Soc 2018 11;21(11):e25204

United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA.

Introduction: Thailand plays a substantial role in global HIV-1 transmission of CRF01_AE. Worldwide, men who have sex with men (MSM) are at elevated risk for HIV-1 infection. Hence, understanding HIV-1 diversity in a primarily Thai MSM cohort with acute infection, and its connections to the broader HIV-1 transmission network in Asia is crucial for research and development of HIV-1 vaccines, treatment and cure.

Methods: Subtypes and diversity of infecting viruses from individuals sampled from 2009 to 2015 within the RV254/SEARCH 010 cohort were assessed by multiregion hybridization assay (MHAbce), multiregion subtype-specific PCR assay (MSSPbce) and full-length single-genome sequencing (SGS). Phylogenetic analysis was performed by maximum likelihood. Pairwise genetic distances of envelope gp160 sequences obtained from the cohort and from Asia (Los Alamos National Laboratory HIV Database) were calculated to identify potential transmission networks.

Results: MHAbce/MSSPbce results identified 81.6% CRF01_AE infecting strains in RV254. CRF01_AE/B recombinants and subtype B were found at 7.3% and 2.8% respectively. Western subtype B strains outnumbered Thai B' strains. Phylogenetic analysis revealed one C, one CRF01_AE/CRF02_AG recombinant and one CRF01_AE/B/C recombinant. Asian network analysis identified one hundred and twenty-three clusters, including five clusters of RV254 participants. None of the RV254 sequences clustered with non-RV254 sequences. The largest international cluster involved 15 CRF01_AE strains from China and Vietnam. The remaining clusters were mostly intracountry connections, of which 31.7% included Thai nodes and 43.1% included Chinese nodes.

Conclusion: While the majority of strains in Thailand are CRF01_AE and subtype B, emergence of unique recombinant forms (URFs) are found in a moderate fraction of new HIV-1 infections. Approaches to vaccine design and immunotherapeutics will need to monitor and consider the expanding proportion of recombinants and the increasing genetic diversity in the region. Identified HIV-1 transmission networks indicate ongoing spread of HIV-1 among MSM. As HIV-1 epidemics continue to expand in other Asian countries, transmission network analyses can inform strategies for prevention, intervention, treatment and cure.
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http://dx.doi.org/10.1002/jia2.25204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282942PMC
November 2018

Retrospective use of next-generation sequencing reveals the presence of Enteroviruses in acute influenza-like illness respiratory samples collected in South/South-East Asia during 2010-2013.

J Clin Virol 2017 09 14;94:91-99. Epub 2017 Jul 14.

Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6, Rajavithi Road, Rajathewi, Bangkok, Thailand. Electronic address:

Background: Emerging and re-emerging respiratory pathogens represent an increasing threat to public health. Etiological determination during outbreaks generally relies on clinical information, occasionally accompanied by traditional laboratory molecular or serological testing. Often, this limited testing leads to inconclusive findings. The Armed Forces Research Institute of Medical Sciences (AFRIMS) collected 12,865 nasopharyngeal specimens from acute influenza-like illness (ILI) patients in five countries in South/South East Asia during 2010-2013. Three hundred and twenty-four samples which were found to be negative for influenza virus after screening with real-time RT-PCR and cell-based culture techniques demonstrated the potential for viral infection with evident cytopathic effect (CPE) in several cell lines.

Objective: To assess whether whole genome next-generation sequencing (WG-NGS) together with conventional molecular assays can be used to reveal the etiology of influenza negative, but CPE positive specimens.

Study Design: The supernatant of these CPE positive cell cultures were grouped in 32 pools containing 2-26 supernatants per pool. Three WG-NGS runs were performed on these supernatant pools. Sequence reads were used to identify positive pools containing viral pathogens. Individual samples in the positive pools were confirmed by qRT-PCR, RT-PCR, PCR and Sanger sequencing from the CPE culture and original clinical specimens.

Results: WG-NGS was an effective way to expand pathogen identification in surveillance studies. This enabled the identification of a viral agent in 71.3% (231/324) of unidentified surveillance samples, including common respiratory pathogens (100/324; 30.9%): enterovirus (16/100; 16.0%), coxsackievirus (31/100; 31.0%), echovirus (22/100; 22.0%), human rhinovirus (3/100; 3%), enterovirus genus (2/100; 2.0%), influenza A (9/100; 9.0%), influenza B, (5/100; 5.0%), human parainfluenza (4/100; 4.0%), human adenovirus (3/100; 3.0%), human coronavirus (1/100; 1.0%), human metapneumovirus (2/100; 2.0%), and mumps virus (2/100; 2.0%), in addition to the non-respiratory pathogen herpes simplex virus type 1 (HSV-1) (172/324; 53.1%) and HSV-1 co-infection with respiratory viruses (41/324; 12.7%).
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http://dx.doi.org/10.1016/j.jcv.2017.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106496PMC
September 2017

Detection of Zika Virus Infection in Thailand, 2012-2014.

Am J Trop Med Hyg 2015 Aug 22;93(2):380-383. Epub 2015 Jun 22.

Zika virus (ZIKV) is an emerging mosquito-borne pathogen with reported cases in Africa, Asia, and large outbreaks in the Pacific. No autochthonous ZIKV infections have been confirmed in Thailand. However, there have been several cases reported in travelers returning from Thailand. Here we report seven cases of acute ZIKV infection in Thai residents across the country confirmed by molecular or serological testing including sequence data. These endemic cases, combined with previous reports in travelers, provide evidence that ZIKV is widespread throughout Thailand.
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http://dx.doi.org/10.4269/ajtmh.15-0022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530765PMC
August 2015

SNPer: an R library for quantitative variant analysis on single nucleotide polymorphisms among influenza virus populations.

PLoS One 2015 13;10(4):e0122812. Epub 2015 Apr 13.

Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

Influenza virus (IFV) can evolve rapidly leading to genetic drifts and shifts resulting in human and animal influenza epidemics and pandemics. The genetic shift that gave rise to the 2009 influenza A/H1N1 pandemic originated from a triple gene reassortment of avian, swine and human IFVs. More minor genetic alterations in genetic drift can lead to influenza drug resistance such as the H274Y mutation associated with oseltamivir resistance. Hence, a rapid tool to detect IFV mutations and the potential emergence of new virulent strains can better prepare us for seasonal influenza outbreaks as well as potential pandemics. Furthermore, identification of specific mutations by closely examining single nucleotide polymorphisms (SNPs) in IFV sequences is essential to classify potential genetic markers associated with potentially dangerous IFV phenotypes. In this study, we developed a novel R library called "SNPer" to analyze quantitative variants in SNPs among IFV subpopulations. The computational SNPer program was applied to three different subpopulations of published IFV genomic information. SNPer queried SNPs data and grouped the SNPs into (1) universal SNPs, (2) likely common SNPs, and (3) unique SNPs. SNPer outperformed manual visualization in terms of time and labor. SNPer took only three seconds with no errors in SNP comparison events compared with 40 hours with errors using manual visualization. The SNPer tool can accelerate the capacity to capture new and potentially dangerous IFV strains to mitigate future influenza outbreaks.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122812PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395159PMC
January 2016

Evidence of West Nile virus infection in Nepal.

BMC Infect Dis 2014 Nov 27;14:606. Epub 2014 Nov 27.

Armed Forces Health Surveillance Center, Silver Spring, Maryland, USA.

Background: Acute febrile illness is common among those seeking medical care and is frequently treated empirically with the underlying illness remaining undiagnosed in resource-poor countries. A febrile illness study was conducted 2009-2010 to identify known and unknown pathogens circulating in Nepal.

Method: Study methods included diagnostic testing and preliminary ELISA screening of acute and convalescent samples for diseases both known and unknown to be circulating in Nepal, including West Nile virus (WNV). The molecular assays including Polymerase Chain Reaction (PCR), Sanger sequencing and ultra deep sequencing on MiSeq Illumina Platform were conducted to further confirm the presence of WNV.

Results: The study enrolled 2,046 patients presenting undifferentiated febrile illness with unknown etiology. Sera from 14 out of 2,046 patients were tested positive for west nile virus (WNV) by nested Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Only two out of 14 cases were confirmed for the presence of WNV by sequencing and identified as WNV lineage 1 phylogentically. The two patients were adult males with fever and no neurological symptoms from Kathmandu and Bharatpur, Nepal.

Conclusion: Two out of 2,046 serum samples contained fragments of WNV genome resembling WNV lineage 1, which is evidence of the continued spread of WNV which should be considered a possible illness cause in Nepal.
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http://dx.doi.org/10.1186/s12879-014-0606-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265323PMC
November 2014

Increasing HIV-1 molecular complexity among men who have sex with men in Bangkok.

AIDS Res Hum Retroviruses 2015 Apr 22;31(4):393-400. Epub 2015 Jan 22.

1 Thailand Ministry of Public Health-U.S. Centers for Disease Control and Prevention Collaboration , Nonthaburi, Thailand .

In Thailand, new HIV-1 infections are largely concentrated in certain risk groups such as men who have sex with men (MSM), where annual incidence may be as high as 12% per year. The paucity of information on the molecular epidemiology of HIV-1 in Thai MSM limits progress in understanding the epidemic and developing new prevention methods. We evaluated HIV-1 subtypes in seroincident and seroprevalent HIV-1-infected men enrolled in the Bangkok MSM Cohort Study (BMCS) between 2006 and 2011. We characterized HIV-1 subtype in 231 seroprevalent and 194 seroincident subjects using the multihybridization assay (MHA). Apparent dual infections, recombinant strains, and isolates found to be nontypeable by MHA were further characterized by targeted genomic sequencing. Most subjects were infected with HIV-1 CRF01_AE (82%), followed by infections with recombinants (11%, primarily CRF01_AE/B recombinants), subtype B (5%), and dual infections (2%). More than 11 distinct chimeric patterns were observed among CRF01B_AE/B recombinants, most involving recombination within integrase. A significant increase in the proportion of nontypeable strains was observed among seroincident MSM between 2006 and 2011. CRF01_AE and subtype B were the most and least common infecting strains, respectively. The predominance of CRF01_AE among HIV-1 infections in Thai MSM participating in the BMCS parallels trends observed in Thai heterosexuals and injecting drug users. The presence of complex recombinants and a significant rise in nontypeable strains suggest ongoing changes in the genetic makeup of the HIV-1 epidemic in Thailand, which may pose challenges for HIV-1 prevention efforts and vaccine development.
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http://dx.doi.org/10.1089/AID.2014.0139DOI Listing
April 2015

Ex vivo activity of endoperoxide antimalarials, including artemisone and arterolane, against multidrug-resistant Plasmodium falciparum isolates from Cambodia.

Antimicrob Agents Chemother 2014 Oct 21;58(10):5831-40. Epub 2014 Jul 21.

Department of Immunology and Medicine, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

Novel synthetic endoperoxides are being evaluated as new components of artemisinin combination therapies (ACTs) to treat artemisinin-resistant Plasmodium falciparum malaria. We conducted blinded ex vivo activity testing of fully synthetic (OZ78 and OZ277) and semisynthetic (artemisone, artemiside, artesunate, and dihydroartemisinin) endoperoxides in the histidine-rich protein 2 enzyme-linked immunosorbent assay against 200 P. falciparum isolates from areas of artemisinin-resistant malaria in western and northern Cambodia in 2009 and 2010. The order of potency and geometric mean (GM) 50% inhibitory concentrations (IC50s) were as follows: artemisone (2.40 nM) > artesunate (8.49 nM) > dihydroartemisinin (11.26 nM) > artemiside (15.28 nM) > OZ277 (31.25 nM) > OZ78 (755.27 nM). Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC50 susceptibility ratios of <2.0. All isolates had P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 (pfmdr1) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC50s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance reported in the literature and pfmdr1 mutations should be examined for their combined contributions to emerging ACT resistance.
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http://dx.doi.org/10.1128/AAC.02462-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187925PMC
October 2014

Hepatitis C genotype distribution and homology among geographically disparate injecting drug users in Afghanistan.

J Med Virol 2013 Jul;85(7):1170-9

Department of Molecular Virology and Pathogenesis, United States Military HIV Research Program (MHRP), Walter Reed Army Institute of Research, Silver Spring, MD, USA.

Hepatitis C virus (HCV) prevalence is high among injecting drug users in Afghanistan, but transmission dynamics are poorly understood. Samples from HCV-infected injecting drug users were sequenced to determine circulating genotypes and potential transmission linkages. Serum samples were obtained from injecting drug user participants in Hirat, Jalalabad, and Mazar-i-Sharif between 2006 and 2008 with reactive anti-HCV rapid tests. Specimens with detected HCV viremia were amplified and underwent sequence analysis. Of 113 samples evaluated, 25 samples (35.2%) were only typeable in NS5B, nine samples (12.7%) were only typeable in CE1, and 37 samples (52.1%) were genotyped in both regions. Of those with typeable HCV, all were Afghan males with a mean age of 31.1 (standard deviation [SD] ± 8.0) years and mean duration of injecting of 3.9 (SD ± 4.3) years. Most reported residence outside Afghanistan in the last decade (90.1%) and prior incarceration (76.8%). HCV genotypes detected were: 1a, (35.2%, n = 25), 3a (62.0%, n = 44), and 1b (2.8%, n = 2). Cluster formation was detected in NS5B and CE1 and were generally from within the same city. All participants within clusters reported being a refugee in Iran compared to 93.5% of those outside clusters. Only 22.2% (4/11) of those within clusters had been refugees in Pakistan and these four individuals had also been refugees in Iran. Predominance of genotype 3a and the association between HCV viremia and having been a refugee in Iran potentially reflects migration between Afghanistan and Iran among IDUs from Mazar-i-Sharif and Hirat and carry implications for harm reduction programs for this migratory population.
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http://dx.doi.org/10.1002/jmv.23575DOI Listing
July 2013

Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform.

J Virol Methods 2013 Nov 12;193(2):394-404. Epub 2013 Jul 12.

Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand. Electronic address:

Active global surveillance and characterization of influenza viruses are essential for better preparation against possible pandemic events. Obtaining comprehensive information about the influenza genome can improve our understanding of the evolution of influenza viruses and emergence of new strains, and improve the accuracy when designing preventive vaccines. This study investigated the use of deep sequencing by the next-generation sequencing (NGS) Illumina MiSeq Platform to obtain complete genome sequence information from influenza virus isolates. The influenza virus isolates were cultured from 6 respiratory acute clinical specimens collected in Thailand and Nepal. DNA libraries obtained from each viral isolate were mixed and all were sequenced simultaneously. Total information of 2.6 Gbases was obtained from a 455±14 K/mm2 density with 95.76% (8,571,655/8,950,724 clusters) of the clusters passing quality control (QC) filters. Approximately 93.7% of all sequences from Read1 and 83.5% from Read2 contained high quality sequences that were ≥Q30, a base calling QC score standard. Alignments analysis identified three seasonal influenza A H3N2 strains, one 2009 pandemic influenza A H1N1 strain and two influenza B strains. The nearly entire genomes of all six virus isolates yielded equal or greater than 600-fold sequence coverage depth. MiSeq Platform identified seasonal influenza A H3N2, 2009 pandemic influenza A H1N1and influenza B in the DNA library mixtures efficiently.
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http://dx.doi.org/10.1016/j.jviromet.2013.07.001DOI Listing
November 2013

Use of dried blood spots for HIV-1 genotyping in Southeast Asia: Thailand experience.

Southeast Asian J Trop Med Public Health 2012 Mar;43(2):333-9

Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

The multi-region hybridization assay (MHAbce) for genotyping HIV-1 subtypes B, C and circulating recombinant form (CRF01_AE) was evaluated on paired plasma and dried blood spots (DBS) collected from 68 HIV-1 infected individuals in Thailand. CRF01_AE was the predominant subtype identified using plasma samples (51/62) and DBS (24/27). There was no discordance in subtype designations between plasma and DBS.
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March 2012

Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates.

Malar J 2012 Sep 13;11:325. Epub 2012 Sep 13.

Department of Immunology and Medicine, US Army Medical Corps, Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS), Bangkok, Thailand.

Background: Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values.

Methods: Performance parameters of the W2 P. falciparum clone (considered artemisinin "sensitive") were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility.

Results: Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72 hours; 6.3 nM) and prolonged (greater or equal to 72 hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy.

Conclusion: The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.
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http://dx.doi.org/10.1186/1475-2875-11-325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489509PMC
September 2012

Pharmacokinetics and pharmacodynamics of oral artesunate monotherapy in patients with uncomplicated Plasmodium falciparum malaria in western Cambodia.

Antimicrob Agents Chemother 2012 Nov 6;56(11):5484-93. Epub 2012 Aug 6.

Department of Immunology and Medicine, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.

Artemisinin-resistant malaria along the Thailand-Cambodian border is an important public health concern, yet mechanisms of drug action and their contributions to the development of resistance are poorly understood. The pharmacokinetics and pharmacodynamics of oral artesunate monotherapy were explored in a dose-ranging trial in an area of emerging artesunate resistance in western Cambodia. We enrolled 143 evaluable subjects with uncomplicated Plasmodium falciparum malaria in an open label study of directly observed artesunate monotherapy at 3 dose levels (2, 4, and 6 mg/kg of body weight/day) for 7 days at Tasanh Health Center, Tasanh, Cambodia. Clinical outcomes were similar among the 3 groups. Wide variability in artesunate and dihydroartemisinin concentrations in plasma was observed. No significant dose-effect or concentration-effect relationships between pharmacokinetic (PK) and parasite clearance parameters were observed, though baseline parasitemia was modestly correlated with increased parasite clearance times. The overall parasite clearance times were prolonged compared with the clearance times in a previous study at this site in 2006 to 2007, but this did not persist when the evaluation was limited to subjects with a comparable artesunate dose (4 mg/kg/day) and baseline parasitemia from the two studies. Reduced plasma drug levels with higher presentation parasitemias, previously hypothesized to result from partitioning into infected red blood cells, was not observed in this population with uncomplicated malaria. Neither in vitro parasite susceptibility nor plasma drug concentrations appeared to have a direct relationship with the pharmacodynamic (PD) effects of oral artesunate on malaria parasites. While direct concentration-effect relationships were not found, it remains possible that a population PK modeling approach that allows modeling of greater dose separation might discern more-subtle relationships.
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http://dx.doi.org/10.1128/AAC.00044-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486599PMC
November 2012

Ex vivo drug sensitivity profiles of Plasmodium falciparum field isolates from Cambodia and Thailand, 2005 to 2010, determined by a histidine-rich protein-2 assay.

Malar J 2012 Jun 13;11:198. Epub 2012 Jun 13.

Department of Immunology and Medicine, US Army Medical Corps, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

Background: In vitro drug susceptibility assay of Plasmodium falciparum field isolates processed "immediate ex vivo" (IEV), without culture adaption, and tested using histidine-rich protein-2 (HRP-2) detection as an assay, is an expedient way to track drug resistance.

Methods: From 2005 to 2010, a HRP-2 in vitro assay assessed 451 P. falciparum field isolates obtained from subjects with malaria in western and northern Cambodia, and eastern Thailand, processed IEV, for 50% inhibitory concentrations (IC50) against seven anti-malarial drugs, including artesunate (AS), dihydroartemisinin (DHA), and piperaquine.

Results: In western Cambodia, from 2006 to 2010, geometric mean (GM) IC50 values for chloroquine, mefloquine, quinine, AS, DHA, and lumefantrine increased. In northern Cambodia, from 2009-2010, GM IC50 values for most drugs approximated the highest western Cambodia GM IC50 values in 2009 or 2010.

Conclusions: Western Cambodia is associated with sustained reductions in anti-malarial drug susceptibility, including the artemisinins, with possible emergence, or spread, to northern Cambodia. This potential public health crisis supports continued in vitro drug IC50 monitoring of P. falciparum isolates at key locations in the region.
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http://dx.doi.org/10.1186/1475-2875-11-198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3403988PMC
June 2012

Two unique recombinant forms identified in incident HIV type 1 infections in Thai blood donors.

AIDS Res Hum Retroviruses 2012 Dec 25;28(12):1703-11. Epub 2012 Jun 25.

Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

HIV-1 genetic diversity of recently seroconverting (<12 months) Thai repeated blood donors attending the National Blood Centre, Thai Red Cross Society (NBC, TRCS) from September 2007 until March 2008 was assessed. Ten HIV-1 recent seroconvertors (10/239,134 donations) were identified during the study period. The estimated median time to seroconversion was 67.3 days (range: 45.5-102.0 days), and viral load ranged from 307 to 341,805 copies HIV-1 RNA/ml. MHAbce, a real-time-based PCR genotyping assay, identified six CRF01_AE, two CRF01_AE/B recombinants, one subtype B, and one CRF01_AE/B dual infection. Nine samples were further characterized by full genome sequencing, identifying CRF01_AE (N=6), unique CRF01_AE/B recombinants (N=2), and subtype B (N=1). One recombinant contained 13 breakpoints located in gag, pol, vif, vpr, env, and nef while the other recombinant contained 10 breakpoints located in pol, vif, env, and nef. This study found two unique CRF01B recombinants circulating in 10 recent HIV-1-positive subjects from a blood donor population in Thailand.
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http://dx.doi.org/10.1089/aid.2011.0339DOI Listing
December 2012

Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection.

PLoS One 2012 30;7(3):e33948. Epub 2012 Mar 30.

South East Asia Research Collaboration with Hawaii, Pathumwan, Bangkok, Thailand.

Background: Limited knowledge exists on early HIV events that may inform preventive and therapeutic strategies. This study aims to characterize the earliest immunologic and virologic HIV events following infection and investigates the usage of a novel therapeutic strategy.

Methods And Findings: We prospectively screened 24,430 subjects in Bangkok and identified 40 AHI individuals. Thirty Thais were enrolled (8 Fiebig I, 5 Fiebig II, 15 Fiebig III, 2 Fiebig IV) of whom 15 completed 24 weeks of megaHAART (tenofovir/emtricitabine/efavirenz/raltegravir/maraviroc). Sigmoid biopsies were completed in 24/30 at baseline and 13/15 at week 24. At baseline, the median age was 29 years and 83% were MSM. Most were symptomatic (87%), and were infected with R5-tropic (77%) CRF01_AE (70%). Median CD4 was 406 cells/mm(3). HIV RNA was 5.5 log(10) copies/ml. Median total blood HIV DNA was higher in Fiebig III (550 copy/10(6) PBMC) vs. Fiebig I (8 copy/10(6) PBMC) (p = 0.01) while the median %CD4+CCR5+ gut T cells was lower in Fiebig III (19%) vs. Fiebig I (59%) (p = 0.0008). After 24 weeks of megaHAART, HIV RNA levels of <50 copies were achieved in 14/15 in blood and 13/13 in gut. Total blood HIV DNA at week 0 predicted reservoir size at week 24 (p<0.001). Total HIV DNA declined significantly and was undetectable in 3 of 15 in blood and 3 of 7 in gut. Frequency of CD4+CCR5+ gut T cells increased from 41% at baseline to 64% at week 24 (p>0.050); subjects with less than 40% at baseline had a significant increase in CD4+CCR5+ T cells from baseline to week 24 (14% vs. 71%, p = 0.02).

Conclusions: Gut T cell depletion and HIV reservoir seeding increases with progression of AHI. MegaHAART was associated with immune restoration and reduced reservoir size. Our findings could inform research on strategies to achieve HIV drug-free remission.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033948PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3316511PMC
November 2012
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