Chadwick Y. Yasuda

Genetic Characterization of Zika Virus Strains: Geographic Expansion of the Asian Lineage

Background Zika virus (ZIKV) is a mosquito-borne flavivirus distributed throughout much of Africa and Asia. Infection with the virus may cause acute febrile illness that clinically resembles dengue fever. A recent study indicated the existence of three geographically distinct viral lineages; however this analysis utilized only a single viral gene. Although ZIKV has been known to circulate in both Africa and Asia since at least the 1950s, little is known about the genetic relationships between geographically distinct virus strains. Moreover, the geographic origin of the strains responsible for the epidemic that occurred on Yap Island, Federated States of Micronesia in 2007, and a 2010 pediatric case in Cambodia, has not been determined. Methodology/Principal Findings To elucidate the genetic relationships of geographically distinct ZIKV strains and the origin of the strains responsible for the 2007 outbreak on Yap Island and a 2010 Cambodian pediatric case of ZIKV infection, the nucleotide sequences of the open reading frame of five isolates from Cambodia, Malaysia, Nigeria, Uganda, and Senegal collected between 1947 and 2010 were determined. Phylogenetic analyses of these and previously published ZIKV sequences revealed the existence of two main virus lineages (African and Asian) and that the strain responsible for the Yap epidemic and the Cambodian case most likely originated in Southeast Asia. Examination of the nucleotide and amino acid sequence alignments revealed the loss of a potential glycosylation site in some of the virus strains, which may correlate with the passage history of the virus. Conclusions/Significance The basal position of the ZIKV strain isolated in Malaysia in 1966 suggests that the recent outbreak in Micronesia was initiated by a strain from Southeast Asia. Because ZIKV infection in humans produces an illness clinically similar to dengue fever and many other tropical infectious diseases, it is likely greatly misdiagnosed and underreported.

Infectious Etiologies of Acute Febrile Illness among Patients Seeking Health Care in South-Central Cambodia

The agents of human febrile illness can vary by region and country suggesting that diagnosis, treatment, and control programs need to be based on a methodical evaluation of area-specific etiologies. From December 2006 to December 2009, 9,997 individuals presenting with acute febrile illness at nine health care clinics in south-central Cambodia were enrolled in a study to elucidate the etiologies. Upon enrollment, respiratory specimens, whole blood, and serum were collected. Testing was performed for viral, bacterial, and parasitic pathogens. Etiologies were identified in 38.0% of patients. Influenza was the most frequent pathogen, followed by dengue, malaria, and bacterial pathogens isolated from blood culture. In addition, 3.5% of enrolled patients were infected with more than one pathogen. Our data provide the first systematic assessment of the etiologies of acute febrile illness in south-central Cambodia. Data from syndromic-based surveillance studies can help guide public health responses in developing nations.

Evidence for avian H9N2 influenza virus infections among rural villagers in Cambodia

BACKGROUND Southeast Asia remains a critical region for the emergence of novel and/or zoonotic influenza, underscoring the importance of extensive sampling in rural areas where early transmission is most likely to occur. METHODS In 2008, 800 adult participants from eight sites were enrolled in a prospective population-based study of avian influenza (AI) virus transmission where highly pathogenic avian influenza (HPAI) H5N1 virus had been reported in humans and poultry from 2006 to 2008. From their enrollment sera and questionnaires, we report risk factor findings for serologic evidence of previous infection with 18 AI virus strains. RESULTS Serologic assays revealed no evidence of previous infection with 13 different low-pathogenic AI viruses or with HPAI avian-like A/Cambodia/R0404050/2007(H5N1). However, 21 participants had elevated antibodies against avian-like A/Hong Kong/1073/1999(H9N2), validated with a monoclonal antibody blocking ELISA assay specific for avian H9. CONCLUSIONS Although cross-reaction from antibodies against human influenza viruses cannot be completely excluded, the study data suggest that a number of participants were previously infected with the avian-like A/Hong Kong/1073/1999(H9N2) virus, likely due to as yet unidentified environmental exposures. Prospective data from this cohort will help us better understand the serology of zoonotic influenza infection in a rural cohort in SE Asia.

Dual Infection of Novel Influenza Viruses A/H1N1 and A/H3N2 in a Cluster of Cambodian Patients

During the early months of 2009, a novel influenza A/H1N1 virus (pH1N1) emerged in Mexico and quickly spread across the globe. In October 2009, a 23-year-old male residing in central Cambodia was diagnosed with pH1N1. Subsequently, a cluster of four influenza-like illness cases developed involving three children who resided in his home and the childrens school teacher. Base composition analysis of internal genes using reverse transcriptase polymerase chain reaction and electrospray ionization mass spectrometry revealed that specimens from two of the secondary victims were coinfected with influenza A/H3N2 and pH1N1. Phylogenetic analysis of the hemagglutinin genes from these isolated viruses showed that they were closely related to existing pH1N1 and A/H3N2 viruses circulating in the region. Genetic recombination was not evident within plaque-purified viral isolates on full genome sequencing. This incident confirms dual influenza virus infections and highlights the risk of zoonotic and seasonal influenza viruses to coinfect and possibly, reassort where they cocirculate.

Influenza virus circulation in Cambodia

Background The Cambodian National Influenza Center (NIC) was established in August 2006 for the purpose of documenting the dynamics of influenza disease and to virologically characterize the circulating strains. To continuously monitor influenza activity, a hospital-based sentinel surveillance system for ILI (influenza-like illness) with a weekly reporting and sampling scheme was initially established in five sites in 2006. In addition, hospitalbased surveillance of acute lower respiratory infection (ALRI) cases was established in 2 sites.

Little evidence of subclinical avian influenza virus infections among rural villagers in Cambodia.

In 2008, 800 adults living within rural Kampong Cham Province, Cambodia were enrolled in a prospective cohort study of zoonotic influenza transmission. After enrollment, participants were contacted weekly for 24 months to identify acute influenza-like illnesses (ILI). Follow-up sera were collected at 12 and 24 months. A transmission substudy was also conducted among the family contacts of cohort members reporting ILI who were influenza A positive. Samples were assessed using serological or molecular techniques looking for evidence of infection with human and avian influenza viruses. Over 24 months, 438 ILI investigations among 284 cohort members were conducted. One cohort member was hospitalized with a H5N1 highly pathogenic avian influenza (HPAI) virus infection and withdrew from the study. Ninety-seven ILI cases (22.1%) were identified as influenza A virus infections by real-time RT-PCR; none yielded evidence for AIV. During the 2 years of follow-up, 21 participants (3.0%) had detectable antibody titers (≥1∶10) against the studied AIVs: 1 against an avian-like A/Migratory duck/Hong Kong/MPS180/2003(H4N6), 3 against an avian-like A/Teal/Hong Kong/w312/97(H6N1), 9 (3 of which had detectible antibody titers at both 12- and 24-month follow-up) against an avian-like A/Hong Kong/1073/1999(H9N2), 6 (1 detected at both 12- and 24-month follow-up) against an avian-like A/Duck/Memphis/546/74(H11N9), and 2 against an avian-like A/Duck/Alberta/60/76(H12N5). With the exception of the one hospitalized cohort member with H5N1 infection, no other symptomatic avian influenza infections were detected among the cohort. Serological evidence for subclinical infections was sparse with only one subject showing a 4-fold rise in microneutralization titer over time against AvH12N5. In summary, despite conducting this closely monitored cohort study in a region enzootic for H5N1 HPAI, we were unable to detect subclinical avian influenza infections, suggesting either that these infections are rare or that our assays are insensitive at detecting them.

Rapid-Test Based Identification of Influenza as an Etiology of Acute Febrile Illness in Cambodia

Influenza can be manifested as an acute febrile illness, with symptoms similar to many pathogens endemic to Cambodia. The objective of this study was to evaluate the Quickvue influenza A+B rapid test to identify the etiology of acute febrile illness in Cambodia. During December 2006–May 2008, patients enrolled in a study to identify the etiology of acute febrile illnesses were tested for influenza by real-time reverse transcriptase PCR (RT-PCR) and Quickvue influenza A+B rapid test. The prevalence of influenza was 19.7% by RT-PCR. Compared with RT-PCR, the sensitivity and specificity of the rapid test were 52.1% and 92.5%, respectively. The influenza rapid test identified the etiology in 10.2% of enrollees and ≥ 35% during peak times of influenza activity. This study suggests that rapid influenza tests may be useful during peak times of influenza activity in an area where several different etiologies can present as an acute febrile illness.

Serotype Distribution of Clinical Streptococcus pneumoniae Isolates before the Introduction of the 13-Valent Pneumococcal Conjugate Vaccine in Cambodia

Abstract. Childhood vaccination with the 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in Cambodia in January 2015. Baseline data regarding circulating serotypes are scarce. All microbiology laboratories in Cambodia were contacted for identification of stored isolates of Streptococcus pneumoniae from clinical specimens taken before the introduction of PCV13. Available isolates were serotyped using a multiplex polymerase chain reaction method. Among 166 identified isolates available for serotyping from patients with pneumococcal disease, 4% were isolated from upper respiratory samples and 80% were from lower respiratory samples, and 16% were invasive isolates. PCV13 serotypes accounted for 60% (95% confidence interval [CI] 52–67) of all isolates; 56% (95% CI 48–64) of noninvasive and 77% (95% CI 57–89) of invasive isolates. Antibiotic resistance was more common among PCV13 serotypes. This study of clinical S. pneumoniae isolates supports the potential for high reduction in pneumococcal disease burden and may serve as baseline data for future monitoring of S. pneumoniae serotypes circulation after implementation of PCV13 childhood vaccination in Cambodia.