Apiradee Theamboonlers

Avian influenza H5N1 in tigers and leopards.

Influenza virus is not known to affect wild felids. We demonstrate that avian influenza A (H5N1) virus caused severe pneumonia in tigers and leopards that fed on infected poultry carcasses. This finding extends the host range of influenza virus and has implications for influenza virus epidemiology and wildlife conservation.

Fatal avian influenza A H5N1 in a dog.

Avian influenza H5N1 virus is known to cross the species barrier and infect humans and felines. We report a fatal H5N1 infection in a dog following ingestion of an H5N1-infected duck during an outbreak in Thailand in 2004. With new reports of H5N1 virus continuing across Asia, Europe, and Africa, this finding highlights the need for monitoring of domestic animals during outbreaks.

Avian Influenza H5N1 in Naturally Infected Domestic Cat

We report H5N1 virus infection in a domestic cat infected by eating a pigeon carcass. The virus isolated from the pigeon and the cat showed the same cluster as the viruses obtained during the outbreak in Thailand. Since cats are common house pets, concern regarding disease transmission to humans exists.

Probable tiger-to-tiger transmission of avian influenza H5N1.

During the second outbreak of avian influenza H5N1 in Thailand, probable horizontal transmission among tigers was demonstrated in the tiger zoo. Sequencing and phylogenetic analysis of those viruses showed no differences from the first isolate obtained in January 2004. This finding has implications for influenza virus epidemiology and pathogenicity in mammals.

Hepatitis B seroprevalence in Thailand: 12 years after hepatitis B vaccine integration into the national expanded programme on immunization

Objectives  To evaluate the impact of the universal hepatitis B (HB) vaccination programme on the prevalence of hepatitis B surface antigen (HBsAg) carriers and immunity to HB virus infection among children <18 years and to determine the HB seroprevalence in the Thai population.

Typing (A/B) and subtyping (H1/H3/H5) of influenza A viruses by multiplex real-time RT-PCR assays.

In this study, a specific and sensitive one-step multiplex real-time RT-PCR was developed in two assays by using primers and a number of specific locked nucleic acid (LNA)-mediated TaqMan probes which increase the thermal stability of oligonucleotides. The first assay consisted of primers and probes specific to the matrix (M1) gene of influenza A virus, matrix (M1) gene of influenza B virus and GAPDH gene of host cells for typing of influenza virus and verification by an internal control, respectively. The other assay employed primers and probes specific to the hemagglutinin gene of H1, H3 and H5 subtypes in order to identify the three most prominent subtypes of influenza A capable of infecting humans. The specificity results did not produce any cross reactivity with other respiratory viruses or other subtypes of influenza A viruses (H2, H4 and H6-H15), indicating the high specificity of the primers and probes used. The sensitivity of the assays which depend on the type or subtype being detected was approximately 10 to 10(3)copies/microl that depended on the types or subtypes being detected. Furthermore, the assays demonstrated 100% concordance with 35 specimens infected with influenza A viruses and 34 specimens infected with other respiratory viruses, which were identified by direct nucleotide sequencing. In conclusion, the multiplex real-time RT-PCR assays have proven advantageous in terms of rapidity, specificity and sensitivity for human specimens and thus present a feasible and attractive method for large-scale detection aimed at controlling influenza outbreaks.

Persistence of antibodies and immune memory to hepatitis B vaccine 20 years after infant vaccination in Thailand

Booster vaccination against hepatitis B (HBV) is not currently recommended, although debate continues on the duration of protection after priming. We assessed antibody persistence and immune memory to hepatitis B 20 years after priming with a recombinant HBV-vaccine during infancy. Infants were vaccinated at birth, 1, 2 and 12 months of age. A subset received a booster dose at Year 5. Antibody persistence was measured approximately yearly until Year 20. Immune memory was assessed by administration of HBV booster dose. At Year 20, anti-HBs seroprotection rates and GMCs tended to be higher in Year 5 boosted than unboosted recipients (83.9% versus 60.5%). After the Year 20 booster dose, anti-HBs anamnestic responses were within the same range 95.8% of subjects in both groups. Primary and booster vaccination with HBV-vaccine in infants induces sustained seroprotection and immune memory against hepatitis B for up to 20 years. Higher persisting seroprotection rates in subjects boosted at Year 5 did not translate into apparent differences in immune memory in a high endemic country.

Hand, Foot, and Mouth Disease Caused by Coxsackievirus A6, Thailand, 2012

Coxsackievirus A6, Thailand

Evidence of protection against clinical and chronic hepatitis B infection 20 years after infant vaccination in a high endemicity region

Summary.  Vaccination against hepatitis B virus (HBV) immediately after birth prevents neonatal infection by vertical transmission from HBV carrier mothers. There is an ongoing debate whether infant vaccination is sufficient to protect against infection when exposed to HBV later in life. We studied 222 Thai infants born to HBsAg −/+ and HBeAg −/+ mothers who were vaccinated with recombinant hepatitis B vaccine at 0‐1‐2‐12 months of age. A subset of 100 subjects received a booster dose at age 5 years. Blood samples collected yearly for 20 years were examined for anti‐HBs antibodies and serological markers of hepatitis B infection (anti‐HBc, HBsAg, and in selected cases HBeAg, anti‐HBe, HBV DNA). During the 20‐year follow‐up, no subject acquired new chronic HBV infection or clinical hepatitis B disease. During the first decade, possible subclinical breakthrough HBV infection (anti‐HBc seroconversion) was only observed in subjects born to HBsAg +/HBeAg + mothers (6/49 [12.2%]). During the second decade, breakthrough HBV infections were detected in all groups (18/140 [12.8%]). Increases in anti‐HBs concentrations that were unrelated to additional HBV vaccination or infection were detected in approximately 10% of subjects in each decade. Primary infant vaccination with a recombinant hepatitis B vaccine confers long‐term protection against clinical disease and new chronic hepatitis B infection despite confirmed hepatitis B exposure. (http://www.clinicaltrials.gov NCT00240500 and NCT00456625)

H5N1 influenza A virus and infected human plasma.

To the Editor: Since January 2004, a total of 22 persons have been confirmed infected with avian influenza A virus (H5N1) in Thailand; 14 of these patients died. Three waves of outbreaks occurred during the past 2 years. The last patient of the third wave was a 5-year-old boy whose symptoms developed on November 28, 2005; he was hospitalized on December 5 and died 2 days later. The child resided in the Ongkharak District, Nakhon Nayok Province, ≈70 km northeast of Bangkok. Villagers informed the Department of Livestock after the patients illness was diagnosed. Five dead chickens had been reported in this area from November 28 to December 1, 2005. Samples from these chickens could not be obtained, thus, no H5N1 testing was performed. The boy had fever, headache, and productive cough for 7 days before he was admitted to the Her Royal Highness Princess Maha Chakri Sirindhorn Medical Center. Clinical examination and chest radiograph showed evidence of lobar pneumonia. He was treated with antimicrobial drugs (midecamycin and penicillin G) and supportive care, including oxygen therapy. On December 7, the patients condition worsened, and severe pneumonia with adult respiratory distress syndrome developed. Laboratory tests showed leukopenia (2,300 cells/mm3), acidosis, and low blood oxygen saturation by cutaneous pulse oximetry (81.6%). Oseltamivir was administered after his parents informed hospital staff about the boys contact with the dead chicken. However, the boy died the same day; no autopsy was performed. On December 9, the cause of death was declared by the Ministry of Public Health to be H5N1 influenza virus. A blood sample was collected from the patient on December 7; anticoagulation was accomplished with ethylenediaminetetraacetic acid (EDTA) for repeated biochemistry analysis and complete blood count. The plasma from the EDTA blood sample was separated 2 days later and stored at –20°C for 12 days. The sample was subsequently given to the Center of Excellence in Viral Hepatitis, Faculty of Medicine, Chulalongkorn University, for molecular diagnosis and then stored at –70°C, where specific precautions implemented for handling highly infectious disease specimens such as H5N1 influenza virus were observed. Plasma was examined by multiplex reverse transcription–polymerase chain reaction (RT-PCR) (1) and multiplex real-time RT-PCR (2), both of which showed positive results for H5N1 virus. The virus titer obtained from the plasma was 3.08 × 103 copies/mL. The plasma specimen was processed for virus isolation by embryonated egg injection, according to the standard protocol described by Harmon (3). Briefly, 100 μL 1:2 diluted plasma was injected into the allantoic cavity of a 9-day-old embryonated egg and incubated at 37°C. The infected embryo died within 48 hours, and the allantoic fluid was shown to contain 2,048 hemagglutinin (HA) units; also, subtype H5N1 was confirmed (1,2). Whole genome sequencing was performed and submitted to the GenBank database under the strain A/Thailand/NK165/05 accession no. DQ 372591-8. The phylogenetic trees of the HA and neuraminidase (NA) genes were constructed by using MEGA 3 (4) for comparison with H5N1 viruses isolated from humans, tigers, and chickens from previous outbreaks in 2004 and 2005 (Figure). The sequence analyses of the viruses showed that the HA cleavage site contained SPQREKRRKKR, which differed from the 2004 H5N1 virus by an arginine-to-lysine substitution at position 341. That finding had also been observed in wild bird species during earlier outbreaks in Thailand in 2004 (5). Similar to the 2004–2005 H5N1 isolates from Thailand, a 20–amino acid deletion at the NA stalk region was observed. Moreover, the amino acid residues (E119, H274, R292, and N294) of the NA active site were conserved, which suggests that the virus was sensitive to oseltamivir. In addition, a single amino acid substitution from glutamic acid to lysine at position 627 of PB2 showed increased virus replication efficiency in mammals (6). Figure Phylogenetic analysis of the hemagglutinin and neuraminidase genes of H5N1 from study patient compared with sequences from previous outbreaks (2004–2005). Observing live influenza virus in human serum or plasma is unusual. However, in 1963, low quantities of virus were isolated from blood of a patient on day 4 of illness (7), and in 1970, the virus was cultivated from blood specimens from 2 patients (8). Recently, a fatal case of avian influenza A (H5N1) in a Vietnamese child was reported. The diagnosis was determined by isolating the virus from cerebrospinal fluid, fecal, throat, and serum specimens (9); viral RNA was found in 6 of 7 serum specimens 4–9 days after the onset of illness (10). In this case, the H5N1 virus could be isolated from plasma on day 10 after symptoms developed. This case showed the virus in the patients blood, which raises concern about transmission among humans. Because probable H5N1 avian influenza transmission among humans has been reported (11), this case should be a reminder of the necessity to carefully handle and transport serum or plasma samples suspected to be infected with H5N1 avian influenza. Because viable virus has been detected in blood samples, handling, transportation, and testing of blood samples should be performed in a biosafety (category III) containment laboratory to prevent the spread of the virus to healthcare and laboratory workers. We express our thanks to the Thailand Research Fund (Senior Research Scholar), Royal Golden Jubilee PhD Program and Center of Excellence in Viral Hepatitis Research, and Prasert Auewarakul for their generous support of our study.