Oseltamivir and inhaled zanamivir as influenza prophylaxis in Thai health workers: a randomized, double-blind, placebo-controlled safety trial over 16 weeks.

OBJECTIVES: Long-term chemoprophylaxis using neuraminidase inhibitors may be needed during influenza epidemics but safety data are limited to several weeks. We sought to assess the tolerability of oseltamivir and zanamivir as primary prophylaxis over 16 weeks. METHODS: We conducted a parallel group, double blind, 2 (active drug) :1 (placebo) randomized trial of oral oseltamivir/placebo or inhaled zanamivir/placebo over 16 weeks in healthy, Thai hospital professionals at two Bangkok hospitals. The primary endpoint was study withdrawal due to drug-related (possibly, probably, definitely) serious or adverse events (AEs) graded ≥ 2. RESULTS: Recruited subjects numbered 129 oseltamivir/65 placebo and 131 zanamivir/65 placebo. A total of 102 grade ≥ 2 AEs were reported or detected in 69 subjects: 23/129 (17.8%) versus 15/65 (23.1%) (P=0.26), and 23/131 (17.6%) versus 8/65 (12.3%) (P=0.28). Intercurrent infections/fevers [26/102 (25.5%)], abnormal biochemistry [25/102 (24.5%)] and gastrointestinal symptoms [18/102 (17.6%)] were the most frequently reported AEs. There were no drug-related study withdrawals. Eight serious AEs were all due to intercurrent illnesses. Laboratory, lung function and ECG parameters were similar between drugs and placebos. CONCLUSIONS: Oseltamivir and zanamivir were well tolerated in healthy hospital professionals. Both drugs can be recommended for primary influenza prophylaxis for up to 16 weeks.

Surveillance of hepatitis A and E viruses contamination in shellfish in Thailand.

AIMS: To survey for hepatitis A virus (HAV) and hepatitis E virus (HEV) contamination in edible bivalve shellfish. METHODS AND RESULTS: A total of 213 shellfish (52 oysters, 69 cockles and 92 mussels) collected from a culture farm and two retailed markets were investigated for HAV and HEV contamination by reverse transcription-polymerase chain reaction (RT-PCR) assay using HA2-HA1 (capsid region) and HE366-HE363 (ORF2/3 overlapping region) primers, respectively. It was found that 3.8% of the shellfish and 2.9 and 6.5% of the cockle and mussel, respectively, showed positive for HAV detection. Nucleotide sequencing of all the 8 HAV-positive shellfish revealed 97-100% similarity to HAV subgenotype IA. Interestingly, viruses were found more frequently in the gills than in digestive tissue (4.5%vs 0.5%, P = 0.045). All the shellfish were negative for HEV. CONCLUSION: Significant contamination of HAV in edible bivalve shellfish was observed. Beside digestive tissue, gills are one of the important samples for viral genome detection. SIGNIFICANCE AND IMPACT OF THE STUDY: HAV-contaminated shellfish can play a role as reservoirs and/or vehicles in faecal-oral transmission in Thailand, and further monitoring of such a contamination is required.

Induction of acute thrombocytopenia and infection of megakaryocytes by Rauscher murine leukemia virus reflect the genetic susceptibility to leukemogenesis.

Acute thrombocytopenia and megakaryocyte infection have been investigated during the preleukemic phase of the disease induced by the Rauscher murine leukemia virus (RMuLV) in mice. Injection of RMuLV, either intravenously or intraperitoneally, rapidly induced thrombocytopenia, possibly as a result of direct interaction between platelets and viral particles. The susceptibility to this acute thrombocytopenia was genetically controlled and was inherited as a dominant trait. Murine strains with H-2d or H-2k haplotype, which are susceptible to the induction of leukemia by RMuLV, developed thrombocytopenia, whereas leukemia-resistant H-2b and H-2q strains of mice failed to develop thrombocytopenia. Using B10 H-2-congenic and intra-H-2-recombinant mice, it was shown that the susceptibility to RMuLV-induced thrombocytopenia was controlled by gene(s) in or closely linked to the D region of the H-2 complex. Megakaryocytes may be one of the first sites for the replication of RMuLV. Indeed, among bone marrow cells, only megakaryocytes expressed viral antigens gp70 and p30 during the initial phase of RMuLV infection. In addition, megakaryocytes from infected mice were able to transfer preleukemic thrombocytopenia as well as leukemia in syngeneic mice. The infection of megakaryocytes by RMuLV appears to be genetically controlled in a manner similar to the induction of thrombocytopenia, since only the megakaryocytes from mice developing thrombocytopenia were infected by RMuLV. These results indicate that the gene(s) governing the induction of thrombocytopenia by RMuLV may be the same gene(s) (or closely linked to the gene) that controls the susceptibility to leukemogenesis, and would be consistent with the expression of the gene product, presumably a receptor-like molecule for RMuLV, on platelet and megakaryocyte membranes.

Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia.

A highly pathogenic avian influenza virus, H5N1, caused disease outbreaks in poultry in China and seven other east Asian countries between late 2003 and early 2004; the same virus was fatal to humans in Thailand and Vietnam. Here we demonstrate a series of genetic reassortment events traceable to the precursor of the H5N1 viruses that caused the initial human outbreak in Hong Kong in 1997 (refs 2-4) and subsequent avian outbreaks in 2001 and 2002 (refs 5, 6). These events gave rise to a dominant H5N1 genotype (Z) in chickens and ducks that was responsible for the regional outbreak in 2003-04. Our findings indicate that domestic ducks in southern China had a central role in the generation and maintenance of this virus, and that wild birds may have contributed to the increasingly wide spread of the virus in Asia. Our results suggest that H5N1 viruses with pandemic potential have become endemic in the region and are not easily eradicable. These developments pose a threat to public and veterinary health in the region and potentially the world, and suggest that long-term control measures are required.

Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants.

Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir-resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir-resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir-resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4-7 days post-treatment delay in emergence of oseltamivir-resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

Comparative analytical sensitivities of six rapid influenza A antigen detection test kits for detection of influenza A subtypes H1N1, H3N2 and H5N1.

BACKGROUND: Rapid and simple methods for diagnosing human influenza A (H5N1) disease urgently needed. The limited data so far suggest that the currently available rapid antigen detection kits have poor clinical sensitivity for diagnosis of human H5N1 disease. OBJECTIVES: To compare the analytical sensitivity of six commercially available rapid antigen detection kits for the detection of "human" (subtypes H1N1, H3N2) and "avian" (subtype H5N1) influenza A viruses. STUDY DESIGN: Six commercially available test kits for the detection of influenza A were investigated. Analytic sensitivity for the detection of two contemporary H1N1, two H3N2 and three H5N1 viruses was determined using virus culture as a reference method. RESULTS AND CONCLUSIONS: Each test kit detected the H5N1 virus subtypes as efficiently as they detected conventional human viruses of subtypes H1N1 or H3N2. However, limits of detection of influenza viruses of all subtypes by antigen detection kits were >1000-fold lower than virus isolation. Thus, the reportedly poor clinical sensitivity of these antigen detection kits for diagnosis of patients with H5N1 disease is not due to a difference of sensitivity for detecting avian influenza H5N1 compared to human influenza viruses.

Typing of Hantaviruses from five continents by polymerase chain reaction.

Hantavirus, a genus in the family Bunyaviridae, is comprised of at least four serologically distinct types: Hantaan, Seoul, Puumala and Prospect Hill. The present communication reports the use of polymerase chain reaction (PCR) for typing 27 independently isolated Hantaviruses from 5 different continents. Total cellular RNA was extracted from virus-infected Vero E6 cell monolayers by the acid guanidium thiocyanate-phenol-chloroform method. We have utilized 5 different sets of oligonucleotide primers ranging from 18 to 22 nucleotides in length; one set was specific for a conserved region of the S genomic segment and used as genus-specific primers, the other 4 sets of primers were designed from unique sequences of the M genomic segment such that each primer set was specific to only one serological type of Hantavirus. The PCR products were analyzed by restriction endonuclease digestion for further confirmation. We typed 10, 12, 3 and 1 isolates into Hantaan, Seoul, Puumala and Prospect Hill respectively. The results of PCR were 100% agreeable with that of serological typing, and thus, PCR can be used as an adjunct test with serological method(s) or an independent test for diagnosis and for typing of new isolates of Hantaviruses.

Comparison of nucleotide sequences among hantaviruses belonging to the same serotype: an analysis of amplified DNA by thermal cycle sequencing.

The hantavirus genus, belonging to the bunyaviridae family, is comprised of at least four serologically distinct types: Hantaan, Seoul, Puumala and Prospect Hill. Previously, we reported the use of the polymerase chain reaction (PCR) for grouping hantavirus isolates by using four sets of primers specific to each serotype. Our PCR typing results agreed with those of serological typing. The present study makes use of thermal cycle sequencing to sequence PCR-amplified DNA products in order to determine the level of similarity among members of the same serotype. We show that members of Hantaan and Seoul serotypes are over 92% homologous, irrespective of their host and geographical origin. Puumala sequences show a degree of homology ranging from 80 to 98%. Despite the variation in sequence at the nucleotide level, amino acids show an even higher level of conservation.

Viral etiologies of encephalitis in Thai children.

A prospective study of childhood encephalitis was performed in Bangkok from 1996 through 1998. The viral agents identifiable in 26 (65%) of 40 children were dengue virus (8), Japanese encephalitis (6), herpes simplex virus (4), human herpes virus type 6 (3), mumps (2), enterovirus (1), varicella-zoster virus (VZV) (1) and rabies (1).

Dengue infection presenting with central nervous system manifestation.

The objective of this study was to investigate the possibility of dengue virus infection causing an abnormal neurologic presentation. Between 1996 and 1998, all pediatric patients with clinical manifestations of encephalitis-like illness who were admitted to the Department of Pediatrics, Siriraj Hospital were prospectively studied for any evidence of dengue virus infection. The diagnosis of dengue virus infection was based on mosquito viral isolation and serologic and polymerase chain reaction (PCR) evidence. Of 44 patients with the preliminary diagnosis of acute viral encephalitis, 8 were diagnosed with dengue infection. All of these 8 patients were diagnosed by serology. In addition to the serologic diagnosis, four also had positive PCR, one had positive viral isolation, and one had both positive PCR and viral isolation. Only two patients were diagnosed by serologic evidence alone. All except one had clinical courses and laboratory findings compatible with typical dengue infection. All had obvious encephalitic clinical manifestations with normal cerebrospinal fluid findings except one patient, who had mildly increased cerebrospinal fluid protein. All of these patients recovered completely and had benign clinical courses except one patient, who developed leakage symptoms. None had liver failure. Dengue virus can cause acute encephalopathy with fever. It can masquerade as other types of acute viral encephalitis. However, its clinical course and prognosis are usually favorable.

Rubella outbreak in Thailand 1983-1984: a study at Siriraj Hospital.

The recent rubella epidemic in Thailand prevailed from September 1983 to August 1984 with its peak in March. Throughout the outbreak, approximately 70% of the cases diagnosed clinical rubella were laboratory proved. In the middle of the outbreak, accuracy of the clinical diagnosis was 75-87%, while it was 25-33% at the beginning and the end. Concerning the clinical findings in rubella, maculopapular rash may be generalized or localized, and lymphadenopathy occurred only in 40% of the clinical cases. Lymphadenopathy and respiratory symptoms appeared 3 times more frequent than those in the non-rubella cases. Rubella inapparent infection occurred in 5.14% of the contact cases. Acquisition of the disease after contact did not depend on the degree of close relationship between the index cases and the contact cases. Outcome of pregnancy and congenital rubella infection after the outbreak have to be further investigated.

Detection of Epstein-Barr virus DNA and HHV-6 DNA in tissue biopsies from patients with nasopharyngeal carcinoma by polymerase chain reaction.

A total of 34 tissue biopsies were collected from nasopharyngeal carcinoma (NPC) patients and 5 controls with non-NPC. Extracted DNA from tissue biopsies were analyzed for presence of specific gene sequences to EBV type A and type B, and HHV-6 by polymerase chain reaction (PCR). The different sequences of EBV type A and B were parts from the highly divergent forms of the EBV nuclear antigen 2 (EBNA 2). The PCR amplified products for EBNA 2A and EBNA 2B were 115 and 119 base pairs respectively whereas that of HHV-6 DNA was 776 base pairs. The results demonstrated that EBV DNA was detected in 32 of 34 cases (94.1%): 28 (82.3%) with type A, 2 (5.9%) with type B, and 2 (5.9%) with both types. EBV DNA of type A could be detected 1 (20%) of 5 controls. HHV-6 DNA was in 5 of 34 samples (14.7%) whereas HHV-6 DNA was not detectable in biopsy tissues from controls. The results show that in the NPC patient group, A type of EBV is predominant. Detection of HHV-6 DNA in patients group only might be resulted from reactivation of a latent infection or association with EBV-induction of NPC.

The abbreviated 2-1-1 schedule of purified chick embryo cell rabies vaccination for rabies postexposure treatment.

During August 1988 to January 1990, the immunogenicity and safety of purified chick embryo cell rabies vaccine (PCEC) given by the conventional and abbreviated regimens in 82 vaccinees moderately to severely exposed to laboratory proven rabid animals were studied. The 16 vaccinees received PCEC six doses as conventional schedule on days 0, 3, 7, 14, 28 and 90, the 11 vaccinees received six doses of PCEC plus human rabies immune globulin (HRIG) on day 0. The 29 vaccinees received an abbreviated schedule of PCEC as two doses on day 0, one dose each on days 7 and 21 and the 26 cases received PCEC abbreviated schedule plus HRIG on day 0. The kinetics of the neutralizing antibodies on days 0, 7, 14, 28, 56, 180 and 365 were studied for comparative purpose. All vaccinees had high antibody levels from day 14 which last longer than a year and were safe after one year follow up. The adverse reactions of the vaccine were mild and self-limited.

Prevalence of human herpesvirus 6 in Thai people and its correlation with human immunodeficiency virus type 1 infection.

Human herpesvirus 6 (HHV-6) is a human herpesvirus isolated from patients with various lymphoproliferative disorders and acquired immunodeficiency syndrome (AIDS). The prevalence of HHV-6 infection and its correlation as a cofactor in pathogenicity of HIV infection was investigated in serum samples from 365 healthy volunteers at various age groups, 50 persons at risk for HIV-1 infection, and 90 HIV-1 seropositive individuals. Sera were screened and titrated for antibodies against HHV-6 by a standard indirect immunofluorescence assay on an acetone fixed HHV-6 infected HSB2 cells. The data show high prevalence of HHV-6 in Thailand (71.7%) and the infection is acquired early in life. Prevalence of anti-HHV-6 IgG antibodies was not strikingly different among people at risk for HIV infection, asymptomatic HIV-1 infected cases, and aged-matched controls with low risk for HIV-1 infection. The AIDS cases showed high titers of anti-HHV-6 IgG antibody and high rates for presence of anti-HHV-6 IgM antibody (33.3%) which suggests higher prevalence of HHV-6 infection by either reactivation of an earlier HHV-6 infection or a new primary infection.

Epstein-Barr virus serological markers for nasopharyngeal carcinoma in Thailand.

The present study reports on the prevalence of specific IgA and IgG antibodies to EBV viral capsid antigen in nasopharyngeal carcinoma (NPC) patients with different histological types of carcinoma and their age-matched controls by the indirect immunofluorescence test, using the B-95-8 lymphoblastoid cell line as source of viral capsid antigen. EBV specific IgG was found in almost all the study cases, and antibody titers were significantly higher in the NPC patients than in non-cancer controls. GMT of anti-EBV IgG in NPC patients, patients with other malignant diseases, and those with non-malignant diseases were 371.5, 97.7 and 35.5, respectively. Anti-EBV specific IgA was more specific to NPC than was IgG, and was present in 86.5% (83 of 96) cases of NPC patients, 6.6% (2 of 30) of patients with other cancers, and 3.1% (3 of 97) cases of non-malignant diseases. A weak correlation between level of anti-EBV IgA in NPC patients was observed (r = 0.3). EBV IgA was found in all histological types of NPC, ie, WHO types 1, 2 and 3, but WHO type 1 was rare among NPC patients in Thailand. Use of anti-EBV IgA for monitoring cancer therapy is to be further investigated.

Incidence of Mycoplasma pneumoniae, Chlamydia trachomatis, and viral infections in pneumonia cases under six months of age, Bangkok, Thailand.

The incidence of infections by Mycoplasma pneumoniae, Chlamydia trachomatis and respiratory viruses was investigated in 76 pneumonic patients aged under 6 months who attended Ramathibodi and Siriraj Hospitals in Bangkok during two study periods. M. pneumoniae infection was not found in any case from either hospital by serological diagnosis. By the isolation method, C. trachomatis infection was found in 7(16.7%) of 42 patients from Ramathibodi Hospital and 5(21.7%) of 23 patients from Siriraj Hospital with the average male:female ratio of 2.6:1; and 91.7% of the infected cases were under 3 months old. Laboratory diagnosis of respiratory virus infection was performed by indirect immunofluorescence (IIF), isolation, and by antibody detection. Data from Ramathibodi Hospital showed that 11 (24.4%), 4 (8.9%), 3 (6.7%) of the 45 patients were infected by respiratory syncytial virus (RSV), adenoviruses, parainfluenza virus type 3, and some other viruses, respectively; infection rates of 10 (32.3%), 4 (12.9%), 1 (3.2%) and 1 (3.2%) by those viruses respectively, were observed in the 31 patients from Siriraj Hospital.

Rabies and post-exposure prophylaxis in Thai children.

Thailand is an endemic area for rabies, with approximately 300 human deaths reported annually. More than half of the rabies patients are children under 14 years of age. This paper reports clinical data of paediatric rabies cases occurring from 1980 to 1986, and the protective efficacies of human diploid cell rabies vaccine (HDCV) and purified Vero cell rabies vaccine (PVRV) in children exposed to rabid animals. The analysis of 120 medical records revealed that rabies in children had incubation periods which ranged from less than fifteen days to more than three months, but generally between one to three months. The most frequent symptoms observed in the patients were hydrophobia, restlessness, fever, vomiting and aerophobia. Most of the rabid children admitted to hospital died within 24 hours. HDCV was administered to 50 children exposed to rabies with the cumulative dosages of 327 ml. All patients survived without serious adverse effects during a-two year follow-up. Mild reactions were seen in 1.5 percent (5/327 doses). Unfortunately, levels of rabies antibody in these vaccinees were not determined. Among another series of children exposed to rabid animals, comprising 27 individuals who received a total of 168 doses of PVRV, only mild local reactions were seen in 6 subjects. No rabies deaths were reported in 2 years of follow-up. The children who received PVRV either with or without human rabies immune globulin developed similar levels of rabies neutralizing (NT) antibody, which reached the high titers on day 30. At one year after the first dose of vaccination, all vaccinees still had NT antibody at titers higher than 0.5 IU/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

V3 peptide enzyme immunoassay for serotyping HIV-1 infected pregnant Thais.

Previous molecular epidemiological studies show that at least 2 subtypes of HIV-1 circulate in Thailand. HIV-1 subtype B or Thai genotype B was associated with an early epidemic and was prevalent in intravenous drug users. Meanwhile, HIV-1 subtype E or Thai genotype A was becoming widespread among heterosexuals. We studied the HIV subtypes of 161 HIV-1 seropositive pregnant women. Of these, 143 pregnant patients (88.8%) tested positive for subtype E alone and 8 women (5.0%) had evidence of infection with subtype B alone. There was serologic evidence of infection with a mixture of subtypes in 7 women while the infecting subtype could not be identified in the remaining 3 women. This result agrees with previous information that subtype E predominates in Thai heterosexuals.

Serological response to respiratory syncytial virus infection in pediatric patients with a comparison to immunofluorescence and virus isolation.

The serological response to respiratory syncytial virus (RSV) in 125 pediatric patients hospitalized with acute lower respiratory infection was investigated by enzyme linked immunosorbent assay (ELISA) for specific immunoglobulin (Ig) A, IgG, and IgM and complement fixation (CF) test. By ELISA, a 4-fold rise in IgG titre in paired sera was most commonly found, followed by a rise in IgA and IgM titres. Investigation by ELISA and CF leads to the suggestion that major CF activity against RSV antigens resides in the IgG and not the IgA and IgM classes. No case with CF activity failed to be diagnosed by ELISA. The youngest infant who could develop seroconversion was one month old, nevertheless two children older than two years could not. When the three diagnostic methods were compared, ELISA serology was the most sensitive followed by indirect immunofluorescence (IIF) for antigen detection and virus isolation, respectively, ELISA could diagnose RSV infection in 45% of the study cases, whereas IIF and virus isolation only diagnosed 26% and 14%, respectively. Half of the cases was diagnosed by all of the three methods together.