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Biomedical subjects

Supaporn Wacharapluesadee

Publications and source records attributed to Supaporn Wacharapluesadee.

At least 19 recordsLinked to original sources
PubMed2006

Duplex nested RT-PCR for detection of Nipah virus RNA from urine specimens of bats.

A method for duplex nested RT-PCR (nRT-PCR) with internal control (IC) for the detection of Nipah virus RNA is described. Incorporation of IC RNA distinguished false and true negative results. The extrinsic RNA was added directly to the PCR master mix and co-amplified with virus specific RNA in a duplex reaction to determine the presence of PCR inhibitor. Limit of detection was affected minimally when IC was added. Of 53 pooled urine samples collected from fruit bats (Pteropus lylei), 16 were validated by the presence of IC band on gel electrophoresis. Seven of these were also Nipah virus RNA positive. The remaining 37 samples were considered invalid. Twenty-two urine samples became valid after dilution of 1:5 and re-examined; two were Nipah virus RNA positive. These nine positive results were confirmed by sequencing of heminested PCR products. The result indicated that at least two different Nipah strains circulated in this bat species from Thailand. This method should be useful for surveillance for Nipah virus infection in animals in a country where a biosecurity level (BSL) 4 laboratory is not available. PCR inhibitors were present in a significant number of bat urine samples. The technique described in this study should improve reliability of surveillance statistics.

Animals↗
PubMed2006

A simple method for detection of rabies viral sequences in 16-year old archival brain specimens with one-week fixation in formalin.

Archival formalin-fixed and paraffin-embedded brain tissues are important source for diagnosis and molecular analysis. However, nucleic acids are particularly vulnerable to degradation during tissue processing. The brain cutting process usually is performed after 1 week of brain storage in formalin followed by embedding of each particular neuro-anatomical specimen in paraffin. A simple method of deparaffinization, proteinase K digestion and RNA extraction using the Boom technique to obtain rabies RNA in unbuffered, formalin-fixed and paraffin-embedded brain tissues kept at 30 degrees C for 16 years is described. Reverse transcription-polymerase chain reaction (RT-PCR) can be used to identify rabies viral N gene sequences of 150 bases in length in all patients, but not from every immunohistochemical (IHC)-positive specimen. Direct sequencing of 301bp of N gene was achieved in 4 of 7 patients. Results of sequencing a single sample of 1432 bases of N gene from a 24h processed formalin-fixed and paraffin-embedded rabies infected brain tissue after 1 month storage were in accord with those from frozen specimen analysis. It is strongly suggested that for further molecular analysis, a piece of fresh brain tissue should be saved prior to the brain sectioning process and stored no longer than 24h in formalin before embedding.

Animals↗
PubMed2006

Rabies.

Despite increases in our understanding of rabies pathogenesis, it remains an inevitably fatal disease. Lack of awareness, low level of political commitment to rabies control, and failure to recognize and correlate clinical, laboratory, and neuroimaging features contribute to continuing deaths. Clinical symptomatology, once believed to be unique, may be variable, even in patients associated with lyssaviruses of the same genotype. This article discusses virus transport, the role of virus and host response mechanisms in relation to protean clinical manifestations, and mechanisms responsible for relative intactness of consciousness in human rabies. Differential involvement of the anterior horn cell in furious rabies and the peripheral nerve in paralytic rabies is summarized. Escape mechanisms from host defenses explain why a fatal outcome is unavoidable regardless of therapy. Neuroprotective treatment, using a coma-induction regimen, proves not to be beneficial. Survival of patients with excellent recovery relies on early innate and adaptive immunity plus adequate intensive care support.

Humans↗
PubMed2006

Failure of therapeutic coma and ketamine for therapy of human rabies.

The recent success in treating a human rabies patient in Milwaukee prompted the use of a similar therapeutic approach in a 33-year-old male Thai patient who was admitted in the early stages of furious rabies. He received therapeutic coma with intravenous diazepam and sodium thiopental to maintain an electroencephalographic burst suppression pattern, which was maintained for a period of 46 h, as well as intravenous ketamine (48 mg/kg/day) as a continuous infusion and ribavirin (48 to 128 mg/kg/day) via a nasogastric tube. He never developed rabies virus antibodies and he died on his 8th hospital day. At least three other patients have been treated unsuccessfully with a similar therapeutic approach. Because of the lack of a clear scientific rationale, high associated costs, and potential complications of therapeutic coma, the authors recommend caution in taking this approach for the therapy of rabies outside the setting of a clinical trial. More experimental work is also needed in cell culture systems and in animal models of rabies in order to develop effective therapy for human rabies.

Adult↗
PubMed2005

Complex genetic structure of the rabies virus in Bangkok and its surrounding provinces, Thailand: implications for canine rabies control.

Dog vaccination and population management have been suggested as priorities in attempts at disease control in canine rabies-endemic countries. Budget limitations and the complexity of social, cultural and religious variables have complicated progress in the developing world. In Bangkok, Thailand, an intensive canine vaccination and sterilization programme has been in place since November 2002. Our objective was to determine if the rabies virus could be mapped according to its genetic variations and geographical location on the small localized scale of Bangkok and its surrounding provinces. Phylogenetic characterization of 69 samples from Bangkok and five neighbouring and two remote provinces, by limited sequence analysis of the rabies virus nucleoprotein gene, distinguished six different clades. Rabies viruses of four clades were intermixed in Bangkok and in the surrounding highly populated regions whereas the other two clades were confined to rural and less populated provinces. Such a complex pattern of gene flow, particularly in Bangkok, may affect the outcome of canine control programmes.

Animals↗
PubMed2005

Mechanisms of escape phenomenon of spinal cord and brainstem in human rabies.

BACKGROUND: Rabies virus preferentially involves brainstem, thalamus and spinal cord in human furious and paralytic rabies beginning in the early stage of illness. Nevertheless, rabies patient remains alert until the pre-terminal phase. Weakness of extremities develops only when furious rabies patient becomes comatose; whereas peripheral nerve dysfunction is responsible for weakness in paralytic rabies. METHODS: Evidence of apoptosis and mitochondrial outer membrane permeabilization in brain and spinal cord of 10 rabies patients was examined and these findings were correlated with the presence of rabies virus antigen. RESULTS: Although apoptosis was evident in most of the regions, cytochrome c leakage was relatively absent in spinal cord of nearly all patients despite the abundant presence of rabies virus antigen. Such finding was also noted in brainstem of 5 patients. CONCLUSION: Cell death in human rabies may be delayed in spinal cord and the reticular activating system, such as brainstem, thus explaining absence of weakness due to spinal cord dysfunction and preservation of consciousness.

Adolescent↗
PubMed2005

Transmission dynamics of rabies virus in Thailand: implications for disease control.

BACKGROUND: In Thailand, rabies remains a neglected disease with authorities continuing to rely on human death statistics while ignoring the financial burden resulting from an enormous increase in post-exposure prophylaxis. Past attempts to conduct a mass dog vaccination and sterilization program have been limited to Bangkok city and have not been successful. We have used molecular epidemiology to define geographic localization of rabies virus phylogroups and their pattern of spread in Thailand. METHODS: We analyzed 239 nucleoprotein gene sequences from animal and human brain samples collected from all over Thailand between 1998 and 2002. We then reconstructed a phylogenetic tree correlating these data with geographical information. RESULTS: All sequences formed a monophyletic tree of 2 distinct phylogroups, TH1 and TH2. Three subgroups were identified in the TH1 subgroup and were distributed in the middle region of the country. Eight subgroups of TH2 viruses were identified widely distributed throughout the country overlapping the TH1 territory. There was a correlation between human-dependent transportation routes and the distribution of virus. CONCLUSION: Inter-regional migration paths of the viruses might be correlated with translocation of dogs associated with humans. Interconnecting factors between human socioeconomic and population density might determine the transmission dynamics of virus in a rural-to-urban polarity. The presence of 2 or more rabies virus groups in a location might be indicative of a gene flow, reflecting a translocation of dogs within such region and adjacent areas. Different approaches may be required for rabies control based on the homo- or heterogeneity of the virus. Areas containing homogeneous virus populations should be targeted first. Control of dog movement associated with humans is essential.

Animals↗
PubMed2005

Pathophysiology of human paralytic rabies.

Furious rabies is a well-recognized clinical disorder in humans but the paralytic form is not as easily identified. The mechanisms responsible for the weakness and longer survival periods are not clear. Several hypotheses have been proposed, including rabies virus variants associated with a particular vector, location of wounds, incubation period, influence of prior rabies vaccination, and virus localization in the central nervous system (CNS). However, none of these have been substantiated. Regarding molecular analyses of rabies viruses isolated from both furious and paralytic rabies patients, only minor genetic variations with no specific patterns in glyco- (G), phospho- (P), and nucleoprotein (N) sequences have been identified and arginine 333 in G protein was present in all samples. Regional distribution of rabies virus antigenin rabies patients whose survival periods were 7 days or less and magnetic resonance imaging (MRI) of the CNS indicated brainstem and spinal cord as predilection sites regardless of clinical presentations. There are clinical, electrophysiological, and pathological indications that peripheral nerve dysfunction is responsible for weakness in paralytic rabies whereas in furious rabies, even in the absence of clinical weakness, abundant denervation potentials with normal sensory nerve conduction studies and proximal motor latencies suggest anterior horn cell dysfunction. The lack of cellular immunity to rabies virus antigen accompanied by an absence of cerebrospinal fluid (CSF) rabies neutralizing antibody in most paralytic rabies patients may argue against role of an immune response against rabies virus-positive axons. Aberrant immune responses to peripheral nerve antigen, in particular those mediated by one or more cellular-dependent mechanisms, may be involved as is supported by the absence of putative anti-ganglioside antibodies commonly found in immune-mediated peripheral nerve diseases. Longer survival period in paralytic rabies may possibly be related to currently unidentified mechanism(s) on neuronal gene expression, required for virus transcription/replication and for maintaining neuronal survival.

Animals↗
PubMed2005

Survey for bat lyssaviruses, Thailand.

Surveillance for lyssaviruses was conducted among bat populations in 8 provinces in Thailand. In 2002 and 2003, a total of 932 bats of 11 species were captured and released after serum collection. Lyssavirus infection was determined by conducting virus neutralization assays on bat serum samples. Of collected samples, 538 were either hemolysed or insufficient in volume, which left 394 suitable for analysis. These samples included the following: Pteropus lylei (n = 335), Eonycteris spelaea (n = 45), Hipposideros armiger (n = 13), and Rousettus leschennaulti (n = 1). No serum samples had evidence of neutralizing antibodies when tested against rabies virus. However, 16 samples had detectable neutralizing antibodies against Aravan virus, Khujand virus, Irkut virus, or Australian bat lyssavirus; all were specifically associated with fruit bats P. lylei (n = 15) and E. spelaea (n = 1). These results are consistent with the presence of naturally occurring viruses related to new putative lyssavirus genotypes.

Animals↗
PubMed2005

Bat Nipah virus, Thailand.

Surveillance for Nipah virus (NV) was conducted in Thailand's bat population. Immunoglobulin G antibodies to NV were detected with enzyme immunoassay in 82 of 1,304 bats. NV RNA was found in bat saliva and urine. These data suggest the persistence of NV infection in Thai bats.

Animals↗
PubMed2005

[Rabies diagnosis in human].

Rabies diagnosis in humans can be made by obtaining a history of exposure particularly to an animal bite, clinical symptoms and signs, neuroimaging such as magnetic resonance imaging (MRI) and laboratory testing. Although diagnosis made by clinical grounds alone can be useful, this can only be limited to furious rabies. Owing to the fact that there is more than one form of rabies (furious, paralytic and atypical rabies) relying on only clinical diagnosis will undoubtedly lead to under reporting. The nonclassic or atypical rabies presents with a non specific pattern lacking pathognomomic signs as found in classic forms (furious and paralytic). A diagnosis of rabies should be considered in any patient who presents with encephalopathy of unknown cause. Diagnosis of rabies requires laboratory confirmation. MRI pattern may differentiate rabies from other viral encephalitides. Laboratory diagnosis can be used for confirmation by demonstration of viral proteins or its genomic RNA. The nucleic acid amplification method is often used as a diagnostic test due to its good sensitivity and specificity and can be applied to wide varieties of specimens. Accurate data on rabies statistics contribute to the success in control and prevention of rabies.

Diagnosis, Differential↗
PubMed2005

[Bat lyssavirus in Thailand].

A study of bat lyssavirus survey was done in Thailand from 2001 to 2003. A total of 932 bats of 11 species were captured in 8 provinces for blood collection and testing for neutralizing antibodies against rabies virus (RABV), Australian bat lyssavirus (ABLV) and broader panel of other lyssaviruses (Irkut, Aravan and Khujand). All Thai bat samples were negative to RABV Sixteen samples of 394 with sufficient volume of serum had detectable neutralizing antibodies against Irkut, Aravan, Khujand and ABL viruses. Another 13 samples were also found to have antibody to ABLV. However, due to insufficient volume, further analysis to other lyssaviruses could not be performed. Nevertheless, this showed that the prevalence of lyssavirus infection in Thai bats could be as high as 7.3% (29/396). The present study showed that natural occurrence of lyssavirus antibodies found in Thai bats were related to newer putative lyssavirus genotype(s) other than those previously described. These data also suggest that several lyssaviruses are in circulation throughout Thailand as well as other Asian countries, such as in the Philippines, Central Asia, and in certain parts of Russia. The present study and preparation of this article was supported by grants from the Thailand Research Fund and the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand.

Animals↗
PubMed2004

Paradigm shift in rabies control: a system approach.

Government centered approach in rabies prevention and control has not led to rabies safe community, both in urban and rural areas. Government services and organizations should recognize that only joint collaboration among households and community can lead to a successful rabies control mission. Dog owners should have been more actively involved in the prevention and solving of rabies problem. Promotion and support of government organizations should aim to strengthen the communities to become self reliance in diseases control and health care services. Only by strengthening community roles result in a more sustainable community disease control program.

Animals↗
PubMed2003

Sequence analysis of rabies virus in humans exhibiting encephalitic or paralytic rabies.

Two distinct clinical patterns, encephalitic (furious) and paralytic (dumb), have been recognized in human rabies. It has been postulated that different rabies virus variants associated with particular vectors may be responsible for these different clinical manifestations. Analysis of the glycoprotein (G), nucleoprotein (N), and phosphoprotein (P) genes of rabies viruses from 2 human cases of encephalitic rabies and from 2 human cases of paralytic rabies demonstrated only minor nucleotide differences. Deduced amino-acid patterns of the N protein were identical in both human and canine samples that came from the same geographic location, regardless of the clinical form. All differences in amino-acid patterns of the G protein were found outside the ectodomain, in either the signal peptide or the transmembrane and endodomains. None of the amino-acid differences of the P protein was within the interactive site with dynein. These findings support the concept that clinical manifestations of rabies are not explained solely by the associated rabies virus variant.

Amino Acid Sequence↗