The potential of RT-PCR to rapidly diagnose dengue infections from both acute and convalescent phase patients' sera was evaluated. The RNA extraction method involved binding of the viral RNA to silica particles in the presence of high concentration of guanidine thiocyanate. The protocol that was established was sensitive enough to detect 40 plaque forming units per 100 microliter of serum and results could be obtained within one day. Results from this study indicate that clinical samples should be collected in the early acute phase of illness when anti-dengue antibodies were undetectable or of low titres to ensure a more reliable diagnosis.
By a combination of PCR and direct-cycle sequencing using consensus primers, we analyzed approximately 400-bp fragments within the NS3 genes of twenty-one dengue virus type 3 strains isolated from five neighboring Southeast Asian countries at different time intervals from 1956 to 1992. The majority of base disparities were silent mutations, with few predicted amino acid substitutions, thus emphasizing the strict conservation of the NS3 gene. Phylogenetic trees constructed on the basis of these nucleotide differences revealed distinct but related clusters of strains from the Philippines, Indonesia, and strains from Singapore and Malaysia of the 1970s and early 1980s, while the Thai cluster was relatively more distant. This genetic relationship was compatible with that proposed by other workers who have studied other dengue 3 virus genes such as E, M and prM. However, we observed that the more recent, epidemic-associated dengue 3 strains from Singapore and Malaysia of the late 1980s and early 1990s were more closely related to the Thai cluster, implying their evolution from the latter, and emphasizing the importance of viral spread via increasing travel within the Southeast Asian area and elsewhere. Nucleotide sequence analysis of the NS3 genes of dengue viruses can serve to advance the understanding of the epidemiology and evolution of these viruses.
Dengue fever (DF) which is caused by four serotypes of dengue virus may in some cases progress into a life threatening situation of dengue haemorrhage fever (DHF) and dengue shock syndrome (DSS). It has been suggested that sequential infection with different dengue virus serotypes predisposes the patient towards DHF/DSS. We report here a primary dengue infection in a 10-year-old boy progressing from DF to DSS while under clinical observation. The report provides unequivocal evidence for the development of DSS in primary dengue infection caused by virus serotype 4. The close relationship between sequential changes in the levels of tumour necrosis factor (TNF), Interleukin 1 and 6 (IL-1 and IL-6) in the serum, to the clinical progression of the disease from DF to DHF/DSS and then to full recovery implicates a pathogenetic role for the inflammatory cytokines. The child also manifested clinical features consistent with Reye's syndrome and this suggests a common pathogenetic origin for DSS and the Reye-like syndrome induced by dengue virus.
This study describes the use of polymerase chain reaction as a diagnostic tool for detecting and typing of dengue virus. PCR was compared against virus isolation. First RT-PCR was done using dengue consensus primers after which positive samples were subjected to RT-PCR using type-specific primers. This study shows that the local strains of the dengue virus could be detected using the chosen primers. Furthermore, RT-PCR was found to be more sensitive than virus isolation in identifying the dengue positive samples.
Involvement of the central nervous system in dengue fever and dengue hemorrhagic fever has always been thought to be secondary to vasculitis with resultant fluid extravasation, cerebral edema, hypoperfusion, hyponatremia, liver failure, and/or renal failure. Thus, the condition has been referred to as dengue encephalopathy. Encephalitis or direct involvement of the brain by the virus was thought to be unlikely. This paper reports on six children who were seen over a period of two years presenting on the second or third day of illness with dengue encephalitis. The diagnosis was based upon a clinical picture of encephalitis and confirmed by cerebrospinal fluid (CSF) microscopy and electroencephalography changes. All six cases were confirmed dengue infections. Dengue 3 virus was isolated from the CSF of four cases and in one case, dengue 2 was detected by the polymerase chain reaction in both the CSF and blood. In the sixth case, virologic evidence was negative but dengue immunoglobulin M was detected in the CSF and blood. Since the onset of encephalitis appears early in the course of illness coinciding with the viremic phase, we postulate that the virus crosses the blood-brain barrier and directly invades the brain causing encephalitis. This study provides strong evidence that dengue 2 and 3 viruses have neurovirulent properties and behave similarly to other members of the Flaviviridae.
Mosquito adults and larvae were collected from dengue high risk areas and transported to the laboratory for identification. Identified mosquitos were pooled according to the species, date and locality and stored at -70 degrees C. A total of 1,385 pools of Aedes albopictus and 267 pools of Ae. Aegypti were collected from major towns in 12 states in Peninsular Malaysia. Virus isolation was carried out using cell culture (C6/36 clone) of Ae. albopictus and detection of dengue virus by the peroxidase anti-peroxidase staining. All positive isolations were further re-confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Most of the pools were negative with PAP staining and RT-PCR. However, 11 mosquito pools were positive with PAP staining. On the other hand, samples from Terengganu, Pulau Pinang and Johor were positive using both methods.
Dengue, an important mosquito-borne flavivirus infection, is endemic in Southeast Asia. We describe two mothers who had acute dengue 4 and 8 days before the births of their infants. One mother had worsening of her proteinuric pregnancy-induced hypertension, liver dysfunction, and coagulopathy and required multiple transfusions of whole blood, platelets, and fresh frozen plasma. Her male infant was ill at birth, developed respiratory distress and a large uncontrollable left intracerebral hemorrhage, and died of multiorgan failure on day 6 of life. Dengue virus type 2 was isolated from the infant's blood, and IgM antibody specific to dengue virus was detected in the mother's blood. The second mother had a milder clinical course; she gave birth to a female infant who was thrombocytopenic at birth and had an uneventful hospitalization. Dengue virus type 2 was recovered from the mother's blood, and IgM antibody specific to dengue virus was detected in the infant's blood. This report highlights not only the apparently rare occurrence of vertical transmission of dengue virus in humans but also the potential risk of death for infected neonates.
The limited sequencing approach was used to study the molecular epidemiology of 24 Malaysian dengue 2 viruses which were isolated between 1968 and 1993. The sequences of a 240-nucleotide-long region across the envelope/non-structural 1 protein (E/NS1) gene junction of the isolates were determined and analysed. Alignment and comparison of the nucleotide and deduced amino acid sequences of the isolates revealed that nucleotide changes occurred mostly at the third position of a particular codon and were of the transition (AG, CU) type. Five nucleotide changes resulted in amino acid substitutions. Pairwise comparisons of the nucleotide sequences gave divergence values ranging from 0 to 9.2%. At the amino acid level, the divergence ranged between 0 and 3.8%. Based on the 6% divergence as the cut-off point for genotypic classification, the isolates were grouped into two genotypes, I and II. Comparison of the nucleotide sequences of the Malaysian dengue isolates with those of the dengue viruses of other regions of the world revealed that members of genotypes I and II were closely related to viruses from the Indian Ocean and Western Pacific regions, respectively.
To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.
Dengue continues to be a major health threat to Malaysia a century after its first reported outbreak in 1902. Examination of the available outbreak data suggested that a major DF/DHF outbreak occurred in Malaysia in a cyclical pattern of approximately every 8 years. All four dengue virus serotypes are found co-circulating in Malaysia, but after the first and only major outbreak involving DEN-4 in 1960's, only DEN-1, DEN-2 and DEN-3 were associated with DF/DHF outbreaks. It is argued that perhaps the spread of the later dengue virus serotypes followed the pattern of spread of the mosquito vector Aedes aegypti, whereas the former was associated with Aedes albopictus, the outdoor and rural area dwelling mosquito. Estimating from the trend and pattern of dengue and the associated dengue virus serotypes, unless there is a major breakthrough in dengue vaccine development, it is likely that dengue outbreaks will continue to occur in Malaysia throughout the 21st century.
Phylogenetic analyses of the envelope (E) gene sequence of five recently isolated dengue virus type 4 (DENV-4) suggested the emergence of a distinct geographical and temporal DENV-4 subgenotype IIA in Malaysia. Four of the isolates had direct ancestral lineage with DENV-4 Indonesia 1973 and showed evidence of intra-serotypic recombination with the other recently isolated DENV-4, MY01-22713. The E gene of isolate MY01-22713 had strong evidence of an earlier recombination involving DENV-4 genotype II Indonesia 1976 and genotype I Malaysia 1969. These results suggest that intra-serotypic recombination amongst DENV-4 from independent ancestral lineages may have contributed to the emergence of DENV-4 subgenotype IIA in Malaysia.
Dengue viral antigens have been demonstrated in several types of naturally infected human tissues, but little is known of whether these same tissues have detectable viral RNA. We studied tissue specimens from patients with serologically or virologically confirmed dengue infections by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively. IHC was performed on specimens obtained from 5 autopsies and 24 biopsies and on 20 blood-clot samples. For ISH, antisense riboprobes to the dengue E gene were applied to tissue specimens in which IHC was positive. Viral antigens were demonstrated in Kupffer and sinusoidal endothelial cells of the liver; macrophages, multinucleated cells, and reactive lymphoid cells in the spleen; macrophages and vascular endothelium in the lung; kidney tubules; and monocytes and lymphocytes in blood-clot samples. Positive-strand viral RNA was detected in the same IHC-positive cells found in the spleen and blood-clot samples. The strong, positive ISH signal in these cells indicated a high copy number of viral RNA, suggesting replication.
The amino acid sequences of the envelope (E) protein of four encephalitogenic and five non-encephalitogenic dengue 3 virus strains isolated in Malaysia were determined and compared. Multiple sequence alignment revealed a high degree of similarity in the E protein of the strains suggesting that neurovirulence of these four encephalitogenic strains is not attributed to this protein.
The rapid detection of dengue infection in mosquito vectors is important for early warning to forestall an outbreak. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) provides a rapid method for dengue detection in man and mosquitoes. An RT-PCR kit developed by the Medical Entomology Unit, Institute for Medical Research to detect dengue infection in mosquitoes, was tested for its shelf life at 3 storage temperatures: room temperature, refrigerator and freezer. Test kits were tested once every 3 days for kits stored at room temperature, and once every week for those stored at refrigerator and freezer temperatures. The results showed that the test kit could only be stored above its recommended storage temperature of -20 degrees C for not more than 3 days. DNA 100 bp markers in the kits appeared to be stable at the tested temperatures and were usable up to the 20th day when stored at 2 degrees C and below.
Dengue and dengue haemorrhagic fever are common and serious arboviral diseases endemic in a number of countries situated in both the tropical and subtropical belts.
During an outbreak from December 2004 to March 2005, 138 isolates of dengue virus were prospectively obtained from acute-phase serum samples of 1,067 patients with the provisional clinical diagnosis of acute dengue illness admitted to the adult wards of Hospital Tengku Ampuan Rahimah, Klang, Malaysia. Of the 138 dengue virus isolates, 87, 11, 24 and 3 were typed as dengue serotypes 1, 2, 3 and 4, respectively, by a commercial dengue virus typing kit using monoclonal antibodies (Mab). 13 dengue virus isolates could not be assigned to any specific serotype by serotyping Mab and molecular typing using dengue-type specific molecular typing primer pairs. We report the associated clinical features and limited molecular genetics of this Mab-escape dengue virus variant.
The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.
The purpose of this study was to examine the extent of dengue infection in Brunei and to determine the predominant serotype circulating in the country. The study generated useful epidemiological data on dengue infection in Brunei. A total of 271 samples from patients suspected of having dengue infections were selected and analyzed. All patients were seen in clinics and hospitals in Brunei. The samples were collected from April 2005 to April 2006 and transported to the WHO Collaborating Centre for Arbovirus Reference and Research, University of Malaya, Malaysia. The following tests were used to achieve the objectives: in-house IgM-capture enzyme-linked immunosorbent assay, virus isolation in mosquito albopictus cell line (C6/36), and viral RNA detection and serotyping by reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that 45 people were positive for dengue-specific IgM (27 males and 18 females), while RT-PCR detected dengue viral RNA in 12 patients, 3 identified as DEN-1 and 9 as DEN-2. Dengue virus was isolated from 6 patients using the C6/36 cell line; 3 were DEN-2 isolates and 3 were DEN-1 isolates. These data show that dengue virus is circulating in Brunei and the predominant infecting serotype for that period was DEN-2 followed by DEN-1. This study is the first to report the detection and isolation of dengue virus from Brunei using RT-PCR and culture in the C6/36 albopictus mosquito cell line.
Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas in the world. Attempts to develop effective vaccines have been hampered by the lack of understanding of the pathogenesis of the disease and the absence of suitable experimental models for dengue viral infection. The magnitude of T-cell responses has been reported to correlate with dengue disease severity. Sixty Malaysian adults with dengue viral infections were investigated for their dengue virus-specific T-cell responses to 32 peptides antigens from the structural and nonstructural regions from a dengue virus isolate. Seventeen different peptides from the C, E, NS2B, NS3, NS4A, NS4B, and NS5 regions were found to evoke significant responses in a gamma interferon enzyme-linked immunospot (ELISPOT) assay of samples from 13 selected patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). NS3 and predominantly NS3(422-431) were found to be important T-cell targets. The highest peaks of T-cell responses observed were in responses to NS3(422-431) and NS5(563-571) in DHF patients. We also found almost a sevenfold increase in T-cell response in three DHF patients compared to three DF patient responses to peptide NS3(422-431). A large number of patients' T cells also responded to the NS2B(97-106) region. The ELISPOT analyses also revealed high frequencies of T cells that recognize both serotype-specific and cross-reactive dengue virus antigens in patients with DHF.
Matched MeSH terms: Dengue/virology; Severe Dengue/virology