A synthetic peptide derived from domain III envelope glycoprotein of Dengue virus induces neutralizing antibody.

Dengue virus (DENV) is an arthropod-borne human pathogen that represents a severe public health threat in both endemic and non-endemic regions. So far, there is no licensed vaccine or specific drugs available for dengue fever. A fifteen-amino-acid-long peptide that includes the NGR motif was chemically synthesized and conjugated with keyhole limpet hemocyanin. A standard immunization protocol was followed for the production of polyclonal antibodies by immunizing rabbits against the synthetic peptide. The immune response elicited high-titer polyclonal antibodies with the reactivity of the anti-peptide antibody against both synthetic peptide and four serotypes of DENV confirmed by DOT-ELISA. Neutralizing activity of anti-peptide antibody was found to be cross-reactive and effective resulting in 60% reduction of infectivity at 1:200 dilution in all four serotypes of DENV. Our findings have the potential to further improve our understanding of virus-host interactions and provide new insights into neutralizing antibodies and could also be used as a drug target.

Imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection from Oman to Thailand, June 2015.

Thailand reported the first Middle East respiratory syndrome (MERS) case on 18 June 2015 (day 4) in an Omani patient with heart condition who was diagnosed with pneumonia on hospital admission on 15 June 2015 (day 1). Two false negative RT-PCR on upper respiratory tract samples on days 2 and 3 led to a 48-hour diagnosis delay and a decision to transfer the patient out of the negative pressure unit (NPU). Subsequent examination of sputum later on day 3 confirmed MERS coronavirus (MERS-CoV) infection. The patient was immediately moved back into the NPU and then transferred to Bamrasnaradura Infectious Disease Institute. Over 170 contacts were traced; 48 were quarantined and 122 self-monitored for symptoms. High-risk close contacts exhibiting no symptoms, and whose laboratory testing on the 12th day after exposure was negative, were released on the 14th day. The Omani Ministry of Health (MOH) was immediately notified using the International Health Regulation (IHR) mechanism. Outbreak investigation was conducted in Oman, and was both published on the World Health Organization (WHO) intranet and shared with Thailand's IHR focal point. The key to successful infection control, with no secondary transmission, were the collaborative efforts among hospitals, laboratories and MOHs of both countries.

Diagnostic accuracy of peripheral venous lactate and the 2009 WHO warning signs for identifying severe dengue in Thai adults: a prospective observational study.

BACKGROUND: Dengue is the most common mosquito-borne viral disease in humans. However, the sensitivities of warning signs (WSs) for identifying severe dengue in adults are low, and the utility of lactate levels for identifying severe dengue in adults has not been verified. Therefore, we aimed to evaluate the diagnostic accuracy of using peripheral venous lactate levels (PVL), as well as WSs established by the World Health Organization, for identifying severe dengue. METHODS: We prospectively evaluated individuals hospitalized for dengue who were admitted to the Hospital for Tropical Diseases in Thailand between May 2013 and January 2015. Blood samples to evaluate PVL levels were collected at admission and every 24 h until the patient exhibited a body temperature of <37.8 °C for at least 24 h. Data were recorded on a pre-defined case report form, including baseline characteristics, clinical parameters, and laboratory findings. RESULTS: Among 125 patients with confirmed dengue, 105 (84.0%) patients had non-severe dengue, and 20 (16.0%) patients had severe dengue. The presence of clinical fluid accumulation as a WS provided high sensitivity (75.0%, 95% confidence interval [CI]: 50.9-91.3%) and specificity (90.5%, 95% CI: 83.2-95.3%). The PVL level at admission was used to evaluate its diagnostic value, and receiver operating characteristic curve analysis revealed an area under the curve of 0.84 for identifying severe dengue. At the optimal cutoff value (PVL: 2.5 mmol/L), the sensitivity and specificity were 65.0% (95% CI: 40.8-84.6%) and 96.2% (95% CI: 90.5-99.0%), respectively. A combined biomarker comprising clinical fluid accumulation and/or PVL of ≥2.5 mmol/L provided the maximum diagnostic accuracy for identifying severe dengue, with a sensitivity of 90.0% (95% CI: 68.3-98.8%) and a specificity of 87.6% (95% CI: 79.8-93.2%). CONCLUSIONS: Clinical fluid accumulation and/or PVL may be used as a diagnostic biomarker of severe dengue among adults. This biomarker may facilitate early recognition and timely treatment of patients with severe dengue, which may reduce dengue-related mortality and hospital burden.

Molecular genotyping of human papillomavirus l1 gene in low-risk and high-risk populations in Bangkok.

BACKGROUND: Human papillomavirus (HPV) infections in Thailand are a public health concern, but information on HPV infection in sex workers and men who have sex with men (MSM) is limited. The aim of this study was to measure the prevalence and genotype distribution of HPV among low- and high-risk, HIV-negative populations. METHODS: A total of 300 participants were categorized as general women, female sex workers, MSM, and MSM sex workers. Human papillomavirus infections were identified by the Papanicolaou test and nested polymerase chain reaction. A phylogenetic analysis of partial HPV L1 genes was performed. RESULTS: Abnormal cytology was found in 5% of general women, 10% of female sex workers, 24% of MSM, and 28% of MSM sex workers. Human papillomavirus was detected in 9% of general women, 13% of female sex workers, and 30% in both MSM and the MSM sex workers. The prevalence of HPV high-risk genotypes was significantly higher in female sex workers and MSM, whereas low-risk genotypes and genital warts were significantly higher in MSM sex workers. Significantly more patients with genital warts and cervical intraepithelial neoplasia I/anal intraepithelial neoplasia I harbored low-risk genotypes, whereas those with cervical intraepithelial neoplasia II/anal intraepithelial neoplasia II harbored high-risk genotypes. CONCLUSIONS: High- and low-risk HPV genotypes persist in high-risk groups in Bangkok. Some genotypes infecting at-risk populations are not vaccine preventable. These findings may help to elucidate the prevalence of HPV infections in Thailand and serve as the basis for additional investigations into risk factors for these populations.

Identification of clinical factors associated with severe dengue among Thai adults: a prospective study.

BACKGROUND: Dengue is the most common mosquito-borne viral disease in humans. Recently, there has been an epidemic shift of dengue from mainly affecting children to affecting more adults with increased severity. However, clinical factors associated with severe dengue in adults have varied widely between studies. We aimed to identify the clinical factors associated with the development of severe dengue according to the World Health Organization (WHO)'s 2009 definition. METHODS: We conducted a prospective study of adults with dengue admitted to the Hospital for Tropical Diseases in Bangkok, Thailand, from October 2012 to December 2014. Univariate and stepwise multivariate logistic regression analyses were performed. RESULTS: Of the 153 hospitalized patients with confirmed dengue viral infections, 132 (86.3 %) patients had non-severe dengue including dengue without warning signs (7 patients, 5.3 %) and dengue with warning signs (125, 94.7 %). The rest (21, 13.7 %) had severe dengue including severe plasma leakage (16, 76.2 %), severe organ involvement (16, 76.2 %), and severe clinical bleeding (8, 38.1 %). Using stepwise multivariate logistic regression, clinical factors identified as independently associated with the development of severe dengue were: (1) being >40 years old (odds ratio [OR]: 5.215, 95 % confidence interval [CI]: 1.538-17.689), (2) having persistent vomiting (OR: 4.817, CI: 1.375-16.873), (3) having >300 cells per µL of absolute atypical lymphocytes (OR: 3.163, CI: 1.017-9.834), and (4) having lactate levels ≥2.0 mmol/L (OR: 7.340, CI: 2.334-23.087). In addition, increases in lactate and absolute atypical lymphocyte levels corresponded with severe dengue (p < 0.05). CONCLUSIONS: Our study identified several clinical factors independently associated with the development of severe dengue among hospitalized adults with dengue. This can aid in the early recognition and prompt management of at-risk patients to reduce morbidity and mortality.

Reduced viral burden in paralytic compared to furious canine rabies is associated with prominent inflammation at the brainstem level.

BACKGROUND: The mechanisms that differentiate rabies infections into furious and paralytic forms remain undetermined. There are no neuropathological features in human brains that distinguish furious and paralytic rabies. This could be due to methodology and/or examination of specimens late in the disease course.In this study, postmortem examination of brain (5 furious and 5 paralytic) and spinal cord (3 furious and 3 paralytic) specimens was performed in 10 rabies-infected dogs, sacrificed shortly after developing the illness. Rabies virus (RABV) antigen (percentage of positive neurons, average antigen area in positive neurons and average antigen area per neuron) and RNA were quantified at 15 different central nervous system (CNS) regions. The distribution and degree of inflammation were also studied. RESULTS: More RABV antigen was detected in furious rabies than paralytic in many of the CNS regions studied. Caudal-rostral polarity of viral antigen distribution was found in both clinical forms in order from greatest to least: spinal cord, brainstem, cerebellum, midline structures (caudate, thalamus), hippocampus, and cerebrum. In contrast, RABV RNA was most abundant in the cerebral midline structures. Viral RNA was found at significantly higher levels in the cerebral cortex, thalamus, midbrain and medulla of dogs with the furious subtype. The RNA levels in the spinal cord were comparable in both clinical forms. A striking inflammatory response was found in paralytic rabies in the brainstem. CONCLUSIONS: These observations provide preliminary evidence that RABV antigen and RNA levels are higher in the cerebrum in furious rabies compared to the paralytic form. In addition, brainstem inflammation, more pronounced in paralytic rabies, may impede viral propagation towards the cerebral hemispheres.

Cloning and application of recombinant dengue virus prM-M protein for serodiagnosis of dengue virus infection.

We studied the use of the precursor to the M structural protein (prM) found only on the surface of mature dengue virus as a target protein to detect dengue virus infection. Recombinant D2-16681 prM-M protein was constructed and tested for immunogenicity with dengue and Japanese encephalitis patient sera by Western blot analysis and indirect ELISA. The sensitivity and specificity of indirect ELISA were 48.1 and 85.5%, respectively, and Western blot assay were 23.1 and 98.7%, respectively, for detection of dengue virus. Although the sensitivity of the indirect ELISA is low, the indirect ELISA using recombinant D2-16681 prM-M proteins as antigen may be used for early detection of dengue virus infection.

Detection of Anti-ZIKV NS1 IgA, IgM, and Combined IgA/IgM and Identification of IL-4 and IL-10 as Potential Biomarkers for Early ZIKV and DENV Infections in Hyperendemic Regions, Thailand.

The frequency of Zika virus (ZIKV)-specific IgA and IgM and the cytokine expression profile of ZIKV-infected patients in hyperendemic areas remain unclear. This study investigated the rates of ZIKV non-structural protein 1 (NS1)-specific IgA and IgM and evaluated serum cytokine levels of ZIKV and Dengue virus (DENV) cases in Thailand to identify potential diagnostic biomarkers, elucidate the immunity against ZIKV and DENV, and investigate the association between cytokine levels and ZIKV symptoms. Low rates of positivity for ZIKV NS1-specific IgA and IgM were detected in our study. ZIKV NS1 IgA/M (11%, 11/101) in combination was more frequently detected than ZIKV NS1 IgM (2%, 2/101) or ZIKV NS1 IgA (4%, 4/96) alone, especially in acute ZIKV cases with previous DENV exposure (14%, 10/72). Cytokine analysis showed that both ZIKV and DENV infections induced polyfunctional immunity, and the latter triggered more prolonged responses. The existence of significant differences in IL-4 and IL-10 levels between acute ZIKV and acute DENV cases suggested that IL-4 (p = 0.0176) and IL-10 (p = 0.0003) may represent biomarkers for acute ZIKV and acute DENV infections, respectively. Analysis of the association between increased cytokine levels and ZIKV symptoms indicated that CXCL10 (p = 0.0029) was associated with exanthema, while IL-5 (p = 0.0496) was linked to headache. The detection of ZIKV NS1 IgA and IgM in combination may enhance the diagnosis of early ZIKV infection, particularly when levels of IgM or IgA alone are low or undetectable. IL-4 and IL-10 may serve as targets for the development of diagnostic tools to detect ZIKV and DENV infections early, respectively, in flavivirus-endemic regions.

Human monoclonal antibodies to neutralize all dengue virus serotypes using lymphocytes from patients at acute phase of the secondary infection.

The global spread of the four dengue virus serotypes (DENV-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, we prepared hybridomas producing human monoclonal antibodies (HuMAbs) against DENV using peripheral blood mononuclear cells (PBMCs) from patients in the acute phase (around 1 week after the onset of illness) or the convalescent phase (around 2weeks after the onset of illness) of secondary infection. Interestingly, a larger number of hybridoma clones was obtained from patients in the acute phase than from those in the convalescent phase. Most HuMAbs from acute-phase infections were cross-reactive with all four DENV serotypes and showed significant neutralization activity to all four DENV serotypes. Thus, secondary DENV infection plays a significant role in stimulating memory cells to transiently increase the number of antibody-secreting plasma cells in patients in the early phase after the secondary infection. These HuMAbs will enable us to better understand the protective and pathogenic effects of DENV infection, which could vary greatly among secondarily-infected individuals.

The serotype-independent but concentration-dependent enhancing antibodies among Thai dengue patients.

Antibody-dependent enhancement of infection (ADE) is central to explaining the development of severe disease at the end of post-dengue virus infection. Non-neutralizing anti-dengue antibodies bound to the dengue virion enhances the virus entrance into the target cells via the Fc receptor. The titer of enhancing antibodies in dengue patients is not determined during dengue virus infection. Sensitive flow cytometry detecting dengue virus-infected K562 cells was used to quantitate enhancing activity among Thai DF and DHF patients against four serotypes and the patient's dengue isolate. The titer was defined as the reciprocal of the final dilution that loses enhancing activity. The serum of Thai patients confirmed to have dengue infection were found to have high titers of enhancing antibodies and increased gradually through the convalescent phase of infection. The enhancing antibody titers were not different among the four serotypes or from the infecting isolate. The anti-dengue antibodies from dengue patients can enhance dengue virus infections in a concentration-dependent, serotype-independent manner.

Dengue virus type 2 recognizes the carbohydrate moiety of neutral glycosphingolipids in mammalian and mosquito cells.

Dengue viruses infect cells by attaching to a surface receptor which remains unknown. The putative receptor molecules of dengue virus type 2 on the surface of mosquito (AP-61) and mammalian (LLC-MK2) cell lines were investigated. The immunochemical detection and structural analysis of carbohydrates demonstrated that the neutral glycosphingolipids, L-3 (GlcNAcß1-3Manß1-4Glcß1-1'Cer) in AP-61 cells, and nLc(4) Cer (Galß1-4GlcNAcß1-3Galß1-4Glcß1-1'Cer) in LLC-MK2 cells were recognized by the virus. These findings strongly suggest that neutral glycosphingolipids share the key determinant for virus binding and that the ß-GlcNAc residue may play an important role in dengue virus binding to the host cell surface.

Molecular detection of Indian Ocean Lineage Chikungunya virus RNA in field collected Culex quinquefasciatus Say from Bangkok, Thailand but no evidence of virus replication.

Following an outbreak of chikungunya virus (CHIKV) infections in Thailand in 2019, numerous cases of CHIKV infection have been diagnosed in Bangkok, the capital of the country. In our previous investigation of the vectors for disease transmission, we found natural infection of CHIKV in both male and female Aedes aegypti mosquitoes collected from the outbreak areas in Bangkok. Some reports mentioned the detection of CHIKV in Culex mosquitoes. In Thailand, the Culex quinquefasciatus Say mosquito is a common species found in urban and rural settings that coexists with Ae. aegypti. However, the role of Cx. quinquefasciatus mosquitoes in the spread of the Indian Ocean Lineage (IOL) of CHIKV in Thailand has never been investigated. In this study, Cx. quinquefasciatus were collected (16 males and 27 females) from an outbreak area in Bangkok. Eight of the 27 in field-caught female Cx. quinquefasciatus were positive for IOL CHIKV RNA, and 99-100% identity and full 100% coverage of sequences similar to CHIKV isolated from female Ae. aegypti in Bangkok, Thailand, whereas viral RNA was not detected in male samples using nested-RT-PCR. To determine whether CHIKV is able to replicate in Cx. quinquefasciatus, the laboratory strain of Cx. quinquefasciatus was allowed to feed on blood containing IOL CHIKV isolated from patient serum. The nested-RT-PCR, virus isolation, and immunofluorescence assay (IFA) were performed for CHIKV detection and replication. The results showed that CHIKV RNA was detected in Cx. quinquefasciatus until day 4 post infection. CHIKV did not produce any remarkable signs of infection, dissemination, or transmission in Cx. quinquefasciatus, and cytopathic effect (CPE) was not observed in C6/36 cells when infected with supernatant obtained from Cx. quinquefasciatus at days 7, 10, 14, and 21 post infection when compared to Ae. aegypti. The data from this study infer that CHIKV may be detected in Cx. quinquefasciatus but that the mosquito is not able to transmit CHIKV in Thailand.

LC-QTOF-MS/MS Based Molecular Networking Approach for the Isolation of α-Glucosidase Inhibitors and Virucidal Agents from Coccinia grandis (L.) Voigt.

Coccinia grandis or ivy gourd is an edible plant. Its leaves and fruits are used as vegetable in many countries. Many works on antidiabetic activity of a crude extract of C. grandis, i.e., in vitro, in vivo, and clinical trials studies, have been reported. Profiles of the antidiabetic compounds were previously proposed by using LC-MS or GC-MS. However, the compounds responsible for antidiabetic activity have rarely been isolated and characterized by analysis of 1D and 2D NMR data. In the present work, UHPLC-ESI-QTOF-MS/MS analysis and GNPS molecular networking were used to guide the isolation of α-glucosidase inhibitors from an extract of C. grandis leaves. Seven flavonoid glycosides including rutin (1), kaempferol 3-O-rutinoside (2) or nicotiflorin, kaempferol 3-O-robinobioside (3), quercetin 3-O-robinobioside (4), quercetin 3-O-ß-D-apiofuranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-ß-D-glucopyranoside (5) or CTN-986, kaempferol 3-O-ß-D-api-furanosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-ß-D-glucopyranoside (6), and kaempferol 3-O-ß-D-apiofuranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-ß-D-galactopyranoside (7) were isolated from C. grandis leaves. This is the first report of glycosides containing apiose sugar in the genus Coccinia. These glycosides exhibited remarkable α-glucosidase inhibitory activity, being 4.4-10.3 times more potent than acarbose. Moreover, they also displayed virucidal activity against influenza A virus H1N1, as revealed by the ASTM E1053-20 method.

Temporal Change of SARS-CoV-2 in Clinical Specimens of COVID-19 Pneumonia Patients.

The quality and type of specimen collection affect the sensitivity of real-time reverse transcriptase-PCR (rRT-PCR) for the diagnosis of SARS-CoV-2. In this report, the course over time of rRT-PCR for SARS-CoV-2 in 26 clinical specimens collected from the upper (nasopharyngeal and throat swabs) and lower (sputum) respiratory tracts of COVID-19 cases with pneumonia was investigated along with the clinical course. The preliminary results revealed that higher SARS-CoV-2 RNA concentration and longer time for detection make self-collected sputum a preferable specimen for the diagnosis and follow-up of COVID-19 pneumonia. Self-collection of sputum can minimize the risk of unnecessary exposure to healthcare workers, preserve the shortage of personal protective equipment, and limit viral transmission to the environment.

Development of single-tube mutiplex RT-PCR for dengue virus typing.

This study evaluated a single-tube multiplex RT-PCR with a primer focusing on nonstructural protein region 5 (NS5) and a highly conserved region for dengue virus serotyping. The method was compared with conventional PCR. This new method had a sensitivity of 96.7% and a specificity of 96.7% for disease detection. The new method also proved suitable for use in the field as it reduces time and decreases risk of contamination.

High prevalence of Hepatitis C virus genotype 6 in Vietnam.

This study aimed to update the prevalence of the various Hepatitis C virus genotypes in Vietnamese blood donors. One hundred and three HCV antibody-positive plasma samples were collected from blood donors at the National Institute of Hematology and Blood Transfusion, Hanoi, Vietnam. All specimens were subjected to RT-PCR of the 5' untranslated region (UTR) to confirm the presence of HCV RNA. The core and NS5B regions of thh positive samples were subsequently amplified by RT-PCR followed by direct sequencing and phylogenetic analysis. Seventy out of 103 samples (68.0%) were RNA positive. Core and NS5B were successfully amplified and sequences were obtained for 70 and 65 samples, respectively. Phylogenetic analysis revealed that genotype 6a was the most predominant among Vietnamese blood donors with a prevalence of 37.1% (26/70), followed by genotype 1a at 30.0% (21/70) and genotype 1b at 17.1% (12/70). The prevalence of two other genotype 6 variants, 6e and 61 was 8.6% and 1.4%, respectively. Further analysis of recent studies showed that the geographic distribution of genotype 6 covered mainly southern China and the mainland of Southeast Asia including Vietnam, Laos, Thailand, and Myanmar. The GenBank accession numbers for the sequences reported in this study are FJ768772-FJ768906.

Molecular Epidemiology and Genetic Diversity of Zika Virus from Field-Caught Mosquitoes in Various Regions of Thailand.

Zika virus (ZIKV) infection is an emerging and re-emerging arbovirus disease that is transmitted to humans through the bite of infected mosquitoes. ZIKV infections were first described in Thailand in 1954 from the sera of indigenous residents and several travelers returning from Thailand in 2014. However, reported cases in Thailand have been increasing since 2015 and 2016, and epidemiological information about the vectors of ZIKV is unclear. We investigated the molecular epidemiology and genetic diversity of ZIKV from mosquitoes collected from different geographic regions experiencing ZIKV outbreaks in Thailand. Polymerase chain reaction was used to amplify the non-structural protein (NS5) gene of ZIKV, which was then sequenced. A total of 1026 mosquito samples (626 females, 367 males, and 33 larvae) were collected from active ZIKV patients' houses. ZIKV was detected in 79 samples (7.7%), including Aedes aegypti (2.24% female, 1.27% male, and 0.19% larvae), Culex quinquefasciatus (1.85% female, 1.66% male, and 0.29% larvae), and Armigeres subalbatus (0.1% female and 0.1% male), whereas no ZIKV was detected in Aedes albopictus. Phylogenetic analysis of the 79 positive samples were classified into two clades: Those closely related to a previous report in Thailand, and those related to ZIKV found in the Americas. This is the first report of the detection of ZIKV in Ae. aegypti, Cx. quinquefasciatus, and Ar. subalbatus mosquitoes, and genetic variations of ZIKV in the mosquitoes collected from several geographic regions of Thailand were examined. Detection of ZIKV in male and larval mosquitoes suggests that vertical transmission of ZIKV occurred in these mosquito species. This study provides a more in-depth understanding of the patterns and epidemiologic data of ZIKV in Thailand; the data could be used for future development of more effective prevention and control strategies of ZIKV in Thailand.

In vitro analysis of synthetic peptides in blocking the entry of dengue virus.

Dengue fever is the most prevalent arthropod-borne viral disease, and no specific therapeutic or promising antiviral drug is available for its treatment. Peptide inhibitors are less toxic than synthetic compounds and have found proven effective against viral infections. Here, three peptides that mimic part of the E protein of the dengue virus (DENV) were synthesized and evaluated for their inhibitory activity against four serotypes of DENV in African green monkey kidney (Vero) and rhesus macaque (Macaca mulatta) monkey kidney (LLC-MK2) cell lines. The three peptides, Pep1, Pep2, and Pep3 are located in domains I, II, and III of the E protein respectively. All three peptides effectively reduced >80% of focus forming units in the virus treated mammalian cell lines than control and exhibited their IC50 in the range of 10-33 µM. Pep1 was found effective against DENV-2, DENV-3, and DENV-4 (IC80 below 50 µM). Pep2 showed the highest inhibitory activity against all four serotypes (IC50 below 20 µM). Pep3 reduced the 80% focus forming units in all serotypes at the concentration of 40 µM. Evaluation of peptides at different time points of viral infection in the mammalian cell lines revealed that the peptides inhibited viral infection by binding to the virus and not by binding to cellular receptors and blocking viral entry. The peptides assumedly exert their inhibitory effects by binding to the E protein and repressing its conformational changes; this prevents the virus from binding to cellular receptors, thereby inhibiting viral entry. Hence, these peptides might limit viral spread and reduce the virus's ability to infect.

Characterization of atypical lymphocytes and immunophenotypes of lymphocytes in patients with dengue virus infection.

To characterize the immunophenotypes of lymphocytes in patients with dengue infection, we performed flow cytometric analysis of peripheral blood mononuclear cells collected from 49 dengue hemorrhagic fever (DHF), 25 dengue fever (DF), and 26 dengue-like syndrome (DLS) cases. The mean total atypical lymphocytes in DHF (916.1 +/- 685.6 cells/microl) and DF (876.2 +/- 801.9 cells/microl) were higher than those of DLS (310.5 +/- 181.4 cells/microl). An atypical lymphocyte count of 10% or higher was a good indicator of dengue infection (sensitivity 50% and specificity 86%). Flow cytometric studies showed that the percentages of atypical lymphocytes correlated with those of CD19+ B lymphocytes and inversely correlated with the percentages of CD69+ lymphocytes. The mean absolute counts of atypical lymphocytes and CD19+ cells on the discharge day were significantly higher than those on the admission day. Low percentages of TdT+ cells were found in all groups of patients. We concluded that atypical lymphocyte and CD19+ cell counts may be a useful diagnostic tool for dengue infection and the recovery from the disease could be judged when numbers of both cell types are significantly elevated.

Rapid detection of dengue viral RNA in mosquitoes by nucleic acid-sequence based amplification (NASBA).

RNA amplification by nucleic acid sequence-based amplification (NASBA) was used to detect serotype specific dengue viruses in artificially-infected female Aedes mosquitoes, in comparison with RT-PCR technique. NASBA could detect dengue virus serotype 2 and 4 below 0.1 PFU, which was more sensitive than RT-PCR, but this technique was as sensitive as RT-PCR when detecting dengue virus serotype 1 and 3. Dengue viruses could be detected at the thorax of mosquitoes at 0, 7, and 14 days after inoculation with dengue virus serotype 2. This method should be useful for virological surveillance of dengue infected Aedes mosquitoes, as an early warning system to predict outbreaks of dengue viruses.