Role of pre-existing immunity in driving the dengue virus serotype 2 genotype shift in the Philippines: A retrospective analysis of serological data.

OBJECTIVE: The invasion of dengue virus (DENV)-2 Cosmopolitan genotype into the Philippines, where the Asian II genotype previously circulated challenges the principle of dengue serotype-specific immunity. Assessment of antibodies in this population may provide a mechanistic basis for how new genotypes emerge in dengue-endemic areas. METHODS: We evaluated the neutralizing antibody (nAb) and antibody-dependent enhancement (ADE) responses against the two genotypes using archived serum samples collected from 333 patients with confirmed dengue in Metro Manila, Philippines, before, during, and after the introduction of the Cosmopolitan genotype. We quantified nAb titers in baby hamster kidney (BHK-21) cells with or without the Fcγ receptor IIA (FcγRIIA) to detect the capacity of virus-antibody complexes to neutralize or enhance DENV. RESULTS: The nAb potency of the archived serum samples against the two genotypes was greatly affected by the presence of FcγRIIA. We found significant differences in nAb titers between the two genotypes in BHK-21 cells with FcγRIIA (P <0.0001). The archived serum samples were incapable of fully neutralizing the Cosmopolitan genotype, but instead strongly promoted its ADE compared to the Asian II genotype (P <0.0001). CONCLUSION: These results reinforce the role of pre-existing immunity in driving genotype shifts. Our finding that specific genotypes exhibit differing susceptibilities to ADE by cross-reactive antibodies may have implications for dengue vaccine development.

Saliva as alternative to naso-oropharyngeal swab for SARS-CoV-2 detection by RT-qPCR: a multicenter cross-sectional diagnostic validation study.

Saliva has been demonstrated as feasible alternative to naso-oropharyngeal swab (NOS) for SARS-CoV-2 detection through reverse transcription quantitative/real-time polymerase chain reaction (RT-qPCR). This study compared the diagnostic agreement of conventional NOS, saliva with RNA extraction (SE) and saliva without RNA extraction (SalivaDirect) processing for RT-qPCR in identifying SARS-CoV-2. All techniques were also compared, as separate index tests, to a composite reference standard (CRS) where positive and negative results were defined as SARS-CoV-2 detection in either one or no sample, respectively. Of 517 paired samples, SARS-CoV-2 was detected in 150 (29.01%) NOS and 151 (29.21%) saliva specimens. The saliva-based tests were noted to have a sensitivity, specificity and accuracy (95% confidence interval) of 92.67% (87.26%, 96.28%), 97.55% (95.40%, 98.87%) and 96.13% (94.09%, 97.62%), respectively, for SE RT-qPCR and 91.33% (85.64%, 95.30%), 98.91% (97.23%, 99.70%) and 96.71% (94.79%, 98.07%), respectively, for SalivaDirect RT-qPCR compared to NOS RT-qPCR. Compared to CRS, all platforms demonstrated statistically similar diagnostic performance. These findings suggest that both conventional and streamlined saliva RT-qPCR are at least non-inferior to conventional NOS RT-qPCR in detecting SARS-CoV-2.

A Preliminary Investigation on the Antiviral Activities of the Philippine Marshmint (Mentha arvensis) Leaf Extracts against Dengue Virus Serotype 2 In Vitro.

In this study, we investigated the antiviral activity of lyophilized crude leaf extracts of the Philippine marshmint (Mentha arvensis L., commonly called yerba buena) against DENV-2 in vitro. The plant specimen was authenticated by DNA barcoding analysis using standard primers for amplification of rbcL, matK, ITS1, ITS2 and trnH-psbA. Aqueous, methanol and ethanol leaf extracts were prepared, and lyophilized prior to testing for its cytotoxicity and antiviral activities. All extracts presented cytotoxic activities against Vero cells in a dose-dependent manner. Half maximal cytotoxicity concentration (CC50) was calculated at 2,889.60 µg/mL for the aqueous extract, 1,928.62 µg/mL for the methanol extract, and 3,380.30 µg/mL for the ethanol extract. Antiviral activities assessed by plaque reduction assay revealed reduced DENV-2 viral infectivity, with the ethanol extract observed to have the strongest activity decreasing plaque numbers by 62% relative to the control. The methanol extract was observed to be most effective when added before infection causing 72% reduction in plaque numbers, whereas none of the extracts inhibited plaque formation by more than 40% when added after infection. DENV-2 NS1 antigen production was significantly reduced by the methanol extract, while viral RNA levels were also decreased as determined by real time RT-PCR. Phytochemical analysis revealed the presence of flavonoids, phenolics, tannins, proteins, reducing sugars and saponins. Our preliminary results are promising, however, it should be interpreted with caution as further studies are needed to establish its potential therapeutic application against dengue infection.

Hybrid Vibrio cholerae El Tor lacking SXT identified as the cause of a cholera outbreak in the Philippines.

UNLABELLED: Cholera continues to be a global threat, with high rates of morbidity and mortality. In 2011, a cholera outbreak occurred in Palawan, Philippines, affecting more than 500 people, and 20 individuals died. Vibrio cholerae O1 was confirmed as the etiological agent. Source attribution is critical in cholera outbreaks for proper management of the disease, as well as to control spread. In this study, three V. cholerae O1 isolates from a Philippines cholera outbreak were sequenced and their genomes analyzed to determine phylogenetic relatedness to V. cholerae O1 isolates from recent outbreaks of cholera elsewhere. The Philippines V. cholerae O1 isolates were determined to be V. cholerae O1 hybrid El Tor belonging to the seventh-pandemic clade. They clustered tightly, forming a monophyletic clade closely related to V. cholerae O1 hybrid El Tor from Asia and Africa. The isolates possess a unique multilocus variable-number tandem repeat analysis (MLVA) genotype (12-7-9-18-25 and 12-7-10-14-21) and lack SXT. In addition, they possess a novel 15-kb genomic island (GI-119) containing a predicted type I restriction-modification system. The CTXΦ-RS1 array of the Philippines isolates was similar to that of V. cholerae O1 MG116926, a hybrid El Tor strain isolated in Bangladesh in 1991. Overall, the data indicate that the Philippines V. cholerae O1 isolates are unique, differing from recent V. cholerae O1 isolates from Asia, Africa, and Haiti. Furthermore, the results of this study support the hypothesis that the Philippines isolates of V. cholerae O1 are indigenous and exist locally in the aquatic ecosystem of the Philippines. IMPORTANCE: Genetic characterization and phylogenomics analysis of outbreak strains have proven to be critical for probing clonal relatedness to strains isolated in different geographical regions and over time. Recently, extensive genetic analyses of V. cholerae O1 strains isolated in different countries have been done. However, genome sequences of V. cholerae O1 isolates from the Philippines have not been available for epidemiological investigation. In this study, molecular typing and phylogenetic analysis of Vibrio cholerae isolated from both clinical and environmental samples in 2011 confirmed unique genetic features of the Philippines isolates, which are helpful to understand the global epidemiology of cholera.

Complete genome sequence of chikungunya virus isolated in the Philippines.

Chikungunya virus is an alphavirus of the Togaviridae family, which causes a febrile illness with arthralgia in humans. We report here on the complete genome sequence of chikungunya virus strain CHIKV-13-112A isolated from a patient in the Philippines who was suspected to have dengue virus. Phylogenetic analysis revealed that the strain is of the Asian genotype.

Association of glutathione S-transferase T1 and M1 genotypes with chronic liver diseases among Filipinos.

The glutathione S-transferase (GST) supergene family is made up of four gene families responsible for the biotransformation of drugs and other xenobiotics. Genetic variations in this supergene family influence individual detoxification levels and may contribute to the development of cancer. A hospital-based case-control study was conducted to evaluate the association between GST polymorphism among Filipino patients positive for hepatitis B virus (HBV DNA) and clinically diagnosed as either with chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma as well as normal individuals negative for HBV infection. Multiplex PCR was used to detect the presence or absence of the GSTT1 and GSTM1 polymorphisms in peripheral blood. DNA sequencing of the S gene region of the virus was used to determine the predominant genotype found among HBV-infected patients. Our results showed that the odds of having a chronic liver disease is only 0.95 (95% CI 0.58-1.57) among those with GSTT1 null genotype compared to those with GSTT1+ genotype. On the other hand, the odds of chronic liver disease is 17.85 times (95% CI 7.34-43.45) for those with GSTM1 null genotype compared to those with GSTM1+ genotype. Using the GSTT1+/GSTM1+ genotype as the reference, both GSTT1+/GSTM1- (OR 16.61; 95% CI 6.69-41.22) and GSTT1-/GSTM1- (OR 11.91; 95% CI 4.48-31.66) genotypes seem to be risk factors for chronic liver disease. From our observations, we conclude that polymorphism in GSTM1 null genotype (OR 17.85; 95% CI 7.34-43.45) seem to be associated with an increased risk of chronic liver disease among Filipinos.

Platelet apoptosis and apoptotic platelet clearance by macrophages in secondary dengue virus infections.

BACKGROUND: The mechanisms of thrombocytopenia and platelet phagocytosis in dengue illness are not fully understood. METHODS: A prospective hospital-based study was conducted to examine the relationships between platelet counts, serum thrombopoietin (TPO) levels, and platelet apoptosis and phagocytosis in 81 patients with secondary dengue virus (DV) infections and 38 healthy volunteers. The apoptosis and phagocytosis of cultured platelets after exposure to DV were also examined. RESULTS: Platelet apoptosis, platelet phagocytosis, and serum TPO levels were increased significantly in patients during the acute and early convalescence phases compared with levels observed in patients during the convalescence phase and in healthy volunteers. A significant correlation between platelet apoptosis and platelet phagocytosis was also observed in these patients. Platelet phagocytosis was inhibited significantly by the D89E mutant, which carries a point mutation in the RGD motif of milk fat globule-epidermal growth factor 8, a phosphatidylserine-recognizing bridge molecule. DV-induced platelet apoptosis and increased phagocytosis of DV-induced apoptotic platelets was confirmed using in vitro assays. CONCLUSIONS: Our data suggest an increased phagocytosis of DV-induced apoptotic platelets by macrophages via a phosphatidylserine-recognizing pathway in secondary DV infection. Accelerated platelet clearance, however, was overcome by TPO-induced enhanced thrombopoiesis in these patients. CLINICAL TRIALS REGISTRATION: UMIN000004835.

Shotgun genome sequence of a Yersinia enterocolitica isolate from the Philippines.

The first shotgun genome sequence of a microbial pathogen from the Philippines is reported. Yersinia enterocolitica subsp. palearctica strain PhRBD_Ye1 is the first Y. enterocolitica strain sequenced from an animal source, swine, which is a natural source of yersiniosis. The closest phylogenetic match is a human clinical isolate from Germany.

Development and evaluation of a fluorogenic real-time RT-PCR for the detection of dengue 3 virus.

A dengue-3-specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was developed using the novel Light Upon eXtension (LUX™) fluorogenic technology. A labeled forward primer and a standard reverse primer that target a conserved region within the non-structural 1 (NS1) gene of dengue 3 strains were designed. The dengue-3-specific assay did not recognize other dengue serotypes and related flaviviruses. Using a tenfold serial dilution of plasmid DNA containing the dengue 3 NS1 gene as standards, the range of dengue virus detection was determined to be from 10(3) to 10(9) copies/ml or from 80 to 8 × 10(7) copies/reaction with an average correlation coefficient of ≥ 0.99. The mean intra-assay coefficient of variation (CV) at 2.01% and the mean inter-assay CV at 2.68% suggest the repeatability of the procedure. Moreover, the fluorogenic assay was evaluated by using clinical specimens and comparing test results with historical data obtained from conventional RT-PCR, which served as the criterion standard. Using patient sera as test samples, the assay demonstrated 95.45% sensitivity and 100% specificity, respectively. These results reveal that the real-time RT-PCR assay may be utilized as a rapid, convenient, and sensitive tool for the detection of dengue 3 in clinical and laboratory specimens.

Flow cytometric analysis of dengue virus-infected cells in peripheral blood.

With the development of permeabilization techniques in flow cytometry and the availability of various monoclonal antibodies (MAbs) that specifically bind with cell surface and intracellular antigens, it is now possible to use flow cytometric assay to identify dengue virus (DEN) infected cells in peripheral blood. Blood samples were analyzed using phycoerythrin (PE) labeled anti-CD3, anti-CD14, anti-CD16, and anti-CD19 antibodies and Alexa Fluor 488 labeled anti-flavivirus monoclonal antibody (MAb) 6B6C-1. The predominant DEN-infected cells were CD19+ in this study. There was dim partial to moderately bright partial expression of CD19 positive cells in the blood samples tested. Virus isolation and serotype-specific RT-PCR revealed the cells were infected with dengue serotype 3 (DEN3). Our results suggest B cells may play an important role in DEN1 and DEN3 replication, and dissemination in vivo.

Evaluation of a dengue IgG indirect enzyme-linked immunosorbent assay and a Japanese encephalitis IgG indirect enzyme-linked immunosorbent assay for diagnosis of secondary dengue virus infection.

To establish a new method for the diagnosis of dengue secondary infection, 187 serum samples from the patients with dengue secondary infection, 40 serum samples from the patients with dengue primary infection, and 44 serum samples from the healthy volunteers were tested using the dengue IgG indirect enzyme-linked immunosorbent assay (DEN IgG ELISA). The results of the test were compared with those from the dengue hemagglutination inhibition (DEN HI) test, which has been recommended as the gold standard by the World Health Organization (WHO, 1997). Japanese encephalitis IgG indirect ELISA (JE IgG ELISA) was also performed to measure anti-flavivirus IgG, which cross-reacts with the Japanese encephalitis virus, to test the possibility of an alternative to DEN IgG ELISA. The results of DEN IgG and JE IgG ELISAs were highly correlated with those of the DEN HI test. In the DEN IgG ELISA, a titer of 1:29,000 was the cut-off value for the diagnosis of dengue secondary infection (91.5% accuracy [95% confidence interval, CI], 90.9% sensitivity [95%CI], and 92.9% specificity [95%CI]). A titer of 1:52,000 was the cut-off value for dengue secondary infection using JE IgG ELISA (95.6% accuracy [95%CI], 98.9% sensitivity [95%CI], and 88.1% specificity [95%CI]). In conclusion, this study confirmed that the results of both DEN IgG and JE IgG ELISAs were highly correlated with the results of DEN HI test. Thus, these ELISAs are simple, rapid, sensitive, and quantitative tests that can be used in the determination of dengue secondary infection.

Increased phagocytosis of platelets from patients with secondary dengue virus infection by human macrophages.

The relationship between the percent phagocytosis of platelets by differentiated THP-1 cells was examined using flowcytometry and the peripheral platelet counts as well as platelet-associated IgG (PAIgG) in 36 patients with secondary dengue virus (DV) infections. The percent phagocytosis and the levels of PAIgG were significantly increased in these patients during the acute phase compared with the healthy volunteers. The increased percent phagocytosis and PAIgG found during the acute phase significantly decreased during the convalescent phase. An inverse correlation between platelet count and the percent phagocytosis (P = 0.011) and the levels of PAIgG (P = 0.041) was found among these patients during the acute phase. No correlation was found, however, between the percent phagocytosis and the levels of PAIgG. Our present data suggest that accelerated platelet phagocytosis occurs during the acute phase of secondary DV infections, and it is one of the mechanisms of thrombocytopenia in this disease.

Antigen sandwich ELISA predicts RT-PCR detection of dengue virus genome in infected culture fluids of Aedes albopictus C6/36 cells.

Antigen detection by sandwich ELISA was evaluated to predict RT-PCR detection of dengue viral genome in infected culture fluid of Aedes albopictus clone C6/36 cells. Serum specimens collected from dengue patients within 5 days from onset of fever in 2 hospitals in Metro Manila, Philippines, were inoculated into C6/36 cells, and incubated at 28 degrees C. A total of 282 infected culture fluid specimens were harvested and examined by sandwich ELISA and RT-PCR to detect dengue viral antigen and genome, respectively. In the sandwich ELISA, the P/N ratio was calculated by dividing optical density (OD) of a given test specimen by the OD of the standard negative specimen. Samples with a P/N ratio > or = 4.001 were positive for viral genome detection by RT-PCR. The sensitivity and specificity of antigen sandwich ELISA with RT-PCR as the standard, were 90.4% and 100%, respectively. Although antigen sandwich ELISA is less sensitive than RT-PCR, its usefulness lies in its capability to screen a large number of samples at a minimum cost, especially during an outbreak. Samples that meet a set cutoff value can undergo confirmation by RT-PCR for further epidemiological studies.

Molecular epidemiology of dengue 2 viruses in the Philippines: genotype shift and local evolution.

The pre-membrane (prM) and envelope (E) genes of 41 viruses isolated from dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) patients from 1995 to 2002 were sequenced to determine the genetic variability of dengue 2 (DENV 2) viruses in the Philippines. The envelope sequence data were compared with a global sample of DENV 2 obtained from GenBank. Phylogenetic analysis revealed that two distinct genotypes, Asian 2 and Cosmopolitan, are currently circulating locally, each with the potential to cause severe hemorrhagic disease. After the initial isolation in 1998, the Cosmopolitan genotype has gradually and effectively replaced Asian genotype 2 in the Philippines. Members of this genotype were closely related to viruses from Australia, Singapore, and Thailand.

Comparison of clinical features and hematologic abnormalities between dengue fever and dengue hemorrhagic fever among children in the Philippines.

To demonstrate the differences of clinical features and hematologic abnormalities between dengue fever (DF) and dengue hemorrhagic fever (DHF), 359 pediatric patients admitted St. Luke's Medical Center in Quezon City, between 1999 and 2001 in Metro Manila, and adjoining provinces the Philippines, with a laboratory-confirmed dengue virus infection were evaluated. One third of the patients had DHF, and most of these patients were without shock. Restlessness, epistaxis, and abdominal pain were more associated with DHF. The platelet count was significantly lower in the DHF group than in the DF group before and after defervescence. In the DHF patients, the hematocrit was significantly increased before defervescence, and decreased the day after due to administration of intravenous fluid. Coagulation abnormalities associated with most DHF patients were thrombocytopenia and an increased fibrinolysis, but not disseminated intravascular coagulation. We present recent data on readily obtained clinical and laboratory data that can be used for early diagnosis and consequently earlier appropriate treatment of dengue virus infections.

Correlation between increased platelet-associated IgG and thrombocytopenia in secondary dengue virus infections.

Although the public health impact of dengue is increasing rapidly, the mechanism of thrombocytopenia in this disease remains unknown. To elucidate this mechanism, the relationship between platelet-associated IgG (PAIgG) and platelet count in 53 patients in the acute phase of secondary dengue virus infection was investigated in a prospective-hospital-based study. A significant inverse correlation between the two parameters was found in these patients, while no correlation was observed in healthy volunteers. The low baseline platelet counts during the acute phase in 12 patients with secondary dengue virus infection significantly increased during the convalescent phase, while the increased PAIgG levels during the acute phase in these patients significantly decreased during the convalescent phase. Anti-platelet IgG autoantibody was detected rarely in the plasma of 53 patients with secondary dengue infection. The involvement of anti-dengue virus IgG was also shown in platelets from all of 8 patients in the acute phase of secondary dengue virus infection. These findings suggest that PAIgG formation involving anti-dengue virus IgG plays a pivotal role in the induction of transient thrombocytopenia during the acute phase of secondary dengue virus infection.