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.

An Epidemic of Dengue Virus Serotype-4 during the 2015 - 2017: the Emergence of a Novel Genotype IIa of DENV-4 in the Philippines.

Volume 72 no 6, p.413-419, 2019. Page 418, Acknowledgments "We would like to thank all staff and members of the Department of Virology, NEKKEN, Nagasaki University, Japan for providing technical support and advice. Our special thanks to the staff of the Pavilion II and the Central Laboratory of San Lazaro Hospital for their kind assistance during patient recruitment and data collection. We are also very grateful for the support of the Senior Vice President and Head of Research and Biotechnology (R&B) Group of St. Luke's Medical Center, Dr. Isaac David E. Ampil II. Finally, our sincere thanks to the members of R&B's dengue research group for kindly preparing the samples to be transported to NEKKEN." should read "This research was supported by grants from the Japan Agency for Medical Research and Development (AMED) under Grant Number JP18fm0108001, JP19fm0108001 (Japan Initiative for Global Research Network on Infectious Diseases (J-GRID)), AMED Research on Emerging and Re-emerging Infectious Diseases (19fk0108035j0003) and e-ASIA Joint Research Program and; Philippine Council for Health Research and Development (PCHRD) of the Department of Science and Technology (DOST), Philippines, with partial support from the Research and Biotechnology of St. Luke's Medical Center (R&B-SLMC), Philippines (Project No. 07-024). Funders have no role in the study design, data collection, and interpretation, or the decision to submit this work for publication. We would like to thank all staff and members of the Department of Virology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Japan, for providing technical support and advice. Our special thanks to the staff of the Pavilion II and the Central Laboratory of San Lazaro Hospital for their kind assistance during patient recruitment and data collection. We are also very grateful for the support of the Senior Vice President and Head of Research and Biotechnology (R&B) Group of St. Luke's Medical Center, Dr. Isaac David E. Ampil II. Finally, our sincere thanks to the members of R&B's Dengue Research Group for kindly preparing the samples to be transported to NEKKEN."

An Epidemic of Dengue Virus Serotype-4 during the 2015 - 2017: the Emergence of a Novel Genotype IIa of DENV-4 in the Philippines.

Dengue remains a major public health problem in the Philippines. In this study, we determined the circulating dengue serotypes in the Philippines during the 2015-2017 outbreaks using a total of 678 serum samples from 537 individual dengue patients. Following an increase in the number of DENV-4 patients in recent years, we conducted a comprehensive molecular and epidemiology analysis on the DENV-4 strains isolated recently in the Philippines. Two genotypes of DENV-4 have been isolated in the Philippines since 1956: GI and GIIa. The GIIa DENV strains that were isolated in the present study were closely related to a distinct group of GIIa strains that were isolated from the Philippines in 2004. A majority of the isolates of this sub-group have been identified in the Philippines, suggesting that this lineage may have been introduced in the Philippines, and evolved to form the distinct sub-group within GIIa strains. The increase in DENV-4 activity also coincided with the appearance of the GIIa subgroup and the phasing-out of the GI lineage in the Philippines. Overall, our study demonstrates a shift in DENV-4 genotype and epidemic dynamics in a hyperendemic region, suggesting the importance of DENV genetic evolution in establishing and sustaining transmission.

Evaluation of a rapid diagnostic test for detection of dengue infection using a single-tag hybridization chromatographic-printed array strip format.

A dipstick DNA chromatography assay, a single-tag hybridization-printed array strip (STH-PAS), was evaluated for its efficacy to detect dengue virus (DENV). Reverse-transcribed DNA was amplified by PCR, and the amplified DNA was detected using the STH-PAS system. The method was evaluated using stored RNA samples previously identified to carry all 4 serotypes of dengue, chikungunya, and influenza viruses. Clinical performance was also assessed in a prospective study using plasma from 269 febrile cases from the Emergency Department of St. Luke's Medical Center, Quezon City, Philippines, and 30 afebrile normal healthy volunteers. A Taqman real-time PCR (RT-PCR) assay and a rapid Dengue NS1 test, SD Bioline, were used for comparison. The STH-PAS system was more sensitive in detecting dengue infection compared to Taqman RT-PCR. For DENV serotypes 1, 2, and 3, the detection was 1 to 2 dilutions (10-fold) higher, and for DENV serotype 4, the detection was 2-4 dilutions higher. In clinical studies, the STH-PAS system showed 100% sensitivity with 88.9% and 86.6% specificities compared to Taqman RT-PCR and SD Dengue Duo NS1 test, respectively. The STH-PAS system was found to have a superior sensitivity than the Taqman system. Further evaluation of its performance in the field may provide important data to extend its usefulness for surveillance and epidemiological research in outbreak situations.

Hepatic steatosis in chronic hepatitis B: a study of metabolic and genetic factors.

Hepatic steatosis is a common finding in liver biopsy and may co-exist with chronic hepatitis B (CHB) infection. The aims of this study were to determine the prevalence of steatosis in CHB patients among Filipinos; determine the factors related to the presence of steatosis among individuals with and without CHB infection; and to investigate the possible association between steatosis and polymorphism in interleukin 28B (IL28B) gene. The presence of steatosis was correlated with clinical, biochemical and histological parameters. Peripheral blood samples of CHB patients with steatosis, CHB patients without steatosis and normal controls were genotyped for IL28B rs8099917 T>G using the TaqMan assay. Of the 46 CHB patients, 41% (19/46) had steatosis. Body mass index (BMI), fasting blood sugar (FBS), lipid profile and alanine transaminase levels were observed to be significantly different between CHB patients with steatosis and normal controls. The serum FBS of CHB patients with steatosis was significantly higher than patients without steatosis. High density lipoprotein cholesterol of patients without steatosis was significantly higher than patients with steatosis. Although not statistically significant, BMI, triglycerides, low density lipoprotein cholesterol and histology activity index in CHB patients with steatosis were found to be higher than those without steatosis. There was no significant association between the stage of fibrosis and severity of steatosis. In conclusion, the prevalence of hepatic steatosis among Filipino patients with CHB is 41%. Steatosis in CHB patients was associated with metabolic factors such as diabetes and dyslipidemia. IL28B rs8099917 T>G polymorphism is not associated with steatosis.

The effect of biological heterogeneity on R-CHOP treatment outcome in diffuse large B-cell lymphoma across five international regions.

Addressing the global burden of cancer, understanding its diverse biology, and promoting appropriate prevention and treatment strategies around the world has become a priority for the United Nations and International Atomic Energy Agency (IAEA), the WHO, and International Agency for Research on Cancer (IARC). The IAEA sponsored an international prospective cohort study to better understand biology, treatment response, and outcomes of diffuse large B-cell lymphoma (DLBCL) in low and middle-income countries across five UN-defined geographical regions. We report an analysis of biological variation in DLBCL across seven ethnic and environmentally diverse populations. In this cohort of 136 patients treated to a common protocol, we demonstrate significant biological differences between countries, characterized by a validated prognostic gene expression score (p < .0001), but International Prognostic Index (IPI)-adjusted survivals in all participating countries were similar. We conclude that DLBCL treatment outcomes in these populations can be benchmarked to international standards, despite biological heterogeneity.

Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score.

As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.

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.

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.