BACKGROUND: The changing nature of dengue epidemiology and control makes dengue one of the challenging infectious disease problems in the present time with certain inadequacies in existing knowledge base becoming apparent.
OBJECTIVE: This quantitative and experimental study was conducted to provide recent local evidence that dengue virus transovarial transmission among field collected Aedes aegypti mosquitoes does occur and presents an important factor in the epidemiology and control of dengue.
METHODS: Households in Quezon City, Philippines, a known dengue infection hotspot in 2011, were randomly selected (H9 and H14) for Aedes aegypti egg and larval collection. Mosquito larvae were captured using standard ovitraps and reared to adulthood in the entomology unit of the Molecular Diagnostics and Genotyping Laboratory at the University of the Philippines (UP), College of Medicine, Manila. Whole organism homogenate of adult mosquitoes were prepared for subsequent dengue virus molecular characterization and virulence testing. Both egg samples and their infection profile for dengue virus was determined by serotype specific RT-PCR.
RESULTS: Molecular test results show that in each household and in each generation (parent, F1 and F2), there were detectable and strong dengue viral presence, predominantly the serotypes DEN-2, DEN-3 and DEN-4 in the Aedes aegypti mosquito homogenates.
CONCLUSIONS: These laboratory evidences indicate that thransovarial transmission of dengue virus does occur in a high urban city like Quezon City where incidence of dengue is high.Thus, it is important to consider the existence of this phenomenon in existing and future dengue control programs to ensure effectiveness of community-based intervention strategies.

Human ; Animal ; Male ; Female ; Dengue Virus ; Aedes ; Serogroup ; Larva ; Entomology ; Pathology, Molecular ; Virulence ; Genotype ; Dengue

Background. Emergence of multidrug-resistant tuberculosis (MDR-TB) poses a major challenge to prevailing disease management. MDR-TB arises from mutations in several genes comprising the resistance determining regions, including rpoB, katG and gyrA. Objective. To detect and characterize mutations in rpoB, katG and gyrA. Methods. Thirty selected Mycobacterium tuberculosis isolates from the IDS-PGH were subjected to PCR amplification and sequencing. Sequences were compared to the wild type strain H37Rv. Results. Mutations were detected in codons 512, 513, 516, 522, 526, 531 and 533 of rpoB, codons 280, 281, 315 and 333 of katG, and codons 90 and 94 of gyrA sequences. The most frequently mutating codons for rpoB, katG and gyrA were 531, 315 and 94, respectively. A clustering analysis of the sequences showed occurrence of seven, four and three clusters for the genes rpoB, katG and gyrA, respectively. The eight clusters obtained from the concatenated sequences of the three genes represent the eight potential genotypes of local strains. One cluster represents the wild type strain genotype, another cluster represents the XDR strain genotype, and six clusters represent the MDR strain genotypes. Conclusion. These findings indicate the utility of multiple RDR sequence analysis in both identifying specific drug resistance mutation and genotyping of various M. tuberculosis isolates.
TUBERCULOSIS ; THERAPEUTICS ; THERAPY

Introduction: The World Health Organization (WHO) reported that special populations are more susceptible to the COVID-19 virus. There is little information on whether COVID-19 affects women of reproductive age and their fertility, pregnancy status, and offspring give: We aimed to assess the knowledge, practices, and perceptions (KPP) on the risk and susceptibility to COVID-19 of women of reproductive age. Methods: We conducted a qualitative exploratory descriptive study using the triangulation method. The study consisted of in-depth interviews (IDIs) with women of reproductive age and their spouses, focus group discussions (FGDs), and key informant interviews (KIIs) with health care professionals (HCP) in University of the Philippines-Philippine General Hospital. We assessed KPP using structured, open-ended interview guides. Interview responses were recorded and transcribed verbatim, and thematic analysis was performed using NVivo 12. Results: Eighty participants, 20 women of reproductive age, 20 spouses, 20 non-pregnant women, and 20 HCPs in UP-PGH were included in the study. All participants were knowledgeable about COVID-19. Pregnant women would consult their physicians when they felt symptoms related to COVID-19. Spouses of pregnant women and non-pregnant women observed health measures to prevent the spread of the virus. Both pregnant and non-pregnant patients with COVID-19 felt shamed and stigmatized while the husbands worried that the exposure to the infection will pose a burden to their families. HCWs provided services through teleconsultation and found it challenging to refer patients to other facilities. Conclusion The study provided insights on KPP of women with reproductive-age to COVID-19 and views of HCWs in providing care to these patients during the pandemic. The effects of COVID-19 are still detrimental and highly evident from the micro to the macro level.
Qualitative Research ; Knowledge ; Perception ; COVID-19

Objective: The Human Leukocyte Antigen (HLA) Class II is the major histocompatibility complex surface glycoproteins of humans responsible for presenting exogenous antigenic peptides which help direct specificity of immune response. In immune-cell therapy, the HLA allelic variants are of particular importance as they determine the successful activation of target cells that results to a desired therapeutic response. However, HLA Class II exhibits high polymorphism and has variable distribution in population, constituting these so-called allelic variants. Specifically, the HLA Class II DRB1 is considered the predominant locus among Filipinos. This research aimed to identify the presence of HLA Class II DRB1 allelic variants in the stem cell samples of ten (10) Filipino cancer patients by reverse transcription polymerase chain reaction (RT-PCR) amplification. Method: This study employed a PCR-based HLA Class II typing to identify the HLA Class II DRB1 allelic variant in Filipino cancer patients. Design of forward and reverse primers for HLA Class II DRB1, optimization of PCR conditions for amplifying HLA Class II DRB1, and identification of HLA Class II DRB1 allelic variants from samples by sequencing and database comparison were conducted. Results: PCR optimization showed that optimum annealing temperature for HLA DRB1 was 58.8°C with 1 mM MgCl2. PCR amplification of HLA DRB1 from ten anonymized cancer patient samples and DNA sequencing revealed that Patients 1, 2, 5, 8, 9, and 10 harbor HLA DRB1 allelic variants, particularly, the HLA DRB1*04:06:01, HLA DRB1*12:01:01, HLA DRB1*0813, HLA DRB1*04:05:01, HLA DRB1*09:01:02, and HLA DRB1*16:02:01, allelic variants, respectively. Conclusion Using the designed primers and optimized RT-PCR protocol, HLA information derived from six out of ten patient samples can be used for further applications in developing personalized or generic antigenic peptides such as dendritic cell cancer vaccine.
HLA Antigens

The ongoing coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is causing major damages in health and economies worldwide. The development of safe and effective vaccines for COVID-19 is of utmost importance yet none have been licensed to date. One of the strategies for vaccine development utilizes dendritic cells which express class I and class II human leukocyte antigen (HLA) molecules. These HLA molecules present the antigenic peptides to T cells which mediate the immune response. Thus, the study aimed to identify SARS-CoV-2 peptides with potential binding to HLA class I and class II molecules using different bioinformatics tools. SYFPEITHI and IEDB were used to predict epitopes for the most common HLA class I and II alleles among Filipinos. The top predicted epitopes were subjected to de novo and template-based molecular docking. Then, binding energies of the generated peptide-HLA complexes to putative T cell receptors were predicted using a homology modeling approach. Several predicted epitopes showed promising MHC and TCR binding, although results varied considerably between the prediction methods used. In particular, the results of de novo and template-based docking methods did not coincide, the latter of which generated complexes that more closely resemble typical peptide-HLA complexes. The results of this study will be validated by the next stage of the vaccine development project which is the in vitro assessment of the T cell responses elicited by dendritic cells pulsed with the candidate peptides.
COVID-19 ; SARS-CoV-2 ; Vaccines ; Molecular Docking Simulation

BACKGROUND: There is a growing interest in the use of Euphorbia hirta Linn. as herbal remedy for dengue, supposedly based on folkloric practice. However, there has been no ethnobotanical documentation of such use in the Philippines. Because of this, the medical community cautions the public against the sole use of E. hirta in treating dengue.

OBJECTIVE: To describe the ethnomedicinal uses of Euphorbia hirta Linn. In selected communities in the Philippines. Specific Objectives. (1) To identify the vernacular names of the plant; (2) to identify the earliest known use of the plant against dengue infection and for other indications; (3) to document the methods of preparation and administration, side effects, and contraindications of use.

METHODS: Cross-sectional descriptive design using the snowball sampling of interviewer-guided key informants for the ethnobotanical interview.

LIMITATIONS: The results of this study may be limited by its convenient sampling design and the use of plant pictures with different magnifications.

RESULTS AND CONCLUSION: Majority of the respondents were female (93%), 41-60 years old (39%), had high school education (43%), and resided in Quezon City (31%). The plant is locally known as tawatawa, butobutonesan, malagatas, and mangagaw. It has been used to treat fever in the Philippines as early as 1948. Its use as a treatment for dengue started only in the 1980s. The plant is either squeezed, crushed, or boiled, and is administered topically or orally. The only reported side-effect is increased urinary frequency.

RECOMMENDATIONS: It is recommended that more comprehensive and large scale studies be conducted, including (1) identification of folkloric uses of E. hirta for the treatment of other diseases; (2) determination of different concentrations of extract (crude or semicrude) using the various reported preparations for optimal outcomes for the different reported medicinal uses.

Plant ; Euphorbia ; Medicine, Traditional ; Philippines

Background and Objective: Retinoblastoma is one of the most common intraocular cancers among children usually caused by the loss of retinoblastoma protein function. Despite being a highly heritable disease, conventional diagnostic and prognostic methods depend on clinical examination, with limited consideration of cancer genetics in the standard of care. CD133, KRT19, and MUC1 are commonly explored genes for their utility in liquid biopsies of cancer including lung adenocarcinoma. To date, there are few extensive molecular studies on retinoblastoma in Filipino patients. To this end, the study aimed to describe the copy number of CD133, KRT19, and MUC1 in retinoblastoma samples from a Filipino patient and quantitate the respective expression level of these genes. Methods: Hematoxylin & Eosin (H&E) staining was utilized to characterize the retinoblastoma tissue while fluorescence in situ hybridization (FISH) using probes specific to CD133, KRT19, and MUC1 was performed to determine the copy number of genes in retinoblastoma samples from a Filipino patient (n = 1). The gene expression of CD133, MUC1, and KRT19 was quantitated using RT-qPCR. Results: The H&E staining in the retinoblastoma tissue shows poorly differentiated cells with prominent basophilic nuclei. CD133 was approximately 1.5-fold overexpressed in the retinoblastoma tissue with respect to the normal tissue, while MUC1 and KRT19 are only slightly expressed. Multiple intense signals of each probe were localized in the same nuclear areas throughout the retinoblastoma tissue, with high background noise. Conclusion These findings suggest that CD133 is a potential biomarker for the staging and diagnosis of retinoblastoma in Filipino cancer patients. However, further optimization of the hybridization procedures is recommended.
Retinoblastoma ; Biomarkers ; In Situ Hybridization

Background and Objective: Endocrine Disrupting Chemicals (EDCs) are ubiquitously found as low-level contaminants and pose serious threat to women’s health. EDCs may result in various reproductive disorders, fetal birth and developmental abnormalities, and endocrine and metabolic disorders. EDCs can be detected in body fluids of exposed individuals including blood and urine. This study aimed to detect four EDCs — Methyl Paraben (MP), 2,4-Dichlorophenoxyacetic acid (2,4-D), Monobutyl Phthalate (MBP), and Bisphenol A (BPA) in urine samples of women using Ultra-Performance Liquid Chromatography – Quadrupole Time-of-Flight (UPLC-QTOF) mass spectrometry. Methods: Sequential steps of enzymatic deconjugation, liquid-liquid extraction, solid phase extraction, and liquid chromatography separation and mass spectrometry detection were optimized in urine samples. The method was used to analyze 70 urine samples from women of reproductive age. Results: The sample preparation method showed a recovery ranging from 86.6% (MBP) to 100 % (2,4-D). The method demonstrated limits of quantitation ranging from 1.52 ng/m(MP) to 6.46 ng/mL(2,4D). Intra-day precisions expressed as relative standard deviation were all below 15% while accuracy was shown to range from 67.10% (2,4-D) to 102.39% (MBP). MP was detected in nine samples (12.86%) with a geometric mean value of 10.15 ng/ml (range: 3.62-52.39 ng/ml). MBP was detected in 68 samples (97.14%) with a geometric mean value of 97.62 ng/ml (range: 15.32-698.18 ng/ml). BPA was detected only once (9.58 ng/ml) while 2, 4-D was not detected in all samples. Conclusion A UPLC-QTOF mass spectrometry method to detect four EDCs at parts per billion level (ng/ml) was adapted and applied for analysis of urine samples. This method can find applicability in routine testing of clinical specimens as well as surveillance and other epidemiological studies.
Endocrine Disruptors

Background and Objectives: Cell lines serve as invaluable tools in studying lung cancer biology and developing new therapies to combat the disease. However, commercially available cell lines are typically of Caucasian origin and may be less representative of the local genetic background. To address this, our lab previously immortalized cells from pleural fluid of a Filipino non-small cell lung cancer (NSCLC) patient via CDK4 transduction. Copy number variations (CNVs) are a type of genetic variation which may affect physiology and disease by disrupting gene function or altering gene expression, and in cancer, these may be associated with patient outcomes. CNV profiling can be valuable for understanding the biology of our immortalized cells and identifying genes that could serve as potential targets for diagnostic, prognostic, and therapeutic interventions. This study aimed to characterize previously immortalized NSCLC-derived cells, GL01, in comparison with an established lung adenocarcinoma (LUAD) cell line, A549, through whole-genome microarray-based copy number profiling. Methods: DNA was extracted from GL01 and A549 cells using a commercially-available silica-based DNA extraction kit. DNA extracts were quantified and normalized for microarray analysis. Whole-genome copy number profiling was done using the OncoScan CNV Plus Assay following the manufacturer’s protocols, and data was analyzed using the Chromosome Analysis Suite software. Functional analysis of genes identified to be involved in copy number aberrations was done using the PANTHER Classification System. Results: Copy number aberrations span 1,592,737,105 bp in GL01 and 1,715,708,552 bp in A549, with a high degree of concordance between the two. Largescale and focal copy number aberrations previously identified to be recurrent in various LUAD cohorts were present in both GL01 and A549. Focal copy number aberrations associated with previously described lung cancer-related genes involve the PDE4D gene in GL01 and the SKIL and CDKN2A/CDKN2B genes in both GL01 and A549. PANTHER Pathway analysis of genes positively correlated with mRNA expression showed that the ubiquitin proteasome pathway was significantly overrepresented in both GL01 (FDR p = 0.000074) and A549 (FDR p = 0.000075), with 20 genes involved. Additionally, the KRAS:p.G12C/S:c.34G>T/A somatic mutation variant was detected in both GL01 and A549. Conclusion This study provides a method for identifying potentially clinically-relevant genes associated with a sample’s copy number aberrations and the pathways they represent, providing personalized mechanistic, prognostic, and therapeutic insights into the cancer biology of our cells.
carcinoma, non-small cell lung ; adenocarcinoma of lung

OBJECTIVES

To validate a method in detecting SARS-CoV-2 via RT-qPCR in pregnant and non-pregnant samples other than nasopharyngeal swabs and/or oropharyngeal swabs such as cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk.

METHODS

We performed a validation experiment using MGI easy extraction kits and BGI PCR kits on non-conventional specimens, including cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk to detect and confirm the presence of SARS-CoV-2. In addition, we tested the validated method on 572 purposively sampled field-collected non-conventional specimens from a cohort of 109 unvaccinated pregnant and 47 unvaccinated non-pregnant women to assess which candidate non-conventional maternal- and fetal-associated specimens may contribute to maternal-fetal viral vertical transmission.

RESULTS

Positive detection of SARS-CoV-2 viral RNA in non-conventional specimens was demonstrated and verified. Of the 572 non-conventional samples tested, 1.8% (10/572) were positively validated by RT-qPCR for SARS-CoV-2 in the maternal-associated specimens particularly the rectal (5), placental (1), and cervical (4) swabs among six pregnant and four non-pregnant individuals. In contrast, no SARS-CoV-2 viral RNA was detected in fetal-associated specimens.

CONCLUSION

The results of the validation study may serve as an additional diagnostic screening layer to support maternal-child care. Furthermore, viral detection in these non-conventional maternal specimens may also be utilized to provide guidance in the clinical management of neonates, and pregnant women during delivery.

Philippines ; Sars-cov-2 ; Pregnant Women ; Umbilical Cord ; Amniotic Fluid ; Polymerase Chain Reaction ; Placenta