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 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 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.

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.

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.

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.

BACKGROUND AND OBJECTIVE

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of COVID-19 has significantly challenged the public health landscape in late 2019. After almost 3 years of the first ever SARS-CoV-2 case, the World Health Organization (WHO) declared the end of this global health emergency in May 2023. Although, despite the subsequent drop of COVID-19 cases, the SARS-CoV-2 infection still exhibited multiple waves of infection, primarily attributed to the appearance of new variants. Five of these variants have been classified as Variants of Concern (VOC): Alpha, Beta, Gamma, Delta, and the most recent, Omicron. Therefore, the development of methods for the timely and accurate detection of viral variants remains fundamental, ensuring an ongoing and effective response to the disease. This study aims to evaluate the feasibility of the application of an in-house approach in genomic surveillance for the detection of SARS-CoV-2 variants using in silico designed primers.

The primers used for the study were particularly designed based on conserved regions of certain genes in the virus, targeting distinct mutations found in known variants of SARS-CoV-2. Viral RNA extracts from nasopharyngeal samples (n=14) were subjected to quantitative and qualitative tests (Nanodrop and AGE). Selected samples were then analyzed by RT-PCR and amplicons were submitted for sequencing. Sequence alignment analysis was carried out to identify the prevailing COVID-19 variant present in the sample population.

The study findings demonstrated that the in-house method was able to successfully amplify conserved sequences (spike, envelope, membrane, ORF1ab) and enabled identification of the circulating SARS-CoV-2 variant among the samples. Majority of the samples were identified as Omicron variant. Three out of four designed primers effectively bound into the conserved sequence of target genes present in the sample, revealing the specific SARSCoV-2 variant. The detected mutations characterized for Omicron found in the identified lineages included K417N, S477N, and P681H which were also identified as mutations of interest. Furthermore, identification of the B.1.448 lineage which was not classified in any known variant also provided the potential of the developed in-house method in detecting unknown variants of COVID-19.

Among the five VOCs, Omicron is the most prevalent and dominant variant. The in-house direct PCR product sequencing surveillance (DPPSS) method provided an alternative platform for SAR-CoV-2 variant analysis which is accessible and affordable than the conventional diagnostic surveillance methods and the whole genome sequencing. Further evaluation and improvements on the oligonucleotide primers may offer significant contribution to the development of a specific and direct PCRbased detection of new emerging COVID-19 variants.

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

Context: Exposure to environmental pollutants (EP) and Endocrine-disrupting chemicals (EDCs) is associated with several general negative health effects which compromise women's reproductive health, maternal, and neonatal outcomes. Unfortunately, many nonpregnant and pregnant women are unaware of their active exposure to these potentially slow-acting toxic substances, EPs, and EDCs. At any stage of life, and in the long-term minute exposures, there is no established safe level of exposure to these substances. Due to the potentially harmful effects on women in general, and to the pregnant and her unborn child in particular, it is important to establish the prevalence of their exposure. Aims: The aim of this study was to determine the magnitude (prevalence) of exposure among nonpregnant and pregnant women aged 18–49 years to common EPs/EDCs such as bisphenol A, pesticides, phthalates, and perfluorinated compounds among others. Settings and Design : This study was conducted at the Philippine General Hospital, University of the Philippines Manila. Subjects and Methods : The study participants were nonpregnant and pregnant women, with low-risk singleton pregnancy, and had a prenatal checkup and eventual delivery at the Philippine General Hospital. After consent, women were asked to answer a survey focused on their sociodemographics and frequency of exposure to EP- and EDC-containing items. Satatistical Analysis Used: Descriptive statistics were used to summarize the demographic and clinical characteristics of the patients. Null hypotheses were rejected at 0.05 α-level of significance. The computer software STATA 13.1 was used for data analysis. Results: One hundred and fifty-nine survey responses by women were analyzed. Possible EDC-containing household items (carpets, linoleum, upholstered, and stain-resistant furniture) are more prevalent in the homes of pregnant women compared to nonpregnant women. Pregnant women are also exposed to wallpapers compared to nonpregnant individuals. Nonpregnant women were 1.5 times more exposed to lotions. Unexposure to hand sanitizers is 1.3 times higher among pregnant individuals. Conclusions There are differences in the prevalence of exposure to household EDC-containing items between pregnant and nonpregnant women, with pregnant women having a higher prevalence of exposure.
Endocrine-disrupting chemicals ; environmental pollutants ; pregnancy