Efficacy Assessment of Autodissemination Using Pyriproxyfen-Treated Ovitraps in the Reduction of Dengue Incidence in Parañaque City, Philippines: A Spatial Analysis.

Dengue is one of the most important vector-borne diseases worldwide and is a significant public health problem in the tropics. Mosquito control continues to be the primary approach to reducing the disease burden and spread of dengue virus (DENV). Aside from the traditional larviciding and adulticiding interventions, autodissemination using pyriproxyfen-treated (AD-PPF) ovitraps is one of the promising methods to complement existing vector control strategies. Our paper assessed the efficacy of AD-PPF in reducing DENV infections in two barangays in Parañaque City. Using saliva samples from the participants from both the control and intervention sites, we collected the seroprevalence data for three months in each of the two years. Spatial analysis was conducted to determine hotspot areas and identify DENV infection distributions across the trial periods. The results showed that the intervention site was identified as having a clustering of DENV infections in Month 0 of Year 1 and shifted to a random dispersion of dengue cases at the end of Month 3 in Year 2. The disappearance of the clustering of the intervention site translates to a decrease in the cases of DENV infection relative to the control site. Furthermore, we also identified that DENV transmission occurred at a small-scale level that did not go beyond 86 m. In conclusion, AD-PPF is suggested to be an effective strategy and may be used as an additional vector control approach, albeit based on this short-term implementation.

Detection of Acanthamoeba spp. in groundwater sources in a rural area in the Philippines.

Research on free-living amoebae (FLA) and its public health implication as an etiologic agent of parasitic infection in humans has recently gained traction in the Philippines. This study aimed to identify potential FLAs in collected groundwater samples from Masinloc, Zambales, Philippines. Fifty-four (54) water samples were collected in 250-mL sterile polyethylene containers by purposive sampling from selected groundwater sources in six (6) barangays of Masinloc. The samples were vacuum filtered through a 1.2-µm pore glass microfiber filter, cultured onto non-nutrient agar (NNA) lawned with Escherichia coli, and observed microscopically for amoebic growth for 14 days using light microscopy. Amoebic growth was observed in 11.1% (6/54) of water samples. DNAs from positive samples were extracted and were made to react with polymerase chain reaction using Acanthamoeba-specific JDP1 (5'-GGCCCAGATCGTTTACCGTGAA-3') and JDP2 (5'-TCTCACAAGCTGCTAGGGAGTCA-3') primers, and universal primer Euk A (5'-AACCTGGTTGATCCTGCCAGT-3') and Euk B (5'-TGATCCTTCTGCAGGTTCACCTAC-3'). The presence of Acanthamoeba genotypes T4, T7, and T11 was confirmed using molecular and phylogenetic analysis. Our results confirmed that groundwater sources from two of six sampling sites (33.3%) in Masinloc, Zambales, were contaminated with potentially pathogenic FLAs. Proper identification of risk factors that may cause contamination consequently leads to the implementation of programs that will prevent future infections.

Assessing Entomological and Epidemiological Efficacy of Pyriproxyfen-Treated Ovitraps in the Reduction of Aedes Species: A Quasi-Experiment on Dengue Infection Using Saliva Samples.

Our study assessed the impact of using ovitraps with pyriproxyfen on mosquito populations and the feasibility of using human saliva samples to test for seroconversion to dengue virus (DENV). We used a quasi-experimental research design by forming the intervention (n = 220) and the control (n = 223) groups in neighboring Taguig City, Philippines, over 4 months. Socio-demographic data, entomological indices, and IgG antibodies against DENV were measured. Associations between the implementation of ovitraps dosed with pyriproxyfen and mosquito densities (percentage positive ovitraps and container indices) and DENV seroconversion were calculated post-intervention in Months 2, 3, and 4. Among the participants recruited at baseline, 17 and 13 were seropositive for dengue (DENV) in the intervention and control groups, respectively. Both entomological indices were lower in the treated area than the control site at post-intervention Months 2, 3, and 4, but not earlier. Dengue seroconversions rates decreased in the treated population, but not significantly so. In conclusion, the use of PPF-treated ovitraps may have impacted the mosquito population, but not seroconversion rates. Compliance in providing saliva samples and the ability to detect IgG antibodies within these samples was encouraging and suggests that further studies on larger populations for longer durations are warranted.

"We Tried to Borrow Money, but No One Helped." Assessing the Three-Delay Model Factors Affecting the Healthcare Service Delivery among Dengue Patients during COVID-19 Surge in a Public Tertiary Hospital: A Convergent Parallel Mixed Methods Study.

Identification of delay barriers to care is essential for an effective and efficient healthcare service delivery. In this study, we described the delay in care among parents of the patients seeking treatment for dengue. We also examined the factors affecting the severity of dengue (dengue with warning signs; severe dengue). A convergent parallel design mixed-method approach using Key Informant Interviews (KII) and a survey guided by the Three-Delay Model were conducted among 24 respondents at the National Children's Hospital (NCH). Coding and thematic analysis using NVIVO and bivariable generalized linear models with a Poisson distribution and robust variance were utilized to analyze the KII transcripts and survey data, respectively. Results showed that financial constraints and previous dengue infection (first delay), mode of transportation, traffic density, and location (second delay), and hospital capacity (third delay) influenced the overall delay uncertainty in seeking care treatment for dengue infection. Furthermore, our bivariable analysis showed that travel time to NCH and place of residency, service given from previous health facilities, and parents' educational background were associated and played a role in the severity of dengue infection. Interventions focused on the identified factors contributing to delayed care should be made to avoid unwanted clinical outcomes.

Antibacterial and COX-2 Inhibitory Tetrahydrobisbenzylisoquinoline Alkaloids from the Philippine Medicinal Plant Phaeanthus ophthalmicus.

Phaeanthus ophthalmicus (Roxb. ex G.Don) J.Sinclair (previously known as P. ebracteolatus (Presl) Merr) is a Philippine medicinal plant occurring as evergreen shrub in the lowland forests of Luzon islands. It is used traditionally by Filipinos to treat bacterial conjunctivitis, ulcer and wound infections. Based on previous investigations where cyclooxygenase-2 (COX-2) functions as immune-linked factor in infectious sensitivities to bacterial pathogens by triggering pro-inflammatory immune-associated reactions, we investigated the antimicrobial and COX inhibitory activities of the extracts and tetrahydrobisbenzylisoquinoline alkaloids of P. ophthalmicus in vitro and in silico to validate its ethnomedicinal uses. Thus, the dichloromethane-methanol (DCM-MeOH) crude extract and alkaloid extracts exhibiting antibacterial activities against drug-resistant bacterial strains such as methicillin-resistance Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Klebsiella pneumoniae + CRE and Pseudomonas aeruginosa + MBL afforded (+)-tetrandrine (1) and (+)-limacusine (2) as the major biologically active tetrahydrobisbenzylisoquinoline alkaloidal constituents after purification. Both tetrahydrobisbenzylisoquinoline alkaloids 1 and 2 showed broad spectrum antibacterial activity with strongest inhibition against the Gram-negative bacteria MßL-Pseudomonas aeruginosa Klebsiella pneumoniae + CRE. Interestingly, the alkaloid limacusine (2) showed selective inhibition against ovine COX-2 in vitro. These results were ascertained by molecular docking and molecular dynamics simulation experiments where alkaloid 2 showed strong affinity in the catalytic sites of Gram-negative bacterial enzymes P. aeruginosa elastase and K. pneumoniae KPC-2 carbapenemase (enzymes involved in infectivity mechanisms), and of ovine COX-2. Overall, our study provides credence on the ethnomedicinal use of the Philippine medicinal plant P. ophthalmicus as traditional plant-based adjuvant to treat bacterial conjunctivitis and other related infections. The antibacterial activities and selective COX-2 inhibition observed for limacusine (2) point to its role as the biologically active constituent of P. ophthalmicus. A limited number of drugs with COX-2 inhibitory properties like celecoxib also confer antibacterial activity. Thus, tetrahydrobisbenzyl alkaloids, especially 2, are promising pharmaceutical inspirations for developing treatments of bacterial/inflammation-related infections.

Neuroprotective effects of vomifoliol, isolated from Tarenna obtusifolia Merr. (Rubiaceae), against amyloid-beta 1-42-treated neuroblastoma SH-SY5Y cells.

Phytochemical investigation on the leaves of Tarenna obtusifolia Merr. (Rubiaceae) led to the isolation and identification of vomifoliol (1), p-coumaric acid (2), and stigmasterol (3) based on spectroscopic analyses and comparison with the literature data. Compound 1 moderately inhibited the aggregation of amyloid-beta (Aß1-42) using the ThT assay (55.71% at 50 µM) and exhibited neuroprotective effects against amyloid-beta (Aß1-42)-induced cytotoxicity in neuroblastoma SH-SY5Y cells at 20 µM concentration. This is the first phytochemical study on T. obtusifolia and the first report on the Aß aggregation activity and neuroprotective potential of vomifoliol (1).

Quantitative ethnopharmacological documentation and molecular confirmation of medicinal plants used by the Manobo tribe of Agusan del Sur, Philippines.

BACKGROUND: The Philippines is renowned as one of the species-rich countries and culturally megadiverse in ethnicity around the globe. However, ethnopharmacological studies in the Philippines are still limited especially in the most numerous ethnic tribal populations in the southern part of the archipelago. This present study aims to document the traditional practices, medicinal plant use, and knowledge; to determine the relative importance, consensus, and the extent of all medicinal plants used; and to integrate molecular confirmation of uncertain species used by the Agusan Manobo in Mindanao, Philippines. METHODS: Quantitative ethnopharmacological data were obtained using semi-structured interviews, group discussions, field observations, and guided field walks with a total of 335 key informants comprising of tribal chieftains, traditional healers, community elders, and Manobo members of the community with their medicinal plant knowledge. The use-report (UR), use categories (UC), use value (UV), cultural importance value (CIV), and use diversity (UD) were quantified and correlated. Other indices using fidelity level (FL), informant consensus factors (ICF), and Jaccard's similarity index (JI) were also calculated. The key informants' medicinal plant use knowledge and practices were statistically analyzed using descriptive and inferential statistics. RESULTS: This study enumerated the ethnopharmacological use of 122 medicinal plant species, distributed among 108 genera and belonging to 51 families classified in 16 use categories. Integrative molecular approach confirmed 24 species with confusing species identity using multiple universal markers (ITS, matK, psbA-trnH, and trnL-F). There was strong agreement among the key informants regarding ethnopharmacological uses of plants, with ICF values ranging from 0.97 to 0.99, with the highest number of species (88) being used for the treatment of abnormal signs and symptoms (ASS). Seven species were reported with maximum fidelity level (100%) in seven use categories. The correlations of the five variables (UR, UC, UV, CIV, and UD) were significant (rs ≥ 0.69, p < 0.001), some being stronger than others. The degree of similarity of the three studied localities had JI ranged from 0.38 to 0.42, indicating species likeness among the tribal communities. Statistically, the medicinal plant knowledge among respondents was significantly different (p < 0.001) when grouped according to education, gender, social position, occupation, civil status, and age but not (p = 0.379) when grouped according to location. This study recorded the first quantitative ethnopharmacological documentation coupled with molecular confirmation of medicinal plants in Mindanao, Philippines, of which one medicinal plant species has never been studied pharmacologically to date. CONCLUSION: Documenting such traditional knowledge of medicinal plants and practices is highly essential for future management and conservation strategies of these plant genetic resources. This ethnopharmacological study will serve as a future reference not only for more systematic ethnopharmacological documentation but also for further pharmacological studies and drug discovery to improve public healthcare worldwide.

Neuroblastoma SH-SY5Y cytotoxicity, anti-amyloidogenic activity and cyclooxygenase inhibition of Lasianthus trichophlebus (Rubiaceae).

The anti-amyloidogenic potential and cyclooxygenase anti-inflammatory activity of Lasianthus trichophlebus extracts were evaluated. The MeOH extract (LTM) and chloroform extract (LTC) exhibited significant cytotoxic inhibition against the neuroblastoma SH-SY5Y cell with an IC50 of 17.52 µg/mL and 12.28 µg/mL, respectively. Thioflavin T assay indicated the LTC extract inhibition (70.56% at 50 µg/mL) to be statistically comparable (p < 0.05) to the positive control. Cyclooxygenase inhibition against COX-1 and COX-2 enzymes gave IC50 values for the LTM extract to be 18.20 and 29.60 µg/mL, respectively; while, the LTC extract showed 4.11 and 2.78 µg/mL, respectively. LC-MS of the LTM extract identified 22 putative compounds, which may prove to be pharmacologically relevant. This study has provided potential insights into the utilization of L. trichophlebus to develop safer plant-based agents for anti-inflammatory or neurodegenerative diseases.

Anti-Amyloidogenic and Cyclooxygenase Inhibitory Activity of Guettarda speciosa.

Guettarda speciosa is known in traditional folk medicine for treating cough, cold, sore throat, fever, wounds, epilepsy, and headaches. To discover the scientific pharmacological potential of G. speciosa, we explore its anti-inflammatory, cytotoxicity, and inhibition of amyloid-beta (Aß) aggregation effects. Cyclooxygenase assay of the G. speciosa CHCl3 (GSC) extract and G. speciosa MeOH (GSM) extract are more selective to COX-1 inhibition with a 50% inhibitory concentration (IC50) of 3.56 µg/mL for the GSC extract and 4.98 µg/mL for the GSM extract. Neuroblastoma SH-SY5Y inhibition and thioflavin T assay amyloid-beta (Aß) aggregate inhibition of the GSM and GSC extracts showed their potential therapeutic effects against Alzheimer's disease. The putative compounds from the LC-MS analysis could be responsible for the observed activities. The results suggest that G. speciosa possesses anti-inflammatory and anti-neurodegenerative properties and a promising lead as a source of pharmacologically active compounds.

Standard reference material (SRM) DNA barcode library approach for authenticating Antidesma bunius (L.) Spreng. (bignay) derived herbal medicinal products.

In the Philippines, the herbal medicinal product (HMP) market is flourishing due to the abundance of pharmacologically important species, and the high level of ethnomedicinal knowledge still widely accepted by the public. As such, herbal products from Antidesma bunius (L.) Spreng., locally known as bignay, are popular as medicine for various ailments of the circulatory and digestive systems. Though efficacy is guaranteed, the authenticity of the marketed products is still in question as several other herbal plants can provide the said benefits. Similar morphology between wild species also hinders species identification and contributes confusion especially to the general consumer. The authenticity of the marketed HMPs was established by means of DNA barcoding techniques which offers quick and reliable species identification by means of (1) the Basic Local Alignment Search Tool (BLASTn) and (2) the establishment of the first Standard Reference Material (SRM) Herbal barcode library for Antidesma spp. A total of 56 gene accessions from matK-psbA-trnH-rbcL sequences of 9 wild Antidesma spp. comprised the SRM which was then used to confirm the identity of 11 randomly sampled bignay-derived HMPs. Following the BLASTn and the SRM (maximum likelihood tree reconstruction) criterion, the subjected sequences revealed that only three of the 11 HMPs were authentic A. bunius-derived products. The other eight HMPs contained substitutes that were either fillers or different herbal medicinal plant not indicated in the product labels. These results indicate that product safety should be reinforced with complete HMP authentication using traditional methods supported by molecular data.

Evaluating the Feasibility of Five Candidate DNA Barcoding Loci for Philippine Lasianthus Jack (Lasiantheae: Rubiaceae).

INTRODUCTION: The pantropical genus Lasianthus Jack is identified for high phenotypic plasticity making traditional taxonomic identification difficult. Having some members with important medicinal properties, a precise complimentary identification through DNA barcoding is needed for species delineation. MATERIALS AND METHODS: In this study, 12 samples representing six Philippine Lasianthus species were used to determine the most efficient barcoding loci among the cpDNA markers (matK, rbcL, rps16, and trnT-F) and nrDNA (ITS) based on the criteria of universality, discriminatory power, and resolution of species. RESULTS: The results revealed that ITS has the recommended primer universality, greatest interspecific divergences, and average resolution of species. Among the cpDNA markers, matK and rbcL are recommended but with minimal resolution of species. While trnT-F showed moderate interspecific variations and resolution of Lasianthus species, rps16 has the lowest interspecific divergence and resolution of species. CONCLUSION: Consequently, ITS is the potential ideal DNA barcode for Lasianthus species. SUMMARY: ITS, matK, and rps16 markers have the excellent amplification and sequence qualityITS marker has the highest interspecific divergence with the maximum values, followed by matK, rbcL, trnT-F, and rps16, respectivelyAll markers except rps16 yielded average resolution to Lasianthus speciesITS marker is the most ideal locus in terms of excellent universality, high interspecific discriminatory ability, and average species resolution. Abbreviations used: ITS: Internal Transcribe Spacer, matK: maturase K, rbcL: ribulose-1,5-biphospahte-carboxylase, rps16: ribosomal protein 16 small subunit gene.

Antitubercular and Cytotoxic Chlorinated seco-Cyclohexenes from Uvaria alba.

Two new chlorine-containing polyoxygenated seco-cyclohexenes, albanols A (1) and B (2), along with the oxepinone metabolite grandiuvarone (3) were isolated from the endemic Philippine Annonaceae plant Uvaria alba. Both new compounds exhibited modest antitubercular activity. Compound 1 showed cytostatic activity (ranging from 1-50 µM) against HeLa cells and weak antiproliferative activity against HUVEC and K-562 cells with GI50 values of 106 and 81 µM, respectively.

Ixora (Rubiaceae) on the Philippines - crossroad or cradle?

BACKGROUND: The Philippine archipelago is globally one of the most important model island systems for studying evolutionary processes. However, most plant species on this archipelago have not yet been studied in sufficient detail. The main aim of this study is to unravel the evolutionary history and biogeographic relationships of the Philippine members of the pantropical genus Ixora. RESULTS: The complex plastid and nuclear divergence patterns in Philippine Ixora, documented using tree and network approaches, reveal a highly dynamic evolution in Ixora, involving several phases of radiation and recolonization. Philippine Ixora comprises at least five lineages, of which one is most closely related to species from Wallacea, and the remaining four to species from Asia. CONCLUSIONS: Our study highlights the importance of Philippine species for understanding phytogeographic patterns in the Indomalayan-Australasian eco-region. The overall genetic differentiation, as well as the incongruence between genealogies based on the biparentally inherited nucleome and the maternally inherited plastome in Ixora, reflect the complex tectonic history of the Philippine archipelago. The Ixora lineage related to Wallacean species supports the delimitation of different ecozones along Huxley's line, because it is absent from Palawan. The remaining four lineages are all allied with Asian taxa, reflecting several waves of colonization. Close relationships between some widespread Philippine species and locally adapted narrow endemics suggest that the widespread, genetically diverse species act as pools for the formation of new species in a process of ongoing speciation. Our results suggest that the species concepts of some of the more widespread taxa need to be revised.

Establishment of a standard reference material (SRM) herbal DNA barcode library of Vitex negundo L. (lagundi) for quality control measures.

The majority of the population in the Philippines relies on herbal products as their primary source for their healthcare needs. After the recognition of Vitex negundo L. (lagundi) as an important and effective alternative medicine for cough, sore throat, asthma and fever by the Philippine Department of Health (DOH), there was an increase in the production of lagundi-based herbal products in the form of teas, capsules and syrups. The efficiency of these products is greatly reliant on the use of authentic plant material, and to this day no standard protocol has been established to authenticate plant materials. DNA barcoding offers a quick and reliable species authentication tool, but its application to plant material has been less successful due to (1) lack of a standard DNA barcoding loci in plants and (2) poor DNA yield from powderised plant products. This study reports the successful application of DNA barcoding in the authentication of five V. negundo herbal products sold in the Philippines. Also, the first standard reference material (SRM) herbal library for the recognition of authentic V. negundo samples was established using 42 gene accessions of ITS, psbA-trnH and matK barcoding loci. Authentication of the herbal products utilised the SRM following the BLASTn and maximum-likelihood (ML) tree construction criterion. Barcode sequences were retrieved for ITS and psbA-trnH of all products tested and the results of the study revealed that only one out of five herbal products satisfied both BLASTn and ML criterion and was considered to contain authentic V. negundo. The results prompt the urgent need to utilise DNA barcoding in authenticating herbal products available in the Philippine market. Authentication of these products will secure consumer health by preventing the negative effects of adulteration, substitution and contamination.

Tetrahydroxanthene-1,3(2H)-dione derivatives from Uvaria valderramensis.

Two tetrahydroxanthene-1,3(2H)-dione metabolites, valderramenols A (1) and B (2), were isolated from the Philippine endemic Annonaceous species Uvaria valderramensis. Planar structures of the rac-xanthene-1,3-(2H)-diones 1 and 2 were established by MS and NMR measurements. Their enantiomers were separated by chiral HPLC, and the absolute configurations of the separated enantiomers were determined by comparison of the HPLC-ECD spectra with computed TDDFT-generated spectra. A TDDFT-ECD study of the known grandiuvarone (3) allowed the revision of its absolute configuration as S. Compound 1 showed antitubercular activity (MIC 10 µg/mL), while 3 and 4 had weaker activities (MIC 32 µg/mL). Oxepinone 3 exhibited cytotoxic activity against KB-562, a chronic myeloid leukemia cell line.

Generic affiliations of Canthium species placed under Pyrostria group B sensu Bridson (Vanguerieae, Rubiaceae) inferred from morphology and molecular data.

BACKGROUND: Pyrostria sensu lato (s.l.) is regarded as one of the polyphyletic group within Vanguerieae formerly comprising of Pyrostria sensu stricto (s.s.), Pyrostria group A and Pyrostria group B delineated by the number of locules and geographical occurrence. Recent molecular phylogenetic studies within the genus have narrowed its circumscription that resulted in the merging of Pyrostria group A and Pyrostria s.s. Although some species of Pyrostria group B were already transferred to Pyrostria s.s. and Psydrax based on morphology, other representatives of the group remain unsettled. RESULTS: Bayesian and parsimony analysis of the combined ITS (nrDNA) and trnL-F (cpDNA) datasets showed a well-supported clade of the whole Vanguerieae containing four Philippine endemic representatives of Pyrostria group B. The placement of Canthium oligophlebium, Canthium obovatifolium and Canthium ramosii within Pyrostria s.s. (PP = 0.99; BS = 85%) is robustly supported likewise the affiliation of C. gynochthodes with Psydrax (PP = 0.94; BS = 85%). Morphological features shared by our species with Pyrostria s.s. and Psydrax further supports our molecular data. CONCLUSION: Our study supports the earlier hypothesis that Canthium oligophlebium, C. obovatifolium and C. ramosii should be placed under Pyrostria s.s. except for C. gynochthodes that grouped with Psydrax. Four new combinations are proposed in this study. The generic affiliations of other species of Pyrostria group B should be reinvestigated towards a more natural classfication in Vanguerieae.

Villarinol, a new alkenoyloxyalkenol derivative from the endemic Philippine Rubiaceae species Villaria odorata.

Villarinol (1), a new alkenoyloxy alkenol metabolite, has been isolated from the dichloromethane extract of Villaria odorata, an endemic Rubiaceae Philippine plant, along with the known compounds stigmasterol and 4-hydroxybenzaldehyde. The structure of 1 was elucidated on the basis of spectroscopic and spectrometric studies. The extracts of V. odorata exhibited moderate inhibition against Mycobacterium tuberculosis H37Rv, based on the colorimetric microplate alamar blue assay.