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Pink pigmented facultative methylotrophic (PPFM) bacteria are ecologically distributed microorganisms. They have been isolated in many types of ecosystems like soil, water, air, in association with plants and even as pathogens in humans. However, a yet unexplored area for PPFM bacteria research is in food. Hence, the objective of this study was to establish the presence of PPFM bacteria in Philippine fermented food, in particular shrimp paste, and characterise them phenotypically and genotypically. A total of 13 PPFM bacteria were obtained from Philippine shrimp paste. Sequencing of the 16S rRNA gene revealed that the PPFM bacterial isolates belong to the genus Methylobacterium. A total of 35 phenotypic characterisations were performed that included morphological, biochemical and physiological tests. Phylogenetic tree was constructed to establish the genetic relatedness of the isolates. Morphological test results showed that all 13 isolates were consistent with the established phenotypic characters of the genus such as pink colony colour, Gram negative and rod-shaped. Biochemically, the use of API® 20 NE tests showed heterogeneity of results and physiological tests exhibited that the isolates are primarily mesophilic and halotolerant, being able to grow at 2% salt. Phylogenetic analysis showed that the isolates are Methylobacterium populi, M. dankookense, M. lusitanum, M. radiotolerans and M. zatmanii. This study confirmed the presence and diversity of PPFM bacteria in Philippine shrimp paste. Further studies are needed to show the functional activity of Methylobacterium in Philippine shrimp paste production.

Fungi are ecologically ubiquitous organisms on earth and regarded as one of the prolific sources of natural products. Fungal endophytes may provide essential prerequisite molecules to plant biochem...

Fecal pollution is a major contributor to the progressive degradation of Laguna Lake, the Philippines’ largest inland lake used for aquaculture, recreation, and as a source of irrigation and domestic water. Consequently, intestinal parasites may be present in this body of water, posing risks to water safety and human health. This study was able to detect the protozoan parasite, Giardia duodenalis, in three Laguna Lake stations and seven tributary rivers in a 13-month monitoring period by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene of Giardia cysts concentrated from water samples. The pathogen was present in 37.7% of tributary samples (n = 69) and 16.7% of lake samples (n = 36). Notable frequencies of detection were observed in four tributary rivers —Bagumbayan, Taguig (66.7%); Santa Rosa, Laguna (55.6%); San Cristobal, Cabuyao, Laguna (44.4%); and Binan, Laguna (42.9%). All test SSU rRNA gene sequences were identified as human-infective genotypes of G. duodenalis predominated by Assemblage A (94.1%). Furthermore, analysis of the glutamate dehydrogenase (gdh) gene revealed the possible presence of mixed genotypes in at least two samples. These results support the pressing need to include protozoan pathogen monitoring in Laguna Lake and its tributaries to prevent Giardia infection in humans and animals. This study also recommends microbial source tracking to identify fecal pollution sources and aid in regulation of waste discharges into the lake and its tributaries.

Leptospira is the causative agent of leptospirosis, which is considered an emerging major threat to public health due to its increasing frequency reported worldwide. In the Philippines, the prevalence of the disease continuously rises, particularly in urban areas. Because leptospirosis is commonly transmitted through contact with contaminated environment, water and soil samples were collected in regions in the Philippines where high incidence of human leptospirosis cases was reported recently. Of the 54 samples screened for the presence of Leptospira, 35% were found positive through 23S rRNA gene PCR-based detection. None were found positive when primers targeting lipL32, lipL41, and ompL1 genes were used. Most of these isolates were collected from rural areas. However, 16S rRNA gene sequencing identified all isolates to be L. yanagawae and L. meyeri, which are nonpathogenic. Despite the lack of evidence of the presence of pathogenic species in the environmental sources, the results still suggest that leptospires persist in these areas. These data are crucial for environmental monitoring and identification of contaminated areas where humans may be at risk of infection.

Contaminated irrigation water may greatly affect not only the quality of produce but also the people exposed to it. In this study, agricultural irrigation waters in Bulacan, Philippines were assessed and found to be contaminated with Escherichia coli (E. coli) ranging from 0.58 to 4.51 log10 CFU/mL. A total of 79 isolates of E. coli were confirmed through polymerase chain reaction (PCR) amplifying the uidA gene and were tested for phenotypic resistance using 10 antimicrobials through the Kirby-Bauer disc diffusion method. Forty-six isolates (58.22%) were noted to be multidrug resistant (MDR) with high resistance rate to cephalothin, tetracycline, streptomycin, ampicillin, trimethoprim, nalidixic acid, and chloramphenicol. Moreover, this study also examined the prevalence of Class I and II integrons accounting to 67.39% and 17.39%, respectively, of the MDR E. coli strains using multiplex PCR. The results imply that the agricultural water used in Bulacan is contaminated with the fecal material of man or other animals present in the area, and the presence of MDR bacteria, which pose a potential threat to individuals in these areas, is alarming. In addition, detection of integrons could be a good marker for the identification of MDR isolates. Lastly, this study could develop strategies for the proper management of farming sites leading to the detection of food-borne pathogens and prevention of infectious diseases.

Background. Metronidazole susceptibility and the presence of Trichomonas vaginalis virus (TVV) are the phenotypes found to be significantly correlated with the microsatellite-based genotypes of T. vaginalis. These phenotypes were assessed in T. vaginalis isolates from select urban areas to determine preliminary “type” of Philippine T. vaginalis. Methods. Culture and microscopy were used to detect T. vaginalis in vaginal swab samples collected from women attending social hygiene clinics in Metro Manila and Angeles City, Philippines. Screening of TVV on T. vaginalis was performed using RNA gel electrophoresis and RT-PCR. A modified protocol for metronidazole susceptibility assay was used to determine the aerobic minimum lethal concentration (MLC) of metronidazole in axenized T. vaginalis isolates. Results. A total of 42 T. vaginalis were screened for the presence of TVV and assayed for metronidazole susceptibility. TVV was detected in 13 of the isolates. All but one of the samples was susceptible to metronidazole. Conclusion. This is the first study to assess the in vitro metronidazole susceptibility of Philippine T. vaginalis isolates. The isolates are generally susceptible to metronidazole even with the presence of TVV. The metronidazole susceptibility and presence of TVV are not enough to classify the isolates into type 1 or type 2.

Background The stranding events of cetaceans in the Philippines provide opportunities for gathering biological information and specimens, especially from the pelagic forms. As part of an effort to monitor the health of wild cetaceans, this study detected Leptospira spp. and Toxoplasma gondii, causative agents of the emerging zoonotic diseases leptospirosis and toxoplasmosis respectively, in their stranded representatives. From October 2016–August 2018, 40 cetaceans (representing 14 species) that stranded nationwide were sampled for brain, cardiac muscle, skeletal muscle, kidney, and blood tissues, urine, and sera. These were subjected to molecular, serological, culture, and histopathological analyses to detect the target pathogens. Results T. gondii was detected in 20 (71%) of the 28 cetaceans with biological samples subjected to either molecular detection through RE gene amplification or IgG antibodies detection through agglutination-based serological assay. On the other hand, Leptospira was detected in 18 (64%) of 28 cetaceans with biological samples subjected to bacterial culture, molecular detection through 16S rDNA amplification, or IgM antibodies detection through ELISA-based serological assay. Conclusions There is the plausibility of toxoplasmosis and leptospirosis in cetacean populations found in the Philippines, however, acute or chronic phases of infections in sampled stranded individuals cannot be confirmed in the absence of supporting pathological observations and corroborating detection tests. Further studies should look for more evidences of pathogenicity, and explore the specific mechanisms by which pelagic cetacean species become infected by Leptospira spp. and T. gondii. As there is growing evidence on the role of cetaceans as sentinels of land-sea movement of emerging pathogens and the diseases they cause, any opportunity, such as their stranding events, should be maximized to investigate the health of their populations. Moreover, the role of leptospirosis or toxoplasmosis in these stranding events must be considered.

Microbial contamination of fresh produce can present a severe risk to public health. By conducting a rigorous survey of irrigation waters, the impacts of fecal contamination on the quality of produce could be assessed. In this study, surface waters were observed to be contaminated with Escherichia coli, Salmonella spp., and somatic coliphages. Culture methods show that out of 373 irrigation water, soil, and vegetable samples collected for a 1-year period, 232 (62.20%) were found positive for E. coli, 213 (57.26%) for somatic coliphages, and 2 (0.53%) for Salmonella spp. Out of 190 water samples, 167 (87.9%) were found to have E.coli, 174 (91.6%) have somatic coliphages, and 1 (0.5%) with Salmonella spp. In soil samples, 36 of 91 (39.6%) have E. coli, 31 (34.0%) have somatic coliphages, and none with Salmonella spp. Lastly, out of 92 vegetable samples, 29 (31.5%), 8 (8.7%), and 1 (1.1%) were found to have E. coli, somatic coliphages, and Salmonella spp., respectively. Molecular analysis confirmed the presence of bacterial contaminants. Seasonal weather conditions were noted to have an effect on the presence and number of these fecal indicator organisms. The observed data suggest that contaminated irrigation water may greatly affect the quality of fresh produce from these agricultural operations.

Foodborne diseases associated with fresh produce consumption have escalated worldwide, causing microbial safety of produce of critical importance. Bacteria that have increasingly been detected in fresh produce are Escherichia coli and Salmonella spp., both of which have been shown to progressively display antimicrobial resistance. The study focused on the assessment of antimicrobial resistance of these enteric bacteria from different kinds of fresh produce from various open air markets and supermarkets in the Philippines. Using the disk diffusion assay on a total of 50 bacterial isolates obtained from 410 fresh produce surveyed, monoresistance to tetracycline was observed to be the most prevalent (38%), followed by multidrug resistance to tetracycline, chloramphenicol, ciprofloxacin, and nalidixic acid (4%), and lastly by dual resistance to tetracycline and chloramphenicol (2%). Using multiplex and simplex polymerase chain reaction (PCR) assays, tetA (75%) and tetB (9%) were found in tetracycline resistant isolates, whereas catI (67%) and catIII (33%) were detected in chloramphenicol resistant isolates. Sequence analysis of gyr and par genes from the ciprofloxacin and nalidixic acid resistant isolates revealed different mutations. Based on the results, fresh produce act as a reservoir of these antibiotic resistant bacteria which may pose health threat to consumers.

A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10-1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis.

Objective To compare the sensitivity and specificity of direct fecal smear microscopy, culture, and polymerase chain reaction in the detection of Blastocystis sp. in human stool. Methods Human stool samples were collected from a community in San Isidro, Rodriguez, Rizal, Philippines. These samples were subjected to direct fecal smear microscopy, culture and polymerase chain reaction to detect the presence of Blastocystis sp. Results Of the 110 stool samples collected, 28 (25%) were detected positive for the presence of Blastocystis sp. by two or more tests. Culture method detected the highest number of Blastocystis -positive stool samples ( n =36), followed by PCR of DNA extracted from culture ( n =26), PCR of DNA extracted from stool ( n =10), and direct fecal smear ( n =9). Compared to culture, the sensitivity of the other detection methods were 66.7% for PCR from culture and 19.4% for both PCR from stool and direct fecal smear. Specificity of the methods was high, with PCR from culture and direct fecal smear having 97.3%, while PCR from stool at 95.9%. Conclusions In this study, in vitro culture is the best method for detecting Blastocystis sp. in human stool samples.

Trichomonads are amitochondrial anaerobic flagellated protists that are either parasites or commensals, generally living in the digestive or genitourinary tract of humans and animals. It has been reported that these protozoa can migrate to other sites in their target host, can adapt to new hosts, and are capable of zoonotic transmission. In this study, 59 trichomonad isolates from different animal hosts in the Philippines were identified and characterized. Primer sets were designed and were successful in amplifying the 18S rRNA gene sequences of the isolates. Phylogenetic trees were constructed using neighbor-joining (NJ), maximum parsimony (MP), maximum-likelihood (ML) and Bayesian inference (BI) analyses. Results showed that BLAST analysis of the isolates corresponded to the clustering of the isolates together with reference sequences in the constructed ML tree. Cattle and pig isolates were most likely Tetratrichomonas buttreyi, which were observed to be commensal in both animals. All duck and rooster isolates were similar with Tetratrichomonas gallinarum. All dog isolates together with single isolates from boa, goat, and owl were identical to Pentatrichomonas hominis. Occurrence of P. hominis in Boa constrictor imperator (boa) and Otus megalotis (Philippine scops owl) suggested the adaptation of the trichomonad to new hosts. Reptile hosts were observed to harbor Trichomitus batrachorum or Hypotrichomonas acosta. Three reptile isolates (Igu2, Igu4, and Liz7) suggest novel species belonging to Class Hypotrichomonadea. Furthermore, iguanas were infected with T. batrachorum or H. acosta. Trichomonads in animal hosts are commensal and the mode of transmission is via fecal-oral route. They are capable of adaptation to new hosts and therefore, zoonotic transmission is possible as well as pathogenesis in host. Thus, trichomonads can pose threats to the health of humans and animals.

Objective To develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Entamoeba histolytica E. histolytica , the causative agent of amebiasis. Methods The LAMP primer set was designed from E. histolytica hemolysin gene HLY6. Genomic DNA of E. histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions. Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye. Results Positive LAMP reactions turned turbid while negative ones remained clear. Upon addition of a fluorescent dye, all positive reactions turned green while the negative control remained orange under ambient light. After elecrophoresis in 1.5% agarose gels, a ladder of multiple bands of different sizes can be observed in positive samples while no bands were detected in the negative control. The sensitivity of the assay was found to be 5 parasites per reaction which corresponds to approximately 15.8 ng/μ L DNA. The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species, Entamoeba dispar 39, and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used. Conclusions The LAMP assay we have developed enables the detection of E. histolytica with rapidity and ease, therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.

Trichomonas vaginalis is a pathogenic protozoon which causes the sexually transmitted infection, trichomoniasis. The absence or non-specificity of symptoms often leads to misdiagnosis of the infection. In this study, 969 samples consisting of vaginal swabs and urine were collected and screened from social hygiene clinics across the Philippines. Of the 969 samples, 216 were used for the comparative analysis of diagnostic tools such as wet mount microscopy, culture, and PCR utilizing universal trichomonad primers, TFR1/2 and species-specific primers, TVK3/7 and TV1/2. PCR demonstrated higher sensitivity of 100% compared to 77% of the wet mount. PCR primer set TVK3/7 and culture had the same and the best expected average performance [receiver-operating characteristic (ROC): 0.98]. Prevalence of infection in the sample population was 6.8%.

Balantidium coli is a ciliated protozoon inhabiting the colon of swine, rodents, horses, nonhuman primates and humans. In association with disease triggered by other infectious agents, B. coli may become a pathogenic opportunist. This study describes the isolation, cultivation, morphological as well as molecular characterization of B. coli isolated from the large intestine of a pig in the Philippines. Based on scanning and transmission electron microscopy, this protozoon presents a dense ciliation in the oral structure and somatic cilia that are arranged in a more transverse field. Oral and somatic monokinetids were identified in the cortex of the organism. The presence of heterokaryotic nuclear condition is evident, and the cell body of the ciliate shows numerous mucocysts, several food vacuoles, mitochondria, endoplasmic reticulum, and contractile vacuoles. Polymerase chain reaction and phylogenetic analysis based on the small subunit ribosomal RNA gene were performed in order to compare our isolate with other previously reported B. coli isolates. The full-length sequence of the SSU rRNA gene of the isolate showed 99% similarity to other B. coli isolates reported in the GenBank. Phylogenetic analysis revealed that the isolate clustered with previously reported B. coli isolates from gorillas, pig, and ostrich. To date, no studies on the ultrastructure and phylogeny of B. coli isolated in the Philippines have been reported. Results from this study may serve as a baseline data for further ultrastructural and phylogenetic studies on this organism. This study also suggests that morphological characteristics along with molecular identification are essential for validating and identifying species of Balantidium.

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral parasitic sexually transmitted infection in the world. Presently, there are no reports on comparative sequence analysis as well as on the identification of phylogenetic positions of T. vaginalis isolates from the Philippines relative to known trichomonads. In this study, 5.8S rDNA and the flanking internal transcribed spacer (ITS) regions of 57 T. vaginalis isolates were sequenced. The phylogenetic positions of the isolates relative to known trichomonads were determined using the model-based (GTR+Gamma+I) neighbor-joining, maximum likelihood, and Bayesian-inference analyses and the nonmodel-based maximum parsimony analysis. Construction of a phylogenetic tree showed the clustering of all the sequences in one branch together with other T. vaginalis strains obtained through basic local alignment search tool search. Sequencing of the 5.8S rDNA gene and the flanking ITS1and ITS2 regions of T. vaginalis isolates from the Philippines demonstrated low genetic polymorphism. However, comparison of the ribosomal DNA sequences may have implications on some phenotypic characteristics of T. vaginalis.

Giardia duodenalis is a flagellated protist that causes gastrointestinal disease throughout the world. In the Philippines, study on G. duodenalis is limited. It is also believed that prevalence rates of this organism in the country are underestimated. In this study, stool samples from residents living in a slum area in Manila were collected. These were examined under microscopy for identification of common helminthic and protistan parasites. Results showed that 22.05% of 2,354 stool samples collected contained Giardia cysts. A fraction of samples (n = 133) positive for Giardia cysts were set aside. Genomic DNA was extracted from these samples and a polymerase chain reaction-restriction fragment length polymorphism procedure based on the organism's triose phosphate isomerase gene was utilized. This particular procedure is capable of distinguishing assemblages or genotypes within G. duodenalis. The highest identified assemblage was Assemblage B (86.47%). The two genotypes of Assemblage A were also detected. This is the first report on the identification of genotypes of G. duodenalis in the Philippines. The results of this study can serve as basis for future control and prevention of giardiasis and parasitism in the country.

The agar-cloning technique of Blastocystis hominis has been observed in both solid and semisolid agar using Iscove's modified Dulbecco's medium. In this study, Philippine isolates of B. hominis were grown by pour-plate method in semisolid agar using Locke's solution. Inoculated plates contained 0.7% agar, 10% horse serum, and 0.1% sodium thioglycollate. Plates were incubated at 37 degrees C in a microaerophilic jar for 7-10 days. Biconvex disk-shaped colonies were seen abound at the bottom half of the medium. Colonies growing at the agar-glass interface were flat and consisted of thin layers of cells. From these colonies, large amoeboid cells were frequently seen on the periphery, whereas smaller cells were concentrated at the core. Analysis of the SSU rDNA genetically established the identity of the clones to be B. hominis. This is the first report on agar cloning of Blastocystis in a compound medium.

Background/Purpose The flagellated protozoon Trichomonas vaginalis that parasitizes the urogenital tract of humans was reported to harbor double-stranded RNA (dsRNA) viruses. These viruses, identified as Trichomonas vaginalis virus (TVV), belong to the genus Trichomonasvirus of the family Totiviridae . Four species, formally recognized by the International Committee on Taxonomy of Viruses (ICTV), have been reported and distinguished by pairwise comparisons of the sequences of genes coding for major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the complimentary DNA of target virus genes coding for CP and RdRp. Sequence analyses confirmed the identity of the TVV isolates from T. vaginalis cultures. Results A total of 35 dsRNA viruses were identified from 18 (19%) T. vaginalis isolates. Multiple TVV species were observed in six of the 18 T. vaginalis cultures. Phylogenetic analyses show monophyly in TVV1 and TVV2 whereas TVV3 and TVV4 appear paraphyletic. The phylogeny of Philippine Trichomonasvirus reflects the global distribution of its host. Conclusion This is the first study in the Philippines and one of the two reports worldwide to detect the four TVVs and their concurrent infection in a single T. vaginalis isolate.

Extensive genomic polymorphism has been demonstrated among morphologically identical Blastocystis isolates. To this end, 32 Blastocystis isolates from the Philippines (12 from humans, 12 from pigs and 8 from chickens) were analyzed genetically by riboprinting or restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified small subunit rDNA. Three distinct riboprint patterns were observed from the HinfI digestion, while four patterns resulted from the RsaI digestion of Blastocystis SSU rDNA. Restriction fragment profiles between Blastocystis isolates from different hosts were generally different from each other. However, Blastocystis isolates within each host group were practically the same. Cluster analysis of the riboprint patterns revealed seven distinct groups of the Blastocystis isolates, including a zoonotic strain. These results demonstrate the genetic heterogeneity of Blastocystis in the Philippines and a support to the idea of the organism's zoonotic potential.

Trichomonas vaginalis, the causative agent of trichomoniasis is generally known to inhabit the genitourinary tract. However, several case reports with supporting molecular and immunological identifications have documented its occurrence in the respiratory tract of neonates and adults. In addition, the reports have documented that its occurrence is associated with respiratory failures. The medical significance or consequence of this association is unclear. Thus, to establish the possible outcome from the interaction of T. vaginalis with lung cells, the cytopathic effects of the parasites were evaluated using monolayer cultures of the human lung alveolar basal carcinoma epithelial cell line A549. The possible effect of association of T. vaginalis with A549 epithelial cells was analyzed using phase-contrast, scanning electron microscopy and fluorescence microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), crystal-violet and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays were conducted for cytotoxicity testing. The results demonstrate that T. vaginalis: (1) adheres to A549 epithelial cells, suggesting a density-dependent parasite-cell association; (2) adherence on A549 is through flagella, membrane and axostyle; (3) causes cell detachment and cytotoxicity (50-72.4%) to A549 and this effect is a function of parasite density; and (4) induces apoptosis in A549 about 20% after 6 h of incubation. These observations indicate that T. vaginalis causes cytopathic effects on A549 cell. To date, this is the first report showing a possible interaction of T. vaginalis with the lung cells using A549 monolayer cultures. Further studies are recommended to completely elucidate this association.

This study is the first in the Philippines to conduct a comprehensive assessment of the prevalence of bacterial pathogens and somatic phages in retailed fresh produce used in salad preparation, namely, bell pepper, cabbage, carrot, lettuce, and tomato, using culture and molecular methods. Out of 300 samples from open air and supermarkets, 16.7% tested positive for thermotolerantEscherichia coli, 24.7% forSalmonellaspp., and 47% for somatic phages. Results show that counts range from 0.30 to 4.03 log10 CFU/g forE. coli, 0.66 to ≥2.34 log10 MPN/g forSalmonellaspp., and 1.30 to ≥3.00 log10PFU/g for somatic phages. Statistical analyses show that there was no significant difference in the microbial counts between open air and supermarkets (α=0.05). TaqMan and AccuPower Plus DualStar real-time polymerase chain reaction (RT-PCR) was used to confirm the presence of these organisms. The relatively high prevalence of microorganisms observed in produce surveyed signifies reduction in shelf-life and a potential hazard to food safety. This information may benefit farmers, consumers, merchants, and policy makers for foodborne disease detection and prevention.

The kinetics of antibody production against Blastocystis hominis, an emerging infectious protozoan parasite which causes intestinal disorder in humans and animals, was studied. Sera and intestinal secretions were collected from B. hominis-immunized Balb/C mice for 8 weeks. Flow cytometry was used to monitor the levels of immunoglobulins A (IgA), G (IgG), and M (IgM) from both types of biological samples. The kinetic profile derived from flow cytometry analysis revealed that IgM led the early immune action against B. hominis infection in immune sera while IgA was the predominant antibody isotype in intestinal secretions. Western blotting revealed an array of antigens recognized by both serum and intestinal secretion antibodies. Immunoreactive B. hominis soluble proteins with molecular weights ranging from 28.2 to 77.6 kDa were detected by serum antibodies and 15.1 to 117.5 kDa by secretory antibodies. These antigens may be cytoplasmic or membrane-bound as determined through indirect fluorescent antibody test. Moreover, two immunogens (39.8 and 77.6 kDa) were commonly recognized by serum antibodies, one (70.8 kDa) by secretory antibodies and two (55.0 and 56.2 kDa) by both serum and secretory antibodies, suggesting a possible target in the further understanding of B. hominis pathogenicity, discovery of virulence factors, and development of immunology-based diagnostic protocols and alternative modes of treatment.

This study was carried out to document the genotypes of Acanthamoeba present in contact lens cases from 50 randomly selected contact lens wearers living in Quezon City, Metro Manila, Philippines. Acanthamoeba species were isolated from eight (16%) in 50 contact lens cases examined. We analyzed partial 18S ribosomal DNA (Rns) sequences of the eight isolates and found that the sequence differences were sufficient to distinguish the genotypes. After the isolates were genotyped, using the Basic Local Alignment Search Tool program, their phylogenetic positions relative to known Acanthamoeba isolates were determined. The model-based (GTR+Gamma+Iota) neighbor-joining, maximum likelihood, and Bayesian inference analyses, as well as the non-model-based maximum parsimony analysis were used. Results showed that of the eight isolates, six were Rns genotype T5 while two were Rns genotype T4. This present study indicates that genotype T5 is also a common contaminant in contact lens storage cases.

Trichomonads found in pigs include the commensal Tritrichomonas suis (more well known because of its synonymy to Tritrichomonas foetus, a trichomonad parasite of cattle and other animals) and Tetratrichomonas buttreyi, which appear similar to Tritrichomonas suis under the light microscope. A trichomonad isolated from pig fecal samples was subjected to scanning and transmission electron microscopy for ultrastructural study. The organism’s ultrastructure revealed features commonly found in trichomonads; however, features such as the number and length of flagella, type of undulating membrane, general body form, and shape and location of organelles such as the nucleus, Golgi complex, and hydrogenosomes indicated that the isolated trichomonad is not Tritrichomonas suis nor Tritrichomonas foetus. Polymerase chain reaction (PCR) corroborated these results. Moreover, the ultrastructure was similar to the ultrastructure of previously described tetratrichomonads. It is especially suggested that the isolate is T. buttreyi. These findings could be of significance in the differentiation among different porcine trichomonads in diagnostic procedures. In addition, this is the first known detailed ultrastructural study of T. buttreyi isolated from pigs; thus, this can serve as an aid for future comparison between porcine and bovine T. buttreyi.

To study the genetic diversity and cross-transmissibility of Blastocystis species in naturally infected hosts, 12 Blastocystis isolates from animal and human hosts in the Philippines were analyzed by sequencing the full-length small subunit ribosomal RNA (SSU rRNA) genes. Each sequence showed very high similarity (from 97% to 100%) to homologous sequences of other Blastocystis isolates reported previously. Phylogenetic analysis revealed that the 12 isolates were classified into 4 genetically distinct subtypes: 1, 2, 3, and 6. Results showed that Blastocystis subtypes 1, 2, and 3 were shared by isolates from varied hosts. This study confirms the remarkable heterogeneity of SSU rRNA gene among different Blastocystis isolates. The findings of this study agree with previous reports that the different Blastocystis subtypes have low host-specificity, comprising isolates from humans and various animal hosts. This study also suggests evidence for zoonotic transmission of the parasite and cross-transmissibility among heterogeneous hosts.

Information on cryptosporidiosis in the Philippines is limited. To date, the disease is not routinely diagnosed in the country's medical institutions. To this end, a total of 53 Filipino cancer patients were surveyed for cryptosporidiosis using an indirect immunofluorescent antibody test. Fifteen patients (28.3%) were found to be positive for antibodies against Cryptosporidium. This study contributes to a better understanding of the incidence of cryptosporidiosis in the country.

A total of 113 mentally retarded patients residing in a mental institution in Metropolitan Manila, Philippines, were screened for the presence of Entamoeba histolytica based on microscopy and polymerase chain reaction (PCR). Anti-E. histolytica antibodies were also screened in 97 serum samples collected using immunofluorescence antibody (IFA) test. Parasitological examination showed E. histolytica/Entamoeba dispar in 43 cases (38.05%), while PCR detected 74 cases (65.48%) positive for E. histolytica and 6 cases (5.30%) positive for E. dispar. Interestingly, these 6 samples were coinfected with E. histolytica. IFA test revealed that 80.41% (78/97) of the respondents possessed significant antibody titers for intestinal infection of E. histolytica. Of this number, there were 5 patients negative in IFA test but positive in PCR. The genetic diversity of E. histolytica isolates was also investigated by analyzing polymorphism in the serine-rich gene by nested PCR on DNA directly extracted from stool specimens. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded six distinct DNA banding patterns among the 74 stool isolates. An apparent clustering of E. histolytica strains was observed in patients living in different residential cottages of the institution. These results indicate the high prevalence of E. histolytica in an institution for the mentally retarded in the Philippines.

Sialidase from Entamoeba histolytica was purified to apparent electrophoretic homogeneity by chromatography on hydroxyapatite, reactive brown agarose, fetuin/agarose and by fast performance liquid chromatography on a MonoQ column. The enzyme had a molecular mass of 65 kDa, as determined by SDS-PAGE. It had an optimum pH of 5.5 and was maximally active at 37 degrees C. It required Ca(2+) and Mg(2+) for activation but was strongly inhibited by Cu(2+), Fe(2+) and Zn(2+). The E. histolytica sialidase exhibited high specificity for Neu5Acalpha2,3lac and methylumbelliferyl-Neu5Ac (4-MU-Neu5Ac) with K(m) values of 0.144 mM and 0.059 mM, respectively. The enzyme was not active against colomic acid and the gangliosides GM(1) and GD(1). It was activated in the presence of lactose and galactose, but was unaffected by glucose, sucrose and mannose. The enzyme was inhibited competitively by 2,3,didehydroneuraminic acid and para-nitro-phenyloxamic acid with inhibition binding constants ( K(i)) of 30 micro M and 185 micro M, respectively. The motility of intact E. hystolytica cells was enhanced about 6-fold in the presence of 0.05-0.1 mM Neu5Acalpha2,3lac, 4-MU-Neu5Ac and fetuin. However, the motility of the parasite was highly diminished when incubated with Neu5Acalpha2en and sialic acid-containing compounds. Lysed E. histolytica trophozoites were found to lack neuraminic acid.

We used the polymerase chain reaction (PCR) to determine the prevalence of Entamoeba histolytica and E. dispar in the wild population of macaque monkeys (Macaca fuscata) in Mt. Takasaki, Oita Prefecture, Japan. Of the 101 samples collected, 41 (42.57%) were found to be positive for E. dispar. However, no E. histolytica was detected from the collected samples. The results of this survey demonstrate the high prevalence of E. dispar in macaque monkeys in the study area. Moreover, they provide additional baseline information on naturally acquired infectious agents of macaque monkeys and offer an accurate tool for detection of E. histolytica and E. dispar, which are needed for biomedical research using nonhuman primate models.

It has been established that two distinct species exist within what was originally known as Entamoeba histolytica. These are E. dispar and E. histolytica, for the nonpathogenic and pathogenic forms, respectively. Differentiation of these two organisms is of great clinical importance since they are morphologically indistinguishable and both forms can infect the human intestinal cavity to different degrees. A simple and rapid DNA-extraction method that can be used directly on formalin-fixed stool specimens has been developed. The extracted DNA was used for the identification of the species existing in the stools by polymerase chain reaction (PCR). A total of 72 randomly collected stool samples from the Philippines were analyzed. In all, 19 samples reacted with E. dispar primers, resulting in the expected 101-bp PCR products; however, none reacted with E. histolytica primers. Furthermore, sensitivity assay suggests that genomic DNA from as few as five cysts can be used as a template for PCR. These observations imply that the use of genomic DNA directly extracted from formalin-fixed stool specimens for PCR amplification is a useful tool for obtaining a sensitive and accurate diagnosis that can be applied even in epidemiology studies.

ObjectiveTo analyze the genotypes of Acanthamoeba species isolated from human nasal swabs in the Philippines.MethodsHuman nasal swabs were collected from two groups: a low exposure group composed of students of the University of the Philippines-Diliman and a high exposure group composed of laborers frequently exposed to garbage, soil and dust. After isolation using non-nutrient agar plate lawned with Escherichia coli and DNA extraction using Chelex-100 resin, the ASA.S1 region of the gene (Rns) coding for nuclear, small subunit ribosomal RNA of Acanthamoeba was amplified through polymerase chain reaction. Purified polymerase chain reaction products were then sequenced. Neighbor-joining, maximum parsimony, and maximum likelihood phylogenetic trees were then constructed.ResultsIn the low exposure group, 1 out of 70 (1.43%) students and 7 out of 110 (6.36%) in the high exposure group were culture-positive. Four soil samples were also obtained for comparison, all of which were tested culture-positive. Of the 12 Acanthamoeba isolates, only 9 were successfully sequenced. The basic local alignment search tool of the US National Center for Biotechnology Information was used to identify most similar sequences. Five isolates were identified as genotype T5, and 3 isolateds were genotype T4. Genotype T11 was also isolated from soil, the first to be reported in the Philippines.ConclusionsGenotype T11 is a possible pathogenic strain and both T4 and T5 can be pathogenic to human, hence, healthy provisions, especially for high exposure groups, should be given more attention and reevaluated.

ObjectiveTo determine the antibacterial, antifungal, antiprotozoal, cytotoxic, and phytochemical properties of ethanol extracts of leaves of Voacanga globosa (Blanco) Merr. (V. globosa).MethodsThe extracts were tested against bacteria and fungus through disc diffusion assay; against protozoa through growth curve determination, antiprotozoal and cytotoxicity assays.ResultsThe extract revealed antibacterial activities, inhibiting the growth of Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Micrococcus luteus, and Salmonella typhimurium. Antifungal assay showed that it inhibited Candida albicans. The antiprotozoal assay against Trichomonas vaginalis and Entamoeba histolytica showed that V. globosa can inhibit the parasites, wherein the action can be comparable to metronidazole. With the in situ cell death detection kit, Trichomonas vaginalis and Entamoeba histolytica exposed to V. globosa leaf extract was observed to fluoresce simultaneously in red and yellow signals signifying apoptotic-like changes. Preliminary phytochemical screening revealed the chemical composition of plant extract containing alkaloids, saponins, 2-deoxysugars, and hydrolysable tannins.ConclusionsThus, this study provides scientific evidence on the traditional use of V. globosa leaf extract in treating microbial diseases. Further, the leaf extract can possibly be used to produce alternative forms of antimicrobials.