Subtype-host patterns and genetic differentiation of Blastocystis sp. in the Philippines.

Blastocystis sp. is a gastrointestinal protozoan commonly encountered in humans and animals. Specificity to certain hosts may be associated with 38 known subtypes (STs) and 8 nonmammalian and avian STs (NMASTs). This can be determined by analyzing ST-host associations, ST-allele data, genetic variability analyses, and fixation index (FST) with sufficient data present. Thus, newly acquired and previously published data on Blastocystis sp. STs and NMASTs from the Philippines were compiled to determine the following: (1) ST-host associations, (2) ST-allele diversity per ST in certain hosts/sources, (3) intrasubtype diversity of certain STs found in different hosts using genetic variability analysis, and (4) comparison of similarities between specific ST populations to determine if these are the same circulating populations using FST. A total of 448 samples subtyped using both sequence-tagged site primers and the 600-bp barcoding region of the Blastocystis sp. SSU rRNA gene were analyzed in this study. Patterns of association for the Philippine samples were similar to those from neighboring Southeast Asian countries and around the world: ST1-ST4 were found in humans but ST3 was the most common, ST5 were found in pigs, and ST6 and ST7 were found in poultry. Blastocystis sp. from humans are mostly the same ST alleles (ST3 allele 34 and ST1 allele 4) while 3-5 ST alleles were found in the most common STs in pigs, macaques, and poultry. Also, ST1, ST3, ST5, and NMAST I are undergoing population expansion according to genetic variability analyses through possible addition of new alleles based on ST-allele diversity. Moreover, FST shows the same circulating population of ST1 in humans, pigs, and water indicating a possible waterborne route of cross-transmission. In contrast, ST3 found in humans possibly come from the same circulating population and is genetically distinct from those in nonhuman sources.

Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples.

Foodborne pathogens compromise food safety and public health, and Salmonella spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent Salmonella outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect Salmonella using the invA gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/µL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 103 cells/mL than 4.8 × 107 cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for invA gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative Salmonella detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.

Phylogenetic relationship of nonmammalian and avian Blastocystis isolates and conventional subtypes.

Blastocystis is an intestinal protist commonly identified in human and animal feces. At present, there are 28 proposed subtypes (STs) identified based on the small subunit rRNA gene, 13 of which are found in both humans and animals. In general, nonmammalian and avian groups are infected by the nonmammalian and avian subtypes (NMASTs). However, NMASTs were also isolated from mammalian hosts, suggesting possible cross-contamination and transmission from nonmammalian and avian hosts to other animals and, potentially, humans. Thus, this study determined the possible relationship between NMAST sequences and conventional STs to provide new insights into Blastocystis classification, identification, and epidemiological significance. Phylogenetic trees were constructed using three statistical models, namely, Maximum Likelihood, Neighbor Joining, and Maximum Parsimony, based on the 30 NMAST sequences. The NMAST sequences formed groups clustered closely with other NMAST subtrees. Most sequences of nonmammalian and avian isolates formed distinct monophyletic clades based on their NMAST classification, with some clustering with mammalian and avian STs. These results indicate the close relationship between Blastocystis isolated from mammalian and avian hosts and nonmammalian and avian hosts. Supplementary Information: The online version contains supplementary material available at (10.1007/s12639-022-01554-7).

Development of a salivary IgA detection method for accurate diagnosis of amebiasis.

An amebiasis detection method was developed based on identifying anti-Entamoeba histolytica IgA in the saliva of infected individuals. The enzyme-linked immunosorbent assay (ELISA)-based detection method was tested along with microscopy and polymerase chain reaction (PCR) on saliva and stool samples from 110 asymptomatic individuals visiting the Manila Health Department - Public Health Laboratory of the City of Manila, Philippines. A receiver operating curve (ROC) was constructed to compare the ELISA results with PCR results. E. histolytica infection was detected in 18 of the 110 individuals. The developed method had an accuracy of 90%, sensitivity of 88.89%, specificity of 90.22%, positive predictive value of 64%, and negative predictive value of 97.65% if a 1:2 dilution of crude saliva sample in phosphate-buffered saline (PBS) was used for diagnosis when compared to PCR. The area under the curve (AUC) of the ROC was 0.9436 if a 1:2 dilution of a crude saliva sample was used. The developed assay presents an easy and accurate method of detecting amebiasis in infected individuals using saliva samples instead of stool or blood samples and has potential applications in both diagnosis and epidemiological studies.

Variations in active proteases of Blastocystis sp. obtained from water and animal isolates from the Philippines.

Blastocystis sp. is a commonly encountered gut protozoan with unclear pathogenicity. The presence of proteases in this organism may be related to its potential pathogenicity or other physiological differences. This study aimed to identify the various proteases that may be present in different subtypes (STs) of Blastocystis sp. using azocasein assay and gelatin zymography. In this study, cysteine, serine, aspartic protease, metalloproteases, and unknown proteases were identified in Blastocystis sp. cultures obtained from animal and water samples belonging to ST1-ST5 and ST7. Azocasein assay and gelatin zymography were conducted on different batches of protease extracts, which showed varying results. Cysteine protease was the most commonly encountered. Protease activity in general is not associated with ST or source (animal or water). However, the presence of cysteine protease alone was associated with animal samples, whereas the presence of more than two protease types was associated with water samples. Azocasein assay and gelatin zymography were conducted on different batches of protease extracts, which showed varying results. Protease activity and the types present may change over time. Blastocystis sp. shows high protease diversity, including possible novel types.

Epidemiology and subtype distribution of Blastocystis in humans: A review.

Blastocystis is a commonly encountered gastrointestinal protozoan in humans and animals with uncertain pathogenicity. Despite its potential public health impact, epidemiological data regarding the prevalence and molecular subtype (ST) distribution of Blastocystis have been rarely reported. Among Blastocystis STs, ST1-ST4 are common in humans, including healthy and immunodeficient populations. According to the Chi-squared (χ2) association based on the data compiled for this cross-sectional study, the presence of ST1 is associated with asymptomatic infection, whereas the presence of ST4 is associated with symptomatic infection. However, cross-sectional studies cannot clarify the potential pathogenicity of Blastocystis, unlike in vivo and in vitro studies. Poor hygiene, poor sanitation and zoonotic transmission are possible factors associated with high Blastocystis prevalence, although this protozoan may be part of the normal healthy human gastrointestinal microbiota. This review covers the prevalence, STs and distribution of Blastocystis infection in humans. Thus, future epidemiological and subtyping studies could reveal new STs in humans as well as possible associations of STs with disease, drug resistance and related mechanisms such as protease activity. These associations with proper ST identification may facilitate the control of potential threats to host health, including the direct pathogenic effects of Blastocystis or alterations of the gastrointestinal microbiome.

Detection of Bartonella infection in pet dogs from Manila, the Philippines.

Dogs can be infected by a wide range of Bartonella spp., but studies regarding the prevalence of Bartonella infection in dogs in the Philippines have not been conducted. This study aimed at determining the prevalence of Bartonella infection in pets dogs from two veterinary clinics in Metro Manila, The Philippines, using both serology and polymerase chain reaction (PCR). Blood samples from 116 dogs from two different groups, one of 60 mainly "healthy dogs" and the other one of 56 dogs enrolled in a tick-borne disease suspect group, were tested for presence of B. henselae antibodies and to detect Bartonella DNA using primers specific for the citrate synthase gene. Seroprevalence for B. henselae was very low (2.6%), as the only three (5%) seropositive dogs (titer 1:64) where among the healthy pet dog group. Following subsequent sequencing, 13 samples, all from the tick-borne disease group, were determined positive for B. henselae (11.2%). This is the first study to report dog infection with B. henselae in the Philippines.

Subtype analysis of Blastocystis sp. isolates from asymptomatic individuals in an urban community in the Philippines

Blastocystis sp. is a commonly reported enteric protistan parasite in faecal specimens with a worldwide distribution afflicting both humans and a wide range of animals. The aim of this study is to characterize the subtypes (STs) of Blastocystis sp. isolates from asymptomatic individuals in an urban community in Pateros, Metro Manila, Philippines. The 600-bp small subunit ribosomal RNA (SSU rRNA) barcoding region of Blastocystis sp. isolates was amplified and sequenced using the primers RD5 and BhRDr. Subtypes were identified by uploading the sequences onto the Basic Local Alignment and Search Tool (BLAST) websites, the Blastocystis Subtype (18S) and Sequence Typing (MLST) Database and by construction of a phylogenetic tree. Twenty-nine (29) out of 35 individuals were detected positive for Blastocystis sp. ST3 is the most common among the three STs detected (65.5%), followed by ST1 (31.0%) and ST4 (3.44%). This study showed that DNA barcoding can serve as a helpful tool to investigate the diversity of Blastocystis sp. in the Philippines.

Loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the sexually-transmitted parasite, Trichomonas vaginalis.

A loop-mediated isothermal amplification (LAMP) assay was developed to detect the sexually-transmitted parasite, Trichomonas vaginalis in vaginal swabs. The presence of T. vaginalis was detected from 121 female sex workers attending a social hygiene clinic in Balibago, Angeles City, Pampanga, Philippines using culture, polymerase chain reaction (PCR), and the developed LAMP assay. The high analytical sensitivity of LAMP detected a higher prevalence of T. vaginalis (42.06%) compared to culture (8.26%) and PCR (7.44%). Additionally, this assay did not cross-react with DNAs of other trichomonads that can infect humans such as Trichomonas tenax and Pentatrichomonas hominis as well as the pathogens, Candida albicans and Staphylococcus aureus. The LAMP assay developed had a limit of detection (0.036 ng/µl) lower than that of PCR using the primers TvK3 and TvK7 (0.36 ng/µl). Prevalence of T. vaginalis in female sex workers in this area of the Philippines may be higher than previously estimated. Discordant results of PCR and LAMP may be due to different reactions to different kinds of inhibitors in the vaginal swabs.

Entamoeba histolytica and E. dispar infections in captive macaques (Macaca fascicularis) in the Philippines.

Entamoeba histolytica is a protozoan parasite that infects man and animals. This parasite has a global distribution and the disease it causes is usually characterized by diarrhea. In order to detect the parasite, it is necessary to differentiate it from Entamoeba dispar. E. dispar appears morphologically similar to E. histolytica but does not cause disease and tissue invasion. This study reports on the prevalence of E. histolytica and E. dispar among captive macaques in a primate facility in the Philippines. PCR was used to correctly identify both Entamoeba species. Indirect fluorescent antibody test (IFAT) was also performed to determine the seroprevalence of amebiasis in the captive macaques. Based on PCR targeting of the peroxiredoxin gene, of the 96 stool samples collected, 23 (24%) contained E. histolytica while 32 (33%) contained E. dispar. IFAT revealed 26 (27%) serum samples positive for antibodies against E. histolytica. Sequence analysis of the 18S rRNA gene showed that the 23 E. histolytica isolates were identical to human E. histolytica isolates deposited in the GenBank and not Entamoeba nuttalli as found in macaques in other recent reports. The Philippines is a major exporter of monkeys for biomedical research purposes, so screening animals before transporting them to other locations lessens the risk of spreading zoonoses to a wider area. This is the first report of the molecular detection of E. histolytica and E. dispar among macaques in the Philippines. This study complements the limited information available on the animal hosts of E. histolytica in the Philippines.

18S ribosomal DNA genotypes of Acanthamoeba species isolated from contact lens cases in the Philippines.

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.