Detection and molecular characterization of Hepatozoon canis, Babesia vogeli, Ehrlichia canis, and Anaplasma platys in dogs from Metro Manila, Philippines

The study of canine vector-borne diseases in the Philippines started in the 1970s but only gained interest in the past decade. Characterization of such diseases in the Philippines remains incomplete, thus, it is necessary to obtain additional information on the prevalence and diversity of canine tick-borne diseases in the country. In this study, blood samples were obtained at two veterinary clinics in Metro Manila, Philippines from 114 dogs suspected of having canine tick-borne pathogens. Polymerase chain reaction (PCR) was performed on whole blood DNA extracts followed by sequencing, and the following pathogens were detected: Hepatozoon (H.) canis (5.26%), Babesia (B.) vogeli (5.26%), Ehrlichia (E.) canis (4.39%), and Anaplasma platys (3.51%). Additionally, a set of multiplex PCR primers were developed to detect H. canis, Babesia spp. (B. canis and B. vogeli), and E. canis in canine blood. Multiplex and conventional single-reaction PCR results for the 114 dog blood samples were similar, except for one H. canis sample. Multiplex PCR is, therefore, a useful tool in screening infected dogs in veterinary clinics. This study's results, together with those of previous studies in the country, show that canine vector-borne pathogens are an emerging veterinary concern in the Philippines.

Metronidazole susceptibility and TVV-infection of trichomonas vaginalis from Metro Manila and Angeles City, Philippines

@#<p><strong>BACKGROUND:</strong> 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.</p><p><strong>METHODS:</strong> 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.</p><p><strong>RESULTS:</strong> 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.</p><p><strong>CONCLUSION:</strong> 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.</p>

Antibacterial activities of ethanol extracts of Philippine medicinal plants against multidrug-resistant bacteria

Objective: To investigate the antibacterial activities of crude ethanol extracts of 12 Philippine medicinal plants. Methods: Crude ethanol extracts from 12 Philippine medicinal plants were evaluated for their antibacterial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, extended spectrum β-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii. Results: The leaf extracts of Psidium guajava, Phyllanthus niruri, Ehretia microphylla and Piper betle (P. betle) showed antibacterial activity against the Gram-positive methicillin- resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. P. betle showed the highest antibacterial activity for these bacteria in the disk diffusion (16-33 mm inhibition diameter), minimum inhibitory concentration (19-156 μg/mL) and minimum bactericidal concentration (312 μg/mL) assays. P. betle leaf extracts only showed remarkable antibacterial activity for all the Gram-negative multidrug-resistant bacteria (extended spectrum β-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-c-lactamase-producing) in the disk diffusion (17-21 mm inhibition diameter), minimum inhibitory concentration (312-625 μg/mL) and minimum bactericidal concentration (312-625 μg/mL) assays. Conclusions: P. betle had the greatest potential value against both Gram-negative and Gram-positive multidrug-resistant bacteria. Favorable antagonistic activities were also exhibited by the ethanol extracts of Psidium guajava, Phyllanthus niruri and Ehretia microphylla.

Genotype analysis of Acanthamoeba isolated from human nasal swabs in the Philippines

OBJECTIVE@#To analyze the genotypes of Acanthamoeba species isolated from human nasal swabs in the Philippines.@*METHODS@#Human 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.@*RESULTS@#In 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.@*CONCLUSIONS@#Genotype 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.

Genotype analysis of Acanthamoeba isolated from human nasal swabs in the Philippines

Objective: To analyze the genotypes of Acanthamoeba species isolated from human nasal swabs in the Philippines. Methods: Human 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. Results: In 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. Conclusions: Genotype 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.

Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica

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 electrophoresis 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.

Comparison of direct fecal smear microscopy, culture, and polymerase chain reaction for the detection of Blastocystis sp. in human stool samples

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.

Profiles of Entamoeba histolytica-specific immunoglobulins in human sera

OBJECTIVE@#To determine the profiles of anti-Entamoeba histolytica (E. histolytica) IgA, IgG, and IgM in sera of diarrheic and non-diarrheic individuals and partially characterize target antigens.@*METHODS@#Serum samples from thirty diarrheic and thirty non-diarrheic individuals were subjected to IgA, IgG, and IgM profiling through enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblot.@*RESULTS@#ELISA titer results showed that both diarrheic and non-diarrheic individuals possess high levels of E. histolytica-specific IgG compared to IgA and IgM. Flow cytometry data showed that diarrheic serum samples had higher mean reaction percentages against E. histolytica cells compared to non-diarrheic samples. Immunoreactive E. histolytica proteins with molecular weights ranging between 7 kDa and 292 kDa were recognized by diarrheic serum IgG, and 170 kDa and 250 kDa by non-diarrheic serum IgG.@*CONCLUSIONS@#Our findings suggest that serum anti-E. histolytica IgG, compared with serum anti-E. histolytica IgA and IgM responses, was generally high in both diarrheic and non-diarrheic sera, indicating a past exposure to the organism both in symptomatic patients as well as in asymptomatic carriers, respectively. In addition, serum IgG from diarrheic and non-diarrheic patients were able to detect immunogenic E. histolytica proteins.

Antimicrobial activity, cytotoxicity, and phytochemical screening of Voacanga globosa (Blanco) Merr. leaf extract (Apocynaceae)

OBJECTIVE@#To determine the antibacterial, antifungal, antiprotozoal, cytotoxic, and phytochemical properties of ethanol extracts of leaves of Voacanga globosa (Blanco) Merr. (V. globosa).@*METHODS@#The extracts were tested against bacteria and fungus through disc diffusion assay; against protozoa through growth curve determination, antiprotozoal and cytotoxicity assays.@*RESULTS@#The 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.@*CONCLUSIONS@#Thus, 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.

Prevalence of Trichomonas vaginalis in vaginal swabs from sex workers in Angeles City, Pampanga, Philippines as detected by PCR

A prevalence study was conducted on <I>Trichomonas vaginalis</I> infection among female sex workers attending the Reproductive Health and Wellness Center of Angeles City, Pampanga in the Central Luzon region of the Philippines. Polymerase chain reaction (PCR) using <I>T. vaginalis</I>-specific primers TV3⁄7 was utilized to detect <I>T. vaginalis</I> in vaginal swabs from the study population. The lower limit sensitivity of <I>T. vaginalis</I> detection by the PCR assay was found to be one trichomonad. The overall prevalence in 377 women was 9.55%. More than half of the study subjects are 23-27 years old. However, the largest proportion of positive cases was found among subjects 18-22 years old, making it the age group with the highest <I>T. vaginalis</I> prevalence (12.84%).