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.

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.

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%).