A New Duplex Reverse Transcription PCR for Simultaneous Detection of Viable Cryptosporidium parvum Oocysts and Giardia duodenalis Cysts

Objective To simultaneously detect viable Cryptosporidium parvum oocysts and Giardia duodenalis cysts for the purpose of reducing time and cost spent. Methods A duplex reverse transcription polymerase chain reaction （RT-PCR） method was newly developed. Results Using duplex RT-PCR method for the hsp70 gene, viable （oo）cyst concentrations of 10^1 and l0^3 （oo）cysts/100 I.tL could be detected for C. parvum and G duodenalis, respectively. However, after heat-shock stimulation the expression of hsp70 mRNAs was detectable at 10^0 and 10^1 （oo）cysts/100 μL concentrations of C. parvum and G. duodenalis, respectively. Thus, the detection sensitivity was significantly increased when the viable （oo）cysts were exposed to heat shock. Conclusion This study describes a new duplex RT-PCR method for hsp70 gene to detect the viable （oo）cysts of the C. parvum and G duodenalis with less time consumed and at a lower cost. This newly developed duplex RT-PCR method may be used to detect these parasites not only in aquatic environments but also in clinical samples.

Giardia duodenalis infection among rural communities in Yemen:A community-based assessment of the prevalence and associated risk factors

Objective: To investigate the prevalence and risk factors of Giardia infection among rural communities among rural communities in Yemen.Methods: In this cross-sectional study, a total of 605 stool samples were collected and screened for the presence of Giardia duodenalis(G.duodenalis) cysts and/or trophozoites by using three different diagnostic methods: direct smear, formalin-ether sedimentation,and trichrome staining.A pre-tested questionnaire was used to collect information on the demographic, socioeconomic, behavioural and environmental characteristics of the participants.Results: Overall, 28.1%(170/605) of the participants were infected by G.duodenalis.The prevalence was significantly higher among male participants compared to female(P = 0.034); however, it was not significant among different age groups(P > 0.05).Univariate and multivariate analyses identified four variables as the significant key risk factors of Giardia infection among the sampled communities.These are, in addition to being of the male gender, using unsafe water sources for drinking water, not washing hands after defecation, presence of other family members infected with Giardia, and close contact with domestic animals.Conclusions: The study reveals that Giardia infection is still prevalent among rural communities in Yemen.The provision of clean and safe drinking water, proper sanitation,and health education regarding personal hygiene practices, particularly handwashing, as well as identifying and treating infected family members is imperative and these interventions should be considered in a strategy to control intestinal parasites among these communities in order to curtail the transmission and morbidity caused by G.duodenalis.

Assemblages of <i>Giardia duodenalis</i>Isolates from Dogs by Amplification and Restriction of tpi and <i>β</i>-Giardin Genes and Sequencing of SSU rDNA Gene

Giardia duodenalis exhibits seven assemblages (A-G) that are distributed in different hosts. The A and B assemblages are commonly found in humans and several mammals, while C and D assemblages are typically found in dogs. The purpose of this study was to determine the assemblage of Giardia duodenalis present in the stool samples of ten canines using an assay based on PCR amplification, restriction analysis, and sequencing of the small subunit ribosomal DNA (SSU-rDNA), β-giardin, and triosephosphate isomerase (tpi) genes in order to establish the similarities or differences between the assemblage obtained with each gene. The results indicated that all positive isolates belonged to assemblage A, and specifically to the sub-assemblage A-I. A comparison of the SSU-rDNA gene sequence revealed the presence of three subgroups of assemblage A. These findings highlight the importance of canine transmission of Giardia in Mexico and its genetic plasticity. They also establish a method for additional and more molecularly extensive epidemiological studies to improve sanitation and hygiene in the most affected areas.

Prevalence of Giardia duodenalis among dogs seized by the Center for Control of Zoonoses (CCZ) of the city of Lages, Santa Catarina, Brazil

The knowledge of the epidemiology of parasitic infections in stray and domestic animals, especially of its incidence and prevalence, is fundamental to adopting effective prophylactic measures. Stray dogs play an important role in environmental contamination favoring the transmission cycle of zoonotic agents. Among the parasitic infections that affect humans, Giardia duodenalis is the most common intestinal protozoa and was designated as a re-emerging infectious disease. This study aimed to determine the prevalence of G. duodenalis in dogs siezed by the Center for Control of Zoonoses (CCZ) of the city of Lages, Santa Catarina, Brazil using two diagnostic techniques. In 357 stool samples analysed, the prevalence of G. duodenalis cysts was 5.3% (19/357) and 4.8% (17/357) detected by floatation and sedimentation techniques, respecttively. No correspondence between gender and age was found among the methods used for analyzing the infected dogs in this study. Our data suggested that two diagnostic techniques should be used in a complementary way to ensure that false negatives are not neglected.

Molecular epidemiology of Giardia duodenalis infection in humans in Southern Ethiopia:a triosephosphate isomerase gene-targeted analysis

Background:Giardia duodenalis is a species complex consisting of multiple genetically distinct assemblages.The species imposes a major public health crisis on developing countries.However,the molecular diversity,transmission dynamics and risk factors of the species in these countries are indeterminate.This study was conducted to determine the molecular epidemiology of G.duodenalis infection in asymptomatic individuals in Southern Ethiopia.Methods:From March to June 2014,fresh stool samples were collected from 590 randomly selected individuals.Socio-demographic data were gathered using a pre-tested structured questionnaire.The genotyping was done using triosephosphate isomerase gene-based nested polymerase chain reaction and DNA sequencing.The genetic identity and relatedness of isolates were determined using the basic local alignment search tool and phylogenetic analysis.Risk factors associated with G.duodenalis infection were analysed using binary and multinomial logistic regression models.Results:The results showed that 18.1%(92/509)of the study subjects were infected by G.duodenalis.Among the isolates,35.9%(33/92)and 21.7%(20/92)were sub-typed into assemblages A and B,respectively,whereas 42.4%(39/92)showed mixed infections of A and B.Most of the assemblage A isolates(94%,31/33)were 100%identical to sequences registered in GenBank,of which the majority belonged to sub-assemblage AII.However,the high genetic variability and frequency of double peaks made sub-genotyping of assemblage B more problematic and only 20%(4/20)of the isolates matched 100%with the sequences.The risk factors of age(P=0.032)and type of drinking water source(P=0.003)both showed a significant association with the occurrence G.duodenalis infection.Conclusions:This study established the endemicity of G.duodenalis in Southern Ethiopia.Infection with assemblage A was more frequent than with assemblage B,and the rate of infection was higher in children and in municipal/tap and open spring water consumers than the other groups.Sub-typing of assemblage B and determining the origin of double peaks were challenging.The present study confirms the need for further inclusive studies to be conducted focusing on sub-types of assemblage B and the origin of heterogeneity.

Sub-assemblages distribution of Giardia duodenalis and its association with clinical symptoms in children from Sancti Spíritus,Cuba

Objective:To determine the prevalence of sub-assemblages of Giardia duodenalis(G.duodenalis)causing infection in preschool children in a central region of Cuba.Methods:A cross-sectional study was conducted on 417 children from Sancti Spiritus from January to June 2013.A PCR amplification of the glutamate dehydrogenase(gdh)gene was performed for all positive samples in direct microscopic examination to enable sub-assemblages identification.Results:The prevalence of G.duodenalis in preschool children was 10.8%.DNA from 39 of 45(86.7%)samples was successfully amplified by PCR-restriction fragment length polymorphism analysis of the gdh gene.Restriction fragment length polymorphism analysis classified the 39 gdh amplicons in sub-assemblages BIII(11,28.2%),AII(8,20.5%)and BIV(4,10.3%).Mixed infections accounted for 41%,with the pattern AII/BIII(12,30.7%)being the predominant.Abdominal pain was statistically associated with infection with sub-assemblage BIII.Conclusions:Sub-assemblage BIII was the most commonly identified in the population of children studied,and significantly associated with abdominal pain in the symptomatic children.More defining tools to discriminate sub-assemblage and genotype levels to correlate with clinical data are needed in further studies.Large studies in endemic settings are required to elucidate the role that assemblage types play in G.duodenalis infections in vulnerable populations,such as children.

Genotyping of Giardia duodenalis isolates from human subjects in Zabul,using PCR-RFLP

Objective:To uncover the molecular prevalence of Giardia duodenalis by PCR-restriction fragment length polymorphism(RFLP)in Zabul city,Iran.Methods:Twenty-four stool samples were collected from 215 patients with suspected giardiasis by microscopic examination.To increase the sensitivity of the PCR,the total genomic DNA from isolates was extracted by applying glass beads and the QIAamp Kit.A one-step PCR-RFLP method,targeting the glutamate dehydrogenase gene,was utilized to differentiate the assemblages A and B among isolates.Results:The PCR fragment was determined from 30 isolates,RFLP assay of 24 isolates showed 24(100)isolates as Genotype B group BIII.Conclusions:The results with the glutamate dehydrogenase gene assay demonstrated that the predominant subtype of Giardia duodenalis in the area is BIII,which showed animals are the main reservoir of the isolates in this area.