Lamblia Assemblage Research Articles

Incidence and Molecular Detection of Genotypes for Giardia lamblia Isolated From Children in Zakho District, Duhok Province, Kurdistan Region, Iraq.

Giardia lamblia is a significant intestinal protozoan in humans worldwide, with a high incidence of infection in developing countries, particularly among children. Molecular analysis has identified eight assemblages (A to H), with A and B more frequently associated with human infections. Regardless of its importance, to the best of our knowledge, this is the first molecular study on assemblages in Giardia lamblia adopted in the Zakho district, province of Duhok, Iraq. The present study aimed to determine the giardiasis infection rate and identify the assemblages of Giardia lamblia in children from four areas in the Zakho district. We collected fecal samples and conducted a microscopic examination. Genomic DNA was extracted, and assemblage identification was done via amplification of the gdhgene using a semi-nested polymerase chain reaction and restriction fragment length polymorphism (RFLP). Out of 31 Giardia-positive samples, 23 were successfully amplified through semi-nested PCR. Nineteen isolates (82.60%) were assigned to assemblage B, and four (17.40%) to assemblage A. Assemblage B was identified as belonging to sub-assemblages B11 and B1V, while assemblage A was identified as sub-assemblages A1 and A11. This study provides insights about Giardia lamblia assemblages in the Zakho district, Duhok province, Iraq, and may serve as a beginning step toward understanding the molecular characterization of Giardia in the studied area.

Giardia lamblia assemblages and their correlation with the severity of diarrhoea: A review article

Background: Giardia lamblia is responsible for a range of clinical conditions, from asymptomatic to acute or chronic symptomatic forms. The genetic variability of Giardia lamblia raises the possibility of a relationship between its assemblages and the clinical severity of giardiasis. We conducted a review of the literature from 2001 to 2023 to explore the association between G. lamblia assemblages and the severity of diarrhea. Material and Methods: A search was performed through databases of PubMed, WHO, Health Protection Agency, the UK and Centre of Disease Control USA websites and Medline. This search yielded 47 studies from 2001 to 2023 as only those articles providing information on the correlation between different assemblages of G. lamblia and the severity of the disease were included. Results: Out of a total of 47 studies that were included, only 26 studies showed a meaningful comparison. 12 studies showed no difference between different G. lamblia assemblages and the severity of giardiasis, while 14 studies demonstrated the link between G. lamblia assemblage and the severity of the disease. Conclusion: Numerous studies have attempted to identify the link between its assemblage profile and the symptoms it causes but with little success. However, the findings of these studies are contradictory and inconclusive, making it difficult to establish a clear association. However, the divergence in findings suggests that a definitive correlation cannot yet be established. To fully understand the link between assemblages and severe diarrhea, further molecular studies are needed, especially in large populations residing in endemic regions. Keywords: Giardia lamblia, Diarrhea, Assemblages

Giardia lamblia-infected preschoolers present growth delays independent of the assemblage A, B or E.

Intestinal parasite Giardia can affect children's physical development mainly stunting even in asymptomatic cases. The protozoa G. lamblia is divided into assemblages A-H. However, it is still unclear whether clinical manifestations and pathogenesis may vary according to the infecting assemblage. To investigate whether G. lamblia assemblages influence differently the physical development of preschoolers from a community of Rio de Janeiro, Brazil. Anthropometric parameters were analysed from children attending a daycare centre and stool samples were obtained for the G. lamblia diagnosis. G. lamblia isolates from positive samples were genotyped. Data were analysed in order to verify whether there is a relationship between G. lamblia infection and the physical development of children according to the assemblage. Herein we demonstrated that although eutrophic, G. lamblia-infected daycare preschoolers from a low-income community presented growth delay compared to non-infected ones. This effect was observed for the three assemblages (A, B or E) found infecting humans. G. lamblia causes growth delays on children independent of infecting assemblage (A, B or E).

Gene migration of giardiasis in Iran; a microevolutionary scale for reflecting transmission patterns of Giardia lamblia assemblages in symptomatic patients

In the microevolutionary scale of Giardia lamblia, the gene migration indicates how G. lamblia assemblages have transmitted between adjacent counties. 33 positive fecal samples were taken from patients suffering gastrointestinal disorders (nausea, bloating, burping constipation and fatty diarrhea) at Tabriz and Ardabil cities, where located in the cold regions of northwest Iran. Following parasitological examinations, DNA samples were extracted, amplified and digested by single-step PCR-RFLP assay, targeting the glutamate dehydrogenase (gdh) locus to distinguish within and between assemblages A and B. PCR products were directly sequenced to reconfirm their heterogeneity traits and phylogenetic analysis. Of the 33 isolates, 81.9% (n: 27), 9% (n: 3) and 9% (n: 3) were successfully identified as assemblages A (genotype AII), B (genotype BIII) and the mixed of genotypes AII and B, respectively. Despite the presence of heterogeneous clinical backgrounds, a low genetic diversity of sub-assemblage AII was identified among symptomatic cases. A low value of pairwise fixation index showed that G. lamblia sub-assemblage AII is not genetically differentiated among northwest regions of Iran. The occurrence of haplotypes TAB-1/ARD-1 between two regional populations indicates that there is a dawn of G. lamblia gene flow due to transfer of alleles through host mobility and/or ecological alterations. To assess the hypothetical evolutionary scenario, further studies are essential for multilocus genotyping of G. lamblia in tropical regions of Iran and neighboring countries.

Genetic Variation in Metronidazole Metabolism and Oxidative Stress Pathways in Clinical Giardia lamblia Assemblage A and B Isolates [Corrigendum

Genetic Variation in Metronidazole Metabolism and Oxidative Stress Pathways in Clinical Giardia lamblia Assemblage A and B Isolates [Corrigendum

Molecular genotyping of Giardia lamblia assemblages by conventional PCR in rural and urban areas in Egypt.

Background Giardiasis is one of the most important parasitic gastro-intestinal infections that affect humans worldwide. Children are the most affected age group either in developing or developed countries. Genotyping of Giardia lamblia by molecular techniques classified it into eight assemblages; of which, assemblages A and B are potentially zoonotic pathogens. This study was done to investigate the prevalence of different Giardia lamblia genotypes by conventional PCR, and to explore the environmental and patientsʼ sociodemographic factors that may affect the disease prevalence. Methods Two hundreds fecal samples were collected at Damanhour General Hospital from patients with gastrointestinal diseases. All samples were examined microscopically, and the positive ones were investigated by conventional PCR. Results Giardiasis was detected in 92 (46%) samples. Eighty-eight samples gave positive results by PCR, 18% of which were assemblage A, 70.5% were assemblage B, and 11.5% were mixed infection of both assemblages. The infection was more prevalent in males, rural patients and the highly educated ones. Conclusions This study presents critical and demonstrative data regarding public health in Egypt. The results reveal that the prevalence of giardiasis is high among both rural and urban patients, particularly in children. It also prevails in patients of all education levels, and patients dealing and not dealing with animals. Moreover, we recommend PCR amplifying the triose phosphate isomerase (tpi) gene in fresh fecal samples as very effective method for the diagnosis and genotyping.

Giardia lamblia assemblages A and B isolated from symptomatic and asymptomatic persons in Hamadan, west of Iran.

Giardia is a very abundant organism bringing about diarrhoea in human beings. The focus of this analysis was the detection of Giardia lamblia assemblages in human stool specimens in Hamadan, west of Iran, as well as the association between obtained assemblages and clinical symptoms. Faecal samples of 4066 individuals admitted tothe medical and health care facilities in Hamadan were inspected microscopically for the existence of Giardia cysts/trophozoites, and the clinical symptoms of the patients were recorded. The DNA of positive sampleswas isolated from and the nucleotide sequences of both glutamate dehydrogenase (gdh) (n = 15) and triose phosphate isomerase (tpi) (n = 8) genes were analyzed. In direct microscopy, a total of sixty-four samples (1.6%), were considered as positive for G. lamblia cysts or trophozoites. The sequence analysis showed that 18 out of 23 sequenced isolates (78.2%) were assemblage A and 5 (21.7%) were assemblage B. Clinical symptoms were observed in 44.4% and 40% of patients with assemblages A and B, respectively. Overall, the predominant assemblage A detected in the tested samples along with bioinformatics analysis suggest a potential zoonotic transmission in the region of the study. Although advanced analyses are necessary to understand the foundation and path of the infection, it seems that more sanitary regulations regarding contact with livestock and pet animals are essential.

The presence of Giardia lamblia assemblage A in dogs suggests an anthropozoonotic cycle of the parasite in Rio de Janeiro, Brazil

BackgroundGiardia lamblia is a zoonotic protozoan that is classified into 8 genotypes and is distributed worldwide. Assemblages A and B were found to infect dogs and humans, whereas assemblages C and D are dog host-specific. Our objective was to investigate the G. lamblia genotypes circulating in a canine population in Rio de Janeiro, RJ. ResultsSixty stool samples positive for G. lamblia from street dogs were characterized. Fragments of the conserved genes encoding beta-giardin (β-gia) and glutamate dehydrogenase (gdh) were used as targets. The sequences from beta-giardin and glutamate dehydrogenase genes obtained from all 60 dog samples were 100% similar to G. lamblia genotype A. ConclusionThe detection of genotype A suggests that G. lamblia transmission in Rio de Janeiro has a predominantly anthropozoonotic cycle.

Occurrence, genotyping, and health risk of Cryptosporidium and Giardia in recreational lakes in Tianjin, China

Cryptosporidium and Giardia are critical parasites in the etiology of diarrhea worldwide, and often cause waterborne outbreaks. The presence of Cryptosporidium and Giardia in recreational lakes was investigated with molecular characterization, and a comprehensive quantitative microbial risk assessment (QMRA) of protozoan infections was performed, considering multiple exposure pathways, differences in age, sex, and disease severity, and the genotypes of the protozoa. Forty-three (82.7%) and 51 (98.1%) water samples were positive for Cryptosporidium oocysts and Giardia cysts, respectively, with average counts of 3.65 oocysts/10 L and 12.58 cysts/10 L, respectively. Six Cryptosporidium species and three Giardia lamblia assemblages were confirmed with molecular analyses. The protozoan concentration was significantly associated with water turbidity, but not with the total coliform numbers. Swimming in the lakes entailed the highest incidence risk of 5.72 × 10−4 per person per year (pppy) (95% confidence interval (CI): 0.03–43.33 × 10−4) for Cryptosporidium and 4.04 × 10−4 pppy (95% CI: 0.01–32.66 × 10−4) for Giardia, whereas wading entailed the lowest risk (2.20 × 10−4 and 1.70 × 10−4 pppy, respectively). The annual burdens attributable to recreational-water-associated cryptosporidiosis and giardiasis were 3.44 (95% CI: 0.04–23.51) and 1.81 (95% CI: 0.01–12.96) disability-adjusted life years per 1,000,000 individuals per year, respectively. Children were more likely to have an individual disease burden than adults, and males were more likely than females. Sensitivity analysis highlighted the great importance of controlling the proportion of exposed individuals and reducing the frequency of exposure. The methodology and results of this study will allow us to better evaluate and reduce the burden of Cryptosporidium and/or Giardia infections associated with recreational water use in China and other countries.

Presence and molecular characterization of Cryptosporidium and Giardia in recreational lake water in Tianjin, China: a preliminary study

Little is known about the occurrence of Cryptosporidium and Giardia in recreational water in China. A total of 52 samples were collected from recreational lakes in Tianjin during a high-occurrence season (June–October) for the waterborne cryptosporidiosis and giardiasis, and the occurrence and genotypes of Cryptosporidium and Giardia were investigated. The results showed that 82.7% (43) and 98.1% (51) of samples were positive for Cryptosporidium oocyst and Giardia cysts, respectively. The mean concentration of parasites was 3.65 oocysts/10 L and 12.58 cysts/10 L, respectively. Molecular characterization revealed that the presence of Cryptosporidium parvum, C. andersoni, C. hominis, C. meleagridis, C. fragile, C. ubiquitum, and Giardia lamblia assemblage A, B and D. The protozoan contamination in the studied lakes may originate from animal feces on ground, which was washed into the lake by stormwater runoff. Nevertheless, there is a potential risk of infection during recreational activities in the lake because the dominant detected protozoan genotypes are common human pathogens. Moreover, microbial indicators analysis does not adequately indicate the protozoan contamination in recreational water. The information from this study will be valuable for future protozoan source tracking, and any further control interventions against Cryptosporidium and/or Giardia infection associated with recreational water.

Molecular characterization of Giardia lamblia in children less than 5 years of age with diarrhoea attending the Bengo General Hospital, Angola.

Background Giardia lamblia is a pathogenic intestinal protozoan with high prevalence in developing countries, especially among children. Molecular characterization has revealed the existence of eight assemblages, with A and B being more commonly described in human infections. Despite its importance, to our knowledge this is the first published molecular analysis of G. lamblia assemblages in Angola.MethodsThe present study aimed to identify the assemblages of G. lamblia in children with acute diarrhoea presenting at the Bengo General Hospital, Angola. A stool sample was collected and microscopy and immunochromatographic tests were used. DNA was extracted and assemblage determination was performed through amplification of the gene fragment ssu-rRNA (175 bp) and β-giardin (511 bp) through polymerase chain reaction and DNA sequencing.ResultsOf the 16 stool samples screened, 12 were successfully sequenced. Eleven isolates were assigned to assemblage B and one to assemblage A. Subassemblage determination was not possible for assemblage B, while the single isolate assigned to assemblage A was identified as belonging to subassemblage A3.ConclusionThis study provides information about G. lamblia assemblages in Bengo Province, Angola and may contribute as a first step in understanding the molecular epidemiology of this protozoan in the country.GenBank accession numbers for the ssur-RNA gene: MF479750, MF479751, MF479752, MF479753, MF479754, MF479755, MF479756, MF479757, MF479758, MF479759, MF479760, MF479761.GenBank accession numbers for the β-giardin gene: MF565378, MF565379, MF565380, MF565381.

Associations of Giardia lamblia assemblages with HIV infections and symptomatology: HIV virus and assemblage B were they born to each other?

Giardia lamblia is an intestinal parasite that has an extensive genetic variation among isolates. This species is divided into eight different assemblages (A–H), but only assemblages A and B have been associated with human infections. Studies on the associations of G. lamblia assemblages and symptoms have been done but were inconclusive. The aim of this study was to correlate G. lamblia assemblages with symptoms in patients with and without HIV/AIDS and its association with the CD4T cell count. The cross-sectional survey was conducted among patients attending the Evandro Chagas National Institute of Infectious Diseases (INI/FIOCRUZ) in Rio de Janeiro from January 2011 to February 2015. Thirty-eight of 65 microscopically positive stool samples for G. lamblia were from HIV positive patients and 27 were from HIV negative patients. Of the HIV infected patients, 19 (55.9%) were genotyped as assemblage B of which 9 (47.4%) had a CD4Tcell count below 200cells/mm3. In addition, we found a greater number of samples belonging to assemblage B in symptomatic cases (11 of 19; 57.9%). Our data suggest that assemblage B is very likely to be found in HIV infected patients and probably the lower CD4T count gives advantages for assemblage B replication. Furthermore, assemblage B seems to be associated with symptomatology, particularly abdominal pain, asthenia, diarrhea, fever, headache and myalgia. This study provides information on G. lamblia assemblages and symptoms in patients with and without HIV/AIDS virus and their association with CD4Tcell counts.

Different risk factors for infection with Giardia lamblia assemblages A and B in children attending day-care centres.

Giardia lamblia is a major cause of diarrhoea in children, especially those attending day-care centres (DCCs). Only Giardia assemblages A and B infect humans. Given the lack of assemblage-specific epidemiological data, we aimed to identify risk factors for infection by assemblages A and B in DCC attendees. During 2010-2013, 5,015 faecal samples from ≤4-year-old children attending 40 DCCs participating in laboratory surveillance in the Netherlands were tested for Giardia using RT-PCR. Giardia-positive samples were typed for identification of assemblages A and B. We compared child- and DCC-level characteristics of Giardia-positive children with those of Giardia-negative children using mixed-effects logistic regression. Overall, 226 samples (4.5%) tested positive for Giardia, and assemblages were determined for 138 of them: 62 (45%) were assemblage A and 76 (55%) were B. The only risk factor for assemblage A infection was attending DCCs with indoor sandpits and cats during spring/summer (odds ratio [OR] 13.5; 95%CI 1.8-101.3). For assemblage B, risk factors were attending DCCs with dedicated diaper-changing (OR 3.6; 95%CI 1.7-7.6) and laundry (OR 2.3; 95%CI 1.1-4.9) areas. Preventing sick children from attending day-care and having cloth-towels at the DCC decreased the risk of assemblage B infection (OR 0.0; 95%CI 0.0-0.5 and OR 0.3; 95%CI 0.1-0.6 respectively). Risk factors for assemblages A and B infection in DCC-attending children were different, with assemblage B being mainly related to anthroponotic transmission, and assemblage A being related to zoonotic transmission. Given these differences, interventions to reduce the burden of childhood giardiasis cannot ignore those assemblage-specific preferred reservoirs and transmission routes.

Molecular Characterization of Giardia lamblia: First Report of Assemblage B in Human Isolates from Rio de Janeiro (Brazil).

BackgroundDespite the high prevalence of giardiasis, the genetic characterization of Giardia lamblia has been poorly documented in Brazil and molecular epidemiology research has only been conducted in the last few years. The aim of this study was to determine the prevalence of different G. lamblia assemblages and detect mixed infections among patients with giardiasis.Methods and Principal FindingsThe cross-section survey was conducted among patients attending the FIOCRUZ in Rio de Janeiro. In order to discriminate the genetic assemblages/sub-assemblages, G. lamblia isolates were characterized by PCR-RFLP and qPCR using four loci genes (bg, gdh, tpi and orfC4). Of the 65 positive samples, 41 (63.1%) were successfully amplified by nested-PCR of bg and gdh genes. Among them, 16 were typed as sub-assemblage AII, 7 as BIII, 4 as BIV and 8 as a mixture of BIII and BIV. After the analysis by qPCR assay, a total of 55 (84.6%) samples were amplified using at least one locus: bg gene was amplified in 38 (58.5%) samples, gdh in 41 (63.1%), tpi in 39 (60%), and orfC4 in 39 (60%). Multilocus genotyping results showed that 29 (52.7%) samples belonged to Assemblage A and 26 (47.3%) samples belonged to Assemblage B. In 2011 and 2012, 20 (74.1%) samples belonged to Assemblage A and 7 (25.9%) belonged to Assemblage B. In subsequent years (2013–2015) there was a predominance of Assemblage B, 19 (67.9%) versus 9 (32.1%) Assemblage A.ConclusionsThis is the first time that Assemblage B of G. lamblia was reported in human clinical samples from Rio de Janeiro (Brazil) and is the first report about genetic characterization using four genes. The qPCR assemblage-specific showed no mixed infections by Assemblages A and B. A switch in genetic profile over the years was observed, firstly predominance of Assemblage A and lastly of Assemblage B.

Immunolocalization of α18- and α12-giardin in Giardia lamblia trophozoites.

To study subcellular localization of α18- and α12-giardin in Giardia lamblia trophozoites, the α18- and α12-giardin genes were amplified from G. lamblia assemblage A, respectively. The PCR products were cloned into the prokaryotic expression vector pET-28a(+), and the positive recombinant plasmids were transformed into E. coli Rosetta (DE3) strain for the expression, and expressed α18- and α12-giardin fusion protein were purified by Ni-Agarose resin, respectively. Mice were immunized with purified fusion proteins for preparation of polyclonal antibody, and then the subcellular localization of α18- and α12-giardin was determined by fluorescence immunoassay. Results showed that the concentrations of purified α18- and α12-giardin fusion proteins were 1.20 and 0.86mg/ml, respectively. The titers of anti-α18- and anti-α12-giardin polyclonal antibody were both as high as 1:25600 dilutions. Immunofluorescent analysis showed that α18- and α12-giardin proteins were mainly localized at four pairs of flagella and the cytoplasm of G. lamblia trophozoites, suggesting that α18- and α12-giardin are the flagella and cytoplasm-associated proteins, respectively. The above information would lay the foundation for research about the crystal structure and biological function of α18- and α12-giardin.

Predominance of Giardia lamblia assemblage A among iron deficiency anaemic pre-school Egyptian children.

Intestinal parasites and nutritional deficiency can coexist and influence each other. This study aimed to clarify the association between Giardia genotypes and presence of iron deficiency anaemia (IDA) among pre-school Egyptian children. Two groups (IDA and non-anaemic) of giardiasis children (44/group) were selected according to their recovery response after treatment of giardiasis. Each group included 24 and 20 gastrointestinal symptomatic and asymptomatic, respectively. Giardia human genotypes were performed by intergenic spacer (IGS) gene based polymerase chain reaction (PCR) with high-resolution melting curve (HRM). PCR/HRM proved that Tms of assemblage A and B ranged from 79.31 ± 0.29 to 84.77 ± 0.31. In IDA patients, assemblages A and B were found among 40/44 (90.9 %) and 4/44 (9.1 %), respectively, while in non-anaemic patients, assemblages A and B were found in 10/44 (22.7 %) and 32/44 (72.7 %), respectively, beside two (4.6 %) cases had mixed infection. The difference was statistically significant. No significant relation was found between symptomatic or asymptomatic assemblages and IDA as assemblage A was found in 21/24 (87.5 %) and 19/20 (95 %) of symptomatic and asymptomatic, respectively, while 3/24 (12.5 %) and 1/20 (5 %) of assemblage B were symptomatic was asymptomatic, respectively. A significant relation was found between assemblage A subtypes distribution among IDA patients as AI and AII were detected on 23 (52.3 %) and 16 (36.4 %) of patients, respectively, while one case (2.3 %) had mixed infection. In conclusion, assemblage A is predominant among IDA giardiasis children suggesting its role in enhancing the occurrence of IDA while B has a protective role.

Giardia lamblia infections in children in Ghana.

IntroductionThough giardiasis is an important public health problem in Ghana, several aspects of its epidemiology, particularly the molecular epidemiology has not been investigated adequately. This could be a major hindrance to effective surveillance and control of giardiasis in the country. The study was carried out to determine the prevalence, risk factors and genotypes of Giardia lamblia infecting children at a paediatric hospital in Ghana.MethodsA total of 485 patients including 365 diarrhoea and 120 non-diarrhoea children were enrolled into the study. Stool samples were collected and analysed for parasite presence using microscopy, ELISA and PCR. Positive samples were subsequently characterized into assemblages by PCR-RFLP, and further confirmed with sequencing of the glutamate dehydrogenase (gdh) gene. Epidemiological data on demographic, clinical and behavioral features of the study subjects were also collected.ResultsPrevalence of G. lamblia infections in diarrhoea and non-diarrhoea children were 5.8% and 5% respectively (P>0.5). Sequence data confirmed Giardia lamblia assemblage B as the predominant genotype in both diarrhoea and non-diarrhoea cases. There was no significant association of G. lamblia infection with any of the epidemiological variables investigated.ConclusionOur findings suggest that assemblage B could be the predominant genotype causing giardiasis in children. Increased public health education focusing on good sanitary practices, particularly among mothers and children, could decrease the risk of G. lamblia infection.

Sequence analysis and prokaryotic expression of Giardia lamblia α-18 giardin gene

To study the genetic variation and prokaryotic expression of α18 giardin gene of Giardia lamblia zoonotic assemblage A and host-specific assemblage F, the α18 genes were amplified from G. lamblia assemblages A and F by PCR and sequenced. The PCR product was cloned into the prokaryotic expression vector pET-28a(+) and the positive recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) strain for the expression. The expressed α18 giardin fusion protein was validated by SDS-PAGE and Western blot analysis, and purified by Ni-Agarose resin. The putative sequence of α18 giardin amino acid was analyzed by bioinformatics software. Results showed that the α18 giardin gene was 861bp in length, encoding 286 amino acids; it was 100% homologous between human-derived and dog-derived G. lamblia assemblage A, but it was 86.8% homologous with G. lamblia assemblage F (cat-derived). Giardin α18 was about 36kDa in molecular weight, with good reactivity. Prediction based on in silico analyses: it had hydrophobicity, without signal peptide and transmembrane domain, and contained 11 alpha regions, 13 beta sheets, 1 beta turn and 7 random coils in secondary structure. The above information would lay the foundation for research about the subcellular localization and biological function of α18 giardin in G. lamblia.

Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.

A real-time RT-PCR method to detect viable Giardia lamblia cysts in environmental waters

Currently, USEPA Method 1623 is the standard assay used for simultaneous detection of Giardia cysts and Cryptosporidium oocysts in various water matrices. However, the method is unable to distinguish between species, genotype, or to assess viability. Therefore, the objective of the present study was to address the shortcomings of USEPA Method 1623 by developing a novel molecular-based method that can assess viability of Giardia cysts in environmental waters and identify genotypes that pose a human health threat (assemblage groups A and B). Primers and TaqMan ® probes were designed to target the beta-giardin gene in order to discriminate among species and assemblages. Viability was determined by detection of de-novo mRNA synthesis after heat induction. The beta-giardin primer/probe sets were able to detect and differentiate between Giardia lamblia assemblages A and B, and did not detect Giardia muris (mouse species) or G. lamblia assemblages C, D, E and F (non-human), with the exception of Probe A which did detect G. lamblia assemblage F DNA. Additionally, DNA or cDNA of other waterborne organisms were not detected, suggesting that the method is specific to Giardia assemblages. Assay applicability was demonstrated by detection of viable G. lamblia cysts in spiked (assemblage B) and unspiked (assemblage A and B) reclaimed water samples.