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Introduction: There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates., Objective: This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii ., Materials and Methods: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced., Results: One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations., Conclusions: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

Introduction: Eight Giardia duodenalis genotypes (A-H) have been described to date. Genotypes A and B have been isolated from humans and a wide range of mammals; however, genotypes C-H have shown greater host specificity., Objective: Identifying G. duodenalis genotypes from cysts in faeces obtained from children attending the Instituto Colombiano de Bienestar Familiar (ICBF) day care centres and from dogs in Ibagué by PCR-RFLP targeting both the b -giardin and glutamate dehydrogenase genes., Materials and Methods: Cysts from G. duodenalis positive samples were concentrated, DNA was extracted and the b -giardin and glutamate dehydrogenase genes were analysed by PCR-RFLP. The MHOM/CO/04/G40 strain was used as positive control (this was obtained from the Grupo de Parasitología at the Instituto Nacional de Salud )., Results: Of the total human samples, 11/23 (48%) were genotyped as A and 12/23 (52%) as B; PCR-RFLP revealed that four canine samples were genotypes C and D, these being host-specific., Conclusions: Only genotypes associated with human infection (AII, BIII and BIV) were found in the children and host-specific genotypes were observed in canines (C and D). No interaction could be established between animal and human transmission cycles due to the small canine sample size and as the former did not come into contact with children attending ICBF day-care centres.

Introduction: Recent studies have suggested an association between the soil-transmitted helminth infections and malaria incidence. However, published evidence is still insufficient and diverging. Since 1977, new ecologic studies have not been carried out to explore this association. Ecologic studies could explore this correlation on a population level, assessing its potential importance on public health., Objectives: The aim of this evaluation is to explore the association between soil-transmitted helminths prevalence and malaria incidence, at an ecologic level in Colombia., Materials and Methods: Using data from the National Health Survey, which was carried out in 1980 in Colombia, we calculated Spearman correlation coefficients between the prevalence of: Ascaris lumbricoides, Trichuris trichiura and hookworm, with the 1980 malaria incidence data of the same year provided from the Colombian Malaria National Eradication Service. A robust regression analysis with least trimmed squares was performed., Results: Falciparum malaria incidence and Ascaris lumbricoides prevalence had a low correlation (R²= 0.086) but this correlation was stronger into the clusters of towns with prevalence of Ascaris lumbricoides infection above 30% were only included (R²= 0.916)., Conclusion: This work showed an ecologic correlation in Colombia between malaria incidence and soil-transmitted helminths prevalence. This could suggest that either there is an association between these two groups of parasites, or could be explained by the presence of common structural determinants for both diseases.

Introduction: Giardia intestinalis is a protozoan parasite that causes a gastrointestinal infection known as giardiosis, which is transmitted primarily through fecal-oral contamination. Genetic studies of axenically cultivated Giardia isolates have identified two major genetic groups distributed throughout the world. In the present study 24 native strains of the parasite were analyzed by the RAPD technique (Random Amplified Polymorphic DNA)., Objective: To determine the level of polymorphism and the complexity of Giardia intestinalis circulating strains in specific areas of Colombia., Materials and Methods: The RAPD method was used, as it allows for a quick, simple and reliable analysis that requires no prior knowledge of the genetics of the parasite. A RAPD analysis was conducted on native isolates collected in Colombia between 1997 and 2001, established in continuous cultures. Several primers were tested separately, in order to enhance the capacity for discrimination of the method., Results: Of the 24 strains that were included in the study, 22 were arranged in independent clusters. The strains that were from the same geographic area and collected at about the same time, generally displayed highly similar but distinguishable RAPD patterns. Clones isolated from a strain were analyzed as well, and it was possible to differentiate them molecularly., Conclusion: The studied strains showed to belong to genotype A . The results suggest that the Colombian strains studied consist of a heterogeneous mixture of closely related populations.

Introduction: The development of polyclonal antibodies requires laboratory animals, as the usually rabbits, which are bled for obtaining the immunoglobulins. Hens are an alternative to developing IgY polyclonal antibodies., Objective: In the current study, hens were used to develop IgY anti-Giardia duodenalis antibodies and to evaluate five protocols for its purification., Materials and Methods: Three hens were immunized intramuscularly immunized with Colombian Giardia duodenalis isolates trophozoites on day 0, 15, 30, 45, 60, 90 and 120. The hen eggs were collected before each immunization, and IgY purified from yolk by delipidation (D) and precipitation (P) using five different protocols: M1: (P: ammonium sulfate/D:dextran sulfate-calcium chloride), M2: (D: dextran sulfate-calcium chloride/P: ammonium sulfate), M3: (D: chloroform/P: ammonium sulfate 50%), M4: (D: solution A/P: solution B) and M5: (D: chloroform/P: ammonium sulfate 30%). The immunochemistry evaluation of IgY anti-Giardia duodenalis was assayed by immunodiffusion, counterimmunoelectrophoresis (CIE) and Western blot. The purity of IgY was assayed by SDS-PAGE under reducing and nonreducing conditions. Immunoglobulin concentration (mg/mL) was estimated by spectrophotometry and densitometry., Results: IgY anti-Giardia duodenalis by CIE had titres up to 1:32. SDS-PAGE, without 2-mercaptoethanol showed a 180 kd band characteristic of the whole to IgY and, with 2-mercaptoethanol, two bands of 68 and 30 kd, characteristic of its light and heavy chains, respectively. The greatest concentrations of immunoglobulin were recovered by method two (M2), producing 4.6 mg of IgY per mL of initial egg yolk., Conclusions: The easy and inexpensive production of IgY anti-Giardia duodenalis in hens is an advantage for using it to develop immunoassays that detect the parasite in fecal eluates.

Introduction: Natural and experimental Giardia infections have been reported from bovines, equines, goats, canines, felines and rodents such as mice, rats and gerbils. The latter have provided successful animal models for Giardia duodenalis and Giardia muris experimental infections., Objective: The gerbil model was used to establish the pattern of infection of Colombian Giardia human isolates., Materials and Methods: Giardia cysts were obtained from stool specimens of symptomatic giardiasis patients by means of sucrose-percoll gradients. Animal inoculation was performed by gastric intubation and injection with 5 x 10(3) Giardia cysts. The course of infection was established by counting cysts every day and trophozoites weekly throughout a period of 30 days., Results: The pattern of cyst excretion was found to be intermittent. Cysts were released during the second and third weeks of infection but not during the first or fourth weeks. The mean minimal number of cysts released per 2-hr collection period was 79 and the mean maximum number was 17,943. Colonization of the small intestine by trophozoites was observed with a mean number ranging from 15,000 to 6,577,778 trophozoites/ml., Discussion and Conclusions: Gerbils inoculated with G. duodenalis isolates obtained from geographical areas outside Colombia resolved the infection between 86 and 114 days after infection, whereas gerbils infected with Colombian G. duodenalis isolates resolved the infection at 30 days. The gerbil proved to be a good animal model for experimental infection with Colombian isolates of G. duodenalis. Experimental Giardia infection of gerbils permit a sufficient yield of cysts and trophozoites to be used as antigens for the immunization of other animals and to obtain Giardia antibodies that could be used for Giardia antigen detection assays in stool specimens.

Little is known about the role of IgA in the immune response against Giardia duodenalis infection. The current study identified the antigens of Colombian G. duodenalis isolates which stimulate the production of IgA anti-G. dudoenalis. Cyst and trophozoite stage proteins were separated by SDS-PAGE and their antigenicity was determined by Western blot. Without 2-mercapto ethanol (2-ME), the protein profile of the cyst stage showed 24 proteins within a molecular weight range of 23-270 kDa; with 2-ME, 35 polypeptides ranging from 22 to 241 kDa were distinguished. The trophozoite stage protein profile without 2-ME was formed by 16 proteins within the range of 24-270 kDa; with 2-ME, 45 proteins were present between 18 and 241 kDa. The identification of 20 and 29 antigens from the cyst and trophozoite stage, respectively, suggested that G. duodenalis stimulates a specific humoral immune response in the human host. The antigens of 31, 57, 110, 133, and 170 kDa recognized by anti-G duodenalis IgA in both cysts and trophozoites corresponded with G. duodenalis isolates from other geographic regions, whereas those of 35, 38, 43, 45, 49, 52, 60, 62, 65, 72, 82, 99, 145, 155, and 185 kDa seemed specific to Colombian isolates. This indicated that antigens of 57, 65, 145, and 170 kDa, recognized by anti-G. duodenalis IgA antibodies in cysts (with frequencies between 82% and 96%) and trophozoites (with frequencies between 86% and 97%) can be considered identification markers for G. duodenalis infections.

Antigen profiles were described for Giardia duodenalis cysts and trophozoites that are recognized by IgG and its anti-G. dudodenalis subclasses (IgG1, IgG2, IgG3, IgG4). Antigens were identified by Western blot from G. duodenalis cyst and trophozoite isolates. Cysts and trophozoites were each subjected to protein separation by SDS-PAGE. The proteins were then transferred to nitrocellulose membranes by electroimmunoblot, and their antigenicity was determined by exposing them to sera from patients with confirmed diagnosis of G. duodenalis infection. The antigen-antibody reaction was revealed by specific alkaline phosphatase antibody conjugates against IgG, IgG2, IgG3, IgG4: bands were visualized by addition of the substrate 5-bromo-4-chloro-3-indolyl-phosphate and the stain nitro blue tetrazolium. The bands were read and analyzed by linear regression using Quantity One software. Thirty two antigens were simultaneously recognized by total IgG anti-G. duodenalis in the cyst and trophozoite stages. The antigens varied in molecular weight from 22 to 185 kDa. Nineteen antigens were identified by both IgG, and IgG3 anti-G duodenalis, with molecular weights ranging from 42 to 180 kDa. IgG2 and IgG4 did not identify any antigen in either stage. The antigens of molecular weights 27, 30, 31, 33, 45, 49, 57, 78, 89 and 170 kDa are shared with G. duodenalis isolates from other geographical regions of Colombia. The recognition of cyst and trophozoite antigens of Colombian G. duodenalis isolates by IgG, IgG1 and IgG3 anti-G. duodenalis suggested that they are involved in the induction of the host immune response.