Description of genotyping.
A total of 104 families were enrolled in the study—43 families in 2002 and 61 families in 2003 (see Table 1). Altogether, they contributed 1531 human and 605 canine samples. Giardia
cysts or trophozoites were detected by microscopy in 313 human samples, and 178 of the 313 positive samples were selected for genotyping (). The 178 were chosen by giving priority to isolates from families with dogs and from those with >1 infected person. They were typed using a nested PCR assay described by Amar et al [25
] that distinguishes genotypes A1, A2, and B. Among the 178 samples chosen for genotyping, there were no A1 isolates, 64 A2 isolates, 78 B isolates, 22 mixed A2/B isolates, and 12 samples that were not successfully amplified ().
In addition, a second approach described by Sulaiman et al [24
] that distinguishes genotypes A, B, C, and D was used for 114 of the 178 isolates, primarily to determine whether any humans were infected with genotypes C or D [24
] (). None of these 114 samples were from genotypes C or D. Although our study was not specifically designed to compare the results of the 2 typing methods, a few comments regarding the comparison are in order. For the isolates that were analyzed by both methods, the concordance was reasonably good (). Of the 32 that were typed as genotype A2 by the Amar approach and tested by the Sulaiman approach, 2 (6%) were classified as genotype B. One of these 2 genotypes was sequenced and confirmed as genotype A2. Of the 63 isolates classified as genotype B by the Amar method and tested by the Sulaiman approach, 1 (2%) was classified as genotype A. The majority of those classified by the Amar approach were successfully sequenced at 1 or both of the chromosome 3 and 5 loci. Of the 66 that were classified as genotype A2 by the Amar method, 59 were sequenced and all were confirmed as genotype A2. Of the 80 that were classified as genotype B, 62 were sequenced and 59 were confirmed as genotype B, whereas 3 were confirmed as genotype A2. Thus, the accuracy of the PCR-based method described by Amar et al [25
] was 100% for the classification of genotype A2 isolates, whereas the accuracy for classification of genotype B isolates was 95%.
Subtyping and evidence for recombination among isolates.
The A2 isolates were also subtyped at 2 sites: (1) adjacent to tpi
(chromosome 5) and (2) adjacent to γ-giardin (chromosome 3) by sequence analysis of uncloned PCR products. The tpi
and γ-giardin genes themselves showed no polymorphism, but these adjacent regions had numerous polymorphisms that allowed accurate sequence-based haplotyping within the genotype A2 isolates () [16
]. There were 5 informative sites at the chromosome 3 locus and 7 informative sites at the chromosome 5 locus. We were able to type a total of 80 samples from 33 individuals at both loci and have named them according to previously reported haplotypes [16
]. When identical samples from individuals were omitted from analysis, there were 27 samples, and when duplicated types within a family were included, there were 19 samples. The 4 subtypes at the chromosome 3 locus and the 5 subtypes at the chromosome 5 locus allow a possible 20 recombination types, and 12 of those 20 were represented in this data set ().
Haplotype Assignment Based on A2 Single-Nucleotide Polymorphism (SNP) Sites
A2 Subtypes Detected in This Study
For the genotype B isolates, there were numerous polymorphisms within the tpi and g-giardin reading frames, so these regions were sequenced. There were 106 polymorphic sites, and in all but one, these were transition substitutions (A/G or C/ T). However, at all but 3 of these polymorphic sites, there were some sequences with double peaks on the chromatogram. These double peaks could result from mixed genotype B infections or from allelic sequence heterozygosity (ASH). We have sequenced multiple cloned PCR products from some samples (data not shown), and in these cases, the data are more consistent with ASH than with mixed infection.
In some cases, different samples from a single individual or a single family varied among each other (eg, A, G, or double-peak purine). For this reason, we considered these sites ambiguous and were unable to assign subtypes for the genotype B isolates. In contrast, there were only 5 potential ASH sites among the genotype A2 isolates, all at the chromosome 5 locus.
The genotype A2 isolates are distinct from genotype B isolates within the tpi and γ-giardin reading frames and demonstrated no polymorphism among themselves. Thus, there is no evidence for recombination between genotype A2 and B isolates at these loci.
Potential for transmission from dogs to humans.
Sixty-seven of the 88 canine fecal samples from 46 dogs that were positive for Giardia
by microscopy were successfully amplified and genotyped by a previously described method that uses sequencing of a portion of the SSU-rDNA [27
] (). Thirty-two dog samples had the D genotype, and 9 samples had the C genotype. Twenty-six had mixed C/D genotype infections that were detected by a double peak in the chromatogram. For 13 of the 19 dogs that were sampled more than once, the genotype changed (C, D, or mixed). Each of the 88 dog samples was also tested with primers designed to amplify and distinguish DNA from A1, A2, B, or mixed A/B infections [25
]. This approach had the advantage of being able to detect coinfections in which the A or B genotypes were present in relatively small quantities in comparison to the C and D genotypes. Using this approach, a single dog sample had a genotype B isolate (see Figure 2, family 95). This dog had samples collected in 3 successive weeks, all with genotype D, and in the middle week had mixed genotypes B and D. The rDNA typing had identified only genotype D, suggesting that D was present in greater quantity. These findings are more indicative of anthropozoonotic transmission with transient genotype B colonization of the dog or of mechanical passage of cysts that had been ingested from human feces than they are of zoonotic transmission from dog to human.
Transmission patterns within families. Twenty-two families representing 120 individuals were chosen for more detailed analysis because they had multiple G. lamblia -infected family members, and 8 of these families had 1 or more dogs (). The infection patterns for 8 of these families are shown in greater detail in Figure 2.
Summary of 22 Families with Multiple Positive Samples
Different members of a family harbor different genotypes. Individual B from family 106 was infected with a genotype B isolate on multiple occasions over both years, whereas individuals C and E were infected with the same subtype of genotype A2. Likewise, the 2 members of family 61 carried distinct subtypes of A2.
Mixed infections or changing genotypes. Family 100 is particular instructive in that 4 individuals were initially infected with genotype B, and then genotype A2 was introduced by individual c. By year 2, the 2 individuals had genotype A2 only, whereas another 2 had genotypes A2+B. Also of note is that 2 subtypes of A2 were found in this family with 335/246 in individual D and 335/335 in individuals E and H. (Individual F could not be typed at locus 2.) The presence of multiple genotypes and subtypes within single households creates an optimal setting for mixed infections to occur, which in turn could facilitate recombination among isolates.
Spatial analysis of G. lamblia infections. A map showing the spatial distribution of families and genotype/molecular type information was generated (). These data indicate that genotypes A2 and B were widely distributed within the population, an observation that is consistent with the frequent observation of mixed A2 and B infections.
Figure 3. Spatial distribution of Giardia genotypes in the study area. The households in which Giardia was identified are shown as circles on this map of the Pampas de San Juan de Miraflores. The households with only genotype A2 are shown with open circles, and (more ...)