Human giardiasis is caused by two distinct genetic groups of Giardia duodenalis
, known as assemblages A and B, which are likely to represent distinct species 
. Both assemblages are found associated with human infection globally, and have been also detected in various animals 
At present, various molecular methods are available to distinguish these assemblages, mainly by nested PCR followed by RFLP or DNA sequencing, or by real-time PCR 
. The majority of these assays are based on the amplification of a gene fragment with primers that bind to DNA sequences that are conserved in the two assemblages (or conserved in all G. duodenalis
assemblages or in Giardia
species). Molecular typing techniques have been extensively used to study the complex epidemiology of giardiasis, including controversial aspects like the extent of zoonotic transmission, the occurrence of mixed infection in humans, the potential for genetic exchanges between parasite isolates, and the correlation between clinical symptoms and the type of assemblage 
. In the course of these studies, it has been observed that a multi-locus typing scheme is needed to address those issues 
In this work, we have developed six novel assays that detect and discriminate G. duodenalis
assemblages A and B using a conventional PCR procedure. Based on fixed differences at these loci between the two assemblages, specific primers were designed to generate amplicons of diagnostic size for each assemblage at each locus. The loci targeted by these assays are highly specific for G. duodenalis
as no significant homology with DNA sequences of other organisms were detected by a BLAST search of the GenBank database (update date: 2012/03/15). Therefore, these assays represent reliable tools to identify G. duodenalis
assemblages in human samples and can be used in combination to perform a multi-locus genotype (MLG) analysis by means of a simple PCR protocol that requires only cheap equipments. We further showed that two of the six assays (4E1-HP and 5C1-P21) are particularly well suited to allow for an effective diagnosis of the parasite in human stools with a simultaneous identification of the assemblage. We demonstrated that these two assays work efficiently in the same reaction tube, producing results that are easy to interpret (), are highly sensitive, detecting the DNA equivalent of a single cyst (), and appropriate to detect mixed assemblage A+B infections even if the ratio between the assemblages is strongly unbalanced (). The two targeted loci correspond to single copy genes that are located on different chromosomes in the WB genome, namely chromosome 4 for 4E1-HP and chromosome 1 for 5C1-P21 , and these chromosomal regions are syntenic in assemblage B (data not shown). The robustness of the 4E1-HP and 5C1-P21 assays was confirmed by testing a panel of 51 human isolates from Sweden to compare the rate of amplification and the genotyping results with those obtained using MLG analysis in the original publication 
. The excellent agreement between the two sets of results () indicates that the two PCR assays have sensitivity and accuracy comparable with the most commonly used genotyping tools.
We therefore believe that these assays will find application in studies aimed at understanding some intriguing aspects of giardiasis, the most important of which are briefly discussed below.
The occurrence of mixed A+B infections in human cases of giardiasis appears to be more common than previously believed 
. Thus, the proper detection of both assemblages is an important aspect for molecular epidemiology studies and for routine screening in clinical settings. The reliable detection of cases of mixed infections is influenced by several factors, including the proportion of each assemblage in the specimen and the extent of preferential amplification of one assemblage over the other. The assays presented in this work can detect the least abundant assemblage even when the other assemblage is 9 times more represented in the sample, at least using deliberately mixed genomic DNAs from axenic cultures (). Moreover, the assemblage-specific primers do not compete for the same template, as they bind to different portions of the genes, and this should help to prevent the amplification of the most abundant template when more than one template is present. It is interesting to note that the differences among the various tests, except for false negatives, always involve samples where at least one of the assay indicates a mixed A+B infection. This fact suggests that assays with different sensitivity for one of the two assemblages not always detect correctly mixed infections. In this respect, the 4E1-HP and 5C1-P21 PCR have a proved reliability also in case of strongly unbalanced templates and demonstrated to be sensitive enough to detect mixed assemblages in the presence of a few copies of the Giardia
genomes. Although these novel assays may be less sensitive than nested PCR in absolute terms, they are better suited to identify mixed infections and, along with previously published procedures 
, will consent to address this important aspect in future studies.
Understanding the relative contribution of the parasite's genetic variability and host factors in the establishment of clinical giardiasis has been the subject of a number of studies in both developed and developing countries 
. So far, these studies have reached controversial conclusions. Assemblage A was associated with diarrhea (and other symptoms) in studies in India, Spain and Turkey, whereas an association with assemblage B was reported in Malaysia, The Netherlands and Ethiopia. No association with either assemblage was found in Cuba, The United Kingdom, Brazil and Albania. Whereas those conflicting results can be explained by differences in the study design, in the population considered (adults versus children), and in the definition of symptoms, is presently unknown. However, since generic PCR assays have been used in those studies, the contribution of mixed infections has not been taken into account.
Albeit the assays have been designed and tested on G. duodenalis
isolates from humans, they can be used to detect assemblages A and B in animal samples. It is important to recall that mixed infections involving both host-specific and zoonotic assemblages of G. duodenalis
have been frequently identified in fecal samples from livestock and domestic animals 
. We noted that the primer pairs used in our assays do not match with the homologous sequences of the assemblage E genome 
, at least as inferred by an in silico
analysis (data not shown). Therefore, it should be possible to use these assays to detect assemblages A and B in livestock, albeit a direct testing will be required to rule out unspecific reactions.
In conclusion, two PCR methods are proposed as valuable tools for the molecular diagnosis of human infections caused by Giardia duodenalis. Since these assays do not require particular expertise or expensive instruments, they can be used in laboratories with basic molecular equipments.