In the present study asymptomatic carriage of G. duodenalis (17%), Blastocystis sp. (13%), and Cryptosporidium spp. (0.9%), but not E. bieneusi, was reported in apparently healthy schoolchildren in Leganés (Madrid); although unadjusted for clustering within schools, this is likely to be a fair estimate. This is the largest PCR and sequencing-based molecular survey conducted in asymptomatic individuals in Spain to date. Molecular data generated here were strengthened by the adoption of a multi-locus genotyping scheme for the molecular characterization of samples positive to G. duodenalis and Cryptosporidium spp.
In Spain, asymptomatic carriage of G. duodenalis has been previously reported in pre-schoolchildren (prevalence: 15%‒25%) and schoolchildren (prevalence: 3%‒36%) populations from rural and urban areas by conventional methods including light and immunofluorescence microscopy, ELISA for the detection of copro-antigens or rapid diagnostic tests [24, 27, 40]. Genotyping data are, however, far scarcer. Assemblage B (57%) was the predominant G. duodenalis genetic variant detected in schoolchildren in the Álava province (northern Spain), followed by sub-assemblage AII (29%) and AII + B mixed infections (14%) [27]. BIV was the only sub-assemblage found in a ensuing survey conducted in the same region [41], and in children attending day care centres in the Madrid province, central Spain [40].
Giardia duodenalis prevalence (17%) and genotyping (AII: 17%; BIV: 79%: BIII/BIV: 4%) data presented here agreed well with the figures reported in the studies mentioned above, corroborating that assemblage B is 2‒3 fold more prevalent than assemblage A in Spanish asymptomatic children populations. This is in sharp contrast with the results obtained in a recent molecular study conducted in G. duodenalis-positive outpatients of all ages attending hospital settings in 10 Spanish provinces showing that asymptomatic infection was more common in individuals with assemblage A than in those with assemblage B (14% versus 1.5%, n = 29) [42]. Strikingly, the G. duodenalis genotype frequencies shown here are very similar to those (AII: 15%; BIV: 62%: BIII/BIV: 1.6%, n = 124) previously reported in patients with gastrointestinal complaints attending two major public hospitals in the Madrid province [38]. Not coincidentally, one of these hospitals was the University Hospital Severo Ochoa located in Leganés, whose catchment area was the very same investigated in the present study. It should be highlighted that these similitudes were also present at the nucleotide level, as gdh and bg sequence variants generated in both studies were observed at similar proportions. Overall, these data seem to indicate that essentially the same G. duodenalis genotypes are circulating in asymptomatic carriers and symptomatic patients in the Madrid province. Different assemblage A/B ratios have been identified in other Spanish regions [9, 43], suggesting that the frequency and diversity of G. duodenalis genotypes may be geographically dependent.
In this study the presence of Cryptosporidium spp. was confirmed in a very low proportion (< 1%) of asymptomatic schoolchildren. Infection rates ranging from 1–10% have been previously estimated by non-molecular methods in children attending day care centres in Madrid [40] and the Salamanca province, western Spain [44], and in schoolchildren in the Álava province [27, 41]. When available, molecular data in these surveys showed the presence of C. hominis only [27]. Our sequence analyses revealed that C. hominis was more prevalent than C. parvum (71% versus 21%, n = 14). Similar proportions have been previously described in clinical samples in different Spanish regions including Barcelona (88% versus 10%, n = 69) [45], Galicia (65% versus 34%, n = 486) [46], La Rioja (82% versus 19%, n = 81) [9], and Madrid (82% versus 13%, n = 108) [47]. These clinical studies also revealed that Ib (predominantly IbA10G2) and IIa (predominantly IIaA15G2R1) were the most frequent gp60 genotype families within C. hominis and C. parvum, respectively. As in the case of G. duodenalis, these data indicate that C. hominis and C. parvum are circulating at similar frequencies in both asymptomatic and symptomatic (clinical) populations in Spain. Absence of canine- (C. canis), feline- (C. felis), livestock (C. andersoni, C. bovis) or avian-specific (C. meleagridis) Cryptosporidium species seem to suggest that the spreading of the infection in the paediatric population under study is mainly through a human transmission cycle.
The Blastocystis sp. carriage rate estimated here (13%) was well in the range of those (8%‒23%) previously reported by microscopy examination in preschool- and schoolchildren in Salamanca province [48]. Infection rates identified in earlier clinical studies in the country ranged from 3%‒7% in symptomatic outpatients to 10% in HIV-infected children [49, 50]. In Europe, ST1 to ST4 are the most common Blastocystis subtypes circulating in humans [51]. This is also the case of the present study, where ST2 (36%), was the most prevalent genetic variant of the parasite, followed by ST1 (23%), ST3 (22%) and ST4 (19%). The finding of ST8 (0.6%) can be considered as sporadic. These data indicate that the spreading of Blastocystis sp. in the paediatric population investigated is primarily through a human transmission cycle. Of note, the same results (although with a higher prevalence rate of 62% for ST2) have been documented in a community survey involving 179 individuals of all ages conducted in Álava province [26]. In both surveys ST4 appeared to be considerably less represented (7.4%‒19%) than ST1-ST3. Interestingly, ST4 has been identified in 94% of Blastocystis mono-infected patients with diarrhoea in Valencia province, central-east Spain [23]. Similar findings have been reported in patients presenting with acute diarrhoea in Denmark [52] and in patients suffering from irritable bowel syndrome (IBS) and chronic diarrhoea in Italy [53]. Additionally, the relative frequency of ST4 seems to be higher than those for ST1-ST3 in patients with diarrhoea in hospital settings in Spain [54]. Taken together, these data suggest that ST4 could be more pathogenic than other Blastocystis STs. Supporting this hypothesis, human acquisition of the ST4 lineage, very likely from wild rodents, has been proposed as a relatively recent evolutionary event [55]. Poor host adaptation may result, therefore, in increased parasite virulence. However, all these lines of evidence should still be considered with caution, as other investigations failed to demonstrate the pathogenic nature of ST4 [56, 57]. Moreover, other research groups have proposed a link between ST1 and the aetiology of IBS [58], or between ST3 and gastrointestinal disorders including diarrhoea [59].
It is well known that ST5 to ST9 are reported in humans only rarely [51]. In the present study ST8 was identified in a 10-year-old female reporting no contact with companion animals and no obvious risk factors for parasite infections. ST8 carriage has been previously documented almost exclusively in captive and free-living non-human primates in Central America [60, 61], South America [62, 63], and Europe [64]. Remarkably, an unexpectedly high prevalence of ST8 was seen among primate handlers in a zoological garden in UK, suggesting that zoonotic transmission of Blastocystis ST8 infections from primates to their handlers had occurred [64]. In our study, the source of infection by ST8 remains unclear.
Intriguingly, the microsporidia E. bieneusi was apparently absent in the surveyed, apparently healthy, schoolchildren population. In the only published study conducted by PCR in asymptomatic healthy individuals, E. bieneusi was detected in 6.0% (23/382) of people of all age groups living in the Czech Republic [65]. In that survey only four out of the 23 detected cases of microsporidiosis by E. bieneusi occurred in children younger than 12 years of age. Taken together, these data are indicative of an age-related pattern of infection, with older individuals being more likely to harbour the parasite than younger ones. To date, E. bieneusi infections in Spain have been only identified in HIV/AIDS patients [50, 66], transplant recipients [67], returning travellers [68], elderly people [69], and immunocompetent clinical populations [70].
This study presents some limitations. For instance, a relatively low proportion of G. duodenalis-positive samples were successfully genotyped at the assemblage and sub-assemblage level. This is a direct consequence of the high Ct values obtained by qPCR in most of the samples tested, a fact that compromised the diagnostic performance of the (single-copy gene) PCR methods used for genotyping purposes. The same is also true for the failure to determine C. hominis/C. parvum subtypes at the gp60 marker. These are somehow expected results, as asymptomatic carriage of intestinal parasites correlates well with light infections. Regarding Microsporidia, this study focuses exclusively on E. bieneusi. However, previous studies have revealed that microsporidia species belonging to the Encephalitozoon genus were frequently found in asymptomatic, apparently healthy individuals [65]. Investigating the presence of Encephalitozoon spp. remains a task to be investigated in future surveys.