Background
Giardia duodenalis is a flagellated protozoan infecting humans and a wide range of animals worldwide, mainly transmitted through food and water contaminated with cysts [
1,
2]. In Asia, Africa, and Latin America, approximately 200 million people have symptomatic giardiasis with some 500,000 new cases being reported each year [
3]. Previous studies on
G. duodenalis had shown that the species comprises eight distinct genetic groups designated as assemblages A to H and which differ on the basis of host occurrence and genomic mutations [
4,
5]. All the assemblages have similar morphology and are indistinguishable using microscopy.
The genotyping of a large number of human
Giardia isolates from different parts of the world revealed that humans are mainly infected with assemblage A or B with assemblage B being the most common [
5]. Moreover these assemblages are found in numerous species of mammals and hence they are considered zoonotic. The assemblages C to H appear to be restricted to animals and are host specific, however occasionally assemblage C and D [
6,
7], E [
8] and F [
9] have been reported in humans.
Three sub-assemblages have been identified within Assemblage A and namely AI, AII and AIII [
5,
10]. The sub-assemblage AI is zoonotic, while subtype AII predominantly occurs in humans [
11] and subtype AIII has solely been identified in animals (mainly wild ungulates) [
12]. Within assemblage B, sub-assemblages BIII and BIV have been identified [
13] and detected in humans, companion animals and wildlife. Studies searching for differences in clinical symptoms between people infected with assemblages A and B have reported varying results. Some studies reported a strong association between intermittent diarrhoea and assemblage A infection while persistent diarrhoea was strongly associated with assemblage B infection, while in others, children infected with assemblage A were more likely to be symptomatic compared with those infected with assemblage B [
14,
15].
The use of multi-locus genotyping approach using
β-giardin,
GDH, Tpi, SSU rRNA,
ef1α, and variant surface protein [
vsp] genes), is the preferred method for studying genetic variability in
G. duodenalis from different hosts [
5,
6]. Moreover the use of primers based on
Tpi marker detected more mixed infections with assemblage A and B than when general PCR primers were used [
16,
17]. In this paper, we report the detection and genetic variability of
G. duodenalis in Human Immunodeficiency Virus infected and/or uninfected children presenting with diarrhoea in outpatient clinics at Mukuru informal settlement on the outskirt of Nairobi, Kenya and those admitted at the Paediatric ward at the Mbagathi district hospital in Nairobi.
Discussion
Giardia duodenalis is among the most common intestinal protozoa and also the most frequent parasitic agent of gastroenteritis especially in the developing countries [
21]. This study provides, for the first time in Kenya, data on prevalence and genetic diversity of
G. duodenalis isolates from children in Kenya.
The genotyping results show that all
Giardia infections in this population are due to
G. duodenalis assemblages A and B. This confirms the results of a number of studies performed elsewhere [
22]. Distribution of different assemblages differs among and within countries, as surveys in several countries showed a diverse prevalence of assemblages A and B [
5]. Here we have shown that children in urban informal settlement in Nairobi, predominantly carry
Giardia assemblage B, which conforms to reports from several other regions of the world [
23‐
28].
Giardia Assemblage B displays high cyst excretion pattern, which in combination with oral-faecal transmission, may contribute to its elevated prevalence rates and broad distribution [
29]. On the other hand, studies carried out in Germany, Uganda, Egypt and Portugal, reported a predominance of assemblage A [
30‐
34]. Although both major
G. duodenalis assemblages A and B have been found in humans throughout the world, their propensity to cause disease might vary.
The predominance of one
G. duodenalis assemblage over another in a particular area has been attributed to biological as well as geographical factors and, in certain endemic areas; all infections due to
Giardia in humans appear to involve just one assemblage [
35]. The reasons behind the geographic variation in the predominance of the
Giardia assemblages are still unclear. It may be explained by the difference in the dynamics of transmission. It has been known that assemblage A is most often responsible for zoonotic transmission with wide range of animals acting as reservoir hosts. Although assemblage B is most likely transmitted from human to human, it has been reported in some animals and may represent a zoonotic potential as well [
5,
25,
35,
36].
In this study 76 % of the samples amplified successfully with
GDH primers. Most samples analysed at this locus were identified as assemblage B, except 2 that were assemblage A. These isolates were further identified as sub assemblages AII, and both occurred as mixed infections with sub- assemblage BIII. Results of previous studies elsewhere have shown that humans are mostly infected with AII, although AI is also seen in some studies, while animals are mostly infected with AI, with AII being occasionally reported [
22,
25,
37]. The AI sub-assemblage and the B assemblage, regardless of the B sub-assemblages, have a broad host range, including pets, wildlife, and livestock while the AII sub-assemblage is more limited to human subjects [
3]. Thus, it is possible the AII infections reported in this study were anthroponotic, while the B infections could have been either zoonotic or anthroponotic. Human to human transmission of
Giardia infection in the study area could have been exacerbated by the water shortage, and poor sanitary conditions in the slum areas, which has a direct effect on hygiene.
Our study identified both sub-assemblages BIII and BIV in the population, with BIV being commonly isolated. Sprong [
11] reported that in Africa, infection with
G. duodenalis assemblage B, sub-assemblage BIII was more prevalent than infection with sub-assemblage BIV, whereas this differed from findings in North-America where more infections were associated with sub-assemblage BIV, and only few with sub-assemblage BIII, with more balanced distribution being found in Europe and Australia [
5]. This however differs with our findings, where BIV is more prevalent. Our study however agrees with findings from Thailand where Assemblage B, sub-assemblage BIV was found to be the most common in preschool children [
38].
Occurrence of mixed infections in human cases of giardiasis involving various assemblages appears to be more common than previously thought [
16,
17].
Tpi assemblage specific primers have proved reliable enough to detect mixed assemblages in the presence of a few copies of the
Giardia genomes [
8,
17,
39]. Co-infection by both
Giardia assemblage A and B which was observed in 6 cases has been previously reported in Ethiopia and Rwanda [
9,
25]. Co-infections with other rare assemblages have also been observed in Ethiopia, where mixed infections with A+ F were reported. In our study mixed infections with sub-assemblage BIII and BIV were frequently observed in 28(56 %) of cases, while AII&BIII was observed in 2 cases. Remarkably, mixtures between BIII and BIV have been previously commonly reported [
11]. The occurrence of mixed infections by several assemblages/sub-assemblages of
G. duodenalis reflects the complex circulation of the parasite in the environment and the exposure of the study population to multiple sources [
40].
Phylogenetic analysis of the isolates after bi-directional sequencing of the
β giardin gene showed that the assemblage B test isolates, formed two clusters. This could be attributed to genetic variation between reference sequences and the test samples, which after comparison of base pair position with the reference B assemblages revealed sequence profile variation within our isolates. A high degree of polymorphism in assemblage B has been observed in other studies [
10,
28,
41], and has been further investigated by cloning [
42,
43]. This feature has been attributed to mixed subtype infections or allelic sequence divergence, or a combination of both. Assemblage B Kenyan isolates, formed two sub-grouping (Assemblage B, Assemblage B, cluster II). This could be attributed to genetic variation between reference sequences (AY072726.1, AY072725.1). Comparison of base pair position between, reference B assemblages, revealed sequence profile variation within the test isolates from the GenBank.
There was good agreement between assignment of assemblages at all three loci, with assemblage swapping (i.e., different assemblages at different loci in the same isolate) not being observed in any of the isolates. Assemblage swapping has been reported by other investigators [
10,
21,
26] and has been attributed to recombination between assemblages or mixed assemblage infection.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MC and KS conceived and designed the study protocol and questionnaires for interviews. MC, GF and ME conducted interviews, performed data and stool collection and provided laboratory analyses of stool samples. WA, WJ and MC did the data analysis. Planning, coordination and supervision of data collection in the field, data entry and cleaning, and writing up of the manuscript was done by MC. KS, ME, WJ, WA and NZK critically revised the manuscript. The final version of the manuscript was reviewed and approved by all authors prior to submission.