Comparison of gdh polymerase chain reaction-restriction fragment length polymorphism and tpi assemblage-specific primers for characterization of Giardia intestinalis in children.

Background: Giardia is a diarrheagenic eukaryotic parasite that consists of at least eight morphologically identical but genetically distinct genotypes. Human giardiasis is caused mainly by A and B assemblages. Aim and objectives: The study aimed to compare the performance of gdh polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and tpi assemblage-specific primers in genotyping of G. intestinalis. Materials and
Methods: Stool samples of 315 children were microscopically screened for G. intestinalis. Positive samples were genotyped using tpi assemblage-specific primers and gdh semi-nested PCR-RFLP techniques.
Results: The prevalence of Giardia was 18.1%. The detected genotypes using tpi and gdh approaches were assemblage A (15.8% vs. 12.7%) and assemblage B (36.8% vs. 74.5%) as single infections and mixed assemblages A and B (47.4% vs. 12.7%). The two approaches showed a moderate agreement (kappa index = 0.413, P < 0.001). PCR-RFLP of gdh gene revealed that sub-assemblages BIII and BIV were equally detected (30.9% each). The remaining samples were equally divided between sub-assemblage AII, mixed BIII and BIV, and mixed AII and BIII (12.7% each). A significant association was detected between the retrieved sub-assemblages and the presence of symptoms. Conclusions: Although both approaches confirmed the predominance of assemblage B, the use of assemblage-specific primers is more effective in elucidating the true picture of mixed assemblage infection. Copyright:

INTRODUCTION

Giardia intestinalis is a frequently identified intestinal parasite that infects humans and other mammals worldwide. The highest prevalence of giardiasis is consistently reported among children aged 1–9 years both in developed and developing countries.[1] Giardiasis is transmitted by the fecal-oral route following exposure to Giardia spp. cysts in contaminated food and water, or through direct contact with infected individuals. It may present by loss of appetite, nausea, flatulence, diarrhea or greasy stool, abdominal cramps, and weight loss. However, most cases are asymptomatic or minimally symptomatic.[2]

It is well documented that G. intestinalis represents a species complex enclosing at least eight major genetic assemblages, each with a different host range and specificity.[3] Based on sequencing data from multiple loci, human infection is mainly caused by assemblages A and B which are subclassified into five sub-assemblages named AI-III and BIII-IV, some of them claimed to have zoonotic potential.[1] The remaining six assemblages (C to H) are specific to animals although assemblages C, D, E, and F have been reported to cause human giardiasis in rare cases.[456]

Polymerase chain reaction (PCR) assays have been widely applied for the diagnosis of Giardia in clinical and environmental samples. They have provided a powerful analytical tool for the identification of different species and genotypes of Giardia for taxonomical and epidemiological research.[78] Several genes are commonly used as markers for detection and genotyping of Giardia including ssu-rRNA, bg, gdh, tpi, ef1α, and vsp. Considering the variation between these genes, some are more conserved (ssu-rRNA) while others are more variable allowing genotyping and subtyping.[9]

The commonly used tool for differentiation of Giardia assemblages has been conventional PCR together with either direct sequencing or analysis of restriction fragment length polymorphism (RFLP).[10] PCR with assemblage-specific primers[11121314] and real-time PCR with assemblage-specific probes[1516] have also been tried. Furthermore, multi-locus genotyping has evolved to be a valuable genotyping method.[17] It has been noted that the sensitivity of different molecular tools relies upon DNA extraction methods, primers used, and targeted genes as certain isolates can be amplified at one locus but not at another.[18]

Considering the different molecular approaches used for identification of G. intestinalis assemblages, this study aimed to compare the performance of gdh PCR-RFLP and tpi assemblage-specific primers in genotyping of G. intestinalis and to study the association of the retrieved assemblages/sub-assemblages with clinical presentations.

MATERIALS AND METHODS

Study population

A total of 315 children aged 2–16 years living in the West Delta Region in Egypt were enrolled in the study during the period from September 2018 to February 2019. Based on clinical presentation, they were categorized as “asymptomatic” if they had no gastrointestinal symptoms or “symptomatic” if they had diarrhea, abdominal pain, nausea/vomiting, or flatulence.

Ethical principles approved by the Research Ethics Committee of the Medical Research Institute, Alexandria University, Egypt (IORG0008812), were followed in the present study.

Collection and examination of stool samples

A single stool sample was collected from the enrolled children in clean, dry containers with tight-fitting lids. Each container was labeled with the child's name and number. Samples were thoroughly homogenized and divided into two parts: one for microscopic examination and the other part for molecular investigations. Stool samples were examined using wet mounts and formalin-ethyl acetate sedimentation technique to detect G. intestinalis and other parasites.[19]

Molecular analysis

Genomic DNA was extracted from fresh Giardia-positive fecal samples using QIAamp® Fast DNA Stool Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions with some modifications: boiling in InhibitEx buffer for 0.5 h at 95°C and incubation with proteinase K for 1 h at 70°C. Extracted DNA was quantified using NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA). The purity was detected by the ratio of absorbance at 260/280 and 260/230. The integrity of DNA was assessed by agarose gel electrophoresis.

A partial sequence of the tpi gene was amplified using assemblage-specific primers described by Bertrand et al. in separate reactions. For assemblage A, tpi A forward primer: 5’-GGA GAC CGA CGA GCA AAG C-3’and tpi A reverse primer: 5’-CTT GCC AAG CGC CTC AA-3’ were used. For assemblage B, tpi B forward primer: 5’-AAT AGC AGC ACA RAA CGT GTA TCT G-3’ and tpi B reverse primer: 5’-CCC ATG TCC AGC AGC ATC T-3’ were used. These primers amplify a 148-bp fragment of assemblage A tpi gene and an 81-bp fragment of assemblage B, respectively.[20] The PCR reaction included 100 ng of template DNA, 10 pmoles of each primer, and 12.5 μl of 2X MyTaq™ Red Mix (Bioline, UK) in a reaction volume of 25 μl. The mixture was then transferred into a thermal cycler (Applied Biosystems, USA). The cycling conditions were initial denaturation at 95°C for 5 min, followed by 50 cycles of 30 s at 94°C, 30 s at 62°C, and 30 s at 72°C, with a final extension at 72°C for 7 min.

A partial sequence of gdh gene (~430 bp) was amplified by the semi-nested PCR described by Read et al.[10] For the primary reaction, the forward primer GDHeF (5’-TCA ACG TYA CGG GYT TCC GT-3’) and the reverse primer GDHiR (5’-GTT RTC CTT GCA CAT CTC C-3’) were used. For the secondary reaction, the forward primer GDHiR (5’-CAG TAC AAC TCY GCT CTC GG-3’) and the reverse GDHiR were used. Primary and secondary PCR reactions were performed in a 25-μL total volume comprising template DNA (100 ng for the primary reaction or 1 μl of the first PCR product for the secondary reaction), 10 pmoles of each primer, and 12.5 μl of MyTaq™ Red Mix. The cycling conditions were initial denaturation at 95°C for 5 min, followed by 35 cycles of 30 s at 94°C, 20 s at 56°C, and 45 s at 72°C with a final extension at 72°C for 7 min.

PCR products were separated by horizontal electrophoresis in 2.5% agarose gels with ethidium bromide staining (0.5 μg/ml). A 50-bp DNA ladder (Thermo Fisher Scientific, UK) was included as a size marker. PCR products were visualized under ultraviolet transilluminator (Major Science, Taiwan). In all the PCR reactions, a Giardia-positive DNA specimen and distilled water were included in each round of PCR as positive and negative controls, respectively, to validate results.

The gdh amplified products of the secondary reactions were purified from agarose gel using FastGene®gel/PCR extraction kit (Nippon Genetics, Europe) according to the manufacturer's instructions. 10 μl of the purified products was separately digested using 10 U of Rsa I and Nla IV (Thermo Fisher Scientific, UK) in 1X Tango buffer in a final volume of 30 μl for 3 h at 37°C. Restriction profiles were interpreted according to the patterns described by Read et al. for different Giardia assemblages.[10]

DNA sequencing

The purified gdh amplicons of six samples typed as assemblage A, B, or mixed A/B were sequenced using Applied Biosystems 3500 DNA Analyzer (Applied Biosystems, USA). The chromatograms and sequences were viewed using BioEdit Sequence Alignment Editor Program (http://www.mbio.ncsu.edu) to detect heterozygous positions that suggest allelic sequence heterozygosity (ASH) or mixed infections. Preliminary similarity comparison with other sequences in GenBank database was made using Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov).

Statistical analysis

Results were analyzed using the Statistical Package for the Social Sciences (SPSS) software version 20.0. (IBM Corp; Armonk, New York). The Chi-square test was used to study the association between categorical variables, and pairwise Z-test was used to compare column proportions. Monte Carlo corrections were used as a correction for Chi-square when more than 20% of the cells have an expected count <5. Kappa statistic was used to determine the degree of agreement between the two methods of genotyping. The kappa value was interpreted as slight agreement (k < 0.2), fair agreement (k = 0.2–0.4), moderate agreement (k = 0.4–0.6), substantial agreement (k = 0.6–0.8), and almost perfect agreement (k > 0.8). P < 0.05 was accepted as statistically significant.

RESULTS

Among the 315 examined children, parasitic infections were detected in 183 samples (58.1%). G. intestinalis was the second most common parasite, being detected in 57 children (18.1%) next only to Blastocystis spp. (46%). Other detected parasites were Entamoeba coli (6.6%), Entamoeba histolytica/dispar complex (2.9%), Ascaris lumbricoides (2.5%), Hymenolepis nana (1.5%), Enterobius vermicularis (0.9%), and Schistosoma mansoni (0.3%).

DNA extraction was performed on the 57 Giardia-positive samples. Tpi gene was detected in all microscopy-positive samples (100%) using assemblage-specific primers [Figure 1a]. Mixed infection with assemblages A and B was detected in 27 samples. This was the most common infection form (47.4%), followed by assemblage B in 21 samples (36.8%) and then assemblage A in 9 samples (15.8%), as shown in Table 1. Gdh gene amplification using semi-nested PCR approach was successful in 55 samples (96.5%) and the remaining 2 samples (3.5%) consistently gave short nonspecific bands. PCR-RFLP of gdh gene revealed the presence of sub-assemblages AII, BIII, BIV, or a mixture of two sub-assemblages. Examples of the obtained patterns are shown in Figure 1b. Each of sub-assemblages BIII and BIV was singly detected in 17 samples (30.9%). The remaining samples were equally divided between sub-assemblage AII, mixed BIII and BIV, and mixed AII and BIII, each in 7 samples (12.7%) [Table 1].

Figure 1

(a) Polymerase chain reaction detection of Giardia assemblages A and B using tpigene. M, 50-bp molecular weight marker (Thermo Fisher, USA); lane 1: TpiA-positive sample; lane 2: Negative control (no target); lane 3: TpiB-positive sample. (b) Polymerase chain reaction-restriction fragment length polymorphism of Giardia gdh gene with Nla IV and Rsa I enzymes. M, a 50-bp DNA marker; lane 1: Crude extracted genomic DNA; lane 2: 430-bp fragment of undigested gdhgene; lanes 3 and 4: Assemblage BIII samples digested with Nla IV (3) and Rsa I (4); lanes 5 and 6: Mixed assemblage AII and BIII samples digested with Nla IV (5) and Rsa I (6); lanes 7 and 8: Assemblage AII samples digested with Nla IV (7) and Rsa I (8)

Table 1

Distribution of Giardia intestinalis assemblages/sub- assemblages based on tpi assemblage-specific primers and gdh polymerase chain reaction-restriction fragment length polymorphism

Assemblages	Tpi assemblages, n (%)	Gdh sub-assemblages, n (%)
Assemblage A	9 (15.8)	AII	7 (12.7)
Assemblage B	21 (36.8)	BIII	17 (30.9
		BIV	17 (30.9)
		Mixed BIII and BIV	7 (12.7)
		Total B	41 (74.5)
Mixed A and B assemblages	27 (47.4)	Mixed AII and BIII	7 (12.7)
Total	57 (100)		55 (100)

Comparison of genotyping results of tpi assemblage-specific primers and PCR-RFLP of gdh gene revealed that mixed assemblage A and B infection was more detected through tpi PCR protocol (47.4%) than PCR-RFLP approach (12.7%). Table 2 shows the agreement between tpi and gdh genes in the detection and genotyping of G. intestinalis. Seven assemblage A samples, 21 assemblage B samples, and 7 mixed assemblage A and B samples gave concordant genotyping results using both approaches. Analysis of 22 discordant results revealed that two samples were missed by gdh approach and that 19 cases were genotyped as assemblage B using gdh approach despite being mixed A and B using tpi assemblage-specific PCR approach. Finally, one sample was genotyped as assemblage B using gdh approach and as assemblage A using the tpi approach. Statistical analysis showed a kappa index of 0.413 indicating a moderate agreement between both approaches.

Table 2

Agreement between polymerase chain reaction-restriction fragment length polymorphism of gdh and tpi assemblage-specific primers in genotyping of Giardia intestinalis

Genotyping approach	Tpi assemblage-specific genotyping				Total
	A	B	Mixed A and B	Negative
Gdh PCR-RFLP genotyping
A	7	0	0	0	7
B	1	21	19	0	41
Mixed A and B	0	0	7	0	7
Negative	1	0	1	0	2
Total	9	21	27	0	57

Kappa index=0.413, P<0.001. PCR-RFLP: Polymerase chain reaction-restriction fragment length polymorphism

Sequencing was successful in five out of six samples which were deposited in the GenBank [Table 3]. The sequence of sample 26 was 99.74% identical to AII reference strain (accession number L40510) from position 4 to 404, the sequence of sample 77 was 99.24% identical to the same reference strain from position 1 to 396, whereas the sequence of sample 112 showed 100% identity from position 7 to 400. Assemblage B isolates (samples 41 and 97) had high nucleotide variations, hindering its classification into sub-assemblages [Table 4]. The failed sequence (sample 36) was omitted as it contained recombinant sequences consistent with both assemblages A and B. Inspection of the chromatograms of samples 41, 77, and 97 revealed the presence of heterogeneous bases in the form of double peaks in certain positions, as depicted in Figure 2.

Table 3

Different genotyping results of sequenced samples and their GenBank accession numbers

Sample code	Genotypic pattern		Virtual digestion of the obtained gdh sequences		GenBank accession numbers
	Tpi approach	Gdh approach	Nla IV	Rsa I
26	A	AII	87, 85, 77, 70, 39, 16	227, 132, 9, 6	MK959769
36	A, B	AII and BIII	No site	No site	Failed
41	B	BIII and BIV	289, 110	No site	MK659644
77	A, B	AII and BIII	87, 85, 77, 70, 39, 16	227, 132, 9, 6	MN018401
97	B	BIV	289, 19	No site	MN018400
112	A, B	AII	109,87, 77, 70, 39,16	251, 132, 9, 6	MK659645

Table 4

Multiple alignments of gdh sequences from this study with the reference sequences of sub-assemblages AI, AII, BII, and BIV obtained from GenBank

Isolates	GenBank accession numbers	Nucleotide position from the start of the gene
Assemblage A		489		492		494		496		497	498		499	826		844	877		889
AI	M84604	G		G		G		G		C	T		C	T		C	G		C
AII	L40510	.		.		.		C		.	.		.	C		T	.		.
AII (26)	MK959769	C		A		.		.		.	.		.	C		T	.		.
AII (77)	MN018401	-		-		-		-		-	.		.	C		T	A		T
AII (112)	MK659645	-		-		A		.		G	C		T	C		T	.		.
Assemblage B		493	494	497	499	502	505	506	507	532	580	583	584	652	670	763	784	820	835	889
BIII (BAH-12)	AF069059	T	G	C	C	C	C	C	A	C	T	G	A	T	T	C	C	C	G	T
BIV (Ad-7)	L40508	.	.	.	.	.	.	.	.	T	.	.	.	C	C	T	T	.	A	.
41	MK659644	A	A	G	T	.	.	.	.	.	C	.	.	.	.	T	.	.	.	C
97	MN018400	N	N	N	T	G	T	T	C	.	C	N	N	C	C	T	.	T	A	C

Numbers in bold represent nucleotide substitutions from the start of the gene. Dots (.) indicate nucleotide identity. Dash (-) indicates unbeginning of the sequence. Accession numbers in bold are reference sequences from the GenBank

Figure 2

Chromatogram showing heterozygous positions (indicated by arrows)

The association of gastrointestinal symptoms with Giardia sub-assemblages (determined by the gdh approach) was statistically significant (P = 0.012). Pairwise comparison revealed that sub-assemblage BIII is significantly associated with asymptomatic infection as it has been found in 58.8% of asymptomatic infection compared to 18.4% of symptomatic infection. Sub-assemblage AII infection alone or even when mixed with sub-assemblage BIII always had symptomatic presentations. Sub-assemblage BIV constituted more or less comparable percentages among asymptomatic and symptomatic children (29.4% and 31.6%, respectively). The same was observed for mixed sub-assemblage BIII and BIV infection which represented 11.8% of asymptomatic and 13.2% of symptomatic infection. Similar findings were observed after excluding samples with discordant genotyping results [Table 5].

Table 5

Association of clinical presentations with Giardia intestinalis assemblages/sub-assemblages in the examined children

Sub-assemblage (gdh) (n=55)	Infection status		P (MC)
	Asymptomatic (n=17), n (%)	Symptomatic (n=38), n (%)
AII (n=7)	0a (0)	7a (18.4)	0.012*
BIII (n=17)	10a (58.8)	7b (18.4)
BIV (n=17)	5a (29.4)	12a (31.6)
BIII- BIV (n=7)	2a (11.8)	5a (13.2)
AII-BIII (n=7)	0a (0)	7a (18.4)
Sub-assemblage concomitant (gdh)# (n=35)	Asymptomatic (n=8), n (%)	Symptomatic (n=27), n (%)	P (MC)
AII (n=7)	0a (0)	7a (25.9)	0.011*
BIII (n=10)	6a (75)	4b (14.8)
BIV (n=8)	1a (12.5)	7a (25.9)
BIII-BIV (n=3)	1a (12.5)	2a (7.4)
AII-BIII (n=7)	0a (0)	7a (25.9)

*Statistically significant (P<0.05), # Samples with discordant gdh and tpi assemblage genotyping were excluded. Similar superscript letters denote a subset of infection categories whose column proportions do not differ significantly. MC: Monte Carlo for Chi-square test

DISCUSSION

Genotyping of Giardia isolates based on two loci was performed in the present study. Application of multiple genes aimed to study the genetic variability among Giardia isolates and to overcome the variation in the efficiency of amplification by different loci observed in previous studies.[2122] Tpi gene was detected in all G. intestinalis microscopy-positive samples, while gdh gene amplification was successful in 55 out of 57 samples. The good performance of the tpi gene in this work coincides with Bertrand et al. who reported successful amplification of the tpi gene in 96% of positive samples, while gdh amplification was successful in only 81% of the same samples.[20]

Mixed infection occurs when a host ingests Giardia cysts of different genetic profiles or with subsequent infection of an infected host by genetically different Giardia cysts. This is especially common in areas where giardiasis is endemic.[23] The use of assemblage-specific tpi primers in the present study allowed the detection of a much higher rate of mixed assemblage A and B infections (47.4%) compared to the gdh approach based on the use of general primers for PCR (12.7%). This finding was also confirmed by Sulaiman et al.[24] Variations would be attributed to the method used with amplification bias toward the most plentiful assemblage, or preferential amplification of a single genotype at a specific locus.[10] The high prevalence of mixed assemblage infection observed in our study among children could be explained by the greater environmental exposure in areas of high endemicity and poor fecal-oral hygiene.

The two approaches applied in the present study showed a moderate agreement in the detection and genotyping of G. intestinalis. In general, the gdh assay is less sensitive for detecting sequences of assemblage A and shows a preference for amplifying isolates of assemblage B. Incongruent assignment of isolates was also reported by other studies which provoked questions about the validity of genotyping relying on a single locus.[12123] Although the underlying mechanisms remain uncertain, inconsistency can be attributed to several factors including retention of ancestral polymorphism, introgression, meiotic recombination, and mixed infections.[325] Moreover, sequenced samples that showed double peaks in certain positions could be explained either by allelic sequence heterozygosity (ASH) or mixed infections. According to de Lucio et al., ASH and mixed infections can present misleading signals toward understanding the status of Giardia taxonomy and epidemiology.[26] The sequence variations isolated from a host might represent the genetic profile of multiple cysts or a single cyst with divergent alleles. As Giardia cyst contains four nuclei, each with full genome content, a single gene could be presented by up to four alleles that carry either similar or different genetic settings. Therefore, ASH occurring between nuclei of a single cyst could represent genetic polymorphism.[2728]

Clinical manifestations of giardiasis are quite variable.[29] The current study showed that most Giardia-positive individuals were symptomatic as previously reported by Ismail et al.[30] Surprisingly, a statistically significant association between clinical presentation and Giardia sub-assemblages was observed, where sub-assemblage BIII infections were mostly asymptomatic. Worldwide, contradictory results were reported by various studies.[313233] A similar study conducted in Iraq reported that sub-assemblage AII was significantly associated with diarrhea, vomiting, and flatulence.[34] This could be attributed to variation in the genotyping method, intra-assemblage variation in virulence, host factors, or a combination of these factors.[2935] Furthermore, it was suggested that in regions where a genotype is endemic, a new genotype might cause particularly severe symptoms when it first appears in the population, and dual infection with two different genotypes might produce a synergistic increase in pathology.[36]

CONCLUSION

The usage of assemblage-specific primers is recommended to elucidate the true picture of mixed assemblage infections in a community. The moderate agreement between tpi and gdh genes in genotyping of G. intestinalis necessitates the usage of both genes for accurate detection of Giardia genotypes. Effective screening and proper identification of G. intestinalis genotypic patterns and their relations to clinical presentations, virulence, drug response, and infectivity are necessary.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.