Dataset Information

Suitability of current typing procedures to identify epidemiologically linked human Giardia duodenalis isolates.

ABSTRACT:

Background

Giardia duodenalis is a leading cause of gastroenteritis worldwide. Humans are mainly infected by two different subtypes, i.e., assemblage A and B. Genotyping is hampered by allelic sequence heterozygosity (ASH) mainly in assemblage B, and by occurrence of mixed infections. Here we assessed the suitability of current genotyping protocols of G. duodenalis for epidemiological applications such as molecular tracing of transmission chains.

Methodology/principal findings

Two G. duodenalis isolate collections, from an outpatient tropical medicine clinic and from several primary care laboratories, were characterized by assemblage-specific qPCR (TIF, CATH gene loci) and a common multi locus sequence typing (MLST; TPI, BG, GDH gene loci). Assemblage A isolates were further typed at additional loci (HCMP22547, CID1, RHP26, HCMP6372, DIS3, NEK15411). Of 175/202 (86.6%) patients the G. duodenalis assemblage could be identified: Assemblages A 25/175 (14.3%), B 115/175 (65.7%) and A+B mixed 35/175 (20.0%). By incorporating allelic sequence heterozygosity in the analysis, the three marker MLST correctly identified 6/9 (66,7%) and 4/5 (80.0%) consecutive samples from chronic assemblage B infections in the two collections, respectively, and identified a cluster of five independent patients carrying assemblage B parasites of identical MLST type. Extended MLST for assemblage A altogether identified 5/6 (83,3%) consecutive samples from chronic assemblage A infections and 15 novel genotypes. Based on the observed A+B mixed infections it is estimated that only 75% and 50% of assemblage A or B only cases represent single strain infections, respectively. We demonstrate that typing results are consistent with this prediction.

Conclusions/significance

Typing of assemblage A and B isolates with resolution for epidemiological applications is possible but requires separate genotyping protocols. The high frequency of multiple infections and their impact on typing results are findings with immediate consequences for result interpretation in this field.

SUBMITTER: Woschke A

PROVIDER: S-EPMC8023459 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Giardia duodenalis is a cryptic protozoan, which has eight assemblages (A-H). Assemblages A and B are the main genotypes reported from humans with probable anthroponotic and zoonotic transmission. The current study aimed to characterize G. duodenalis assemblages in tuberculosis (TB) patients and healthy subjects using multilocus genotyping (MLG). Thirty Giardia-positive stool samples, which were obtained from TB patients and healthy subjects were included in the study. After total DNA extraction, three β-giardin (bg), triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh) genes were amplified and sequenced. Obtained sequences were compared to the GenBank database to characterize assemblages. Phylogenetic analysis using Maximum Likelihood (ML) and Tamura 3-parameter was performed for each gene. From 30 Giardia-positive subjects, 17 (57%) and 13 (43%) were from healthy and TB-infected subjects, respectively. There was no significant co-existence of Giardia and tuberculosis (P-value = 0.051). In addition, 14 (46.7%) and 16 (53.3%) of Giardia isolates were from asymptomatic and symptomatic subjects, respectively. PCR amplification was successful in 25 single samples (83.3%) consisted of 20 for tpi, 15 for bg, and 13 for gdh genes. Accordingly, 13/25 (52%) and 8/25 (32%) belonged to assemblage A and assemblages B, respectively, whereas 4/25 (16%) were either assemblage A or B with different genes at the same time. Significant correlation between assemblages and TB, age, and symptoms was not seen. The phylogenetic analyses represented no separation based on TB and gastrointestinal symptoms. Assemblage A was the predominant genotype in samples. The high frequency of assemblage AII indicated importance of anthroponotic transmission of Giardia in both healthy and TB patients. In addition, considering the exclusive reports of sub-assemblage AIII in wild ruminants, the presence of AIII in the current study have to be carefully interpreted. The inconsistency between the assemblage results of either bg or gdh loci with tpi gene signifies the insufficiency of single gene analysis and the necessity for MLG in molecular epidemiology of G. duodenalis.