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

• Published in: PLoS neglected tropical diseases 2021-03, Vol.15 (3), p.e0009277-e0009277
• Main Authors: Woschke, Andreas, Faber, Mirko, Stark, Klaus, Holtfreter, Martha, Mockenhaupt, Frank, Richter, Joachim, Regnath, Thomas, Sobottka, Ingo, Reiter-Owona, Ingrid, Diefenbach, Andreas, Gosten-Heinrich, Petra, Friesen, Johannes, Ignatius, Ralf, Aebischer, Toni, Klotz, Christian
• Format: Article
• Language: English
• Subjects: Ashes; Biology and Life Sciences; Causes of; Cysts; Deoxyribonucleic acid; DNA; DNA polymerase; Epidemiology; Gastroenteritis; Gene loci; Genetic aspects; Genetic testing; Genotype; Giardia; Giardia lamblia; Identification and classification; Infections; Laboratories; Medicine and Health Sciences; Multilocus sequence typing; Nucleotide sequence; Open source software; Parasites; Parasitological research; Patients; PCR; Phylogenetics; Polymerase chain reaction; Procedures; Protocols; Research and Analysis Methods; Samples; Statistical analysis; Strains; Tropical diseases
• ISSN: 1935-2735; 1935-2727; 1935-2735
• DOI: 10.1371/journal.pntd.0009277

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. 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. 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.