Molecular Epidemiology and Assemblage Typing of Giardia duodenalis in School-Age Children Situated along the Southern Shoreline of Lake Malawi, Malawi

Abstract

Almost all human giardiasis infections are caused by Giardia duodenalis assemblages A and B. Differentiation between human infections with these assemblages, as well as between single-assemblage (A or B) and mixed-assemblage (A and B) infections, is therefore needed to better understand the pathological impact of infection with either, or both, assemblages. We assessed the prevalence of G. duodenalis assemblages A and B using 305 fecal samples provided by school-age children situated along the southern shoreline of Lake Malawi. Concurrently, intestinal pathology data were also collected to test for association(s) between assemblage infection status and intestinal health. Prevalence of G. duodenalis infection was 39.3% by real-time polymerase chain reaction. Of all identified infections, 32% were single G. duodenalis assemblage A and 32% were single G. duodenalis assemblage B, whereas 33% were mixed-assemblage infections. Fifteen unique G. duodenalis assemblage A and 13 unique G. duodenalis assemblage B β-giardin haplotypes were identified. There was a positive association between single infection with G. duodenalis assemblage B and both self-reporting of abdominal pain (odds ratio [OR]: 3.05, P = 0.004) and self-reporting of diarrhea (OR: 3.1, P = 0.003). No association between single infection with assemblage A and any form of intestinal pathology was found. Additionally, there was a positive association between mixed-assemblage infections and self-reporting of abdominal pain (OR: 3.1, P = 0.002). Our study highlights the importance G. duodenalis assemblage typing and reaffirms the need for improved access to water, sanitation and hygiene infrastructure in rural areas of low- and middle-income countries.

Figure 1.

Samama, Mchoka, Sungusya, and Malinde (St. Martin’s) primary schools, situated along the southern shoreline of Lake Malawi, Mangochi District, Malawi. Malawi’s country border can be seen within figure inset (upper left corner). Within figure inset, study area is highlighted in red box. Figure generated using the “map view” package version 2.10.0 within R Studio version 2021.09.0, build 351 (Posit).

Figure 2.

Templeton Crandall and Sing haplotype network of Giardia duodenalis β-giardin (bg) locus (forward direction sequences). Each node (circle) represents a unique haplotype. Nodes colored red denote G. duodenalis assemblage A haplotypes. Nodes colored blue donate G. duodenalis assemblage B haplotypes. Nodes colored black denote a missing but predicted ancestral haplotype. Size of nodes is proportional to the frequency of each haplotype. Hatched lines denote the number of single nucleotide polymorphisms between nodes. MAL = Malinde (St. Martin’s) school; MCH = Mchoka school; SAM = Samama school; SUN = Sungusya school.

Figure 3.

Giardia duodenalis infection status correctly identified (%) when targeting triosephosphate isomerase (tpi) loci using G. duodenalis assemblage-specific real-time polymerase chain reaction PCR compared with when targeting β-giardin (bg) locus using nested PCR with subsequent Sanger sequencing, relevant to diagnostic 18S real-time PCR Ct values. Plot generated using ggplot2 package version 3.4.0.

Figure 4.

Bar chart showing number of participants infected with Giardia duodenalis according to diagnostic 18S real-time polymerase chain reaction across all participant ages, as well as number of participants harboring single infections with G. duodenalis assemblage A, single infections with G. duodenalis assemblage B, and mixed infections with both G. duodenalis assemblages A and B, across all participant ages when targeting the β-giardin locus. Plot generated using ggplot2 package version 3.4.0.

Figure 5.

Scatterplot showing Giardia duodenalis diagnostic 18S real-time polymerase chain reaction values against QUIK CHEK RDT band strength. Regression line (Spearman’s rank coefficient) is plotted in blue, and 95% ICs can be seen shaded gray. Plot generated using ggplot2 package version 3.4.0.