High Diversity of Giardia duodenalis Assemblages and Sub-Assemblages in Asymptomatic School Children in Ibadan, Nigeria

Giardia duodenalis is a significant contributor to the burden of diarrheal disease in sub-Saharan Africa. This study assesses the occurrence and molecular diversity of G. duodenalis and other intestinal parasites in apparently healthy children (n = 311) in Ibadan, Nigeria. Microscopy was used as a screening method and PCR and Sanger sequencing as confirmatory and genotyping methods, respectively. Haplotype analyses were performed to examine associations between genetic variants and epidemiological variables. At microscopy examination, G. duodenalis was the most prevalent parasite found (29.3%, 91/311; 95% CI: 24.3-34.7), followed by Entamoeba spp. (18.7%, 58/311; 14.5-23.4), Ascaris lumbricoides (1.3%, 4/311; 0.4-3.3), and Taenia sp. (0.3%, 1/311; 0.01-1.8). qPCR confirmed the presence of G. duodenalis in 76.9% (70/91) of the microscopy-positive samples. Of them, 65.9% (60/91) were successfully genotyped. Assemblage B (68.3%, 41/60) was more prevalent than assemblage A (28.3%, 17/60). Mixed A + B infections were identified in two samples (3.3%, 2/60). These facts, together with the absence of animal-adapted assemblages, suggest that human transmission of giardiasis was primarily anthroponotic. Efforts to control G. duodenalis (and other fecal-orally transmitted pathogens) should focus on providing safe drinking water and improving sanitation and personal hygiene practices.This research was funded by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, grant number PI19CIII/00029.S

<i>Babesia divergens</i> Shows Equal Predilection for Human ABO Blood Types in an In Vitro Erythrocyte Preference Assay

Babesia is spread to humans via ticks or blood transfusions. Severity of Plasmodium falciparum malaria is strongly correlated to the ABO blood group of the patient. Babesia divergens is an intraerythrocytic parasite with many similarities to malaria, but the impact of ABO on the susceptibility to and progression of the infection in humans is unknown. We have now cultured B. divergens in human group A, B and O erythrocytes in vitro and measured rates of multiplication. The predilection for the different erythrocyte types was also determined using an in vitro erythrocyte preference assay when the parasites were grown in group A, B or O erythrocytes over time and then offered to invade differently stained erythrocytes of all the blood types at the same time. The results showed no difference in multiplication rates for the different blood types, and the parasite exhibited no obvious morphological differences in the different blood types. When cultured first in one blood type and then offered to grow in the others, the preference assay showed that there was no difference between the A, B or O blood groups. In conclusion, this indicates that individuals of the different ABO blood types are likely to be equally susceptible to B. divergens infections

Factors influencing the induction of high affinity antibodies to Plasmodium falciparum merozoite antigens and how affinity changes over time

Understanding the functional characteristics of naturally acquired antibodies against P. falciparum merozoite antigens is crucial for determining the protective functions of antibodies. Affinity (measured as kd) of naturally acquired antibodies against two key targets of acquired immunity, EBA175 and PfRh2, was determined using Surface Plasmon Resonance (SPR) in a longitudinal survey in Nigeria. A majority of the participants, 79% and 67%, maintained stable antibody affinities to EBA175 and PfRh2, respectively, over time. In about 10% of the individuals, there was a reciprocal interaction with a reduction over time in antibody affinity for PfRh2 and an increase for EBA175. In general, PfRh2 elicited antibodies with higher affinity compared to EBA175. Individuals with higher exposure to malaria produced antibodies with higher affinity to both antigens. Younger individuals (5-15 years) produced comparable or higher affinity antibodies than adults (>15 years) against EBA175, but not for PfRh2. Correlation between total IgG (ELISA) and affinity varied between individuals, but PfRh2 elicited antibodies with a higher correlation in a majority of the participants. There was also a correlation between antibody inhibition of erythrocyte invasion by merozoites and PfRh2 affinity. This work gives new insights into the generation and maintenance of antibody affinity over time

Subclass responses and their half-lives for antibodies against EBA175 and PfRh2 in naturally acquired immunity against Plasmodium falciparum malaria

Background: Plasmodium falciparum EBA175 and PfRh2 belong to two main families involved in parasite invasion, and both are potential vaccine candidates. Current knowledge is limited regarding which target antigens and subclasses of antibodies are actually important for protection, and how naturally acquired immunity is achieved. Methods: Repeated blood samples were collected from individuals in Nigeria over a period of almost one year. ELISA was used to analyse subclasses of IgG responses. Results: For both EBA175 (region III-V) and (a fragment of) PfRh2, the dominant antibody responses consisted of IgG1 and IgG3 followed by IgG2, while for PfRh2 there was also a relatively prominent response for IgG4. High levels of IgG1, IgG2 and IgG3 for EBA175 and total IgG for PfRh2 correlated significantly with a lower parasitaemia during the study period. Children with HbAS had higher levels of some subclasses compared to children with HbAA, while in adults the pattern was the opposite. The half-lives of IgG2 and IgG4 against EBA175 were clearly shorter than those for IgG1 and IgG3. Conclusion: EBA175 and PfRh2 are potential targets for protective antibodies since both correlated with lower parasitaemia. The shorter half-lives for IgG2 and IgG4 might explain why these subclasses are often considered less important in protection against malaria. Triggering the right subclass responses could be of critical importance in a successful vaccine. Further studies are needed to evaluate the role of haemoglobin polymorphisms and their malaria protective effects in this process

Acquisition, maintenance and adaptation of invasion inhibitory antibodies against Plasmodium falciparum invasion ligands involved in immune evasion

Erythrocyte-binding antigens (EBAs) and P. falciparum reticulocyte-binding homologue proteins (PfRhs) are two important protein families that can vary in expression and utilization by P. falciparum to evade inhibitory antibodies. We evaluated antibodies at repeated time-points among individuals living in an endemic region in Nigeria over almost one year against these vaccine candidates. Antibody levels against EBA140, EBA175, EBA181, PfRh2, PfRh4, and MSP2, were measured by ELISA. We also used parasites with disrupted EBA140, EBA175 and EBA181 genes to show that all these were targets of invasion inhibitory antibodies. However, antigenic targets of inhibitory antibodies were not stable and changed substantially over time in most individuals, independent of age. Antibodies levels measured by ELISA also varied within and between individuals over time and the antibodies against EBA181, PfRh2 and MSP2 declined more rapidly in younger individuals (15 years) compared with older (>15). The breadth of high antibody responses over time was more influenced by age than by the frequency of infection. High antibody levels were associated with a more stable invasion inhibitory response, which could indicate that during the long process of formation of immunity, many changes not only in levels but also in functional responses are needed. This is an important finding in understanding natural immunity against malaria, which is essential for making an efficacious vaccine

Longitudinal invasion inhibitory activity profiles of representative individuals.

<p>Individuals exhibited diverse invasion inhibitory activity against the 3D7 WT relative to the EBA knockout parasites overtime. Individual AD23 inhibited the EBA knockout parasites more than the parental parasite; individual AD50 inhibited the parental parasite more than the EBA knockout parasite lines.</p

Seasonal variation in overall differential inhibition of 3D7 WT and the knockout lines.

<p>There were no differences between the overall inhibition between the 3D7 WT and EBA knockout parasites through the seasons except for EBA175 knockout parasites in the months of July, October, and January. Values represent mean of all samples ± SEM.</p

Kaplan- Meier survival curves showing the proportion of individuals with antibody levels of >50% of the day 0 antibody level for all antigens among the different age groups and among individuals with different levels of exposure to <i>P</i>. <i>falciparum</i>.

<p>Kaplan- Meier survival curves showing the proportion of individuals with antibody levels of >50% of the day 0 antibody level for all antigens among the different age groups and among individuals with different levels of exposure to <i>P</i>. <i>falciparum</i>.</p

Pattern of differential inhibition of <i>P</i>. <i>falciparum</i> lines exhibited by individuals over time.

<p>The pie charts show the proportion of individuals that differentially inhibited 3D7WT invasion compared with 3D7 lines with genetically disrupted EBA genes (KO). The proportions of individuals with all observed outcomes are represented with various shades as shown. For example, “KO>WT only over time” means that the knock-out parasite was inhibited more than the wild-type and this was a stable pattern over time in the studied individuals.</p

Relationship between invasion inhibitory antibodies to invasion pathway ligands parasitaemia and age.

<p>(A) Effects of antibodies directed against SA-dependent or SA-independent pathways on the mean parasitaemia. “<b>A</b>”, “<b>B</b>” represent individuals with only antibody responses to SA-dependent or SA-independent pathway invasion ligands, respectively. “<b>A and B”</b> represents individuals that presented a response to both pathways. (B) Effect of age on the acquisition of antibody responses to the two invasion pathways.</p