Microscopic and submicroscopic infection by Plasmodium falciparum: Immunoglobulin M and A profiles as markers of intensity and exposure

Abstract

Assessment of serological Plasmodium falciparum–specific antibodies in highly endemic areas provides valuable information about malaria status and parasite exposure in the population. Although serological evidence of Plasmodium exposure is commonly determined by Plasmodium-specific immunoglobulin G (IgG) levels; IgM and IgA are likely markers of malaria status that remain relatively unexplored. Previous studies on IgM and IgA responses have been based on their affinity for single antigens with shortage of immune responses analysis against the whole Plasmodium proteome. Here, we provide evidence of how P. falciparum infection triggers the production of specific IgM and IgA in plasma and its relationship with parasite density and changes in hematological parameters. A total of 201 individuals attending a hospital in Breman Asikuma, Ghana, were recruited into this study. Total and P. falciparum–specific IgM, IgA, and IgG were assessed by ELISA and examined in relation to age (0–5, 14–49, and ≥50 age ranges); infection (submicroscopic vs. microscopic malaria); pregnancy and hematological parameters. Well-known IgG response was used as baseline control. P. falciparum–specific IgM and IgA levels increased in the population with the age, similarly to IgG. These data confirm that acquired humoral immunity develops by repeated infections through the years endorsing IgM and IgA as exposure markers in endemic malaria regions. High levels of specific IgA and IgM in children were associated with microscopic malaria and worse prognosis, because most of them showed severe anemia. This new finding shows that IgM and IgA may be used as diagnostic markers in this age group. We also found an extremely high prevalence of submicroscopic malaria (46.27% on average) accompanied by IgM and IgA levels indistinguishable from those of uninfected individuals. These data, together with the observed lack of sensitivity of rapid diagnostic tests (RDTs) compared to PCR, invoke the urgent need to implement diagnostic markers for submicroscopic malaria. Overall, this study opens the potential use of P. falciparum–specific IgM and IgA as new serological markers to predict malaria status in children and parasite exposure in endemic populations. The difficulties in finding markers of submicroscopic malaria are highlighted, emphasizing the need to explore this field in depth.

Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC–MS/MS from Dried Serum Spots

“…Different P. falciparum-specific IgM responses are shown to various malaria disease , or protection , stages, agreeing with the distinct IgM antigen recognition patterns revealed in the separate clustering of MI and NI individuals. Thus, our developed methodology also shows a potential to functionally characterize the IgM antigenome according to the circulating IgM status.…”

“…This finding shows that the designed immunoprecipitation system specifically retains antigens of the pathogen (in our case, P. falciparum) that have infected the individuals from which the serum is derived, and which consequently have specific circulating IgM. Different P. falciparum-specific IgM responses are shown to various malaria disease 42,44 or protection 45,46 stages, agreeing with the distinct IgM antigen recognition patterns revealed in the separate clustering of MI and NI individuals. Thus, our developed methodology also shows a potential to functionally characterize the IgM antigenome according to the circulating IgM status.…”

“…We had focused on discovering IgM antigen collections from infectious diseases and chose malaria as a model for its development. The wealth of exposure to the malaria parasite in people living in endemic areas ensures access to circulating IgM to assess the potency and potential of the method. Although established techniques such as gel-based proteomics and Western blot allow the identification of antigenic proteins, − our shotgun technique upon specific immunoprecipitation with IgM from serum shows clear advantages, as it allows the identification of a much larger collection of antigens in a high throughput, more reliable, and simpler way, while only single protein spots can be analyzed by 2D PAGE .…”

“…To validate the conditions suggested in the previous study in a real situation of elution of pathogen antigens with IgM reactivity within the human sera and to compare the number of antigens detected by the two elution methods, a test was performed with extracts of P. falciparum and human sera from a malaria endemic area where contact with the parasite is ensured. 42 The selection of an appropriate elution method optimizes the content of different antigens recovered and their concentration 28 and minimizes the leaching of antibodies in the eluate. 29 To this end, the selected pair of elution procedures (acid and boiling) was tested at a serum/bead ratio of 0.4 in the presence of P. falciparum extracts to compare the number of pathogen and human proteins identified with each.…”

“…The result found in groups MI and NI is consistent with the fact that repeated P. falciparum infections in malaria hyperendemic regions modify the circulation of specific IgM antibodies over time, even in people not infected at the time of sampling, thus sharing some persistent antibodies but showing distinctiveness in others due to different infection states. 12,42,44,45 In immunoprecipitation controls C2 (with no anti-IgM antibody added), C3 (with serum from non-malaria-exposed individuals), and C4 (with no serum added), we identified a total of 20 different P. falciparum proteins, 10 of them also detected in endemic malaria samples but the other 10 unique to these controls (Figure 6C). Moreover, most of the identified P. falciparum proteins in those controls were in the high abundance range (>20, Figure 6C), perhaps suggesting overrepresentation of the proteins in extracts.…”