Dynamics of anti-malarial antibodies in non-immune patients during and after a first and unique Plasmodium falciparum malaria episode

Malaria is the most important disease caused by protozoa in tropical and subtropical regions with an estimated burden of about 220 million cases in 2017 and about 430,000 deaths worldwide. The vast majority of cases is caused by Plasmodium falciparum [1]. Malaria is also a major topic in travel medicine and should be considered in all febrile patients after return from endemic areas. About 6200 imported malaria cases are reported in Europe each year [2], and in addition many travellers report a history of malaria during their stay abroad.

In endemic countries, malaria microscopy is often inaccurate or simply unavailable, and malaria remains a clinical and, therefore, presumptive diagnosis in many cases, although in recent years immune chromatographic, rapid diagnostic tests, much easier to perform and to read, not requiring a laboratory, have gradually replaced microscopy across most African endemic countries, often allowing a prompt diagnosis and management of malaria [3, 4].

Malaria serology is of no value in diagnosing acute malaria and may be still negative during a first, acute malaria episode. However, the presence of antibodies is witness of a previous malaria episode, and in non-endemic countries, particularly in Europe, serological testing of blood donors is recommended to all people having visited malaria-endemic countries. Positive donors are excluded from donation until negativization. However, recent research has questioned this approach to donor screening [5, 6].

Antibody detection can also be useful for the retrospective differential diagnosis of fever in non-immune travellers presenting after a journey to endemic areas and who report an episode of fever that was diagnosed as malaria [7, 8]. In the experience of this as well as of other centres dealing with travel medicine, many diagnoses of malaria (typically P. falciparum malaria) reported by returning travellers are unreliable, either because clinically based without testing, or because the quality of local diagnosis is often poor outside the few reference centres. If it would possible to show that: a) anti-malarial (anti-trophozoite, P. falciparum) antibody tests are invariably positive after an acute P. falciparum malaria episode, and b) positivity persists for at least some weeks in absence of further exposure, then the test could be retrospectively used to confirm or exclude P. falciparum malaria in a non-immune, recently returned traveller. However, little is known of the dynamic of anti-malarial antibodies after a first malaria episode in non-immune patients. The main purpose of this study was to retrospectively retrieve all available data on P. falciparum malaria antibody tests (IFAT, Bio-Mérieux) carried out on non-immune patients presenting with a first malaria episode, in order to describe the dynamic of anti-malarial antibodies. The underlying hypothesis is that, in all non-immune subjects with a first P. falciparum malaria episode, a detectable antibody titre invariably appears within the first few days after the onset of fever and remains detectable for at least some weeks. If this is so, then the test would be useful for the retrospective diagnosis (or exclusion) of malaria in non-immune travellers reporting a recent treatment for malaria before returning home. Moreover, serology is currently the method of choice for the screening of blood donors. Understanding the duration of the persistence of a positive titre after a single malaria episode, as well as the dynamics of the antibody titre evolution with time, may help to better inform the current policy.

The primary objective of this study was to describe the evolution of anti-malarial antibody titre during and after a first malaria episode in non-immune patients. Secondary objectives were:

b) To discuss the implications of the study results on the policy of anti-malarial antibody testing as a screening tool in transfusional medicine.

Primary endpoint: results of all available anti-malarial antibody tests (IFAT BioMérieux) performed over time on patients responding to the inclusion criteria, since the first determination during the acute clinical malaria.

Medical records of patients admitted with P. falciparum malaria to the Department of Infectious – Tropical Diseases and Microbiology (DITM), IRCCS Sacro Cuore - Don Calabria Hospital, Negrar, Verona, Italy, in a 14-year period between January 2004 and December 2017, were retrospectively analysed in order to identify non-immune patients presenting with a first malaria episode caused by P. falciparum. If anti-malarial serology was performed at least once during hospitalization, the patient was included in the study and the essential demographic, clinical and laboratory data were recorded in an anonymous database. For each recruited subject, any subsequent determinations of anti-malarial antibody titre (during admission and at any follow-up visit) was also recorded, with no time limits. History of any subsequent malaria episode as reported at follow-up visits was also recorded. The dynamic of the evolution of the antibody titre over time was analysed.

This study analysed the evolution of the anti-malarial (P. falciparum anti-trophozoites) antibody titre in non-immune patients suffering from their first P. falciparum malaria episode, which was confirmed by a negative antibody titre in almost all of them. While there is a paucity of papers describing the natural evolution of anti-malarial antibodies after a natural infection, relevant research has been done on experimental infection, often in the context of studies aimed at malaria vaccine development [9].

Recently, Boyle et al. interestingly compared the dynamics of anti-merozoite IgM and IgG following experimentally induced as well as natural malaria infection [10]. The latter, differently from this study population, was mainly composed of children and adults with repeated malaria exposure. Among other interesting findings, they discovered that IgM had a long persistence in both groups, similar to that of IgG, and that IgM in particular were likely to play a protective role by blocking merozoite invasion of erythrocytes in a complement-dependent manner. They also studied a small cohort of 10 Australian returning travellers with a (self-reported) first malaria episode, showing again persistence of IgM antibodies in all of them up to 6 months after the infection and no difference in the decline of the latter, compared with IgG, up to 300 days after infection [10].

To the authors’ knowledge this is the first such study published. Virtually all patients became positive during the acute malaria episode, and the positivity, after a sharp increase in titre, persisted for at least 1 month in the 19 patients who had a follow-up after discharge. Thus, a negative test result would virtually rule out a previous P. falciparum malaria in a returning traveller with a recent (i.e. 1 month or less) diagnosis while travelling, while a positive test would retrospectively confirm malaria. Five patients had already a detectable antibody titre at their first observation, four at the lowest titre of 20, and one at titre 40. One of them was a (VFR) patient from Africa (Nigeria) who reported no previous malaria history, although a past exposure might have escaped recollection. Unfortunately, the last available antibody titre for this patient (at the very high value of 5120) was recorded during hospitalization and no follow-up was available. Although it has been shown that immigrants, even if with a malaria history, tend to lose their semi-immunity after a long stay abroad [11, 12], the dynamic of the antibody response in this group, after a new malaria episode, would be probably different from that in completely naïve patients.

One patient remained negative until the last observation before discharge (5 days after admission). This was a patient with an exceedingly low parasite density, and given the strong negative correlation between the parasite density and the days to positivization, had the patient had a sufficient observation time a positive antibody titre would probably eventually have appeared. This cannot be stated with certainty, though, as he was unfortunately lost to follow-up after discharge. As for the five patients who had already a positive (albeit very weak) antibody titre at their first determination, a possible explanation is a previous, unreported malaria episode, or alternatively this may represent an early positivization (at least for two of them who had both a very high parasite density on admission).

In all patients with a sufficiently long follow-up, the antibody titre reached a peak in about a couple of months and then started declining, with no case of rebound, tending to negativization. This could open interesting perspectives in transfusional medicine. Current rules tend to exclude a donor who has a positive serology, irrespective of the titre. However, a declining titre most probably indicates that malaria infection is no longer present and might be taken as sufficient to re-admit a subject to donation, best if in combination with a sensitive molecular test, and without waiting for a complete negativization that would take a comparatively long time. It is reasonable to assume that chronic carriers of a very low or undetectable parasitaemia (who might represent a theoretical risk of transmission through blood donation) would maintain a high antibody titre. This is typically observed in chronic malaria infection [13]. Subjects with a persistently high antibody titre would be automatically excluded from blood donation, moreover they might benefit from an effective anti-malarial treatment, even in case of failure to detect circulating Plasmodium parasites, antigen or DNA [13]. Of course, a change of the current policy would need more evidence to be suggested, but a periodical serologic testing might be suggested especially for donors with particularly requested blood groups, also considering that serologic tests are available at a reasonable cost.