In this study, we assessed two indicators of P. falciparum
invasiveness (PMR and SI) and determined the relationship of these factors to malaria severity. The PMR measures parasite multiplication under standard conditions in the first cycle of in vitro
culture, whereas the SI is determined from blood smears taken directly from patients. There was no significant difference in the PMR and SI of isolates from severe malaria patients compared with isolates from uncomplicated malaria patients in two diverse sites in Africa. This lack of association with severe malaria is in contrast to previous studies on PMR and SI from Thailand.7,8
We were able to exclude technical differences as the source of this discrepancy in results because a small, blinded study of Thai isolates in our laboratory confirmed the previously published data.7
The PMR of the African isolates were mostly in the range of 1–6 fold (), and are comparable to those of uncomplicated malaria isolates from Thailand.7
However, isolates with high PMR in the 10–22-fold range characteristic of severe malaria cases in Thailand were absent from Africa. It seems extraordinary that many isolates in both Africa and Thailand should have such low PMR when every schizont produces approximately 24 merozoites. We only used healthy parasites that matured well for one cycle in culture for the PMR assays; however, we do not know how well the in vitro
assay reflects PMR in vivo
. The high PMR seen in Thai severe malaria isolates are in agreement with estimates of PMR in vivo
from recent studies with the P. falciparum
laboratory-adapted clone 3D7 infecting European volunteers21–23
and from retrospective analysis of data collected during neurosyphilis treatment programs.24,25
To our knowledge, there are no comparable data available for in vivo
PMR using fresh clinical isolates, or from any parasite clones in semi-immune individuals.
Why should there be a difference between Asia and Africa in the association of PMR and SI with parasite virulence? In Thailand (and most of southeast Asia), malaria transmission levels tend to be low, there is little acquired immunity in the population, and severe malaria affects all age groups.26
In contrast, in sub-Saharan Africa, malaria transmission tends to be moderate to high, older children and adults show partial immunity to malaria, and severe disease mainly occurs in children less than five years of age. It is possible that the different levels of immunity to malaria in the two regions are responsible for the diverse results highlighted here. Partial immunity in African patients could delay the course of infection and lead them to present at hospitals later than Asian patients, by which time differences between isolates in PMR and SI may no longer be apparent. If such a scenario were true, one might expect African children experiencing their first malaria infections after maternally acquired immunity has waned to show higher PMR more typical of those seen in Thailand. However, there was no correlation between age and PMR in our study, and isolates from children between 6 and 12 months of age who are unlikely to have had many previous malaria infections did not have higher PMR than isolates from older children (Kenya: infants, n = 5, median PMR = 1.5; older children, n = 25, median PMR = 2.3, P
= 0.52, by Mann-Whitney U test; Mali: infants, n = 4, median PMR = 2.7; older children, n = 57, median PMR = 2.6, P
= 0.79, by Mann-Whitney U test). There was also no significant correlation between age and SI, and isolates from children in the 6–12-month age group did not show significantly higher SI than isolates from older children (Kenya: infants, n = 13, geometric mean SI = 1.77, 95% CI = 1.38–2.16; older children, n = 66, geometric mean SI = 1.86, 95% CI = 1.55–2.17, F1,75
= 0.15, P
= 0.70, by ANOVA; Mali: infants, n = 8, geometric mean SI = 1.70, 95% CI = 0.06–3.34; older children, n = 96, geometric mean SI = 1.60, 95% CI = 1.08–2.12, F1,100
= 0.05, P
= 0.82, by ANOVA). The small number of children in the 6–12-month age group at both sites reflects the relative rarity of severe malaria in children less than one year of age in these areas. A prospective study with active surveillance would be required to determine conclusively whether parasites from African children experiencing their first malaria infection show higher PMR and lower SI than parasites from older children.
An alternative explanation of our results is that parasites adapted to grow in semi-immune populations in sub-Saharan Africa have evolved different mechanisms of virulence compared with those adapted to grow in non-immune populations in Asia. The growth of parasites within the host during the asexual blood stage depends upon the multiplication potential of the parasite and ability of the host to remove infected RBCs. It is plausible that in Asia, in the absence of significant levels of immunity, virulent parasites reach high parasite burdens within the host by having high PMRs. In Africa, parasites with high PMRs may not be effective in reaching high parasite burdens because of the presence of invasion-blocking antibodies in many hosts. In this case, parasite properties contributing to avoidance of the host’s immune system clearance mechanisms may be more important in helping a parasite to achieve high parasite burdens, produce high levels of sexual stages, and maximize its transmission and fitness.
Could the relationship between PMR and in vivo parasitemia explain the differences between African and Thai isolates? The interaction between parasitemia and PMR was not commented upon in the Thai study, and the parasitemias of the patients were not shown. However, high parasite burdens equivalent to those seen in Africa are not uncommon in southeast Asia; therefore, we believe it is unlikely that differences in parasitemia levels explain the diverse results from the two areas. In the current study we have carried out additional analysis on the subset of samples with parasitemias less than 5%, and on all data after adjustment to take into account host parasitemia effects. In both cases, there was no significant association between disease severity and PMR and SI in African children.
Whatever the explanation for the discrepancies between sub-Saharan Africa and Asia indicated by this study, our results raise the possibility that different pathogenic mechanisms of severe malaria may operate in parts of the world with different malaria transmission levels. This possibility is supported by studies of another potential virulence factor, rosette formation, which is associated with severe disease in multiple sites in Africa,19,27,28
but not in Asia.29,30
The hypothesis that Asian and African parasites differ in virulence properties could be tested in the absence of confounding effects of host immunity and host genetics by studying isolates infecting non-immune travelers.
One striking finding from our study was that isolates from Malian patients with hyperparasitemia had significantly lower multiplication rates than other Malian isolates ( and ). Additionally, there was a negative correlation between in vivo
parasitemia and PMR across all isolates from both Kenya and Mali (). These results seem counterintuitive because one might expect that isolates reaching high parasitemias in vivo
would have the highest multiplication rates rather than the lowest. Density-dependent mechanisms are thought to occur in vivo
to prevent excessive expansion of parasite populations, and can be determined by the availability of host RBCs, the presence of host immunity, or down-regulation of parasite invasiveness.31–34
Our data are consistent with P. falciparum
having the ability to down-regulate its multiplication rate to avoid overwhelming the host. We are not aware of any other experimental evidence to support such an idea; however, it is plausible that switching of invasion pathways35–39
could potentially underlie such a process. It would be of great interest to examine the relationship between particular invasion pathways and parasite PMR and SI, and to determine whether the PMR and SI are stable properties of each isolate or can vary in response to external stimuli. An initial study found no association between invasion pathways and SI in Gambian field isolates.40
An alternative interpretation of the finding that isolates from hyperparasitemic patients have very low PMRs is that slowly multiplying parasites may do less damage to their hosts and therefore are able to reach high parasitemias without causing the type of life-threatening disease manifestations seen in the severe malaria group.
One similarity between Thai and African isolates is that RBC selectivity is highest at low parasitemias, with isolates from patients with parasitemias less than 2% having significantly higher SI than other groups ( and ). Parasites with restricted invasion (high SIs) may be unable to reach high parasitemias. A parallel to this occurs in P. yoelii
, where virulent strains that invade normocytes reach higher parasitemias than avirulent strains that have a strong reticulocyte preference.41,42
An alternative explanation for the high SI at low parasitemia is that the presence of host antibodies to merozoites could lead to partial blocking of invasion and keep the parasitemia low, while also agglutinating merozoites and increasing the number of multiple invasions.9
In this study we have found no evidence to support a role for high parasite multiplication or lack of RBC selectivity as virulence factors in African children, a finding that contrasts sharply with previous work from southeast Asia.7,8
Other virulence factors such as rosette formation19,27,28
and platelet-mediated clumping43
may play a major role in parasite virulence in sub-Saharan Africa, whereas PMR and RBC selectivity are more important in Asia. If distinct pathogenic mechanisms underlie severe malaria in different parts of the world, this could have implications for the development of new strategies to treat and prevent severe disease. The differences in pathogenic mechanisms of severe malaria in regions of varying malaria transmission in both Africa and Asia are therefore worthy of further study.