Stress, drugs and the evolution of reproductive restraint in malaria parasites

Life-history theory predicts that sexually reproducing organisms have evolved to resolve resource-allocation trade-offs between growth/survival versus reproduction, and current versus future reproduction. Malaria parasites replicate asexually in their vertebrate hosts, but must reproduce sexually to infect vectors and be transmitted to new hosts. As different specialized stages are required for these functions, the division of resources between these life-history components is a fundamental evolutionary problem. Here, we test how drug-sensitive and drug-resistant isolates of the human malaria parasite Plasmodium falciparum resolve the trade-off between in-host replication and between-host transmission when exposed to treatment with anti-malarial drugs. Previous studies have shown that parasites increase their investment in sexual stages when exposed to stressful conditions, such as drugs. However, we demonstrate that sensitive parasites facultatively decrease their investment in sexual stages when exposed to drugs. In contrast to previous studies, we tested parasites from a region where treatment with anti-malarial drugs is common and transmission is seasonal. We hypothesize that when exposed to drugs, parasites invest in their survival and future transmission by diverting resources from reproduction to replication. Furthermore, as drug-resistant parasites did not adjust their investment when exposed to drugs, we suggest that parasites respond to changes in their proliferation (state) rather the presence of drugs

A comparative study of single Theileria lestoquardi and mixed infections with Theileria ovis

Abstract Background Epidemiological surveys in Oman have revealed a high prevalence of the co-occurrence of the pathogenic Theileria lestoquardi and the non-pathogenic Theileria ovis among sheep in the Barka region, Oman. Our most recent data illustrated an interaction and reduced mortality risk in animals co-infected with T. lestoquardi and T. ovis, suggesting that the latter confers protection against pathogenicity of T. lestoquardi. The present study extends the above findings and examines disease outcomes; clinical markers, hematological parameters, and parasite density in mixed and single T. lestoquardi infections. Methods A total of 390 blood samples were collected from 16 sheep pens located in Barka, Oman between July and November 2019. Theileria spp. were detected and quantified using qPCR assay targeting 18S rRNA, and the extent of genetic diversity was estimated by a panel of T. lestoquardi specific micro- and mini-satellites. The association of some disease markers with the presence of Theileria spp. and genetic diversity was tested. Results Theileria spp. were detected in 75 (19.2%) sheep; of these 65 (86.7%) had mixed infections (T. lestoquardi plus T. ovis), 8 (10.6%) were infected with T. lestoquardi alone, and 2 (2.7%) with only T. ovis. Exotic breeds had a higher risk for Theileria spp. infection. The density (18S rRNA gene copies) of both parasites was higher in single infection against mixed infection, and there was a relatively lower density of T. lestoquardi in mixed infections. However, there was no difference in hematological indices between single T. lestoquardi and mixed infections. High genetic diversity was observed among T. lestoquardi in Barka, with no differences of T. lestoquardi in single and mixed infections. The extent of diversity seen in Barka was higher (He = 0.772) than that reported in Oman in 2019 (He = 0.582), with distinct T. lestoquardi genotypes. Conclusion The lower density of T. lestoquardi as mixed infection with T. ovis compared to single infection supports the hypothesis that T. ovis confers protection against lethal T. lestoquardi infection. However, there were no differences in disease correlations (clinical markers, hematological parameters, and density of parasites) or the extent of diversity of T. lestoquardi between the two types of infection. The presence of distinct T. lestoquardi genotypes in Barka, compared to that reported earlier in Oman, likely reflects movement of carrier animals and highlights the need for further analysis of the parasite populations to inform novel approaches for controlling malignant ovine theileriosis. Graphical Abstrac

Gametocytes: insights gained during a decade of molecular monitoring

In vertebrate hosts, malaria parasites produce specialized male and female sexual stages (gametocytes). Soon after being taken up by a mosquito, gametocytes rapidly produce gametes and, once mated, they infect their vector and can be transmitted to new hosts. Despite being the parasite stages that were first identified (over a century ago), gametocytes have remained elusive, and basic questions remain concerning their biology. However, the postgenomic era has substantiated information on the specialized molecular machinery of gametocytogenesis and expedited the development of molecular tools to detect and quantify gametocytes. The application of such highly sensitive and specific tools has opened up novel approaches and provided new insights into gametocyte biology. Here, we review the discoveries made during the past decade, highlight unanswered questions and suggest new directions

Real-time PCR assays for detection and quantification of early P. falciparum gametocyte stages

Introduction The use of reverse transcription, quantitative qRT-PCR assays for detection and quantification of late gametocyte stages has revealed the high transmission capacity of the human malaria parasite, Plasmodium falciparum. A full understanding how the parasite adjusts its transmission in response to varying in-host environmental conditions during natural infections requires simultaneous quantification of early and late gametocytes. Here, we describe qRT-PCR assays that are specific for detection and quantification of early-stage gametocytes of P. falciparum. Methods The assays are based on expression of known early gametocyte genes (pfpeg4, pfg27, pfge1, pfge3 and pfgexp5). The specificity of the qRT-PCR assays was tested using purified stage II and stage V gametocytes. These validated assays were used with qRT-PCR assays targeting late stage (pfs25) and all-stage (pfs16) gametocyte-specific transcripts to quantify gametocytes in natural P. falciparum infections and in a controlled human clinical infection study. Results The relative expression of pfpeg4, pfg27 and pfge3, but not of pfge1 and pfgexp5, was significantly higher in purified stage II compared to stage V gametocytes, indicating early gametocyte specificity. In natural infections, 71.2% of individuals had both early and late gametocyte transcripts (pfpeg4/pfg27 plus pfs25), 12.6% harboured only early gametocytes transcripts (pfpeg4/pfg27), and 15.2% had only late gametocytes transcripts (pfs25). In natural infections, the limit of detection was equivalent to 190 and 390 gametocytes/mL blood for pfpeg4 and pfg27, respectively. In infected volunteers, transcripts of pfpeg4 and pfg27 were detected shortly after the onset of blood stage infection, demonstrating the specificity of the assays. Conclusion The pfpeg4 and pfg27 qRT-PCR assays can be used specifically to quantify circulating immature gametocytes. Quantification of early gametocytes will improve understanding of epidemiological processes that modulate P. falciparum transmission and enhance the evaluation of transmission blocking interventions

Influx of diverse, drug resistant and transmissible Plasmodium falciparum into a malaria-free setting in Qatar

Background: Successful control programs have impeded local malaria transmission in almost all Gulf Cooperation Council (GCC) countries: Qatar, Bahrain, Kuwait, Oman, the United Arab Emirates (UAE) and Saudi Arabia. Nevertheless, a prodigious influx of imported malaria via migrant workers sustains the threat of local transmission. Here we examine the origin of imported malaria in Qatar, assess genetic diversity and the prevalence of drug resistance genes in imported Plasmodium falciparum, and finally, address the potential for the reintroduction of local transmission. Methods: This study examined imported malaria cases reported in Qatar, between 2013 and 2016. We focused on P. falciparum infections and estimated both total parasite and gametocyte density, using qPCR and qRT-PCR, respectively. We also examined ten neutral microsatellites and four genes associated with drug resistance, Pfmrp1, Pfcrt, Pfmdr1, and Pfkelch13, to assess the genetic diversity of imported P. falciparum strains, and the potential for propagating drug resistance genotypes respectively. Results: The majority of imported malaria cases were P. vivax, while P. falciparum and mixed species infections (P. falciparum / P. vivax) were less frequent. The primary origin of P. vivax infection was the Indian subcontinent, while P. falciparum was mostly presented by African expatriates. Imported P. falciparum strains were highly diverse, carrying multiple genotypes, and infections also presented with early- and late-stage gametocytes. We observed a high prevalence of mutations implicated in drug resistance among these strains, including novel SNPs in Pfkelch13. Conclusions: The influx of genetically diverse P. falciparum, with multiple drug resistance markers and a high capacity for gametocyte production, represents a threat for the reestablishment of drug-resistant malaria into GCC countries. This scenario highlights the impact of mass international migration on the reintroduction of malaria to areas with absent or limited local transmission

Theileria lestoquardi in Sudan is highly diverse and genetically distinct from that in Oman

Malignant ovine theileriosis is a severe tick-borne protozoan disease of sheep and other small ruminants which is widespread in sub-Saharan Africa and the Middle East. The disease is of considerable economic importance in Sudan as the export of livestock provides a major contribution to the gross domestic product of this country. Molecular surveys have demonstrated a high prevalence of sub-clinical infections of Theileria lestoquardi, the causative agent, among small ruminants. No information is currently available on the extent of genetic diversity and genetic exchange among parasites in different areas of the country. The present study used a panel of T. lestoquardi specific micro- and mini-satellite genetic markers to assess diversity of parasites in Sudan (Africa) and compared it to that of the parasite population in Oman (Asia). A moderate level of genetic diversity was observed among parasites in Sudan, similar to that previously documented among parasites in Oman. However, a higher level of mixed-genotype infection was identified in Sudanese animals compared to Omani animals, consistent with a higher rate of tick transmission. In addition, the T. lestoquardi genotypes detected in these two countries form genetically distinct groups. The results of this work highlight the need for analysis of T. lestoquardi populations in other endemic areas in the region to inform on novel approaches for controlling malignant theileriosis

Dynamics and within-host interaction of Theileria lestoquardi and T. ovis among naive sheep in Oman

Mixed species infections of Theileria spp. are common in nature. Experimental and epidemiological data suggest that mixed species infections elicit cross-immunity that can modulate pathogenicity and disease burden at the population level. The present study examined within-host interactions, over a period of 13 months during natural infections with two Theileria spp., pathogenic (T. lestoquardi) and non-pathogenic (T. ovis), amongst a cohort of naive sheep in Oman. In the first two months after exposure to infection, a high rate of mortality was seen among sheep infected with T. lestoquardi alone. However, subsequently mixed-infections of T. lestoquardi and T. ovis prevailed, and no further death occurred. The overall densities of both parasite species were significantly higher as single infection vs mixed infection and the higher relative density of pathogenic T. lestoquardi indicated a competitive advantage over T. ovis in mixed infection. The density of both species fluctuated significantly over time, with no difference in density between the very hot (May to August) and warm season (September to April). A high degree of genotype multiplicity was seen among T. lestoquardi infections, which increased with rising parasite density. Our results illustrate a potential competitive interaction between the two ovine Theileria spp., and a substantial reduction in the risk of mortality in mixed parasite infections, indicating that T. ovis confers heterologous protection against lethal T. lestoquardi infection

Genetic complexity of Plasmodium falciparum in two ethnic groups of Burkina Faso with marked differences in susceptibility to malaria.

We have characterized Plasmodium falciparum genotypes among the Mossi and Fulani sympatric ethnic groups in villages in Burkina Faso during the rainy season. Differences in clinical malaria presentation and in immune responses to malaria occur between the two groups. Asexual parasite rate, density, and gametocyte rate were higher among the Mossi than the Fulani. There was no difference in frequencies of alleles of the P. falciparum merozoite surface protein 1 (msp-1), msp-2, and glutamate-rich protein (glurp) genes among the parasites in each group. However, there were significant differences in the mean number of P. falciparum clones in the two populations, with there being more in the Mossi than in the Fulani. This effect was especially marked in older children. These differences can most probably be attributed to genetic differences in immune responsiveness to malaria between the two ethnic groups

A Randomized Open-Label Trial of Artesunate- Sulfadoxine-Pyrimethamine with or without Primaquine for Elimination of Sub-Microscopic P. falciparum Parasitaemia and Gametocyte Carriage in Eastern Sudan

In areas of seasonal malaria transmission, treatment of asymptomatic carriers of malaria parasites, whose parasitaemia persists at low densities throughout the dry season, could be a useful strategy for malaria control. We carried out a randomized trial to compare two drug regimens for clearance of parasitaemia in order to identify the optimum regimen for use in mass drug administration in the dry season.A two-arm open-label randomized controlled trial was conducted during the dry season in an area of distinct seasonal malaria in two villages in Gedarif State in eastern Sudan. Participants were asymptomatic adults and children aged over 6 months, with low-density P. falciparum infection detected by PCR. Participants were randomized to receive artesunate/sulfadoxine-pyrimethamine (AS+SP) combination for three days with or without a dose of primaquine (PQ) on the fourth day. Parasitaemia detected by PCR on days 3, 7 and 14 after the start of treatment and gametocytes detected by RT-PCR on days 7 and 14 were then recorded. 104 individuals who had low density parasitaemia at screening were randomized and treated during the dry season. On day 7, 8.3% were positive by PCR in the AS+SP+PQ group and 6.5% in the AS+SP group (risk difference 1.8%, 95%CI -10.3% to +13.8%). At enrolment, 12% (12/100) were carrying gametocytes. This was reduced to 6.4% and 4.4% by day 14 (Risk difference 1.9% (95%CI -9.3% to +13.2%) in AS+SP+PQ and AS+SP groups, respectively.Addition of primaquine to artemisinin combination treatment did not improve elimination of parasitaemia and prevention of gametocyte carriage in carriers with low-density parasitaemia in the dry season.ClinicalTrials.gov NCT00330902

Theileria lestoquardi displays reduced genetic diversity relative to sympatric Theileria annulata in Oman

The Apicomplexan parasites, Theileria lestoquardi and Theileria annulata, the causative agents of theileriosis in small and large ruminants, are widespread in Oman, in areas where cattle, sheep and goats co-graze. Genetic analysis can provide insight into the dynamics of the parasite and the evolutionary relationship between species. Here we identified ten genetic markers (micro- and mini-satellites) spread across the T. lestoquardi genome, and confirmed their species specificity. We then genotyped T. lestoquardi in different regions in Oman. The genetic structures of T. lestoquardi populations were then compared with previously published data, for comparable panels of markers, for sympatric T. annulata isolates. In addition, we examined two antigen genes in T. annulata (Tams1 and Ta9) and their orthologues in T. lestoquardi (Tlms1 and Tl9). The genetic diversity and multiplicity of infection (MOI) were lower in T. lestoquardi (He = 0.64–0.77) than T. annulata (He = 0.83–0.85) in all populations. Very limited genetic differentiation was found among T. lestoquardi and T. annulata populations. In contrast, limited but significant linkage disequilibrium was observed within regional populations of each species. We identified eight T. annulata isolates in small ruminants; the diversity and MOI were lower among ovine/caprine compared to bovine. Sequence diversity of the antigen genes, Tams1 and Ta9 in T. annulata (π = 0.0733 and π = 0.155 respectively), was 10-fold and 3-fold higher than the orthologous Tlms1 and Tl9 in T. lestoquardi (π = 0.006 and π = 0.055, respectively). Despite a comparably high prevalence, T. lestoquardi has lower genetic diversity compared to sympatric T. annulata populations. There was no evidence of differentiation among populations of either species. In comparison to T. lestoquardi, T. annulata has a larger effective population size. While genetic exchange and recombination occur in both parasite species, the extent of diversity, overall, is less for T. lestoquardi. It is, therefore, likely that T. lestoquardi evolved from an ancestor of present day T. annulata and that this occurred either once or on a limited number of occasions