Microscopic examination of blood smears remains the gold standard for laboratory inspection and diagnosis of malaria. Smear inspection is, however, time-consuming and dependent on trained microscopists with results varying in accuracy. We sought to develop an automated image analysis method to improve accuracy and standardization of smear inspection that retains capacity for expert confirmation and image archiving. Here, we present a machine learning method that achieves red blood cell (RBC) detection, differentiation between infected/uninfected cells, and parasite life stage categorization from unprocessed, heterogeneous smear images. Based on a pretrained Faster Region-Based Convolutional Neural Networks (R-CNN) model for RBC detection, our model performs accurately, with an average precision of 0.99 at an intersection-over-union threshold of 0.5. Application of a residual neural network-50 model to infected cells also performs accurately, with an area under the receiver operating characteristic curve of 0.98. Finally, combining our method with a regression model successfully recapitulates intraerythrocytic developmental cycle with accurate lifecycle stage categorization. Combined with a mobile-friendly web-based interface, called PlasmoCount, our method permits rapid navigation through and review of results for quality assurance. By standardizing assessment of Giemsa smears, our method markedly improves inspection reproducibility and presents a realistic route to both routine lab and future field-based automated malaria diagnosis.

Four species of malaria parasite, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi infect humans living in the Khanh Phu commune, Khanh Hoa Province, Vietnam. The latter species also infects wild macaque monkeys in this region. In order to understand the transmission dynamics of the three species, we attempted to detect gametocytes of the three species in the blood of infected individuals, and sporozoites in the salivary glands of mosquitoes from the same region. For the detection of gametocyte-specific mRNA, we targeted region 3 of pfg377, pvs25, pmg and pks25 as indicators of the presence of P. falciparum, P. vivax, P. malariae and P. knowlesi gametocytes, respectively. Gametocyte-specific mRNA was present in 37, 61, 0 and 47% of people infected with P. falciparum (n = 95), P. vivax (n = 69), P. malariae (n = 6) or P. knowlesi (n = 32), respectively. We found that 70% of mosquitoes that had P. knowlesi in their salivary glands also carried human malaria parasites, suggesting that mosquitoes are infected with P. knowlesi from human infections.

Gametocytes are the specialized form of Plasmodium parasites that are responsible for human-to-mosquito transmission of malaria. Transmission of gametocytes is highly effective, but represents a biomass bottleneck for the parasite that has stimulated interest in strategies targeting the transmission stages separately from those responsible for clinical disease. Studying targets of naturally acquired immunity against transmission-stage parasites may reveal opportunities for novel transmission reducing interventions, particularly the development of a transmission blocking vaccine (TBV). In this review, we summarize the current knowledge on immunity against the transmission stages of Plasmodium. This includes immune responses against epitopes on the gametocyte-infected erythrocyte surface during gametocyte development, as well as epitopes present upon gametocyte activation in the mosquito midgut. We present an analysis of historical data on transmission reducing immunity (TRI), as analysed in mosquito feeding assays, and its correlation with natural recognition of sexual stage specific proteins Pfs48/45 and Pfs230. Although high antibody titres towards either one of these proteins is associated with TRI, the presence of additional, novel targets is anticipated. In conclusion, the identification of novel gametocyte-specific targets of naturally acquired immunity against different gametocyte stages could aid in the development of potential TBV targets and ultimately an effective transmission blocking approach.

A population of the sipunculan worm Aspidosiphon muelleri, located in temperate waters of the western Mediterranean Sea, was monitored monthly for a year. Some aspects related to its ecology and reproductive biology are shown in the present work. The sex-ratio for this population was close to 1:1 (54% females vs 46% males), thus indicating a dioecious reproduction, although showing a lack of sexual dimorphism. Oocytes were detected in females from 4–5 mm; this size might be reached by A. muelleri in a few months. The population density increased notably during the summer (June–August). The spawning event date was indirectly estimated from the average size of the oocytes and the percentage of females with free oocytes in the coelom. Both variables were significantly correlated to shallow water temperature (Pearson correlation; P = 0.003 and P = 0.001, respectively). Oocyte size was also significantly correlated to average irradiance level (Pearson correlation; P = 0.044). Spawning took place between August and September (when the water temperature is getting close to its annual maximum) and may last only a few weeks in these temperate waters. The abundance of A. muelleri decreased drastically in September, likely as a consequence of the spawning event effort. Bivalves of the species Epilepton clarkiae were collected together with specimens of A. muelleri living inside the polychaete tubes used as shelters by the sipunculans, with a prevalence of 11.64%. To a lesser extent some Foraminifera were also reported attached to the body of the sipunculans.

Drugs that kill or inhibit the sexual stages of Plasmodium in order to prevent transmission are important components of malaria control programmes. Reducing gametocyte carriage is central to the control of Plasmodium falciparum transmission as infection can result in extended periods of gametocytaemia. Unfortunately the number of drugs with activity against gametocytes is limited. Primaquine is currently the only licensed drug with activity against the sexual stages of malaria parasites and its use is hampered by safety concerns. This shortcoming is likely the result of the technical challenges associated with gametocyte studies together with the focus of previous drug discovery campaigns on asexual parasite stages. However recent emphasis on malaria eradication has resulted in an upsurge of interest in identifying compounds with activity against gametocytes. This review examines the gametocytocidal properties of currently available drugs as well as those in the development pipeline and examines the prospects for discovery of new anti-gametocyte compounds.

Eimeria maxima has been used as a model apicomplexan parasite to study sexual stage development and oocyst wall formation. A complete understanding of the wall's biochemical and biophysical properties is of great interest in research on all apicomplexan parasites. Purified gametocytes, zygotes and oocysts were analysed by three-dimensional confocal microscopy, and wide-field fluorescent microscopy was used to investigate the appearance and spatial organization of the 2 types of wall-forming bodies (WFBs). In addition, a variety of staining procedures and immunoassays were used to assess the biosynthesis, metabolic activity, intactness and molecular composition of the WFBs in situ. WFBs were extracted from gametocytes/zygotes and their composition was assessed by microscopy and SDS-PAGE analysis. It was concluded that isolated gametocytes are intact and metabolically active. Additionally, it was observed that the Type 1 WFBs are aligned at the periphery of the parasite and fuse together producing neutral lipid rich patches that appear to be inserted into the space between 2 parasite-specific membranes. Finally, it was shown that the WFBs extracted from purified gametocytes had the same shape, size and staining properties as those observed in situ, and contained the major glycoprotein antigens known to be present in these organelles.

The most important function of any parasite is to secure transmission to new hosts. The gametocyte, the stage which has become developmentally committed to the sexual cycle, provides a critical link in the transmission of Plasmodium falciparum from the human host to the anopheline mosquito vector. It is therefore imperative that our determination to understand the biology of the gametocyte is greater than the technical obstacles which have resulted in the gametocyte being left very much out of the limelight by the intensive investigation of the asexual bloodstream parasite. Here we explore the areas of gametocyte biology which by nature of their relevance to control and pathology as well as basic biology, are the subjects of investigation in our laboratory. We also point out areas in need of particular attention.

Sex ratio theory usually predicts an equilibrium sex ratio and equal proportions of males and females in a population, including the progenitors of the reproductive cells of protozoans. This proposal was tested with three species of malarial parasites of lizards, Plasmodium mexicanum of the western fence lizard, and P. agamae and P. giganteum of the African rainbow lizard, using single samples from naturally infected lizards, repeated samples from free-ranging lizards (P. mexicanum only), and repeated samples from laboratory maintained animals. Macrogametocytes were usually more abundant than microgametocytes, and were slightly larger, revealing a typically greater investment of resources by the progenitors of female reproductive cells. However, the proportion of microgametocytes varied among the three species and among infections within each species of Plasmodium. The sex ratio of gametocytes often remained constant within infections followed over time even if the absolute number of gametocytes was changing. However, the equilibrium sex ratio of gametocytes varied among those infections that had an unchanging microgametocyte proportion. Thus, although an equilibrium sex ratio apparently occurs for most infections, there appears to be no characteristic proportion of microgametocytes for any of the species. Potential explanations for this conflict with theory are presented.

In an area of holoendemic malaria in Northern Tanzania, Anopheles gambiae s.l. females were age-graded by Polovodova's method and dissected for sporozoites. Age-specific sporozoite rates implied that mosquitoes acquired new infections at all ages. The extrinsic period lasted just over 3 gonotrophic cycles (9–11 days). Very high sporozoite rates in the oldest females implied the absence or rarity of genetic refractoriness to infection. A method is described for estimating the proportion of bloodmeals which result in mosquito infection. This method makes relatively few assumptions about mosquito behaviour, and could be useful for evaluating transmission-blocking interventions. Overall, it is estimated that about 21% of meals are infectious. This is much higher than previous estimates derived either from experimental mosquito feeding studies or from similar age-grading data collected from the same area in 1962. Various alternative explanations are considered, and it is concluded that there has been a 2.5-fold increase in human infectiousness in the last 25 years. This is partly attributable to suppression of human infectiousness by widespread chloroquine usage during the 1960s, followed by removal of this effect by drug resistance. It is argued that chloroquine would be expected to select for increased infectivity in the parasite, and this may also have contributed to the observed increase.

Up to 200 laboratory reared Anopheles dirus mosquitoes were fed on each of 496 symptomatic Thai men who had patent, naturally acquired Plasmodium vivax gametocytaemia. Mean gametocyte densities were 455/mm3 (range: 0–3281), geometric mean oocyst number was 9 (0–142), mean frequency of infection was 43% (0–100%), and mean sporozoite number in salivary glands was 9525 (0–285 000). There was little relation between gametocyte density and either oocyst number or frequency of mosquito infection. There were, however, statistically strong positive correlations between oocyst numbers and frequency of infection, and between number of oocysts and number of salivary gland sporozoites. The data suggest that each oocyst contributed about 850 sporozoites to a gland infection.

Various factors that may influence routine and high levels of mosquito infection with cultured Plasmodium falciparum gametocytes are considered in this paper. One of the most important is the choice of an appropriate isolate, with facilities for cryopreservation and a good technique for initiation of cultures. The use of automated culture systems with strict adherence to detail and routine has eliminated much of the variability. The quality of the serum used for the culture of gametocytes and inclusion in the feed material for mosquitoes is of the highest importance. Blood collection for culture purposes must preferably involve alcohol as an antiseptic for cleaning donor skin or suitable receptacles. Mosquito blood meals should not include plasma with citrate phosphate dextrose or sera collected in microtainer tubes or from volunteers on proguanil-chloroquine prophylaxis. Sera of individuals on chloroquine alone do not influence transmission. Haemato-crits of from 5 to 10% permit the culture of equally infective gametocytes. It was impossible to predict the outcome of an infection in mosquitoes based on the number of female gametocytes or gametes. Within any experiment, the oocyst load initially increased, followed by a decline with progressively lower numbers of gametocytes accompanied by a progressive increase in the efficiency of transmission. Some of the variability of mosquito infection within an experiment was due to individual differences in the speed of blood digestion of the mosquitoes. A new membrane feeder is described with three different sizes to accommodate a variety of goals. The new design permitted more mosquitoes to feed within a limited period compared to the older more widely used feeder of equivalent capacity, and mosquitoes feeding on the new-type feeder were frequently better infected than those fed on the older type.

Most mathematical models of malaria infection represent parasites as replicating continuously at a constant rate whereas in reality, malaria parasites replicate at a fixed age. The behaviour of continuous-time models when gametocytogenesis is included, in comparison to a more realistic discrete-time model that incorporates a fixed replication age was evaluated. Both the infection dynamics under gametocytogenesis and implications for predicting the amount parasites should invest into gametocytes (level of investment favoured by natural selection) are considered. It is shown that the many malaria models with constant replication rates can be represented by just 3 basic types. For these 3 types, it is then shown that under gametocytogenesis (i) in 2 cases, parasite multiplication and gametocyte production is mostly much too low, (ii) in the third, parasite multiplication and gametocyte production is mostly much too high, (iii) the effect of gametocyte investment on parasite multiplication is mostly too high, (iv) the effect of gametocyte investment on gametocyte production is nearly always too low and (v) with a simple approximation of fitness, the predicted level of gametocyte investment is mostly much too low. However, a continuous model with 48 age-compartments compares well to the discrete model. These findings are a further argument for modelling malaria infections in discrete time.

We have examined Plasmodium falciparum gametocyte prevalence, density and their genetic complexity among children of 2 sympatric ethnic groups (Mossi and Fulani) in villages in Burkina Faso. The 2 groups are known to have distinct differences in their susceptibility and immune responses to malaria. We used RT-PCR and sequence-specific probes to detect and type RNA of the gametocyte-specific protein Pfs48/45. There were no differences in detection rates of asexual forms and gametocytes among the 2 groups, using PCR and RT-PCR, respectively. However, there were significant differences in densities of asexual forms and gametocytes, which were both higher among Mossi than Fulani. Both asexual forms and gametocyte densities were influenced by age and ethnicity. Multiple-clone infections with more than 1 gametocyte genotype were equally prevalent among Fulani and Mossi. These differences can most probably be attributed to genetic differences in malaria susceptibility in the 2 ethnic groups.

The life-cycle of Plasmodium depends on transmission of the parasite from the vertebrate host into its vector when the insect takes a bloodmeal. Transmission success may depend in part on the parasite's gametocyte density and sex ratio in the blood. P. mexicanum, a parasite of fence lizards in California, USA, exploits the sandfly Lutzomyia vexator as its vector. In experimental transmissions using naturally infected lizards as donors of blood, transmission success (measured as percentage of vectors infected and number of parasite oocysts on the insect's midgut) was positively related to gametocyte density, although density above 20/1000 erythrocytes did not improve transmission. Sex ratio (proportion of microgametocytes in the infection) was positively correlated with gametocyte density. Transmission improved with higher proportion of microgametocytes, but partial correlations revealed that this was a result only of higher gametocyte densities. These results agree with the theory of virulence and sex ratios because single clone infections should produce a more female-biased sex ratio and grow to the minimum parasitaemia that would maximize clonal transmission, whereas multiple clone infections will be closer to a 1[ratio ]1 sex ratio and yield a higher parasitaemia when each clone competes for transmission to the vector.

Interactions between parasite genotypes sharing a host are poorly understood, but have important consequences for the epidemiology and evolution of the parasite. In mixed-genotype malaria infections, patterns of asexual replication and transmission favoured by natural selection may be different from those in single-genotype infections. The infectivity to mosquitoes of mixed-genotype and single-genotype infections were compared using 2 clones of Plasmodium chabaudi inoculated into mice either together or alone. Mice given mixed-clone infections received the sum of the inocula given to the single-clone controls. Mosquitoes were fed on the mice and the numbers of oocysts which developed were counted to assess transmission intensity. For 3 combinations of starting inocula and feed days, mixed-clone infections produced more oocysts per mosquito than the sum of the 2 single-clone infections. This effect was correlated with an increase in gametocyte density, but was less clearly related to asexual infection parameters. The results show that interactions between clones in mixed-clone infections can profoundly affect transmission.

Examination of Giemsa stained smears made from the peripheral blood, liver and spleen of hand-caught lizards from West Pokot area showed the presence of seven out of the nine Plasmodium species of lizards which have been previously described in Africa. These species are redescribed using a set of biological characters and procedure which enabled a large quantity of each stage of any species to be studied, measured and compared. The utilization of these characters and procedure was a reaction to the multiple infections in individual lizards and a wide host range of each species among lizard species. The adoption of the criteria is recommended for future taxonomie work on lizard Plasmodia in Africa.

A key which brings together all the previously described species of lizard Plasmodia and the new species encountered in the present investigation is presented. The key is simplified so that it can be of use to epidemiologists and pathologists who may work on saurian malaria in Africa.

During investigations on the malaria parasites of lizards in the West Pokot district of Kenya, haemoproteids, haemogregarines, pirhemocyton-like and anaplasma-like organisms were found in the blood. Microscopic examination showed that some of these are new species and they are described in this paper. The new described species are Haemoproteus mungutii (haemoproteid), Karyolysus poleensis, Schellakia mabuyai (haemogregarines), Pirhemocyton kongelai, Haematractidium omogoi, Sauroplasma kachelibaensis (Pirhemocyton-like organisms), Eperythrozoon ngokai and Aegyptianella elgonensis (anaplasma-like organisms). K. poleensis and S. mabuyai are regarded as the first report of the genera in lizards in Africa while A. elgonensis is regarded as the first report of the genus Aegyptianella in lizards.

During investigations into the epidemiology of malaria of lizards in Kenya, Giemsa-stained smears made from the peripheral blood, liver and spleen of hand-caught lizards from West Pokot district were examined for the presence of Plasmodium species. Ten new species were found and described. These are Plasmodium sapaaensis, P. kyaii, P. kachelibaensis, P. icipeensis, P. gloriai, P. odhiamboi, P. bowiei, P. kaninii, P. kadogoi and P. adunyinkai.

P. sapaaensis was found to be related to P. mabuiae, P. kadogoi to P. rhadinurum, P. kaninii to P. minasense (s. Telford) and P. adunyinkai to P. maculilabre. P. kyaii and P. kachelibaensis have distinct characteristics which distinguish them from the Plasmodium species in the tropiduri group reported so far.

Four species i.e. P. icipeensis, P. odhiamboi, P. gloriai and P. bowiei, exhibited peculiar characteristics most especially their effect on the white cells. They were therefore constituted into the “Kenyan” group.