Plasmodium falciparum: Development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer

Reliable analytical techniques to test growth-promoting and antimalarial efficacy on plasmodia are very important. Flow cytometry (FCM) offers the possibility to study developmental stages of intraerythrocytic growth of malaria parasites using nucleic acid staining. To analyze the growth of Plasmodium falciparum SYBR Green I was introduced as an intercalating dye with FCM for the 488 nm line of an argon laser. Procedures employing FCM, including fixatives, dye concentrations, dilution buffer, and staining period, were optimized to simplify the method. FCM as described here allows parasitemia and parasites of different stages to be quantified according to the DNA content. The proportion of parasitized erythrocytes estimated by FCM and the Giemsa method agreed with determination by parasite lactate dehydrogenase. The protocol was extended to merozoite counting as a sensitive assay of growth inhibition of the parasite.     

Flow cytometry to evaluate Theileria sergenti parasitemia using the fluorescent nucleic acid stain, SYTO16

BACKGROUND: Although the infection of Theileria sergenti is demonstrated by intraerythrocytic localization of this parasite, much time and labor are necessary in order to determine this. We applied flow cytometry to evaluate T. sergenti parasitemia using the fluorescent nucleic acid stain method. METHODS: Peripheral blood samples from cattle infected with T. sergenti were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16, and hydroethidine (HE). Stained parasitized erythrocytes were measured by a flow cytometer equipped with a single argon laser operating at 488 nm. RESULTS: SYTO16-stained intraerythrocytic parasites were detected on the FL1 (525 nm) and parasitized cells were separated completely from unparasitized cells. However, HE-stained erythrocytes could not be divided clearly into parasitized and unparasitized cells. SYTO16-stained parasites were reproducibly detected at a percentage above 0.1%. Contaminating leukocytes, which were indicated by CD18-positive cells, were eliminated from the analysis by narrowing the light scatter gate of the erythrocyte fraction. A correlation (r = 0.983) between the percentage of SYTO16-positive cells and parasitemia in grazing cattle was observed. CONCLUSIONS: Flow cytometric detection using SYTO16 is a rapid and reliable method of monitoring parasitemia in T. sergenti-infected cattle.     

Accurate quantitation of Leishmania infection in cultured cells by flow cytometry

BACKGROUND: Leishmaniases are major parasitic diseases caused by protozoans that are obligate intracellular parasites during the mammalian phase of their life cycle. Quantitation of experimental mammalian cell infections is usually performed by time-consuming microscopic examination. In this report a flow cytometry (FCM)-based assay suitable for studying in vitro infections by L.amazonensis is presented. METHODS: Intense fluorescence staining of the amastigote forms with a stage- and species-specific monoclonal antibody was obtained after permeabilization of both the host-cell cytoplasmic membrane and the parasitophorous vacuole membrane by saponin treatment. RESULTS: Upon flow cytometry (FCM) analysis, parasitized cells separated sharply from the auto-fluorescence of the mammalian host cells, giving the assay a high degree of sensitivity and specificity. Ninety to 98% of cells in the more fluorescent population harbored parasites visible by phase-contrast and UV-light microscopy, while no parasites were observed in more than 95% of the cells in the population with background fluorescence. Comparisons of the FCM results with those from microscope counting and analysis of various dilutions of parasitized cells confirmed the reliability of the method. CONCLUSIONS: The FCM assay provided rapid quantitation of Leishmania infection either in mouse macrophages, the natural host cell in murine leishmaniasis, or in Chinese hamster ovary (CHO) cells, a non-macrophage cell line proposed as an in vitro model for studying host-parasite interactions. The protocol described here should be adaptable to studies involving other parasites residing in nucleated cells.     

Ultrastructure of erythrocytes parasitized by Haemobartonella felis.

Experimental Haemobartonella felis infections were studied in 3 mature, intact cats by examining peripheral blood, lung, and spleen by electron microscopy. Coccoid, rod, or ring forms of the organism were found on or close to the erythrocytic membrane, and adjacent parasitized erythrocytes often were attached. Intracytoplasmic crystalloid inclusions occupying most of erythrocytic cytoplasm were seen in the 3 infected cats. The cat with the highest parasitemia had inclusions in about 10% of the erythrocytes. Less than 0.01% of the erythrocytes of a control cat contained inclusions. Parasitized erythrocytes, with and without inclusions, were seen in capillaries of the lung and spleen of infected cats. Macrophages in the lung and spleen of infected cats contained parasitized erythrocytes, either with or without inclusions. Some macrophages contained erythrocyte-free organisms in phagocytic vacuoles.     

Lipid peroxidation in Plasmodium falciparum-parasitized human erythrocytes.

cis-Parinaric acid (PnA) was used as a fluorescent probe to study lipid peroxidation in nonparasitized and Plasmodium falciparum-parasitized erythrocytes, upon challenge by cumene hydroperoxide and tert-butyl hydroperoxide. Parasitized erythrocytes were less susceptible toward lipid peroxidation than nonparasitized erythrocytes with which they had been cultured. Furthermore, nonparasitized erythrocytes cultured together with parasitized cells, and thereafter isolated on a Percoll gradient, were less susceptible toward lipid peroxidation than erythrocytes kept under the same experimental conditions but in the absence of parasitized cells. We concluded, therefore, that the intracellular development of the parasite leads to an increase in the resistance against oxidative stress, not only of the host cell membrane of the parasitized erythrocyte, but also in the plasma membrane of the neighboring cells. The erythrocyte cytosol of parasitized cells and/or the intraerythrocytic parasite was required for the increased protection of the host cell membrane, since ghosts prepared from parasitized erythrocytes were more susceptible to lipid peroxidation than those prepared from nonparasitized ones. Vitamin E content of parasitized erythrocytes was lower than that of nonparasitized cells. However, parasitized erythrocytes promoted extracellular reduction of ferricyanide at higher rates, which might be indicative of a larger cytosolic reductive capacity. It is suggested that the improved response of intact erythrocytes is due to an increased reduction potential of the host-erythrocyte cytosol. The role of vitamin C as a mediator of this process is discussed.     

Phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection

The phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection has been studied. Parasitized and nonparasitized erythrocytes from malaria-infected blood were separated and pure erythrocyte membranes from parasitized cells were isolated using Affi-Gel beads. In this way, the phospholipid content and composition of the membrane of nonparasitized cells, the erythrocyte membrane of parasitized cells and the parasite could be determined. The phospholipid content and composition of the erythrocyte membranes of nonparasitized and parasitized cells and erythrocytes from chloroquine-treated monkeys cured from malaria, were the same as in normal erythrocytes. The phospholipid content of the parasite increased during its development, but its composition remained unchanged. Three independent techniques, i.e., treatment of intact cells with phospholipase A2 and sphingomyelinase C, fluorescamine labeling of aminophospholipids and a phosphatidylcholine-transfer protein-mediated exchange procedure have been applied to assess the disposition of phospholipids in: erythrocytes from healthy monkeys, nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and erythrocytes from monkeys that had been cured from malaria by chloroquine treatment. The results obtained by these experiments do not show any abnormality in phospholipid asymmetry in the erythrocyte from malaria-infected (splenectomized) monkeys, neither in the nonparasitized cells, nor in the parasitized cells at any stage of parasite development. Nevertheless, a considerable degree of lipid bilayer destabilization in the membrane of the parasitized cells is apparent from the enhanced exchangeability of the PC from those cells, as well as from their increased permeability towards fluorescamine.     

Sensitivity of the erythrocyte micronucleus assay: dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., < or =0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

Oxygen consumption in Plasmodium berghei-infected murine red cells: a direct spectrophotometric assay in intact erythrocytes

A spectrophotometric assay has been devised to measure oxygen consumption non-invasively in intact murine red cells parasitized by Plasmodium berghei. The method uses oxyhemoglobin in the erythrocytes both as a source of oxygen and as an indicator of oxygen consumption. Spectra of intact cells show broad peaks and sloping baselines due to light-scattering. In order to ascertain the number of varying components in the 370-450 nm range, the resolution of the spectra was enhanced using Fourier transforms of the frequency domain spectra. Calculation of oxygen consumption was carried out for two-component systems (oxyhemoglobin, deoxyhemoglobin) using absorbances at 415 and 431 nm. Samples prepared from highly parasitized mice (greater than 80% parasitemia, 5% hematocrit) showed oxygen consumption rates of (4-8) X 10(-8) microliter/cell per h. This rate was not attributable to the presence of white cells or reticulocytes. The rate of oxygen consumption in the erythrocytes is shown to be modulated by various agents: the respiratory inhibitors NaN3 and KCN (1 mM) reduced oxygen consumption 2-3-fold; salicylhydroxamic acid (2.5 mM) caused a 20% reduction in rate and 10 mM NaN3, completely blocked deoxygenation. Antimalarial drugs and metal-chelating agents were also tested. Chloroquine, EDTA and desferal (desferoxamine mesylate) did not decrease the deoxygenation rate of hemoglobin in parasitized cells. Quinacrine, quinine and primaquine reduced the rate of formation of deoxyhemoglobin but also produced substantial quantities of methemoglobin. The lipophilic chelator, 5-hydroxyquinoline, decreased the rate of deoxygenation one-third. The spectrophotometric assay provides a convenient means to monitor oxygen consumption in parasitized red cells, to test the effects of various agents thereon, and potentially to explore possible mechanisms for oxygen utilization.     

Sensitivity of the erythrocyte micronucleus assay: Dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., ≤0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

Enhanced uptake and metabolism of riboflavin in erythrocytes infected with Plasmodium falciparum

Riboflavin deficiency inhibits the growth of malaria parasites both in vitro and in vivo in infected animals and humans. Although the precise mechanisms underlying this inhibition are unknown, they may involve enhanced requirements for riboflavin by parasites. To investigate this possibility, the rate of uptake of [14C]riboflavin and the biosynthesis of FMN and FAD from riboflavin were studied in infected (5-8% parasitemia) and uninfected human erythrocytes. All cells were incubated for 0-3 h at 37 degrees C in phosphate buffered saline containing MgCl2, glucose, and [14C]riboflavin (2.5-7.5 microM). At hourly intervals, samples were removed, centrifuged, washed twice with cold buffer, and lysed before counting the radioactivity. The rate of in vitro biosynthesis of FMN and FAD from riboflavin in erythrocytes was measured by ion exchange chromatography and reverse isotope dilution techniques. Results showed that the rate of riboflavin uptake and the biosynthesis of FMN and FAD were enhanced in erythrocytes with parasitemia as compared with results in unparasitized erythrocytes. Riboflavin uptake in erythrocytes was proportional to the extent of parasitemia and especially to percent of schizonts present in erythrocytes. These studies indicate that the requirement for riboflavin may be greater in the parasite than in the host erythrocyte. This increased riboflavin requirement may be due to rapid multiplication, higher metabolic rate, and extreme vulnerability to oxidative stress of malaria parasites compared with that of host erythrocytes. The differential requirement of riboflavin by the host and the malaria parasite may hold important potential for developing new strategies for malaria chemotherapy.     

Plasmodium falciparum: increased and multiple invasion during short periods of time

We describe how to obtain an increased merozoite invasion of Plasmodium falciparum into human erythrocytes during short periods of time. Using this procedure, infected erythrocytes show multiple invasions (2-4 merozoites per erythrocyte), amplifying, several times, the effects of parasite entry into host cells. The procedure yields synchronous cultures (2-h age range) with parasitemia as high as 15%. It is possible to reach parasitemia of 50% or higher allowing for a 6-h invasion period.     

Erythrocytes infected by Plasmodium falciparum activate human platelets

Blood platelets are involved in Plasmodium falciparum malaria pathology as shown by thrombocytopenia and increased plasma level of two alpha granule proteins: beta thromboglobulin (beta TG) and platelet factor 4 (PF4). In this study we demonstrate that Plasmodium falciparum parasitized erythrocytes activate directly the secretion of beta TG and PF4 by human platelets. This secretion is related to parasitemia and occurs immediately after contact. Treatment of parasited erythrocytes by trypsin and diffusion chamber experiments suggest that platelet activation is triggered by parasitic substances shed on erythrocyte membrane and released in the culture medium.     

Characterization of a modified red cell membrane protein expressed on erythrocytes infected with the human malaria parasite Plasmodium falciparum: possible role as a cytoadherent mediating protein

Infections with the human malaria Plasmodium falciparum are characterized by the retention of parasitized erythrocytes in tissue capillaries and venules. Erythrocytes containing trophozoites and schizonts attach to the endothelial cells that line these vessels by means of structurally identifiable excrescences present on the surface of the infected cell. Such excrescences, commonly called knobs, are visible by means of scanning or transmission electron microscopy. The biochemical mechanisms responsible for erythrocyte adherence to the endothelial cell are still undefined. In an attempt to identify the cytoadhesive molecule on the surface of the infected cell, we have prepared monoclonal antibodies to knob-bearing erythrocytes infected with the FCR-3 strain of P. falciparum. One of these monoclonal antibodies, designed 4A3, is an IgM that reacts (by means of immunofluorescence) with the surface of unfixed erythrocytes bearing mature parasites of the knobby line; it does not react with knobless lines or uninfected erythrocytes. By immunoelectron microscopy the monoclonal antibody 4A3 was localized to the knob region. In an in vitro cytoadherence assay, the monoclonal antibody partially blocked the binding of knob-bearing cells (FCR-3 strain) to formalin-fixed amelanotic melanoma cells. The monoclonal antibody was used to immunoprecipitate a protein from extracts of knobby erythrocytes that had been previously surface iodinated. By a two-dimensional peptide mapping technique, the antigen recognized by the monoclonal antibody was found to be structurally related to band 3 protein, the human erythrocyte anion transporter.     

Plasmodium falciparum: Increased and Multiple Invasion During Short Periods of Time

We describe how to obtain an increased merozoite invasion of Plasmodium falciparum into human erythrocytes during short periods of time. Using this procedure, infected erythrocytes show multiple invasions (2–4 merozoites per erythrocyte), amplifying, several times, the effects of parasite entry into host cells. The procedure yields synchronous cultures (2-h age range) with parasitemia as high as 15%. It is possible to reach parasitemia of 50% or higher allowing for a 6-h invasion period.     

Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

BACKGROUND: Naturally induced antibodies binding to surface antigens of Plasmodium falciparum-infected erythrocytes can be detected by direct agglutination of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously been shown to recognise Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This protein is inserted by the parasite into the host cell membrane and mediates the adhesion to the venular endothelium of the host organism in vivo. METHODS: Erythrocytes infected at high parasitaemias with ethidium-bromide-labelled mature forms of P. falciparum parasites were sequentially exposed to immune plasma, goat anti-human immunoglobulin (Ig) G, and fluorescein-isothiocyanate-conjugated rabbit anti-goat Ig. Plasma antibodies recognising antigens exposed on the surface of parasitised erythrocytes were subsequently detected by two-colour flow cytometry. RESULTS: Binding of human antibodies to the surface of erythrocytes infected with adhesive strains of Plasmodium falciparum can be measured by the two-colour flow cytometry (FCM) assay described. In addition, we demonstrate that the adhesive capacity of a parasite isolate correlates with the capacity of human immune plasmas to label the isolate as detected by FCM. We also show that the antigens recognised by the labelling antibodies are strain specific and that their molecular weights are in the range previously described for PfEMP1 antigens. CONCLUSIONS: Our FCM assay predominantly detects antibodies that recognise PfEMP1 and thus constitutes a convenient assay for the analysis of acquisition, maintenance, and diversity of anti-PfEMP1-specific antibodies and for the examination of class and subclass characteristics.     

Enhanced Uptake and Metabolism of Riboflavin in Erythrocytes Infected with Plasmodium falciparum

Riboflavin deficiency inhibits the growth of malaria parasites both in vitro and in vivo in infected animals and humans. Although the precise mechanisms underlying this inhibition are unknown, they may involve enhanced requirements for riboflavin by parasites. To investigate this possibility, the rate of uptake of [¹⁴C]riboflavin and the biosynthesis of FMN and FAD from riboflavin were studied in infected (5–8% parasitemia) and uninfected human erythrocytes. All cells were incubated for 0–3 h at 37° C in phosphate buffered saline containing MgCl₂, glucose, and [¹⁴C]riboflavin (2.5–7.5 μM). At hourly intervals, samples were removed, centrifuged, washed twice with cold buffer, and lysed before counting the radioactivity. The rate of in vitro biosynthesis of FMN and FAD from riboflavin in erythrocytes was measured by ion exchange chromatography and reverse isotope dilution techniques. Results showed that the rate of riboflavin uptake and the biosynthesis of FMN and FAD were enhanced in erythrocytes with parasitemia as compared with results in unparasitized erythrocytes. Riboflavin uptake in erythrocytes was proportional to the extent of parasitemia and especially to percent of schizonts present in erythrocytes. These studies indicate that the requirement for riboflavin may be greater in the parasite than in the host erythrocyte. This increased riboflavin requirement may be due to rapid multiplication, higher metabolic rate, and extreme vulnerability to oxidative stress of malaria parasites compared with that of host erythrocytes. The differential requirement of riboflavin by the host and the malaria parasite may hold important potential for developing new strategies for malaria chemotherapy.     

Erythrocyte membrane-associated immunoglobulins during malaria infection of mice.

Immunoglobulin (Ig) is associated with erythrocyte membranes during infection of A/J mice with Plasmodium berghei. It was detected by agglutination of the cells with rabbit antibodies to mouse IgG and by a radioimmunoassay with 125I-labelled rabbit antibodies to mouse IgG. As shown by the degree of agglutination and of binding of radiolabeled antibodies to the erythrocyte membranes, the amount of Ig increased with time after infection and paralleled parasitemia and reticulocytosis. The erythrocyte-associated immunoglobulins are mainly IgG but IgM was also present on the cells of some mice. A large proportion of the Ig could be eluted at 37 degrees C and was analyzed by immunoprecipitation and acrylamide gel electrophoresis. A radioautographic study with 125I-labeled anti-mouse IgG revealed that both parasitized and nonparasitized reticulocytes of infected mice had much larger amounts of membrane-bound immunoglobulin than did mature, nonparasitized erythrocytes. The nature of the bonds between the Ig and the surface membrane of the reticulocytes is not known. The Ig could be part of immune complexes nonspecifically bound to the cell surface. However, since we have not detected Fc or C3d receptors on reticulocytes, it is possible that the Ig constitutes autoantibodies against reticulocytes or antibodies against parasitic antigens present on the cell membrane.     

Plasmodium knowlesi induces alterations in phosphatidylcholine and phosphatidylethanolamine molecular species composition of parasitized monkey erythrocytes

Using high performance liquid chromatography and gas-liquid chromatography, we have characterized the phosphatidylcholine and phosphatidylethanolamine molecular species composition of trophozoite and schizont forms of Plasmodium knowlesi parasitized erythrocytes. Similarly, we determined these parameters in the erythrocyte membranes of trophozoite parasitized cells, unparasitized erythrocytes from infected monkeys before and after a chloroquine treatment and erythrocytes from monkeys that had never been infected. Plasma phosphatidylcholine molecular species composition was also studied. P. knowlesi parasitized erythrocytes presented higher amounts of 16:0/18:2-phosphatidylcholine than the various control cells, which appeared to be compensated for by a decrease in 18:0/20:4-, 16:0/20:3-, 16:0/18:1-, 18:0/18:2-, 18:0/20:3-, 16:0/16:0- and 16:0/18:0-phosphatidylcholines. In the case of phosphatidylethanolamine, the alterations were quantitatively of greater importance and consisted of an increase in, again, 16:0/18:2-phosphatidylethanolamine and a decrease in several species containing 20:4, namely 16:0/20:4-, 18:0/20:4- and 18:1/20:4-phosphatidylethanolamine; also the levels of alkoxy-phosphatidylethanolamines were markedly decreased. P. knowlesi development within monkey erythrocytes therefore appears to be associated with changes in phosphatidylcholine and phosphatidylethanolamine molecular species in the whole parasitized cell. These alterations are also exhibited by the host cell membrane, which provides the first experimental evidence that the parasite is able to manipulate the erythrocyte membrane lipid species composition. The consequences of these alterations on membrane physiology are discussed, as well as the implications that these data may have on the trafficking of phosphatidylcholine and phosphatidylethanolamine in the erythrocytes of P. knowlesi infected monkeys.     

Numbers of rosette formine cells in human peripheral blood.

Thymus-derived (T-cell) and “bursal” derived (B-cell) lymphocytes in human peripheral blood were quantitated by assaying percentages of cells forming erythrocyte rosettes. T-cell rosettes were formed with neuraminidase treated sheep erythrocytes. B-cell rosettes were formed with complement coated sheep erythrocytes. Large differences in the percentages of T-rosette forming cells were noted depending on the method used to assay these cells. When rosette forming cells (RFC) and non-RFC were counted concurrently the percentage of T-cell rosettes were 53–75% whereas methods involving the separate counting of RFC and total cells gave T-cell RFC percentages of 23–40%. These differences were due to the “co-rosetting” of non-RFC into the T-cell rosette clusters. This occurred because of the gentleness required to resuspend the fragile T-cell rosettes. “Co-rosetting” was demonstrated by forming stable complement receptor rosettes with complement-coated human erythrocytes and resuspending them either gently or vigorously. Significantly higher percentages of rosettes were noted with gentle cell suspension than with vigorous resuspension. The percentages of rosette forming T-cells in human peripheral blood are therefore lower than previously estimated.     

Quantification of hematozoa in blood smears

Ten thin blood smears from mourning doves (Zenaida macroura) infected with Haemoproteus maccallumi were examined by each of two observers using identical techniques and microscopy in an attempt to delineate the factors necessary to provide an accurate estimate of the number of parasites/n erythrocytes. The number of erythrocytes examined must be actually counted, not estimated from extrapolated partial counts or from the number of fields of view examined. Doubling the number of erythrocytes counted (1) decreased the overdispersed frequency distribution patterns in only 25% of the replicate counts for numbers of H. maccallumi/100 erythrocytes for a series of 2,000 versus 4,000 erythrocytes counted; and (2) did not significantly increase the accuracy for determining parasite intensities. Thus, the number of erythrocytes that must be counted to determine parasite intensities could be considerably reduced from the 10,000 or 20,000 estimated for most studies, and still provide an accurate determination of the number of parasites/n erythrocytes in datasets collected from hosts with moderate to high levels of parasitemia. This resulted in a decreased amount of time expended by the observer on each blood smear examined. With two equivalently trained individuals, differences between observers examining the same blood smears were minimal. This study suggests an approach by which a more standardized methodology for quantifying blood parasite intensities could be developed.