A novel semi-automatic image processing approach to determine <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> parasitemia in Giemsa-stained thin blood smears

Background  

Malaria parasitemia is commonly used as a measurement of the amount of parasites in the patient's blood and a crucial indicator for the degree of infection. Manual evaluation of Giemsa-stained thin blood smears under the microscope is onerous, time consuming and subject to human error. Although automatic assessments can overcome some of these problems the available methods are currently limited by their inability to evaluate cases that deviate from a chosen "standard" model.     

Premature release of Plasmodium falciparum from swollen erythrocytes induced by some antimalarials

Qinghaosu and chloroquine, but not pyrimethamine, treatment of Plasmodium falciparum cultures resulted in the formation of swollen red blood cells (RBCs) and the expulsion of degenerate trophozoites and schizonts, but not ring-stage parasites, from the infected RBCs. The parasite release resulted in the formation of RBCs with holes, that had otherwise retained their structural integrity. Membranes of swollen RBCs and their ghosts associated with parasites were efficiently visualized by Giemsa staining of thin smears for 18-24 hr but not by standard Giemsa staining for 20 min.     

Evaluation of a Novel Magneto-Optical Method for the Detection of Malaria Parasites

Improving the efficiency of malaria diagnosis is one of the main goals of current malaria research. We have recently developed a magneto-optical (MO) method which allows high-sensitivity detection of malaria pigment (hemozoin crystals) in blood via the magnetically induced rotational motion of the hemozoin crystals. Here, we evaluate this MO technique for the detection of Plasmodium falciparum in infected erythrocytes using in-vitro parasite cultures covering the entire intraerythrocytic life cycle. Our novel method detected parasite densities as low as ∼40 parasites per microliter of blood (0.0008% parasitemia) at the ring stage and less than 10 parasites/µL (0.0002% parasitemia) in the case of the later stages. These limits of detection, corresponding to approximately 20 pg/µL of hemozoin produced by the parasites, exceed that of rapid diagnostic tests and compete with the threshold achievable by light microscopic observation of blood smears. The MO diagnosis requires no special training of the operator or specific reagents for parasite detection, except for an inexpensive lysis solution to release intracellular hemozoin. The devices can be designed to a portable format for clinical and in-field tests. Besides testing its diagnostic performance, we also applied the MO technique to investigate the change in hemozoin concentration during parasite maturation. Our preliminary data indicate that this method may offer an efficient tool to determine the amount of hemozoin produced by the different parasite stages in synchronized cultures. Hence, it could eventually be used for testing the susceptibility of parasites to antimalarial drugs.     

食蟹猴疟原虫感染恒河猴的长期观察

本项研究证明,食蟹猴疟原虫感染恒河猴后出现的红细胞内期是一个长期的寄生过程,不论血传或子孢子感染,于原虫密度高峰后,均有较长期的低原虫密度阶段。恒河猴均可耐受食蟹猴疟原虫高密度的感染。     

Detection of different developmental stages of malaria parasites by non-radioactive DNAin situ hybridization

Summary A highly sensitive non-radioactive DNAin situ hybridization procedure is described that enables detection and unequivocal identification of various developmental stages of human and rodent malaria parasites. Using biotinylated species-specific DNA probes, erythrocytic parasites can be specifically stained in blood smears. Similarly exoerythrocytic stages can be visualized in cell culture and in sections of paraffin-embedded liver. In blood smears, the hybridization procedure provides a rapid detection of (low) parasitemia and species-determination for experienced microscopists at 100 to 400x magnification. Moreover, the procedure can be applied even after previous Giemsa staining of the preparation, enabling revision of patient smears which were difficult to read after routine Giemsa staining.     

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.     

A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis

Measures of parasitemia by intraerythrocytic hematozoan parasites are normally expressed as the number of infected erythrocytes per n erythrocytes and are notoriously tedious and time consuming to measure. We describe a protocol for generating rapid counts of nucleated erythrocytes from digital micrographs of thin blood smears that can be used to estimate intensity of hematozoan infections in nonmammalian vertebrate hosts. This method takes advantage of the bold contrast and relatively uniform size and morphology of erythrocyte nuclei on Giemsa-stained blood smears and uses ImageJ, a java-based image analysis program developed at the U.S. National Institutes of Health and available on the internet, to recognize and count these nuclei. This technique makes feasible rapid and accurate counts of total erythrocytes in large numbers of microscope fields, which can be used in the calculation of peripheral parasitemias in low-intensity infections.     

Achievable standards, Benchmarks for reporting, and Criteria for evaluating cervical cytopathologySecond edition including revised performance indicators

Endocervical cells are not essential for an adequate smear, except where the previous abnormality was seen in endocervical cells. When three consecutive smears are reported as inadequate, the recommendation for colposcopy should be made at the discretion of the pathologist in the light of a review of the relevant slides and the clinical history of the woman concerned. The cellularity of previous sequential smears should not be combined in order to judge the present smear test as negative. There should be no more than three abnormal smears (including borderline) over any 10-year period without a recommendation for colposcopy. At least three negative smears, at least 6 months apart, should be reported before a woman is returned to routine recall following a smear showing mild dyskaryosis or borderline nuclear change. There is no evidence that demonstrates that selective double screening is any more effective in preventing false-negatives than rapid review and this practice cannot therefore be justified. Sensitivity should be based on all abnormalities detected on primary screening rather than on moderate dyskaryosis or worse. Ranges for reporting rates are based on the 10-90th percentiles of the range for laboratories reporting over 10000 screening smears per year in KC61 returns, but apply to all laboratories reporting screening smears.     

Plasmodium berghei: Isolation and maintenance of an irradiation attenuated strain in the nude mouse

An attenuated strain of malaria causing limited parasitemia in mice was derived from a highly virulent strain of Plasmodium berghei (NK65) which produced 100% lethality in mice. A pool of mouse blood infected with the original highly virulent P. berghei was exposed to 40 Krad irradiation and parasites were inoculated into nude mice as well as into thymus competent normal littermates. Thymus competent mice showed no parasitemia, while one out of the five nude mice inoculated with the irradiated parasites developed a slow and progressive parasitemia. These parasites induced a self-limiting parasitemia in thymus competent mice, even when a large inoculum was administered. Maintenance of the low virulence strain required passage through nude mice. After 50 passages at two weekly intervals, reversion to virulence did not occur. A single vaccination with the attenuated strain induced immunity in mice against a challenge inoculation with the original virulent strain. Specific IgG persisted at high titer for more than 9 weeks in mice receiving a single inoculation of the attenuated strain.     

Blood parasites of two Costa Rican amphibians with comments on detection and microfilaria density associated with adult filarial worm intensity

The 2 objectives of this study were: (1) to compare parasite detectability in blood smears obtained from toe-clips versus the heart from amphibian hosts; and (2) to test whether microfilariae density is correlated with adult filarial worm intensity. We examined blood parasites of 2 species of amphibians, Rana vaillanti (n = 45) and Eleutherodactylus fitzingeri (n = 36), from Costa Rica collected during the summer of 2003. Separate blood smears were obtained from toe-clips and the heart during necrospy. Eight species of blood parasites were identified from R. vaillanti and 1 from E. fitzingeri. Each parasite species was counted in a 2 x 2.2-cm2 area on each blood smear, and the density of host red blood cells (RBCs) was estimated using a sub-sampling approach, allowing parasite infections to be expressed as individuals per RBC. The detection failure rate for toe-cut smears ranged from 71-100% (x = 92.3%) and from 0-9% (x = 2.4%) for heart smears, depending on parasite species. The density of RBCs was significantly higher in smears produced from heart samples and may explain the differences in detectability. Foleyellides striatus microfilariae densities (per RBC) were significantly correlated with adult female worm intensity (R2 = 0.32, P = 0.011).     

First report of avian malaria in a Manx shearwater (Puffinus puffinus)

This study reports the case of a Manx shearwater (Puffinus puffinus) that died from avian malaria while under care at a rehabilitation center in Espírito Santo, Brazil. The bird was rescued on October 2018, and remained under care until it died suddenly on January 2019. A blood smear produced 8 days before death was negative for parasites, whereas a blood smear produced post-mortem revealed a high parasitemia by a parasite resembling Plasmodium cathemerium. The sequence of a 412 bp segment of the cyt-b gene was identical to that of lineage PADOM09, and phylogenetic analysis corroborated that this parasite was closely-related to known lineages of P. cathemerium. The acuteness and severity of the infection documented in this case suggest that seabirds of the order Procellariiformes might be highly susceptible to Plasmodium infections, raising the concern that avian malaria may present a significant threat to their conservation.     

Parasitemia in PCR‐detected Plasmodium and Haemoproteus infections in birds

Most comparative studies of avian blood parasites based on visual inspection of smears have reported Haemoproteus infections to be more prevalent than Plasmodium infections in both tropical and temperate locations. Recently, molecular techniques have increased our ability to detect infections often missed on blood smears. Here we quantify the bias in prevalence resulting from unrecognized infections by examining blood smears of infected passerine birds from the West Indies (312 individuals) and the Ozark Mountains of southern Missouri (134 individuals) for which we could identify parasites based on cytochrome b sequences. In the West Indian sample, 63 of 179 Haemoproteus infections (35%) and 121 of 133 Plasmodium infections (91%) were not detected among ca. 2,800 red blood cells examined per smear. In the Missouri sample, 19 of 77 Haemoproteus infections (25%) and 31 of 57 Plasmodium infections (54%) were not detected among ca. 10,000 red blood cells examined. Clearly, visual inspection of blood smears at this level of effort fails to recognize many malaria parasite infections ascertained by PCR screening, and this bias for Plasmodium parasites exceeds that for Haemoproteus parasites. The lower prevalence of Plasmodium compared to Haemoproteus reported in comparative studies based on blood smears likely reflects differences in detection rather than infection rates. Estimates obtained from visual inspection of blood smears would appear to be more indicative of parasite virulence and how well host individuals control infections than of the prevalence of infections in host populations.     

Measurement of erythrocytes on diagnostic slides by scanning electron microscopy

OBJECTIVE: To determine whether the measured sizes of erythrocytes in both paraffin-embedded sections and air-dried blood smears differ from values published in standard texts. STUDY DESIGN: Routinely prepared surgical pathology slides as well as an air-dried blood smear were viewed with a scanning electron microscope. Erythrocytes were measured using the instrument software. RESULTS: Erythrocyte size in the peripheral blood smear correlated well with textbook values, 7.2-7.9 microns. However, red blood cells within sectioned material from several laboratories showed a prominent decrease, ranging from 25% to 35%, as compared to textbook values, about 7 microns. CONCLUSION: Since cytologists and surgical pathologists often use the erythrocyte as a convenient marker on diagnostic slides, attention should be given to these observations in making sizing judgments.     

Prevalence and spatial distribution of intraerythrocytic parasite(s) in Puget Sound rockfish (Sebastes emphaeus) from the San Juan Archipelago, Washington (USA)

Two morphologically distinct forms of an intraerythrocytic parasite(s) were detected by microscopic observation of Giemsa-stained blood films in 45.7% of 119 rockfish (Sebastes emphaeus) from the San Juan Archipelago (Washington State, U.S.A.). Infection prevalence for both forms was 53% in males, 44% in females, and 33% in fish of undetermined gender. A binucleate "ring-stage" was present at all 4 geographic sites, with a mean prevalence of 45.7%, while mean prevalence of a larger gamont-like form from the same sites was 5.1%. The relationship of the 2 forms to each other could not be determined. Neither schizogony nor binary fission was evident in any of the infected erythrocytes and the parasites contained no obvious pigment. The possibility of the 2 morphologic forms being 2 distinct species is supported by the observation that no difference in parasitemia was seen in the binucleate form among sites (1.6-1.9%), while parasitemia of the gamont-like form varied significantly among sites, ranging from a high of 4% to a low of 0.1%. Taxonomic status of either form could not be determined at this time based on limited existing morphologic data.     

Performance Evaluation of Biozentech Malaria Scanner in Plasmodium knowlesi and P. falciparum as a New Diagnostic Tool

The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2–6.7% for Plasmodium knowlesi and 0.3–4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson’s correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r²=0.99, P<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.     

Hepatocystis Parasitemia in Wild Kenya Vervet Monkeys (Cercopithecus aethiops)

Blood smears of 159 vervet monkeys from three sites in Kenya were stained with Giemsa and examined for Hepatocystis parasites. The populations differ in incidence of parasitemia, ranging from 0–62% affected individuals. These differences are probably due to altitude and local environmental conditions.     

A study of the effect of introducing a restrictive cervical screening policy on laboratory workload and cervical cancer detection rates

This study examines the effect of a change in screening policy on the detection rate of severe dyskaryosis. During 1987 a total of 423 cases of severe dyskaryosis were identified by the Avon Screening Programme. Eleven per cent of these abnormal smears were repeat smears taken without clinical indication within the recommended 5 year recall period (interval smears). In a comparable control group of negative smears 31% were interval smears. Twenty-five per cent of the dyskaryotic interval smears (3% of the total severely dyskaryotic smears) were taken within 3 years of the previous negative smear, compared with 50% of the control group. By discouraging opportunistic smears within 5 years of the previous smear, the laboratory workload could be reduced by 30%, or within 3 years of the previous smear by 15%. There is, however, a risk of 11% and 3% respectively of missing a significant lesion (severe dyskaryosis).     

Computer-assisted primary screening of cervical smears using the PAPNET method: comparison with conventional screening and evaluation of the role of the cytologist

The PAPNET method is an interactive computer-assisted screening procedure. the diagnostician selects the abnormal video tiles out of the 128 and decides which smears need additional light microscopy. the original diagnoses of 1494 archival smears were compared with the PAPNET analysis of the same smears. the general trend observed was that the PAPNET-assisted diagnoses were of a higher grade than those assigned by the primary screener, thus less cases were signed out as negative. In addition, the PAPNET method was used for primary screening of 2971 randomly selected smears, whilst in the same period 5797 smears were conventionally screened. Using the PAPNET method, significantly fewer smears were signed out as negative. Seventy-three percent of the cases were diagnosed on the basis of the information provided by the 128 video tiles, 11% had to be screened completely by the light microscope, and the remaining cases needed additional light microscopy of a part of the smear. As a result, PAPNET-assisted screening was approximately two times faster. the great advantage of the method is that it is much less tiring for the eyes than conventional screening, making fatigue-related errors less likely, and if a smear contains only a few abnormal cells, these are easier to find.