Rapid diagnosis of malaria by fluorescence microscopy

The routine procedure for detection of blood stages of Plasmodium spp involves Giemsa staining of thin and thick blood smears. This procedure, although simple, is time consuming, and its interpretation is dependent upon the training and experience of the observer. New methods for malaria diagnosis still require considerable financial outlay for specialist equipment and re-training of staff In this article, Hiko Kawamoto and Peter Billingsley discuss an efficient method for the detection of malaria parasites using low-cost, paired filters adapted for standard light microscopes and acridine orange staining.     

Triton X-100 in Giemsa Staining of Blood Parasites

The effects of a surface active agent, Triton X-100, were studied in the routine Giemsa staining of seven blood parasites: Plasmodium vivax, Trypanosoma cruzi, T. lewisi, Leishmania donovani, Toxoplasma gondii, and microfilariae of Dirofilaria immitis and of Wuchereria bancrofti. Concentrations of Triton X-100 ranging from 0.01% to 0.5% were used in staining (a) both thick and thin blood films of all organisms except L. donovani, (b) tissue smears of L. donovani, and (c) tissue and peritoneal fluid smears of T. gondii. In general, the addition of the detergent to the Giemsa solution resulted in cleaner preparations and better stained organisms. Morphological details were more distinct, thus facilitating microscopical detection and identification of species. The most beneficial concentration of Triton X-100 was found to be 0.1%. Since it has a hemolytic effect on erythrocytes, concentrations above 0.01% cannot be used in staining thin blood films. It is suggested also that the use of Triton-Giemsa may help prevent transfer of some of these organisms from one slide to another during mass staining procedures as it has been demonstrated to do with malaria parasites (Brooke and Donaldson, 1950).     

Triton X-100 in Giemsa Staining of Blood Parasites

The effects of a surface active agent, Triton X-100, were studied in the routine Giemsa staining of seven blood parasites: Plasmodium vivax, Trypanosoma cruzi, T. lewisi, Leishmania donovani, Toxoplasma gondii, and microfilariae of Dirofilaria immitis and of Wuchereria bancrofti. Concentrations of Triton X-100 ranging from 0.01% to 0.5% were used in staining (a) both thick and thin blood films of all organisms except L. donovani, (b) tissue smears of L. donovani, and (c) tissue and peritoneal fluid smears of T. gondii. In general, the addition of the detergent to the Giemsa solution resulted in cleaner preparations and better stained organisms. Morphological details were more distinct, thus facilitating microscopical detection and identification of species. The most beneficial concentration of Triton X-100 was found to be 0.1%. Since it has a hemolytic effect on erythrocytes, concentrations above 0.01% cannot be used in staining thin blood films. It is suggested also that the use of Triton-Giemsa may help prevent transfer of some of these organisms from one slide to another during mass staining procedures as it has been demonstrated to do with malaria parasites (Brooke and Donaldson, 1950).     

The use of percoll gradients, elutriator rotor elution, and mithramycin staining for the isolation and identification of intraerythrocytic stages of plasmodium berghei

Intraerythrocytic parasites of Plasmodium vinckei and Plasmodium berghei were separated according to their developmental stages using discontinuous Percoll gradients. Contaminating nucleated blood cells such as leukocytes were removed by elutriation centrifugation. The stages were unequivocally identified in smears using a newly developed DNA-specific staining procedure with mithramycin and fluorescence microscopy. This stain can also be used to detect parasites in human blood of very low parasitemias. The combination of methods described has many possible applications in immunologic and biochemical parasite research.     

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.     

Babesia gibsoni: Detection in blood smears and formalin-fixed, paraffin-embedded tissues using deoxyribonucleic acid in situ hybridization analysis

In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis.     

Plasma Levels of Zinc,Copper, Copper/Zinc Ratio,and Activity of Carbonic Anhydrase in Equine Piroplasmosis

We have determined the plasma concentrations of copper, zinc, copper/zinc ratio, and carbonic anhydrase activity in horses infected with Babesia equi. The study was conducted in 14 horses with the disease and 10 healthy animals that served as controls. The infection was confirmed by the clinical manifestations of the disease and by Giemsa staining of thin blood smears showing the parasites inside red blood cells. The horses with piroplasmosis had lower plasma levels of zinc, elevated copper, and increased activity of carbonic anhydrase. Consequently, the copper/zinc ratio was also higher than in the healthy controls.     

Reduced susceptibility to pyrethroid insecticide treated nets by the malaria vector Anopheles gambiae s.l. in western Uganda

Background

Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is time-consuming, whereas the more sensitive parasite lactate dehydrogenase assay involves multiple steps and requires fresh reagents. A simple PCR-based method was therefore tested for parasite detection that can be adapted to high throughput studies.

Methods

Approximately 1 μL of packed erythrocytes from each well of a microtiter cloning plate was directly used as template DNA for a PCR reaction with primers for the parasite 18s rRNA gene. Positive wells containing parasites were identified after rapid separation of PCR products by gel electrophoresis.

Results

The PCR-based method can consistently detect a parasitaemia as low as 0.0005%, which is equivalent to 30 parasite genomes in a single well of a 96-well plate. Parasite clones were easily detected from cloning plates using this method and a comparison of PCR results with Giemsa-stained blood smears showed that PCR not only detected all the positive wells identified in smears, but also detected wells not identified otherwise, thereby confirming its sensitivity.

Conclusion

The PCR-based method reported here is a simple, sensitive and efficient method for detecting parasite clones in culture. This method requires very little manual labor and can be completely automated for high throughput studies. The method is sensitive enough to detect parasites a week before they can be seen in Giemsa smears and is highly effective in identifying slow growing parasite clones.     

A Quick and Standardized Giemsa Stain for Wet-Fixed Cytological Material

The Giemsa stain is one of the most widely used staining techniques in cytology, especially in hematology. A standardized Romanowsky-Giemsa staining procedure using pure cationic azure B (C.I. 52010) and anionic eosin (C.I. 45380) has been described by Wittekind et al (1982). A revised standard Giemsa staining procedure was recently published (Wittekind and Kretschmer 1987). Usually the Romanowsky-Giemsa stain is applied to air dried and methanol fixed cytological material, e.g. blood smears and bone marrow films (ICSH 1984).     

A simple staining method for cryptosporidian oocysts and sporozoites

A useful staining method for detection of cryptosporidian oocysts and sporozoites was developed and described. The modified Kohn's one-step staining technique with an additional modification, i.e., longer staining time and higher staining temperature than those originally described, was tested on fecal smears from cats infected with Cryptosporidium sp. By this improved staining procedure, the oocyst appeared as a slightly oval body containing four internal sporozoites colored blue to blue-gray. The oocyst wall was stained dark green to black. The morphological feature of the oocyst was recognized much more clearly by this staining technique than by such currently used techniques as acid-fast and Giemsa staining. This staining procedure proved to be simple and less costly and to secure a good preservation.     

Dientamoeba fragilis is more prevalent than Giardia duodenalis in children and adults attending a day care centre in Central Italy

Giardia duodenalis is a well recognised enteropathogen, while Dientamoeba fragilis is rarely detected and consequently it is not recognised as an important human pathogen. In 2002-2003, a survey has been carried out on enteroparasites in faecal samples of outpatients attending a day care centre in the town of Perugia (Central Italy). To improve the detection level, at least three samples from each patient were collected at different days and within two hours from defecation. The coproparasitological examination has been carried out by direct microscopic examination, faecal concentration, and Giemsa and modified Ziehl-Nielsen stainings of faecal smears. The genotypes of Giardia duodenalis isolates were determined by PCR of the beta-giardin gene. Of 1,989 enrolled people (966 children, 1,023 adults), 165 persons (8.3%; 153 adults, 15.0%; 12 children, 1.2%), were positive for parasites, but only 1 12 adults (73.2% of those infected) and eight children (66.7% of those infected) harboured D. fragilis and G. duodenalis. Both the Assemblages A and B were detected in 18 G. duodenalis isolates examined at the beta-giardin gene. The higher prevalence of D. fragilis infections than that of G. duodenalis is probably related to the method used, a procedure, which is rarely followed in laboratories for the diagnosis of enteric parasites. These epidemiological data suggest that when faecal samples are examined after a period of time and without Giemsa staining, most D. fragilis infections goes undetected.     

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.     

The diagnosis of malaria and identification of plasmodium species by polymerase chain reaction in Turkey

More than half of the world's population is exposed to malaria in approximately 100 countries. Rapid diagnosis and correct treatment of cases are the main objectives of control programs in malaria endemic areas. We have developed a PCR method to determine the presence of plasmodium DNA in blood. The method can also identify the species of the plasmodium by restriction enzyme analysis of the amplified product. We evaluated the performance of this method in the diagnosis of malaria suspected cases in Turkey by comparing to microscopy of the blood smears: blood samples were obtained from 114 patients with malaria symptoms, including fever and/or chills lasting for several days, before starting treatment. Thin and thick blood smears were prepared immediately in the region of specimen collection. After isolation of DNA from blood samples, DNA was amplified by PCR and digested by restriction enzyme AluI. The obtained fragments were analyzed by agarose gel electrophoresis. The number of parasites in the thick and thin smears of the blood samples was evaluated microscopically after staining by Giemsa and results were compared by PCR results. Among 114 plasmodium positive cases detected by microscopy, 100 were also detected by PCR. There were 14 false negatives and no false positive by PCR. Compared to microscopy, the sensitivity, specificity and Positive Predictive Value (PPV) of PCR were determined as 76%, 100% and 100%, respectively.     

A Simple Staining Procedure for Detecting the true Acrosome Reaction in Buffalo (Bubalus bubalis) Spermatozoa

A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.     

A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum

We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2–3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages.     

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.     

Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas

Malaria is a responsible for approximately 600 thousand deaths worldwide every year. Appropriate and timely treatment of malaria can prevent deaths but is dependent on accurate and rapid diagnosis of the infection. Currently, microscopic examination of the Giemsa stained blood smears is the method of choice for diagnosing malaria. Although it has limited sensitivity and specificity in field conditions, it still remains the gold standard for the diagnosis of malaria. Here, we report the development of a fluorescence in situ hybridization (FISH) based method for detecting malaria infection in blood smears and describe the use of an LED light source that makes the method suitable for use in resource-limited malaria endemic countries. The Plasmodium Genus (P-Genus) FISH assay has a Plasmodium genus specific probe that detects all five species of Plasmodium known to cause the disease in humans. The P. falciparum (PF) FISH assay and P. vivax (PV) FISH assay detect and differentiate between P. falciparum and P. vivax respectively from other Plasmodium species. The FISH assays are more sensitive than Giemsa. The sensitivities of P-Genus, PF and PV FISH assays were found to be 98.2%, 94.5% and 98.3%, respectively compared to 89.9%, 83.3% and 87.9% for the detection of Plasmodium, P. falciparum and P. vivax by Giemsa staining respectively.     

Peroxidase Staining Combined with Autoradiography for Study of Eosinophilic Granules

Giemsa staining and a peroxidase reaction were applied to blood films in conjunction with autoradiography to establish the types of granulocytes that stain differentially with the benzidine-peroxidase reaction. Differential counts made on Ciemsa-stained and peroxidase-stained autoradiograms were compared. In T. spiralis-infected rats with an elevated eosinophil count, as judged by Giemsa staining, the percentage of granulocytes that stained more intensely with peroxidase was increased. The results suggested that the eosinophils were the intensely peroxidase-positive cells. Blood smears were stained for peroxidase before being coated with NTB₂ liquid emulsion. Although the blue color of the peroxidase reaction faded during photographic development, the color redeveloped when peroxidase-stained autoradiograms were stained once again after photographic development. It was found necessary to stain for peroxidase both before and after autoradiography. The correlation of Giemsa-stained and peroxidase-stained autoradiograms indicated that the peroxidase stain can be combined with autoradiography to obtain authentic results.     

Rapid detection of malaria and other bloodstream parasites by fluorescence microscopy with 4'6 diamidino-2-phenylindole (DAPI).

DAPI is a fluorescent dye which appears to complex specifically with DNA. We have used this probe to detect and identify malarial infections by fluorescence microscopy. Experiments were conducted using Plasmodium berghei yoeli--infected mouse blood, P. lophurae--infected duck blood, and P. vivax--infected human blood. Infected avian blood was used to detect parasites within nucleated erythrocytes. Control blood smears from uninfected hosts revealed fluorescence only in the leukocytes of mammalian blood or in nuclei of leukocytes and erythrocytes of avian blood. Cytoplasmic staining of red blood cells was absent in all controls. In contrast, the cytoplasm of infected red blood cells was stippled with fluorescence centers. Ring forms, trophozoites, segmenters, and merozoites frequently were observed. This simple procedure can be applied directly to routine clinical analysis, as well as experimental procedures, DAPI can also be used to stain other parasites, including nuclei in microfilariae.     

Automated detection of working area of peripheral blood smears using mathematical morphology.

The paper presents a technique to automatically detect the working area of peripheral blood smears stained with May-Grünwuald Giemsa. The optimal area is defined as the well spread part of the smear. This zone starts when the erythrocytes stop overlapping (on the body film side) and finishes when the erythrocytes start losing their clear central zone (on the feather edge side). The approach yields a quick detection of this area in images scanned under low magnifying power (immersion objective x 25 or x 16). The algorithm consists of two stages. First, an image analysis procedure using mathematical morphology is applied for extracting the erythrocytes, the centers of erythrocytes and the erythrocytes with center. Second, the number of connected components from the three kinds of particles is counted and the coefficient of spreading rho(s) and the coefficient of overlapping rho(o) are calculated. The data from fourteen smears illustrate how the technique is used and its performance. Colour figures can be viewed on http://www.esacp.org/acp/2003/25-1/angulo.htm.