Giemsa-stained Blood Smears Research Articles

Comparison of Grayscale Conversion Methods for Malaria Classification

Malariaparasitemia is used for measuring the degree of infection by detecting the plasmodium (commonly known as the malaria parasite) in the blood of an infected patient. The most commonly used method of malarial diagnosis is counting the number of malariainfected red blood cells in a Giemsa-stained blood smear by using a microscope. This method requires expert knowledge and is prone to inter-investigator variability. Therefore, a number of studies have been conducted on automated classification techniques that can measure malarial infection rapidly and accurately. In order to detect malaria parasites by analyzing plasmodium-infected blood smear images, conversion processing is required to make the images insensitive to the luminance contrast of microscopy and staining intensity. This paper aims to identify a grayscale conversion method optimal for plasmodium-infected blood smear images by comparing the performances of various grayscale conversion methods. The grayscale conversion methods selected for the comparison are colorimetric conversion, luma coding, conversion using the green channel only, and principal component analysis (PCA)based conversion. We used 20 malaria-infected red blood cells and 20 normal red blood cells to compare the performances of these methods by obtaining thearea under the receiver operating curve (AUC) as the minimum histogram intra-class variance value for each cell image. With the AUC value of 0.9225, the PCA-based grayscale conversion method outperformed all other methods.

The effect of Insecticide Treated Nets (ITNs) on Plasmodium falciparum infection in rural and semi-urban communities in the south west region of Cameroon.

Insecticide Treated Nets (ITNs) have been shown to reduce morbidity and mortality, but coverage and proper utilization continues to be moderate in many parts of sub-Saharan Africa. The gains made through a nationwide free distribution were explored as well as the effect on malaria prevalence in semi-urban and rural communities in south western Cameroon. A cross sectional survey was conducted between August and December 2013. Information on net possession, status and use were collected using a structured questionnaire while malaria parasitaemia was determined on Giemsa-stained blood smears by light microscopy. ITN ownership increased from 41.9% to 68.1% following the free distribution campaign, with 58.3% (466/799) reportedly sleeping under the net. ITN ownership was lower in rural settings (adjusted OR = 1.93, 95%CI = 1.36–2.74, p<0.001) and at lower altitude (adjusted OR = 1.79, 95%CI = 1.22–2.62, p = 0.003) compared to semi-urban settings and intermediate altitude respectively. Conversely, ITN usage was higher in semi-urban settings (p = 0.002) and at intermediate altitude (p = 0.002) compared with rural localities and low altitude. Malaria parasitaemia prevalence was higher in rural (adjusted OR = 1.63, 95%CI = 1.07–2.49) compared to semi-urban settings and in those below 15 years compared to those 15 years and above. Overall, participants who did not sleep under ITN were more susceptible to malaria parasitaemia (adjusted OR = 1.70, 95%CI = 1.14–2.54, p = 0.009). Despite the free distribution campaign, ITN ownership and usage, though improved, is still low. As children who reside in rural settings have greater disease burden (parasitemia) than children in semi-urban settings, the potential gains on both reducing inequities in ITN possession as well as disease burden might be substantial if equitable distribution strategies are adopted.

English

The aim of this study was to determine the seroprevalence of Anaplasma phagocytophilum infections in rural areas of Bursa Province, where vector ticks and blood-sucking flies are commonly found. In total, 150 blood samples were collected from people living in four different locations where vector ticks are common, and 45 blood samples were collected from people living in non-tick-infested areas. Giemsa-stained blood smears were prepared from the blood samples and examined by light microscopy. The blood samples were also serologically examined using the indirect fluorescent antibody test (IFAT). One sample (0.7%) from a patient with a tick bite history was positive, as determined by the IFAT; however, the same sample was negative when its Giemsa-stained smear was examined. The co-seroprevalence of A. phagocytophilum and Borrelia burgdorferi was not observed. Key words: Bursa, Anaplasma phagocytophilum, human, serology, prevalence.

Vector identification and clinical, hematological, biochemical, and parasitological characteristics of camel (Camelus dromedarius) theileriosis in Egypt.

The objectives of the present study were to identify a possible tick vector and to determine the prevalence of camel theileriosis in Egypt using blood smears stained with Giemsa's stain and PCR assay. Hemogram and serum biochemical constituents were also investigated. A total of 243 camels, aged 3-5years, were examined. The results revealed that 75 (30.86%) camels were infected with Theileria spp. of Giemsa-stained blood smears. Hyalomma dromedarii was identified as the carrier tick of Theileria spp. Multinucleated sporoblast and free sporozoite were observed in the salivary gland smears from collecting ticks. PCR result revealed that Theileria annulata was the most abundant in camels (60%) followed by Theileria spp. (10%). Macrocytic hypochromic anemia was recorded in the infected camels with T. annulata. Leukocytosis, neutrophilia, eosinophilia, and lymphopenia were also observed in the infected group. In the serum of infected camels, total proteins, albumin, β-globulin, and A/G ratio were significantly decreased (P < 0.05); however, total globulins and α- and γ-globulins were markedly increased (P < 0.05). The activity of aspartate aminotransferase and the levels of glucose, creatinine, and high-density lipoprotein cholesterol were markedly increased (P < 0.05) in the infected group. In contrast, triglycerides and total cholesterol concentrations were significant decreased (P < 0.001) in the infected group. In conclusion, a high prevalence of camel theileriosis was recorded in apparently healthy camels. H. dromedarii commonly infested these camels and were found infected with the transmissible forms of Theileria, indicating a role in transmission. Camels infected with T. annulata induced alterations in the cellular and biochemical constituents.

Improved Detection of Sleeping Sickness Cases by LED Fluorescence Microscopy: Evidence from a Prospective Multi-Centric Study in the Democratic Republic of the Congo

Background: Confirmatory diagnosis of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) is based on demonstration of parasites by microscopy. However, the sensitivity of routine microscopy methods is very low, and many cases are missed and left untreated. A clinical study was conducted in the Democratic Republic of the Congo to evaluate the accuracy of improved microscopy methods in diagnosis of HAT. These included examination by fluorescence microscopy (FM) of acridine orange (AO) stained smears of whole blood and smears made following a new procedure for concentrating trypanosomes by selective lysis of red blood cells (RBC). Methodology/Principal Findings: Venous blood was collected from 213 HAT cases, 101 HAT suspects and 95 controls and used to determine the accuracy of four microscopy methods: bright field microscopy of Giemsa-stained thick blood smears, FM of AO-stained thick blood smears, FM of AO-stained thick blood smears prepared after RBC lysis and concentration, and FM of AO-stained thin blood smears prepared after RBC lysis and concentration. The sensitivity of FM using thick blood smears stained with AO was 3 times higher than bright field microscopy using Giemsa-stained thick blood smears [19.7% (95% CI: 14.9% - 25.6%) versus 6.1% (95% CI: 3.6% - 10.2%)]. When the RBC lysis and concentration procedure was included, sensitivity of the test was further enhanced to 23.0% (95% CI: 17.9% - 29.1%) with thick blood smears and 34.3% (95% CI: 28.2% - 40.9%) with thin blood smears. Specificity of all four microscopy methods was 100% (95% CI: 96.1% - 100.0%). However, the miniature anion exchange chromatography technique (mAECT) and capillary tube centrifugation (CTC) method remained more sensitive. Conclusions: These new methods have practical advantages, including shorter staining time, ease of demonstration of parasites, and the possibility of archiving slides. They could, therefore, be alternative methods to improve case detection where concentration procedures such as mAECT or CTC are not performed.

SD Bioline malaria antigen Pf (HRP-2/pLHD) for assessing efficacy of artemisinin combination therapy against Plasmodium falciparum in pediatric patients in the Democratic Republic of the Congo.

IntroductionThe emergence of Plasmodium falciparum resistance to artemisinin combination therapy (ACT) is a worrying development. It calls for close surveillance to monitor the efficacy of the drugs. The objectives of this study were to determine the performance of SD Bioline malaria AgPf(HRP-2/pLDH) 3 band Rapid Diagnostic Test (RDT) against Giemsa-stained blood smear and evaluate the suitability of this test in assessing the therapeutic efficacy of ACT in pediatric malaria patients in the Democratic Republic of the Congo (DRC).MethodsFive hundred and one patients with malaria symptoms were screened for P. falciparum in Kinshasa, DRC. Of the 166 patients who tested positive for P. falciparum at recruitment (day 0), 103 consented to participate in this study and were followed up and retested for P. falciparum on day 3, day 7, day 14, day 21 and day 28.ResultsSensitivity and specificity of the test were significantly high on day 0 and so were their positive and negative predictive values. Higher proportions of false positive cases were observed on the HRP-2 band irrespective of patient parasite densities during the follow up but these were barely seen on the pLDH band. Some patients turned positive during follow up but pLDH readings remained consistent with blood smear readings.ConclusionSD Bioline malaria AgPf(HRP-2/pLDH) RDT demonstrated high performance in DRC. Thus, the test can be employed to assess the efficacy of ACT in pediatric malaria patients and prioritize areas that require the deployment of advanced testing like polymerase chain reaction (PCR).

Assessment of the First Commercial ELISA Kit for the Diagnosis of Theileria annulata.

The present study assesses the efficacy of SVANOVIR Theileria annulata-Ab, the first commercial ELISA kit for the diagnosis of Theileria annulata infection in cattle based on a recombinant protein known as T. annulata surface protein (TaSp). As a reference test, a polymerase chain reaction (PCR) assay depending on T. annulata merozoite surface antigen (Tams-1) was applied. A total of 468 blood samples as well as serum samples were randomly collected from cattle and tested in the PCR as well as in the ELISA developed in this study. Moreover, all samples were also analyzed by conventional Giemsa-stained blood smear. The results of this study revealed a good correlation between the results obtained by PCR and the ELISA, whereas all PCR positive samples scored correctly positive in the ELISA and 73 of the 125 PCR negative samples scored correctly negative. Taken together, a sensitivity of 91.25% and a specificity of 78.4% were recorded, when compared to the PCR data. In conclusion, the SVANOVIR Theileria annulata-Ab is a suitable diagnostic assay for use in the diagnosis and epidemiological surveys of Theileria annulata infection in chronic and carrier animals.

Babesiosis of small ruminants in Sulaimani city Kurdistan – Iraq

Babesiosis was studied in sheep and goats in Sulaimani city, Kurdistan -Iraq from June to September 2012. A total of 160 blood samples (135 of sheep and 25 of goats) were collected from different farms. Giemsa stained blood smears were applied to study babesiosis, also ticks were collected from external animals body surfaces. Results of this study showed that Babesia species was recorded among the small ruminants as 92/160 (57.5 %), 76/135 (56.3%) of these from sheep and 16/25 (64%) of goats. Statistically, there are no significant differences between sheep and goats infection by Babesia species at (P&lt; 0.05). According to the morphological characters four Babesia species were recorded in the study which was B. ovis, B. motasi, B. foliata, and B. taylori. One species of tick Hyalomma anatolicum was seen

The performance of an Histidine rich protein-2 rapid diagnostic test (RDT) against the standard microscopy in the diagnosis of malaria parasitaemia among febrile under-five children at Nnewi

Background : Malaria remains a major cause of morbidity and mortality, thus there is need for quick, reliable inexpensive diagnostic tool to facilitate its prompt treatment especially in resource poor settings. Objectives : To compare the sensitivity of a locally available Histidinerich protein-2 based rapid diagnostic test (RDT) with the standard microscopy. Methods : This study was carried out to test the performance of an histidine rich protein -2 rapid diagnostic test (RDT) against the standard microscopy in the diagnosis of malaria among febrile under-five children attending Paediatric Clinic of NAUTH Nnewi. A total of 200 children under the age of five years were recruited for the study. Data on socio-demographic characteristics and symptoms were collected through an interviewer administered questionnaire. Blood sample was collected in EDTA bottle after observing universal precautions. All of them were tested with both Giemsa stained blood smear and Histidine rich protein-2 (HRP-2) rapid diagnostic test (RDT). Results : There were 118 males and 82 females, giving a male: female ratio of 1.44:1. Their ages ranged from 3-59 months and the average age was 27+17.49 months. Average number of days the subjects had fever before presentation were 3.78+1.95 days with a range of 1- 14 days. Body temperature ranged from 35.9-40.40C with average of 37.7+0.80C. Forty (20%) were positive by microscopy while 42 (21%) were positive by rapid diagnostic test. Twenty-percent of those positive by microscopy (n=8) were negative by RDT while 23.8% of those positive by RDT (n=10) were negative by microscopy. Using microscopy as a gold standard, the sensitivity of the RDT was 80%, the specificity was 93.8%. The positive and negative predictive values were 76.2% and 94.9% respectively. Conclusion : Based on these findings, the RDT demonstrated reasonable concordance with microscopy and was recommended for use at every level of healthcare in the diagnosis of malaria. Keywords : Malaria, RDT, Microscopy, under- five.

Distribution patterns of Babesia gibsoni infection in hunting dogs from nine Japanese islands.

Canine babesiosis constitutes a major global veterinary medical problem caused by tick-borne hemoparasites Babesia gibsoni and Babesia canis. Babesia gibsoni induces more severe clinical signs and is mainly transmitted by the ixodid Haemaphysalis longicornis. In Japan, B. gibsoni is primarily found in the western districts, with few records in the eastern parts. The aim of the current investigation was to evaluate distribution patterns of B. gibsoni infection in 9 Japanese islands and peninsulas using direct microscopy and PCR. Therefore, 196 hunting dogs were randomly sampled during the period from March to September 2011. Ages and sexes of dogs were identified. Direct microscopy of Giemsa-stained blood smear revealed pear-shaped piroplasms of B. gibsoni in 3 (1.6%) dogs. PCR was done initially with the universal primer set (B18S-F and B18S-R) amplifying the 1,665-bp portion of the 18S rRNA gene, followed by the specific primer set (Bg18F1 and Bg18R2) amplifying 2,363-bp fragments of the same gene. Accordingly, 84 (42.9%) and 8 (4.1%) dogs were positive, respectively. The current investigation shows that canine babesiosis was recorded in all islands except for Sado Island, Atsumi Peninsula, and Tanegashima Island. The highest infection rate was detected in the main island of Okinawa, while the lowest was on Ishigaki Island. Both sexes were non-significantly infected. However, the diversity of infection in islands was significantly different (P < 0.05). Although B. gibsoni has been previously found in western and eastern Japan, the present work highlights the prevalence of infection in many Japanese districts, including islands and peninsulas, giving realistic data that can facilitate treatment and control.

First Molecular Identification and Genetic Characterization of Theileria lestoquardi in Sheep of the Maghreb Region.

Theileria lestoquardi is the most prominent Theileria species in small ruminants that causes malignant theileriosis of sheep in Africa and Asia. In the present survey, blood samples and ticks were collected in Kebili (southern Tunisia) from 166 Queue Fine de l'Ouest sheep. Giemsa-stained blood smears, immunofluorescent antibody test (IFAT) and PCR were performed. The DNA was extracted from blood and analysed by PCR targeting 18S rRNA gene of Theileria spp. and then sequenced. A total number of 140 ticks were collected from a total number of 166 sheep during the four seasons. The ticks belonged to two genera and 4 species; the most frequent tick was Hyalomma excavatum 84.3% (118/140) and then Rhipicephalus spp. 15.7% (22/140). Only two animals had positive Giemsa-stained blood smears, and they were also positive by IFAT. The amplicons had 99.3 and 99.6% homology with the BLAST published T.lestoquardi amplicons. To our knowledge, this is the first report of T.lestoquardi in small ruminants within the Maghreb region.

Age-related pattern and monocyte-acquired haemozoin associated production of erythropoietin in children with severe malarial anaemia in Ghana.

BackgroundMalaria continues to be a global health challenge, affecting more than half the world’s population and causing approximately 660,000 deaths annually. The majority of malaria cases are caused by Plasmodium falciparum and occur in sub-Saharan Africa. One of the major complications asscociated with malaria is severe anaemia, caused by a cycle of haemoglobin digestion by the parasite. Anaemia due to falciparum malaria in children has multifactorial pathogenesis, which includes suppression of bone marrow activity. Recent studies have shown that haemozoin, which is a by-product of parasite haemoglobin digestion, may play an important role in suppression of haemoglobin production, leading to anaemia. In this study we correlated the levels of erythropoietin (EPO), as an indicator of stimulation of haemoglobin production, to the levels of monocyte acquired haemozoin in children with both severe and uncomplicated malaria. There was a significantly negative correlation between levels of haemozoin-containing monocytes and EPO, which may suggest that haemozoin suppresses erythropoiesis in severe malaria. A multiple linear regression analysis and simple bar was used to investigate associations between various haematological parameters.MethodsTo examine the levels of erythropoietin in the age categories, the levels of erythropoietin was measured using a commercial Enyme-Linked Immunosorbent Assay (ELISA). Giemsa-stained blood smears were used to determine percentage pigment containing monocytes. The haemozoin containing monocytes was expressed as a percentage of the total number of monocytes. To obtain the number of haemozoin containing monocytes/μL the percentage of haemozoin containing monocytes was multiplied by the absolute number of monocytes/μL from the automated haematology analyzer.ResultsThe levels of erythropoietin in younger children (<3 years) was significantly higher than in older children with a similar degree of malaria anaemia (Hb levels) (p < 0.005). Haemozoin-containing monocytes were relatively higher in severe malaria anaemia patients compared to those with uncomplicated malaria (p < 0.001).ConclusionsAge purportedly has a direct effect on background levels of erythropoietin. With corresponding decreased levels of erythropoietin in older children with the same degree of severe malarial anaemia, conceivably, the bone marrows of younger children with acute malaria may be less sensitive to erythropoietin.

Malaria in Hadhramout, a southeast province of Yemen: prevalence, risk factors, knowledge, attitude and practices (KAPs).

BackgroundYemen is a Mediterranean country where 65% of its population is at risk of malaria, with 43% at high risk. Yemen is still in the control phase without sustainable reduction in the proportion of malaria cases. A cross-sectional household survey was carried out in different districts in the southeast of the country to determine malaria prevalence and identify factors that impede progress of the elimination phase.MethodsBlood specimens were collected from 735 individuals aged 1–66 years. Plasmodium species were detected and identified by microscopic examination of Giemsa-stained thick and thin blood smears. A household-based questionnaire was used to collect demographic, socioeconomic and environmental data.ResultsThe overall prevalence of malaria was 18.8% with Plasmodium falciparum as the predominant species (99.3%), with a low rate of Plasmodium vivax detected (0.7%). The infection rate was higher in Al-Raydah and Qusyer districts (21.8%) compared to Hajer district (11.8%). Fifty-two percent of the persons positive for Plasmodium were asymptomatic with low parasite density. The adults had a higher infection rate as compared to children. Univariate analysis identified those whose household’s head are fishermen (OR = 11.3, 95% CI: 3.13 – 40.5) and farmers (OR = 4.84, 95% CI: 1.73 – 13.6) as high-risk groups. A higher number of positive smears were observed in people living in houses with uncemented brick walls (OR = 2.1, 95% CI: 1.32 – 3.30), without access to toilets (OR = 1.6, 95% CI: 1.05 – 2.32), without a fridge (OR = 1. 6, 95% CI: 1.05 – 2.30), or without TV (OR = 1. 6, (95% CI: 1.05 – 2.30). People living in houses with water collection points located less than 200 meters away were also at higher risk of acquiring malaria (OR = 1.6, 95% CI: 1.05 – 2.30). Knowledge about the importance of using insecticide-treated mosquito nets (ITNs) and indoor residual spraying (IRS) for prevention of malaria was 7% and 2%, respectively.ConclusionsSeveral environmental, socioeconomic and behavioral issues were discovered to be the contributing factors to the high prevalence of malaria in southeast Yemen. Novel strategies adapted to the local situations need to be established in order to improve the effectiveness of malaria control.

Acute phase proteins in Andalusian horses infected with Theileria equi

Clinical and laboratory findings were determined in 23 Andalusian horses in southern Spain that were positive for Theileria equi by PCR, including 16 mares at pasture (group A1) and seven stabled stallions (group B1). Five healthy mares at pasture (group A2) and five stabled stallions (group B2), all of which were negative for T. equi in Giemsa stained blood smears and by PCR, were used as controls. The most frequent clinical signs were anorexia, anaemia, depression and icterus (group A1), along with loss of performance or failure to train and depression (group B1). Thrombocytopoenia was evident in 5/7 horses in group B1. Lower serum iron concentrations were observed in both diseased groups compared with their respective control groups. There were no significant differences in APP concentrations between diseased and control groups; all affected horses had APP concentrations within reference limits. Serum haptoglobin, serum amyloid A and plasma fibrinogen concentrations were higher than the reference limits in 5/23, 3/23 and 1/23 diseased horses, respectively. It was concluded that horses with theileriosis exhibited only a mild systemic inflammatory response.

The epidemiology of malaria in Bursa--2009-2012

Malaria is a common disease in many tropical and subtropical areas, which may threaten life. In this study, we examined the epidemiology of malaria in Bursa province using the data provided by the Province Health Directorate, collected over 2009 to 2012. The data include a total of 29.683 blood samples taken by active and passive surveillance. Giemsa-stained thin and thick blood smears were examined with a 100X oil immersion objective using a standard microscope. A total of 21 (0.07%) malaria cases were detected. Of these, 20 (95.2%) cases were male and 1 (4.8%) case was female, with highest rates occurring in June and September. All of the cases were imported, of whom 10 (47.6%) were caused by Plasmodium vivax and 11 (52.4%) by P. falciparum. All P. falciparum cases were found to be imported cases that traveled to African nations (Côte d'Ivoire, Sudan, Equatorial Guinea, Nigeria, Senegal, Mali, Somalia). Malaria cases were mainly observed in the 15-to-44-year-old range. We believe that these results will lead to better-targeted and more effective malaria control programs.

Clinical and laboratory findings associated with naturally occurring babesiosis in dromedary camels

Abstract Clinical, haematological, and biochemical changes induced by naturally occurring babesiosis in dromedary camels were described. Of 258 dromedary camels studied, 34 camels suffered from fever, appetite loss, weakness, depression, and reluctant movement; abortion and/or infertility were also observed. Parasitological blood examinations were performed using Giemsastained blood smears. The clinically affected animals were diagnosed with babesiosis, with 13.17% overall morbidity. Camels that suffered from babesiosis were subjected to haematological and biochemical analyses and the affected group was compared with a control group containing 34 healthy camels. The affected animals showed a highly significant (P&lt;0.001) reduction of the total red blood cell (RBC) count, haemoglobin (HGB) concentration, and mean corpuscular volume (MCV) as well as a highly significant reduction (P&lt;0.01) of haematocrit (HCT) and a significant reduction of (P&lt;0.05) mean corpuscular haemoglobin (MCH). Additional, highly significant increases (P&lt;0.01) in white blood cell (WBC) count and plateletcrit (PCT) percentage were detected. However, other haematological parameters were not significantly altered. There was a very significant reduction (P&lt;0.001) of the blood iron level and a very significant increase (P&lt;0.001) in blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) in the affected camels. Additionally, significant increases in total protein, albumin, γ-glutamyltransferase (GGT), aspartate aminotransferase (AST), and total bilirubin were observed in the affected camels. It was concluded that babesiosis highly affects the haematobiochemical parameters of dromedary camels, including the liver, kidney, and muscle functions. These results represent novel findings concerning natural babesiosis in camels.

Flow cytometry-based analysis of artemisinin-resistant Plasmodium falciparum in the ring-stage survival assay.

The ring-stage survival assay (RSA) is a powerful tool for phenotyping artemisinin-resistant Plasmodium falciparum but requires experienced microscopists to count viable parasites among 10,000 erythrocytes in Giemsa-stained thin blood smears. Here we describe a rapid flow cytometric assay that accurately counts viable parasites among 250,000 erythrocytes in suspension. This method performs as well as light microscopy and can be used to standardize the collection of RSA data between research groups in laboratory and field settings.

Phenotypic characterization of Plasmodium berghei responsive CD8+ T cells after immunization with live sporozoites under chloroquine cover

BackgroundAn effective malaria vaccine remains elusive. The most effective experimental vaccines confer only limited and short-lived protection despite production of protective antibodies. However, immunization with irradiated sporozoites, or with live sporozoites under chloroquine cover, has resulted in long-term protection apparently due to the generation of protective CD8+ T cells. The nature and function of these protective CD8+ T cells has not been elucidated. In the current study, the phenotype of CD8+ T cells generated after immunization of C57BL/6 mice with live Plasmodium berghei sporozoites under chloroquine cover was investigated.MethodsFemale C57BL/6 mice, C57BL/6 mice B2 macroglobulin −/− [KO], or invariant chain−/− [Ic KO] [6–8 weeks old] were immunized with P. berghei sporozoites and treated daily with 800 μg/mouse of chloroquine for nine days. This procedure of immunization is referred to as “infection/cure”. Mice were challenged by inoculating intravenously 1,000 infectious sporozoites. Appearance of parasitaemia was monitored by Giemsa-stained blood smears.ResultsBy use of MHC I and MHC II deficient animals, results indicate that CD8+ T cells are necessary for full protection and that production of protective antibodies is either CD4+ T helper cells dependent and/or lymphokines produced by CD4 cells contribute to the protection directly or by helping CD8+ T cells. Further, the phenotype of infection/cure P. berghei responsive CD8+ T cells was determined to be KLRG1high CD27low CD44high and CD62Llow.ConclusionThe KLRG1high CD27low CD44high and CD62Llow phenotype of CD8+ T cells is associated with protection and should be investigated further as a candidate correlate of protection.

A rapid sensitive, flow cytometry-based method for the detection of Plasmodium vivax-infected blood cells

BackgroundPlasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation. These features complicate detection and quantification by flow cytometry (FC) using standard nucleic acid-based staining methods. A simple antibody-based FC method was developed for rapid parasite detection along with simultaneous detection of other parasite and erythrocyte markers.MethodsClinical samples were collected from patients diagnosed with P. vivax at a district Malaria Clinic in Kanchanaburi, Thailand. One μL of infected blood was washed, fixed, stained with a Plasmodium pan-specific anti-PfBiP antibody conjugated with Alexa Fluor 660, and analysed by FC. Additional primary conjugated antibodies for stage-specific markers of P. vivax for late trophozoite-early schizonts (MSP1-Alexa Fluor 660), late-stage schizonts (DBP-Alexa Fluor 555), and sexual stages (Pvs16) were used to differentiate intra-erythrocytic developmental stages.ResultsThe percentages of P. vivax-infected cells determined by the FC method and manually by microscopic examination of Giemsa-stained thick blood smears were positively correlated by Spearman’s rank correlation coefficient (R 2 = 0.93843) from 0.001 to 1.00% P. vivax-infected reticulocytes.ConclusionsThe FC-based method is a simple, robust, and efficient method for detecting P. vivax-infected reticulocytes.

Molecular detection of Babesia bigemina infection in apparently healthy cattle of central plain zone of Punjab.

Bovine babesiosis is an important tick-borne disease caused by the parasites belonging to the genus Babesia, distributed worldwide and infecting a wide range of domesticated and wild cattle, occasionally man. The present study was conducted to determine the prevalence of B. bigemina infection in apparently healthy cattle from central plain zone of Punjab, India. Examination of Giemsa-stained peripheral thin blood smears revealed 2.45% (5/204) animals to be positive for piroplasms of B. bigemina. However, genomic DNA isolated from these blood samples when subjected to primary PCR revealed a positivity of 7.35% (15/204) as detected by the amplification of a 278-bp product in the agarose gel. PCR products obtained from the primary PCR of B. bigemina, when employed as template in nested PCR produced the amplicons of desired size (170bp) was detected in 30.39% (62/204) of the samples. It can thus be concluded that B. bigemina infection is prevalent in apparently healthy cattle population of this region and PCR assays can serve as a valuable tool for epidemiological studies in endemic areas.