Hydrophilic-treated plastic plates for wide-range analysis of Giemsa-stained red blood cells and automated Plasmodium infection rate counting.

BACKGROUND: Malaria is a red blood cell (RBC) infection caused by Plasmodium parasites. To determine RBC infection rate, which is essential for malaria study and diagnosis, microscopic evaluation of Giemsa-stained thin blood smears on glass slides ('Giemsa microscopy') has been performed as the accepted gold standard for over 100 years. However, only a small area of the blood smear provides a monolayer of RBCs suitable for determination of infection rate, which is one of the major reasons for the low parasite detection rate by Giemsa microscopy. In addition, because Giemsa microscopy is exacting and time-consuming, automated counting of infection rates is highly desirable. RESULTS: A method that allows for microscopic examination of Giemsa-stained cells spread in a monolayer on almost the whole surface of hydrophilic-treated cyclic olefin copolymer (COC) plates was established. Because wide-range Giemsa microscopy can be performed on a hydrophilic-treated plate, the method may enable more reliable diagnosis of malaria in patients with low parasitaemia burden. Furthermore, the number of RBCs and parasites stained with a fluorescent nuclear staining dye could be counted automatically with a software tool, without Giemsa staining. As a result, researchers studying malaria may calculate the infection rate easily, rapidly, and accurately even in low parasitaemia. CONCLUSION: Because the running cost of these methods is very low and they do not involve complicated techniques, the use of hydrophilic COC plates may contribute to improved and more accurate diagnosis and research of malaria.

Wright-Giemsa staining to observe phagocytes in Locusta migratoria infected with Metarhizium acridum.

Hemocytes are the first line of defense in the invertebrate immune system. Understanding their roles in cellular immunity is important for developing more efficient mycoinsecticides. However, the exact classification of hemocytes has been inconsistent and the various types of phagocytes in Locusta migratoria are poorly defined. Herein, the Wright-Giemsa staining method and microscopy were employed to characterize the hemocytes of L. migratoria following infection by Metarhizium acridum. Hemocytes were classified into four types, including granulocytes, plasmatocytes, prohemocytes, and oenocytoids, based on size, morphology, and dye-staining properties. Each type of hemocyte was classified into several subtypes according to different ultrastructural features. At least four subtypes of granulocytes or plasmatocytes, including small-nucleus plasmatocytes, basophil vacuolated plasmatocytes, homogeneous plasmatocytes, and eosinophilic granulocytes, carried out phagocytosis. The percentage of total phagocytes increased two days after infection by M. acridum, then gradually declined during the next two days, and then increased sharply again at the fifth day. Our data suggested that plasmatocytes and granulocytes may be the major phagocytes that protect against invasion by a fungal pathogen in L. migratoria. Total hemocytes in locusts significantly increased in the initial days after infection and decreased in the late period of infection compared to controls. In the hemocoel, hyphal bodies were recognized, enwrapped, and digested by the phagocytes. Then, the broken hyphal pieces were packaged as vesicles to be secreted from the cell. Moreover, locusts might have a sensitive and efficient cellular immune system that can regulate phagocyte differentiation and proliferation before fungi colonize the host hemolymph.

Eosinophils in hereditary and inflammatory myopathies.

It is not known whether eosinophilic myositis is a specific histopathological feature of limb girdle muscular dystrophy 2A (LGMD2A). Number and location of eosinophils in skeletal muscle biopsies (n=100) was analysed by Giemsa and modified hematoxylin/eosin staining in patients with genetically confirmed myopathies (LGMD2A, LGMD2B, LGMD2L, facioscapulohumeral muscular dystrophy, dystrophinopathy), histologically confirmed idiopathic inflammatory myopathies (sporadic inclusion body myositis (sIBM), dermatomyositis (DM), polymyositis), amyotrophic lateral sclerosis (neurogenic control), and normal controls. The number of eosinophils/mm² was significantly higher in LGMD2A, PM, DM, and sIBM compared to controls but not significantly higher than other myopathies. A large overlap in the number of eosinophils/mm2 between all groups was seen. In all disease groups eosinophils were mainly found endomysially (46- 88%) and intra- and perivascularly (4-37%). There was no correlation between the numbers of eosinophils/mm² and (i) age at biopsy and (ii) the duration of the disease. The extent of myopathic, fibrotic, and inflammatory changes did not differ in samples with high and low eosinophil count. Eosinophils seem to represent an unspecific histological finding in hereditary and inflammatory myopathies, but also amyotrophic lateral sclerosis.

Flow cytometric detection and microscopic observation of activated eosinophils in peripheral blood.

Eosinophils possess highly electron-dense granules with crystal-like structures and are characterized as high side scatter (SSC) areas by flow cytometry analysis. Eosinophils with low SSC features have been noted in extremely rare cases; however, the underlying cause remains unclear. Eosinophils in the low SSC area were analyzed using microscopy. A transmission electron microscope revealed the loss of crystal-like structures in granules with low electron density and piecemeal degranulation, which was undetectable by May-Grünwald-Giemsa staining. Based on the results of flow cytometry, May-Grünwald-Giemsa staining and transmission electron microscopy, SSC values could help potentially detect crystal-like structures and piecemeal degranulation eosinophils.

Prevalence of camel babesiosis in southeast of Iran.

Babesiosis is a globally distributed zoonotic parasitic disease in a broad range of vertebrates with great importance in the veterinary field. The standard diagnostic test for Babesiosis in animals is microscopic identification of the parasite in a venous blood smear stained with Giemsa combined with assessment of clinical manifestations throughout the acute phase of the disease. The present study was planned to determine the presence of Babesia species in camels from the southeastern regions of Iran. A total of 140 blood samples of camels were randomly collected in four selected cities including Qaen, Nehbandan, Iranshahr, and Zahedan from March to August 2019. Blood smears of each case were also examined by the Giemsa staining method and extracted DNA samples were subjected to internal transcribed spacers (ITS1) polymerase chain reaction (PCR) amplification. The prevalence rates using microscopically and molecular examinations were 10% and 19.28%, respectively. The prevalence rates significantly vary between the selected regions (p = 0.003). PCR technique showed higher sensitivity than microscopy. We found that all infected camels were positive for Babesia caballi. The rate of infection with Babesia among the camel in Zahedan is remarkable. Early diagnosis and early treatment can prevent further spread of the disease in this area.

Comment on Skrebinska et al. Who Could Be Blamed in the Case of Discrepant Histology and Serology Results for Helicobacter pylori Detection? Diagnostics 2022, 12, 133.

In their article, Skebrinska and colleagues analysed the potential pitfalls of detecting Helicobacter pylori (H. pylori) by serology, histological (Giemsa) and immunohistochemical (IHC) staining. However, in the Introduction, the authors state: "…IHC is recommended only in individuals with active gastritis without H. pylori identification by histochemistry". Although this is a widely-held view, it does not seem to hold up in view of the results of the study by Kocsmár et al., which showed that the diagnostic sensitivity of Giemsa in the absence of activity is only 33.6%, but it is 92.6% in the presence of active gastritis, which is close to the 99.4% sensitivity of IHC. Considering that chronic active gastritis with the features of H. pylori gastritis is also common in other entities, if active inflammation is present in the sample, there is a very small chance that a Giemsa-negative case will be confirmed as H. pylori-positive by IHC. Based on this, the use of IHC is more reasonable in Giemsa-negative cases with no activity in which the etiological role of H. pylori is suggested by clinical, anamnestic or other data. However, it may also be reasonable to routinely use IHC as the primary staining method instead of Giemsa.

Malaria Detection Accelerated: Combing a High-Throughput NanoZoomer Platform with a ParasiteMacro Algorithm.

Eradication of malaria, a mosquito-borne parasitic disease that hijacks human red blood cells, is a global priority. Microscopy remains the gold standard hallmark for diagnosis and estimation of parasitemia for malaria, to date. However, this approach is time-consuming and requires much expertise especially in malaria-endemic countries or in areas with low-density malaria infection. Thus, there is a need for accurate malaria diagnosis/parasitemia estimation with standardized, fast, and more reliable methods. To this end, we performed a proof-of-concept study using the automated imaging (NanoZoomer) platform to detect the malarial parasite in infected blood. The approach can be used as a steppingstone for malaria diagnosis and parasitemia estimation. Additionally, we created an algorithm (ParasiteMacro) compatible with free online imaging software (ImageJ) that can be used with low magnification objectives (e.g., 5×, 10×, and 20×) both in the NanoZoomer and routine microscope. The novel approach to estimate malarial parasitemia based on modern technologies compared to manual light microscopy demonstrated 100% sensitivity, 87% specificity, a 100% negative predictive value (NPV) and a 93% positive predictive value (PPV). The manual and automated malaria counts showed a good Pearson correlation for low- (R2 = 0.9377, r = 0.9683 and p < 0.0001) as well as high- parasitemia (R2 = 0.8170, r = 0.9044 and p < 0.0001) with low estimation errors. Our robust strategy that identifies and quantifies malaria can play a pivotal role in disease control strategies.

Effect of 80% ethanol or 10% formalin fixation, freezing at -20 °C and staining on Myxobolus (Myxosporea) spores to be deposited in parasitological collections.

The preparation of myxosporeans for the description of myxospores and their preservation as type material in parasitological collections show great variations. Most frequently, formalin and ethanol are used for fixation and Giemsa solution for staining spores. In this work, authors studied the effect of 80% ethanol and 10% formalin fixation, freezing at -20 °C and staining on the size and transparency of two Myxobolus species of cyprinid fishes, M. bramae and M. bliccae spore, and recommended a new method for the deposition of type material to parasitological collections in museums. The studies have commended that fresh spores from mature plasmodia are the best material for measuring the size and studying the inner structures, the number of polar tubules in polar capsules and the morphological characters of the intercapsular appendix. The obtained quantitative data suggest that cryo- and chemical preservation do not have a notable negative effect on spores compared to fresh samples but they decrease the transparency of spores. Staining the spores with Ziehl-Neelsen has proved to be a useful method for studying the fine structure without size reduction, while Giemsa staining induced a shrinkage of spores so it seems to be not ideal for description of a new species. When treating spores of Myxobolus spp. with Lugol's solution, iodinophilous vacuoles in the sporoplasm were not recognised but visualisation of the coils of polar tubules was enhanced. As a type material for newly described species, authors suggest phototypes and spores fixed in 80% ethanol to be deposited into collections, as this preservation method is suitable for subsequent research, such as re-measurements and molecular analysis.

Haemocyte variations in 35 species of grasshoppers and locusts.

INTRODUCTION: Grasshoppers and locusts are widely distributed worldwide, causing significant losses in agriculture. The origin and functions of their haemocytes are not entirely understood. OBJECTIVES: Insect haemocytes arbitrate cellular defence and participate in humoral defences. Due to their importance, the haemocytes of 35 species of grasshoppers and locusts from China were morphologically examined in this study. We aim to highlight a simple method for the morphological examination of insect haemocytes. METHODS: The haemocytes were observed, counted and compared under a light microscope after Wright-Giemsa staining. RESULTS: High complexity in form and shape were observed in the haemocytes. These include prohaemocytes, plasmatocytes, granulocytes, vermicytes, podocytes and megakaryocytes. No clear relationship was seen between the haemocyte type and their phylogenetic relationship among the three families examined. The high abundance of plasmatocytes and granulocytes suggests their importance in the immunity of grasshoppers and locusts. The minor haemocyte populations including prohaemocytes, vermicytes and podocytes may not be always present in individuals. CONCLUSION: All examined species shared similarities in their haemocyte types. Wright-Giemsa staining is a simple and efficient method for evaluating haemocytes.

Hydrogel-Based Stamping Technology for Solution-Free Blood Cell Staining.

An accurate microscopical analysis of blood smears requires a reproducible and convenient method of staining. Solution-based staining procedures can be cumbersome. Especially in low- and middle-income countries, the lack of skilled technicians and adequate laboratory facilities, as well as insufficient water and reagent quality, often become confounding factors. To overcome these obstacles, we developed a new cell staining method based on sequential stamping of agarose gel patches that contain eosin, methylene blue/oxidized methylene blue, Azure B, and buffer, respectively. Our method, termed "hydrogel staining", provides a simple, reproducible, solution-free, and inexpensive approach to stain blood cells. We have optimized incubation times to achieve the optimal transfer of dyes to fixed blood cells on a glass slide, with outcomes comparable to conventional solution-based methods for white blood cells and malaria-infected red blood cells. This hydrogel staining method does not require special skills to produce excellent quality stained blood film slides. The new method could enhance the accuracy of microscopical examination of blood smears, especially in resource-limited settings.

Cytopathological Findings of Secretory Carcinoma of the Salivary Gland and the Diagnostic Utility of Giemsa Staining.

Secretory carcinoma is a salivary gland neoplasm first described as a mammary analogue secretory carcinoma by Skalova and redesignated as a secretory carcinoma in the 2017 World Health Organization Classification of Head and Neck Tumors. Secretory carcinoma diagnosis is reliant on specific cytological and histological findings and the detection of an ETV6-NTRK3 fusion gene. Here, we examined the clinical and cytopathological features of four cases of secretory carcinoma occurring in three males and a female, aged between 39 and 74 years. All four tumors involved the parotid gland, and were found to have the ETV6-NTRK3 fusion gene. Fine-needle aspiration-based cytology smears of all tumors displayed papillary and/or dendritic pattern clusters, some of which were associated with blood vessels. The neoplastic cells displayed enlarged nuclei with fine chromatin and small, distinct, single nucleoli. Furthermore, several neoplastic cells with a characteristic vacuolated cytoplasm were identified in each specimen. Giemsa staining revealed cytoplasmic vacuolation, intracytoplasmic metachromatic secretions and/or various sized metachromatic granules, and a background of metachromatic mucin in all four specimens. Given this, we conclude that these cytological findings, especially those of the Giemsa staining, might be helpful in the diagnosis of secretory carcinoma.

Presence of Trichomonas tenax and Entamoeba gingivalis in peri-implantitis lesions.

OBJECTIVE: The aim was to investigate the presence of Entamoeba gingivalis and Trichomonas tenax in peri-implantitis lesions. METHOD AND MATERIALS: A total of 141 individuals were included in this study, of which 40 had clinically healthy implants (group H); the remaining were associated with peri-implantitis (group P). Gingival crevicular fluid was collected using absorbent paper, followed by a dental plaque sample from the peri-implant sulcus/pocket using a titanium curette. The samples were transferred into an Eppendorf tube. Each specimen was divided into two parts. One part was examined under a light microscope at a 10 × and 40 × magnification to detect parasites. The other part was spread on a microscope slide, stained with Giemsa stain, and examined under a microscope at 100 × magnification. Pearson chi-square test was used in the statistical analysis of data, with a significance level of P < .05. RESULTS: Although there was no presence of parasite around the healthy implants, two parasites were detected in peri-implantitis lesions. Out of 101 lesions, 31 (30.7%) showed E gingivalis, and 34 (33.6%) presented with T tenax. There was a statistically significant difference between the presence of E gingivalis and demographic data including gender, education status, frequency of dental visits, and brushing frequency. Presence of T tenax in lesions was correlated with frequency of dental visits (P < .05). It was observed that E gingivalis and T tenax were mostly detected in the mandible (P = .004 and .014, respectively) in comparison with the maxilla. CONCLUSION: This study showed that peri-implantitis lesions were involved with E gingivalis and T tenax, in contrast to the healthy areas.

Macrophages in Giemsa-stained cerebrospinal fluid specimens predict carcinomatous meningitis.

Carcinomatous meningitis is a condition in which tumor cells spread to the subarachnoid space. Leukocyte counting and typing of cerebrospinal fluid (CSF) cell components are performed manually or using flow cytometry. However, a detailed analysis of these variables using cytological specimens has not yet been reported. The present study analyzed cytological specimens using Giemsa staining and whole slide imaging with computer-assisted image analysis (CAIA) to clarify the characteristics of the leukocyte population in CSF, especially in carcinomatous meningitis. Manual evaluation was performed using 280 Giemsa-stained cytological CSF specimens. For 49 samples, CAIA was used for the whole area of Papanicolaou (Pap) staining, and Giemsa-stained specimens of the same samples were imaged using a virtual slide scanner. The nuclear morphology of the leukocytes was assessed, and the total leukocyte and leukocyte subset (lymphocytes, neutrophils and macrophages) counts were evaluated. Then, the number and percentage of each leukocyte subset population were evaluated. The total leukocyte count was significantly higher in Giemsa-stained specimens compared with in Pap-stained specimens. The percentage of macrophages was significantly higher in samples from patients with non-hematological tumors compared with in samples from patients without tumors, which was confirmed by manual evaluation of the specimens. In addition, the cut-off value of the percentage of macrophages that could discriminate between the tumor history negative cases and cytologically tumor positive cases was determined, revealing that a higher proportion of macrophages reflected the existence of atypical/malignant epithelial tumor cells in CSF samples. Thus, atypical cell screening and analysis of the background characteristics of the leukocyte population should be the focus of cytological specimen screening, especially not to miss carcinomatous meningitis.

Cellular response to bacterial infection in the grasshopper Oxya chinensis.

Oxya chinensis is one of the most widespread grasshopper species found in China and one of the most common pests against rice. In view of the importance of haemocytes in insect immunity in general, and the lack of information on the haemocytes of O. chinensis, we examined the haemocytes of this species in detail. We challenged the cellular response of this grasshopper with the bacteria Escherichia coli, Staphylococcus aureus, and Bacillus subtilis Haemocyte morphology was observed using light, scanning electron and transmission electron microscopy, which revealed distinct morphological varieties of haemocytes. Granulocytes and plasmatocytes responded to the bacterial challenge by phagocytosis. Histochemical staining indicated the presence of acid phosphatase in plasmatocytes and granulocytes. We also observed non-phagocytic prohemocytes and vermicytes, but their functions in the circulation are unclear. Insect haemocytes play a crucial role in cellular immunity, and further research is needed for a comprehensive understanding.

Dose-response curves for analyzing of dicentric chromosomes and chromosome translocations following doses of 1000 mGy or less, based on irradiated peripheral blood samples from five healthy individuals.

In terms of biological dosimetry at the time of radiation exposure, the dicentric chromosome (Dic) assay (DCA) is the gold standard for assessing for the acute phase and chromosome translocation (Tr) analysis is the gold standard for assessing the chronic phase. It is desirable to have individual dose-response curves (DRCs) for each laboratory because the analysis criteria differ between laboratories. We constructed the DRCs for radiation dose estimation (with three methods) using peripheral blood (PB) samples from five healthy individuals. Aliquots were irradiated with one of eight gamma-ray doses (0, 10, 20, 50, 100, 200, 500 or 1000 mGy), then cultured for 48 h. The number of chromosome aberrations (CAs) was analyzed by DCA, using Giemsa staining and centromere-fluorescence in situ hybridization (centromere-FISH) and by chromosome painting (chromosome pairs 1, 2 and 4) for Tr analysis. In DCA, there was large variation between individuals in the frequency of Dics formed, and the slopes of the DRCs were different. In Tr analysis, although variation was observed in the frequency of Tr, the slopes of the DRCs were similar after adjusting the background for age. Good correlation between the irradiation dose and the frequency of CAs formed was observed with these three DRCs. However, performing three different biological dosimetry assays simultaneously on PB from five donors nonetheless results in variation in the frequency of CAs formed, especially at doses of 50 mGy or less, highlighting the difficulty of biological dosimetry using these methods. We conclude that it might be difficult to construct universal DRCs.

Persistence of radiation-induced aberrations in patients after radiotherapy with C-ions and IMRT.

BACKGROUND AND PURPOSE: Chromosomal aberrations in peripheral blood lymphocytes are a biomarker for radiation exposure and are associated with an increased risk for malignancies. To determine the long-term cytogenetic effect of radiotherapy, we analyzed the persistence of different aberration types up to 2.5 years after the treatment. MATERIALS AND METHODS: Cytogenetic damage was analyzed in lymphocytes from 14 patients that had undergone C-ion boost + IMRT treatment for prostate cancer. Samples were taken immediately, 1 year and 2.5 years after therapy. Aberrations were scored using the multiplex fluorescence in situ hybridization technique and grouped according to their transmissibility to daughter cells. RESULTS: Dicentric chromosomes (non-transmissible) and translocations (transmissible) were induced with equal frequencies. In the follow-up period, the translocation yield remained unchanged while the yield of dicentrics decreased to ≈40% of the initial value (p = 0.011 and p = 0.001 for 1 and 2.5 years after compared to end of therapy). In 2 patients clonal aberrations were observed; however they were also found in samples taken before therapy and thus were not radiotherapy induced. CONCLUSION: The shift in the aberrations spectrum towards a higher fraction of translocations indicates the exposure of hematopoietic stem and progenitor cells underlining the importance of a careful sparing of bone marrow during radiotherapy to minimize the risk for secondary cancers.

Nyctotherus sp. infection in pet turtle: a case report.

The present clinical case represents the successful therapeutic management of Nyctotherus sp. protozoan infection in turtles. Two pet turtles were presented with history of diarrhea, dehydration, weight loss and passage of undigested food in the faeces. Direct faecal examination and Giemsa staining revealed the presence of cysts of Nyctotherus sp. Treatment was initiated with antiprotozoal drug, Metronidazole at the rate of 25 mg/kg body weight per-oral consecutively for 5 days, and Vimeral suspension at rate of 10-20 mg/kg per-oral for 8 days. After 10 days of intensive care and management with antiprotozoal drug, multivitamin under proper sanitary measures, the case showed gradual improvement in condition. During medication, the fecal samples were checked on every alternative day for the presence of ciliated trophozoites of Nyctotherus sp. After 4th day, there was no trophozoites observed by both the techniques examined and the animal was quite healthy and active and started taking food. Improvement was noticed with resumption of normal appetite. Incessant patient monitoring and initiation of suitable need based therapeutic strategy is most important in management and control of Nyctotherus sp. infection.

Prevalence of Helicobacter Pylori Infection and Stress, Anxiety or Depression in Functional Dyspepsia and Outcome after Appropriate Intervention.

INTRODUCTION: The association between psychological factors and non-ulcer dyspepsia remains controversial. AIM: To determine the prevalence of Helicobacter pylori (HP) and Stress/Anxiety/Depression (SAD) in patients with Functional Dyspepsia (FD) and assess the outcome at three months after appropriate intervention. MATERIALS AND METHODS: This prospective non-randomized interventional study was conducted on 120 patients with FD. Initial workup included upper gastrointestinal endoscopy to confirm HP infection with either of two tests, the urease test or histopathology. Patient Health Questionnaire-9 scale (PHQ-9) was used to assess depression, General Anxiety Disorder-7 scale (GAD-7) for anxiety and Perceived Stress Scale (PSS) for stress. Patients were considered positive when they had significant scores on one or more of the questionnaires (SAD+). The subjects were then classified into four groups: Group A (positive for HP and SAD, n=35), Group B (positive for HP and negative for SAD, n=31), Group C (negative for HP and positive for SAD, n=33) and Group D (negative for HP and SAD, n=21). The groups were then treated as follows: Group A: HP eradication plus psychiatric intervention, Group B: HP eradication alone, Group C: psychiatric intervention alone and Group D: proton pump inhibitors. Modified Glasgow Dyspepsia Symptom Score (Mod. GDSS) was used to assess the severity of dyspepsia at baseline and to monitor the change in score over three months. Statistical analysis was done using the Statistical Package for the Social Sciences version 16.0. Non-parametric data like proportions of response in different groups to treatment was analysed using the Chi square test and quantitative data using ANOVA. Gender wise distribution and response to treatment was calculated using the z-test and unpaired t-test. RESULTS: Overall 120 patients were recruited across four groups. A 55% of the subjects were positive for HP and 56.7% for SAD and 29.2% for both. In all three groups with psychiatric comorbidity, females exceeded males in a proportion of 3:1. Mod. GDSS was not significantly different at baseline between HP+ and HP- patients (p=0.1278) except when HP positivity was also associated with SAD (p<0.001), whereas SAD positivity alone significantly increased the baseline Mod. GDSS (p=0.006). Mod. GDSS declined in all four groups at three months. When a fall of four or more was considered as an indicator of significant response to intervention, it was seen that overall 74.2% responded to intervention with the best response in Group B and the poorest was in Group C. CONCLUSION: There is a significant prevalence of HP and SAD in FD. Appropriate intervention is beneficial except in those who are HP negative and SAD positive. This latter group requires further investigation and or drug intervention for SAD.

Comparative evaluation of polymerase chain reaction assay with microscopy for detection of asymptomatic carrier state of theileriosis in a herd of crossbred cattle.

AIM: This study aims to develop and to standardize a polymerase chain reaction (PCR) assay that will diagnose clinical as well as carrier state of the disease and to compare the results with conventional microscopy technique. MATERIALS AND METHODS: A herd of crossbred cattle with the previous history of theileriosis in village Lahli, district Rohtak, Haryana, was selected for this study. A total of 29 blood samples were collected randomly from cows including five clinically ill cattle. Blood smears from all animals and lymph node biopsy smears from animal with swollen lymph nodes were examined microscopically after conventional Giemsa staining. Phenol chloroform isoamyl alcohol method was used for extracting DNA from blood. Previously published primers targeting cytochrome b gene sequence of Theileria annulata were used in the PCR assay that was standardized to use in the laboratory. RESULTS: Out of 29 samples tested,18 (62.06%) were found positive for theileriosis by PCR assay, whereas only 10 (34.48%) samples were detected positive by conventional microscopic technique using Giemsa staining method. CONCLUSIONS: On the basis results of comparative studies, it can be concluded that PCR assay is a more sensitive than microscopic examination for detection of theileriosis. This can be attributed to the ability of PCR assay to detect small amounts of genomic DNA of T. annulata or low parasitemia in cows. Therefore, PCR assay can serve as a more sensitive tool to detect Theileria for detection of theileriosis even in asymptomatic carrier cattle which is important for the implementation of successful control programs.