Blood Smear Examination Research Articles

Implementation and validation of a new qPCR assay to detect imported human Plasmodium species.

Diagnosis of imported malaria is based on microscopic examination of blood smears (BS), detection of circulating plasmodial antigen by immunochromatography (ICT), or detection of Plasmodium spp. DNA by loop mediated isothermal amplification. We have developed duplex (Plasmodium spp. and Plasmodium falciparum) real-time PCR (qPCR) and species-specific qPCR assays for the identification and quantification of human Plasmodium species. Whole nucleic acids from 523 samples prospectively collected from 410 patients suspected of malaria between June 2016 and March 2021 were tested by qPCRs and compared to standard diagnostic procedures. All qPCRs were designed on 18S ribosomal ribonucleic acid. The limit of detection of duplex qPCR was 8 copies/reaction, and analytical specificity of species-specific qPCRs was 100%. Seventy-nine patients diagnosed for single species malaria involving P. falciparum, P. vivax, P. ovale, and P. malariae were positive with duplex and species-specific qPCRs. P. knowlesi qPCR detected DNA from a P. knowlesi culture. Of eight cases of mixed Plasmodium species infection, five were identified with our qPCR assays with better sensitivity as compared to BS and ICT. Eight out of 323 patients with negative BS were hospitalized for symptoms suggestive malaria after malaria-endemic area travel or known with history of malaria had a low positive duplex qPCR. Between day of diagnosis and post-treatment follow-up at D2-D4 of malaria, a 3.1-log P. falciparum load decrease was observed by qPCR. These new Plasmodium qPCRs allowing detection of human plasmodial species have excellent species specificity and allow rapid detection of P. falciparum cases, malaria with low parasitaemia, and mixed Plasmodium species infection.IMPORTANCEMalaria is a disease transmitted by a Plasmodium parasite genus. Most cases are caused by Plasmodium falciparum. Despite a significant drop of incidence and mortality since 2000, 249million cases and 608,000 deaths have been estimated in 2022, mainly in Africa. Due to the increasing number of travels to endemic areas, incidence of imported malaria is rising in Europe. Various techniques are used in European laboratories, such as microscopic examination of thin and thick smears and rapid diagnostic tests. However, these techniques require skilled operators to differentiate plasmodial species and have limited sensitivity. Actually, molecular diagnosis is carried out using point-of-care test for rapid results with excellent sensitivity but is unabled to determine involved species and assess parasitaemia. In this study, we developed a combined molecular tool based on both detection of all human plasmodial species (Plasmodium spp.) and P. falciparum. We have also developed specific qPCRs for each human plasmodial species.

Post-mortem diagnosis of malaria in decomposed and embalmed body.

The diagnosis of malaria during the autopsy of a decomposed corpse may prove challenging. Macroscopic changes are non-specific and may include, among others, cerebral oedema, pulmonary oedema, hepatosplenomegaly and, on occasion, the presence of petechiae. The most effective diagnostic tools for malaria are the examination of blood smears and the use of rapid immunochromatographic tests. As a result of the progressive putrefaction of the corpse and blood hemolysis, classical tests are no longer viable. Consequently, the sole remaining option is the utilisation of real-time reaction (RT-PCR) to ascertain the presence of plasmodium DNA in specific organs. This study concerns the diagnosis of a fatal form of cerebral malaria in a 23-year-old Caucasian male who had travelled to Africa. The autopsy was conducted at a local hospital, after which the body was embalmed and stored in cold storage for a period of 8.5 months. Subsequently, the corpse was transported to Poland, where a further forensic autopsy was conducted. A significant challenge was to confirm the presence of malaria in a corpse that had been embalmed several months prior to the investigation. Samples were obtained from internal organs for genetic analysis to determine the presence of parasite DNA. An RT-PCR test was conducted on genetic material obtained from the brain, heart, lungs, kidney, liver, and spleen. The presence of Plasmodium falciparum genetic material was identified in samples obtained from the brain, lungs, kidney, liver, and spleen. These findings substantiated the post-mortem diagnosis of a severe form of cerebral malaria, which was the underlying cause of death.

Prevalence and seasonal patterns of vector borne diseases in patients presenting with acute febrile illness in a tertiary care hospital in Puducherry- a prospective observational study.

Vector-borne diseases (VBD) are a major public health concern. Globalization, urbanization & climate change are reasons for the emergence and re-emergence of VBDs. In our study, we looked into the prevalence of VBD infections around our tertiary care hospital in South India. The objective was to determine the prevalence of common VBDs like Malaria, Dengue, Japanese encephalitis (JE), Chikungunya and Scrub typhus in patients with acute febrile illness (AFI). This was a prospective laboratory based observational study. Blood samples from patients with AFI were tested for Dengue NS1 Antigen, IgM and IgG; and IgM antibodies for JE, Chikungunya and Scrub typhus using ELISA tests. Peripheral blood smear examination was performed for malarial parasite detection. Total 802 samples were analysed. The sample positivity rate for VBD was 63.6% (510/802 samples) On diving the positive results across seasons in the study period, the VBD positivity rates were 66.3%, 49.1%, 61.2% and 67.3% for the first post-monsoon, summer, monsoon and the second post-monsoon seasons respectively- a trend of increased rates noted during the post-monsoon seasons. 192 samples (23.9%) were positive for scrub typhus alone, 189 samples (23.6%) were positive for dengue infection alone, six samples (0.7%) were positive for chikungunya infection alone, 121 samples (15.1%) were positive for dengue plus scrub typhus co-infection, two samples (0.2%) were positive for dengue plus chikungunya co-infection, while 292 samples (36.4%) showed negative results. None of the samples were positive for malaria and Japanese encephalitis. Scrub typhus and dengue were the most prevalent VBDs in concordance with the prevalence pattern noted in other studies in South India. Increasing awareness and surveillance of the VBDs, developing stringent control policies, easy access to testing and initiating early appropriate therapy can help reduce the incidence of VBDs.

Literature Study: Babesiosis in Dogs in The Period of 2013-2023

Background: Babesiosis is an infectious disease caused by Babesia spp. with tick vectors Rhipicephalus sanguineus and Dermacentor margniatus (a tick-borne disease). Babesiosis transmission occurs through tick bites or blood transfusions. Diagnostic techniques involve the microscopic examination of blood smears, hematology, blood chemistry, and PCR laboratory analysis. Purpose: To determine the clinical symptoms, confirmation of diagnosis, and therapy used for babesiosis conditions in dogs. Method: Analysis was conducted on 22 literature cases of babesiosis in dogs with a publication period of 2013-2023. The analysis was carried out using the descriptive analysis method, a problem-solving procedure that describes the condition of the subject or object based on facts, characteristics, and any relationships between the phenomena being investigated. Qualitative research methods were used to examine natural object conditions, as a key instrument. Data source sampling was done purposively, the data collection techniques were combined, the data analysis was either inductive or qualitative, and qualitative research results emphasize meaning. Result: Dog patients with babesiosis have the highest prevalence of clinical symptoms, namely anorexia (62.5%), vomiting (33.3%), pyrexia (54.16%) lethargy (45.83%), brownish urination (29.16%), and ectoparasite infestation (29.16%). Confirmation of the diagnosis carried out included the microscopic examination of blood smears (100%), hematology (91.66%), blood chemistry (41.66%), and Polymerase Chain Reaction (PCR) (20.83%). The therapeutic management used included 3 combinations of antibiotics in 7 cases (31.81%), combination therapy of 2 antibiotics and an antiparasitic in 3 cases (13.63%), and combination therapy of 1 antibiotic and antiparasitic in 3 cases (13.63%). Conclusion: Antiparasitic therapy that can be used is imidocarb dipropionate, which works by inhibiting inositol in the erythrocytes infected with Babesia spp. and diminazene aceturate, which works by disrupting parasite DNA synthesis.

“Morphological Typing Of Anemia Based On Peripheral BloodSmear Examination And Automated CellCounter Generated RBCIndices.

“Morphological Typing Of Anemia Based On Peripheral BloodSmear Examination And Automated CellCounter Generated RBCIndices.

Effects of environmental factors on host-parasite interaction patterns in backyard-tethered goats of Kerala, India

The environment is the most important stratum in the epidemiological triad of a parasitic disease and any variations in the environmental factors may decide the dynamic occurrence and existence of different lifecycle stages of these parasites. The present study investigated the correlations between key biometeorological and demographical parameters with the incidence of different gastrointestinal parasites and hemoparasites among goats. Four hundred and thirty-two goats were screened for parasitic infection in a yearlong survey conducted from July 2022 to June 2023 in the Teaching Veterinary Clinical Complex, Mannuthy, Kerala, India. The weather parameters (Tmax, Tmin, RH, THI, and bright sunshine hours), air quality parameters [AQI, PM2.5, and PM10], and demographic parameters (gender and age) were recorded along with the test positivity of different categories of gastrointestinal parasites and hemoparasites in goats by routine fecal sample examination and blood smear examination, respectively. The infection level was ranked based on the severity of the infection. The mean and daily variations in biometeorological parameters were calculated and the data were statistically analyzed to figure out the pertinent correlations in host-parasite-environment interaction patterns. High levels of parasitic infections with significant month-wise variations and climate-correlated peak infection patterns were noticed. The incidence of parasites was negatively correlated to all parameters except humidity, indicating more severe parasitism during monsoon months. The significant variations in the host-parasite interaction dynamics point towards the need for detailed explorations concerning the lifecycle of each specific parasite with a focus on the possible environment-favourable and resistant lifecycle stages. Future studies may be designed from a biometeorological perspective to develop a crucial understanding of host-parasite-environment interactions in goats ensuring sustainable goat farming.

Utility of digital cell imaging as an aid to enhance pathologist peripheral blood smear review for routine and on call practice: A verification study

Abstract Introduction/Objective Microscopic examination of peripheral blood smear (PBS) provides important information to support the diagnosis of hematological diseases. However, manual microscopic examination of PBS is labor-intensive and cannot be done remotely. CellaVision is digital cell imaging technology that provides pre-classification and digital imaging of the red blood cells (RBCs), white blood cells (WBCs), and platelets, myeloid, lymphoid and erythroid precursors. Several studies have evaluated the performance of this technology in the hematology laboratory as tool to assist laboratory technologists; our study evaluates its use for pathologist review of peripheral blood smears both during routine and on call practice. Methods/Case Report Our study included 30 PBS and two hematopathologists with greater than 10 years’ experience in performing PBS review. We compared pathologist’s own impression of microscopic PBS review to their own impression using CellaVision software (intraobserver comparison). Additionally, pathologist to pathologist impression was compared for both methods (interobserver comparison). All 30 cases revealed abnormal findings including, but not limited to: pancytopenia, leukocytosis, leukopenia, anemia, thrombocytopenia, and thrombocytosis. Of the 30 cases, 5 “on-call” were cases when the pathologist had to come on–site for pathologist PBS review. Results (if a Case Study enter NA) The pathologists own PBS interpretation (intraobserver) by microscopy and CellaVision had a concordance of 96.7%. The pathologist to pathologist interpretation of both methods had a concordance of 96.7%.The one discrepant case was a smear with platelet clumping and normal platelet count. On chart review of this case, this discrepancy would have minimal impact on patient care. Conclusion Pathologist use of CellaVision in their routine practice may result in speedier PBS review, particularly in cases with leukopenia, and better communication with laboratory technologists. CellaVision is less reliable in identifying platelet clumps and should not be used in isolation in evaluating thrombocytopenia. Used with other relevant data, CellaVision has the potential to preempt in person microscopic review of PBS when on-call in certain situations.

Rare Adult acute leukemia with Coexisting myeloblastic (basophilic differentiation) & B-lymphoblastic subpopulations Case Study

Abstract Introduction/Objective Acute basophilic leukemia is a very rare subgroup of acute myeloid leukemia that is defined in the 2022 WHO classification by presence of at least 20% blast cells in peripheral blood or bone marrow with immature and maturing cells of basophil lineage. The simultaneous occurrence of two hematological malignancies in a middle- aged adult patient is quite a rare event as evidenced by the rare cases reported in the literature. To our knowledge, this is the first case to be reported with concomitant acute basophilic and B-lymphoblastic leukemia in adult patients. these findings necessitates the comprehensive systematic approach to attain accurate diagnosis and plan the appropriate treatment protocol. Methods/Case Report Bone marrow aspiration & biopsy procedure was performed under local anesthesia and marrow samples were subjected to: lieshman staining of marrow films and direct examination. Immunophenotypic analysis of marrow aspirate by FACS Canto II flow cytometer. Cytogenetics studies were done as karyotyping and gene mutation detection by FISH. Results (if a Case Study enter NA) NA Conclusion Morphology of marrow films: 50% Blast cells, group of them are medium to larg-cells with high nucleocytoplasmic ratio and immature chromatin and the other group shows blast cells with sparse basophilic granulations, plus 20% immatue and maturing basophils. IPT shows two populations: the Myeloblast population (basophilic differentiation) is Positive for CD45dim, CD34het, CD13, CD33, CD117prt, CD25het, CD123het, cyt MPO part, HLA-DR partial. the B lineage lymphoblast population is Positive for CD45dim, CD34het, CD19, CD79a, TdT, CD20het, CD38. Cytogenetics studies: hyperploidy 56, (X, Y, 9, 10, 12, 13, 15, 18, 22, mar+ extra-chromosomes) FISH: +ve for FLT3-ITD mutation. the picture is by morphology & IPT consistent with biclonal leukemia, myeloid clone (acute basophilic leukemia) & ProB-ALL. The systematic laboratory approach from careful morphological examination of peripheral blood and bone marrow smears to confirmatory immunophenotypic analysis of marrow specimens by flow cytometry, and the comprehensive cytogenetics analysis, has a vital role in diagnosing such rare and aggressive phenomenon and provide a good chance for planning the appropriate treatment protocol with critical timesaving and cost effectiveness.

Limited Alleviation of Lysosomal Acid Lipase Deficiency by Deletion of Matrix Metalloproteinase 12.

Lysosomal acid lipase (LAL) is the only known enzyme that degrades cholesteryl esters and triglycerides at an acidic pH. In LAL deficiency (LAL-D), dysregulated expression of matrix metalloproteinase 12 (MMP-12) has been described. The overexpression of MMP-12 in myeloid lineage cells causes an immune cell dysfunction resembling that of Lal knockout (Lal KO) mice. Both models develop progressive lymphocyte dysfunction and expansion of myeloid-derived suppressor (CD11b+ Gr-1+) cells. To study whether MMP-12 might be a detrimental contributor to the pathology of LAL-D, we have generated Lal/Mmp12 double knockout (DKO) mice. The phenotype of Lal/Mmp12 DKO mice closely resembled that of Lal KO mice, while the weight and morphology of the thymus were improved in Lal/Mmp12 DKO mice. Cytological examination of blood smears showed a mildly reversed lymphoid-to-myeloid shift in DKO mice. Despite significant decreases in CD11b+ Ly6G+ cells in the peripheral blood, bone marrow, and spleen of Lal/Mmp12 DKO mice, the hematopoietic bone marrow progenitor compartment and markers for neutrophil chemotaxis were unchanged. Since the overall severity of LAL-D remains unaffected by the deletion of Mmp12, we conclude that MMP-12 does not represent a viable target for treating the inflammatory pathology in LAL-D.

Malaria and dengue fever in febrile children entering healthcare facilities in Mwanza, Tanzania.

Plasmodium spp. infections and cases of malaria are a long-standing public health problem for children living in middle- and low-income countries. Dengue virus causes an emerging under-recognized disease burden. A cross sectional study was conducted between March 2020 and December 2021 to determine the status of malaria and dengue fever, and the associated factors in children living in Mwanza, Tanzania. Clinical features were recorded; blood samples were analyzed using dengue NS1 rapid diagnostics test (NS1-RDT), malaria rapid diagnostic test (MRDT) and PCR and microscopy for malaria parasites. Descriptive analysis was based on infection status; odds ratio and confidence interval were used to determine the factors associated with dengue fever and malaria. The prevalence of malaria in the 436 children included in the final analysis was 15.6%, 8.5%, and 12.1% as determined by MRDT, blood smear examination and PCR, respectively. The prevalence of dengue fever determined by the NS1-RDT was 7.8%. Body rash, muscle and joint/bone pain were associated with a positive rapid dengue test result. Retro-orbital pain characterized Plasmodium spp. and dengue virus co-infections. Clinical signs and symptoms could not readily differentiate between malaria and dengue fever patients or patients co-infected with both causative agents underscoring the urgent need for the accurate laboratory diagnostics. Additional large-scale studies are required to assess the epidemiological burden of acute febrile illness in developing countries and to produce data that will guide empirical treatment.

SCKansformer: Fine-Grained Classification of Bone Marrow Cells via Kansformer Backbone and Hierarchical Attention Mechanisms.

The incidence and mortality rates of malignant tumors, such as acute leukemia, have risen significantly. Clinically, hospitals rely on cytological examination of peripheral blood and bone marrow smears to diagnose malignant tumors, with accurate blood cell counting being crucial. Existing automated methods face challenges such as low feature expression capability, poor interpretability, and redundant feature extraction when processing highdimensional microimage data. We propose a novel finegrained classification model, SCKansformer, for bone marrow blood cells, which addresses these challenges and enhances classification accuracy and efficiency. The model integrates the Kansformer Encoder, SCConv Encoder, and Global-Local Attention Encoder. The Kansformer Encoder replaces the traditional MLP layer with the KAN, improving nonlinear feature representation and interpretability. The SCConv Encoder, with its Spatial and Channel Reconstruction Units, enhances feature representation and reduces redundancy. The Global-Local Attention Encoder combines Multi-head Self-Attention with a Local Part module to capture both global and local features. We validated our model using the Bone Marrow Blood Cell FineGrained Classification Dataset (BMCD-FGCD), comprising over 10,000 samples and nearly 40 classifications, developed with a partner hospital. Comparative experiments on our private dataset, as well as the publicly available PBC and ALL-IDB datasets, demonstrate that SCKansformer outperforms both typical and advanced microcell classification methods across all datasets.

Molecular and microscopic detection of haemoprotozoan diseases in dogs from Haryana, India.

Haemoparasitic infections are frequently observed in dogs from tropical regions, including India. The present investigation combined microscopic blood smear examination and PCR assays to assess the occurrence of canine tick-borne diseases (CTBD) from suspected dogs in and around Hisar, Haryana. Using the Giemsa-stained peripheral thin blood smear examination, 15 (12.5%) of the 120 dogs were infected with CTBD, with 5.8%, 3.3%, 2.5%, and 0.8% dogs testing positive for Hepatozoon canis, Ehrlichia canis, Babesia vogeli, and Babesia gibsoni, respectively. Using the PCR assay, CTBD was found to be 64.16% (77/120) in examined dogs. Of the 77 PCR-positive canines, 56 were infected with a single haemoparasite, while 21 were infected with two or more species. H. canis was the most abundant tick-borne pathogen, representing 35%, followed by E. canis 25.8%, B. vogeli 20%, and B. gibsoni 2.5%. The most common co-infection was with H. canis along with E. canis (7.5%). The PCR assay was proven to be more efficient for detecting haemoparasites in dogs compared to blood smear examinations. The study suggests that canine tick-borne diseases are common in Haryana and recommends using PCR-based molecular tests in addition to conventional microscopic examination to diagnose these infections for effective treatment and management of infected canines.

Histidine-rich protein (hrp) 2-based RDT false-negatives and Plasmodium falciparum hrp 2 and 3 gene deletions in low, seasonal and intense perennial transmission zones in Cameroon: a cross - sectional study.

False negative rapid diagnostic tests (RDTs) accruing to the non-detection of Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) is threatening the diagnosis and management of malaria. Although regular monitoring is necessary to gauge the level of efficacy of the tool, studies in Cameroon remain limited. This study assessed Plasmodium spp. prevalence and Pfhrp2/3 gene deletions across ecological and transmission zones in Cameroon. This is a cross-sectional, multi-site, community- and hospital- based study, in 21 health facilities and 14 communities covering all five ecological settings in low seasonal (LS) and intense perennial (IPT) malaria transmission zones between 2019 and 2021. Participants were screened for malaria parasite using Pfhrp2 RDT and light microscopic examination of thick peripheral blood smears. DNA was extracted from dried blood spot using chelex®-100 and P. falciparum confirmed using varATS real-time quantitative Polymerase Chain Reaction (qPCR), P. malariae and P. ovale by real-time qPCR of Plasmepsin gene, and P. vivax using a commercial kit. Isolates with amplified Pfcsp and Pfama-1 genes were assayed for Pfhrp 2/3 gene deletions by conventional PCR. A total of 3,373 participants enrolled, 1,786 Plasmodium spp. infected, with 77.4% P. falciparum. Discordant RDT and qPCR results (False negatives) were reported in 191 (15.7%) P. falciparum mono-infected samples from LS (29%, 42) and IPT (13.9%, 149). The Pfhrp2+/Pfhrp3 + genotype was most frequent, similar between LS (5.5%, 8/145) and IPT (6.0%, 65/1,076). Single Pfhrp2 and Pfhrp3 gene deletions occurred in LS (0.7%, 1/145 each) and IPT (3.6%, 39/1,076 vs. 2.9%, 31/1,076), respectively. Whilst a single sample harboured Pfhrp2-/Pfhrp3- genotype in LS, 2.4% (26/1,076) were double deleted at IPT. Pfhrp2+/Pfhrp3- (0.3%, 3/1,076) and Pfhrp2-/Pfhrp3+ (1.2%, 13/1,076) genotypes were only observed in IPT. Pfhrp2, Pfhrp3 deletions and Pfhrp2-/Pfhrp3- genotype accounted for 78.8% (26), 69.7% (23) and 63.6% (21) RDT false negatives, respectively. Plasmodium falciparum remains the most dominant and widely distributed Plasmodium species across transmission and ecological zones in Cameroon. Although the low prevalence of Pfhrp2/3 gene deletions supports the continued use of HRP2-based RDTs for routine malaria diagnosis, the high proportion of false-negatives due to gene deleted parasites necessitates continued surveillance to inform control and elimination efforts.

Classification of Malaria Using Convolutional Neural Network Method on Microscopic Image of Blood Smear

Malaria, a critical global health issue, can lead to severe complications and mortality if not treated promptly. The conventional diagnostic method, involving a microscopic examination of blood smears, is time-consuming and requires extensive expertise. To address these challenges, computer-assisted diagnostic methods have been explored. Among these, Convolutional Neural Networks (CNN), a deep learning technique, has shown considerable promise for image classification tasks, including the analysis of microscopic blood smear images. In this study, we employ the NIH Malaria dataset, which consists of 27,558 images, to train a CNN model. The dataset is divided into parasitized (malaria-infected) and uninfected. The CNN architecture designed for this study includes three convolutional layers and two fully connected layers. We compare the performance of this model with that of a pre-trained VGG-16 model to determine the most effective approach for malaria diagnosis. The proposed CNN model demonstrates high accuracy, achieving a value of 96.81%. Furthermore, it records a recall of 0.97, a precision of 0.97, and an F1-score of 0.97. These metrics indicate a robust performance, outperforming previous studies and highlighting the model's potential for accurate malaria diagnosis. This study underscores the potential of CNN in medical image classification and supports its implementation in clinical settings to enhance diagnostic accuracy and efficiency. The findings suggest that with further refinement and validation, such models could significantly improve the speed and reliability of malaria diagnostics, ultimately aiding in better disease management and patient outcomes.

Detection of Bartonella henselae in feline erythrocytes in Egypt by using Giemsa staining, transmission electron microscopy, and polymerase chain reaction.

Bartonella species (Bartonella spp.) have gained recognition as a significant human pathogen, implicated in a wide range of diseases. Among these, Bartonella henselae infection has been extensively studied for its primary occurrence in cats and its role in the development of cat-scratch disease in humans. While light microscopy and transmission electron microscopy (TEM) have traditionally played crucial roles in identifying causative agents of infectious diseases, including Bartonella spp., the accuracy of these methods in identifying Bartonella spp. remains undefined. Therefore, this study aims to bridge this gap by employing both light microscopy and TEM to detect Bartonella in feline blood samples and to confirm B. henselae with polymerase chain reaction (PCR). Examination of blood smears stained with Giemsa and toluidine blue semithin sections by using light microscopy revealed the presence of intraerythrocytic corpuscles, suggesting Bartonella infection in six out of 33 examined cat blood samples. TEM findings corroborated these observations, showcasing the engulfment of bacteria by the erythrocyte membrane, along with the presence of some Bartonella spp., adhering to the erythrocyte wall. PCR-based molecular detection confirmed the presence of B. henselae in these six samples. It is concluded that light microscopy and TEM are considered valuable in the screening of cats' blood for the potential presence of Bartonella. However, further molecular techniques are essential for precise identification and confirmation of specific Bartonella spp. RESEARCH HIGHLIGHTS: Giemsa-stained blood smear and semithin section showed potential intraerythrocytic Bartonella spp. corpuscles. TEM demonstrated the engulfment of Bartonella spp. by the erythrocyte membrane, along with the presence of some Bartonella spp. adhering to the erythrocyte wall. Molecular analysis of blood samples from cats by PCR unveiled that six out of 33 (18.18%) samples tested positive for B. henselae infection.

A Case Report: Treatment of Anaplasmosis and Ectoparasitic Infestation in Mixed-Breed Golden Retriever Dog

Anaplasmosis is a disease in dogs caused by intracellular gram-negative bacteria belonging to the Anaplasmataceae family. A 6-month-old mixed-breed golden retriever was examined at the Laboratory of Veterinary Internal Medicine, Faculty of Veterinary Medicine, Udayana University, with complaints of itching, tick infestation, weakness, and decreased appetite. The clinical examination revealed pale mucous membranes in the mouth and eye conjunctiva, as well as an infestation of Rhipicephalus ticks on the skin. Routine hematological examination indicated the presence of anemia and thrombocytopenia. Blood smear examination confirmed the presence of Anaplasma sp. Treatment was provided in a causative and supportive manner. Causative therapy involved the administration of doxycycline at a dose of 10 mg/kg body weight orally for twenty-eight days, ivermectin at a dose of 0.2 mg/kg body weight injected subcutaneously every two weeks for four weeks. Supportive therapy included the daily administration of Fufang E'jiao Jiang ®(Dong E Ejiao Co, Ltd., Shandong, China) at 2 ml/day and Sangobion® (PT. Merck Tbk, Jakarta, Indonesia) at one tablet per day for fourteen days. Treatment with doxycycline, ivermectin, Fufang E'jiao Jiang® (Dong E Ejiao Co, Ltd., Shandong, China), and Sangobion® (PT. Merck Tbk, Jakarta, Indonesia) resulted in a positive outcome for the dog, with improved activity, hair growth, good appetite, and the absence of ticks.

Assessment of Reticulocyte Count as a Prognostic Indicator for Canine Ehrlichiosis: Implications for Diagnostic and Therapeutic Strategies

Canine ehrlichiosis is increasingly recognized in India, with significant implications for canine health. It is a tick-borne disease, caused by Ehrlichia canis. It is prevalent in various regions, particularly in areas with high tick populations. Anaemia is one of the most common and important manifestation of this disease. Anemia results from the destruction of red blood cells and bone marrow suppression. The prevalence of canine ehrlichiosis correlates with the density of tick vectors and can be exacerbated by environmental factors. Canine anemia can be classified as either regenerative or non-regenerative. Objective of the study was, investigating the prognostic value of reticulocyte counts in dogs with ehrlichiosis. The study involved 212 dogs screened between February 2023 and March 2024. Risk factors such as age, sex, breed, and season were recorded. Canine ehrlichiosis was diagnosed by finding morula stages in monocytes and neutrophils in thin blood smear examination. The pathogen was confirmed through PCR-based molecular examination. Reticulocyte count was performed through supravital stain new methylene blue (NMB). Reticulocytes contain ribosomal RNA, which is less condensed than in mature erythrocytes. NMB has a high affinity for ribosomal RNA, resulting in a blue coloration of these immature cells. This staining reaction makes reticulocytes clearly distinct compared to mature red blood cells under a microscope, facilitating their identification and quantification. The study found that the haematological parameters of infected dogs were significantly lower than healthy dogs. The mean ±SE values of Hb, TEC, PCV, and corrected reticulocyte were significantly lower (p<0.001) in infected dogs compared to healthy dogs. These findings suggest that significantly lower values of Hb, TEC, PCV, MCV, MCH, MCHC, and corrected reticulocyte indicate anaemia, normocytic, normochromic anaemia, and non-regenerative anaemia. It can be concluded than in canine ehrlichiosis, anemia is one of the most common finding accounting aprox 28% of total diagnosed cases. Accurate anemia characterization is key for diagnosing and treating ehrlichiosis.

Molecular evidence of hepatozoonosis in tigers of Vidarbha region of Maharashtra State of India

BackgroundHepatozoonosis has been reported in many species around the world. Few incidences have been reported in various species of wild felids. Tigers are endangered large cats and are protected under the Wildlife Protection Act, 1972 under Schedule I. The study was carried out to estimate the positivity rate of hepatozoonosis in tigers of the Vidarbha region of Maharashtra, India.MethodsBlood (n = 21) or tissue samples (n = 5) were collected from 26 wild captured / zoo-born or dead tigers during the quarantine period/post-mortem examination. Blood smear examination along with Polymerase Chain Reaction (PCR) studies were conducted for the detection of hepatozoonosis. All the amplicons from the positive samples were purified and sequenced, and the sequences were subjected to nBLAST analysis to detect the species of Hepatozoon. The sequences were deposited into public domain database of National Center for Biotechnology Information (NCBI) and accession numbers were allotted. A phylogenetic study was undertaken to understand the evolutionary lineage of the pathogen. Tissue distribution studies were carried out on tissue samples received during post mortem. A clinical case in a tiger cub was managed and sub-clinical cases were monitored for relapse. Age-wise, sex-wise, region-wise and captive time-wise positivity rate was estimated. The data was analyzed using statistical tools.ResultsA total of 12 tigers were found positive for H. felis during the screening. A clinical case was diagnosed and successfully treated. The age group of 0–3 years reported a positivity rate of 66.66%, and all the cases found positive were reported between the age group of 0–7 years. Males reported a positivity rate of 58.33 per cent, while females reported 35.71%. Taboba and Andhari Tiger Reserve of the state had a positivity rate of 52.94 per cent. However, the statistical analysis for blood parameters and positivity rate by ‘t’ test and Chi-squared test were found to be non-significant.ConclusionsAn overall positivity rate of 46.15% indicates the wide distribution of hepatozoonosis among wild tigers of the Vidarbha region of Maharashtra, India, which is strategically important considering the gene flow and migration of tigers. Hepatozoonosis can progress to clinical outcomes in young animals and require veterinary intervention. Molecular tools and phylogenetic studies can supplement important data on circulating species of Hepatozoon in the field. Further studies on the clinical management and epidemiology of the infection in wild felids will comprehend the cause of wildlife conservation.

Clinical manifestation and management of hypomagnesemia in Theileria spp. infected periparturient Malabari goats

BackgroundGrass tetany or hypomagnesemia is a metabolic syndrome causing acute neurological manifestations in periparturient ruminants grazing on rapidly grown green pastures or cereal crops with high potassium or nitrogen content and low magnesium levels. Clinical reports of naturally occurring grass tetany in goats are least described in the literature and it is considered to be a rare disease. The present study documents the clinical manifestation and management of hypomagnesemia-associated excitatory neuropathy in two Theileria spp. infected Malabari goats presented to the small ruminant medicine OP unit of the University Veterinary Hospital and Teaching Veterinary Clinical Complex, Mannuthy, Thrissur, Kerala, India. MethodsThe first goat was in the last trimester of pregnancy and was showing signs of hyperesthesia, limping, difficulty in standing, and inappetence for the last six hours. The second goat was kidded six weeks back and was showing recurrent epileptic seizures and hyporexia from the previous day. Both the goats were kept on extensive grazing from the forest areas with unidentified lush vegetation. On clinical evaluation, both the goats were showing persistent excitatory neurological signs. Fecal examination, peripheral blood smear examination, PCR screening panel for hemoparasites, Complete Blood Count, and serum levels of calcium, magnesium, and glucose were evaluated. ResultsA significant reduction in serum magnesium levels was noted in both cases along with the varied glucose levels. Theileria spp. infection was noticed in blood smear examination and confirmed by PCR. The goats responded well to the therapy using MIFEX™ and other supportive medications and showed uneventful recovery. ConclusionIt is suggested that the farmers should be vigilant enough while allowing goats to graze in unknown vegetation and prevent indiscriminate fodder intake during periods of drought. This is the first documented report of hypomagnesemic excitatory neurologic symptoms in Theileria spp. infected periparturient Malabari goats and showing response to appropriate therapy.

Storage Induced Alterations in Erythrocyte Morphology and Platelet Count

Background: Examination of blood smears and various hematologic parameters is the first step in assessment of hematologic function and diagnosis of possible underlying diseases. The aim of this study was to identify artefacts in stored red blood cells and classify the occurrence of such alterations according to the age of the stored sample. This studyalso aimed to note the changes in platelet count dispatched by automated hematology coulter in such stored blood samples. Material and Methods: A prospective cross sectional study was conducted from the EDTA anticoagulated blood samples of 100 patients received in the department of Pathology after receiving ethical approval from the institutional review committee. All the samples were analyzed in 4 occasions i.e. within 2 hours, at 24 hours, at 48 hours and at 96 hours. Analysis was carried out by Sysmex 5 part analyzer for platelet counts and the Leishman stained blood films were examined to note changes in the erythroctyte morphology. Results: There was alterationsin platelet counts in 22% cases between 2-hour samples and other samples. The red blood cell morphology was not different among 2 hours samples and 24 hours sample. However, the red blood cell morphology was altered in 48 hours samples and 96 hours samples. Conclusion: Storage of EDTA anticoagulated blood even upon refrigeration can cause changes in platelet counts as well as morphology of red blood cells. Changes in red blood cells are not significant till 24 hours while the platelet counts may alter within 24 hours.