Examination Of Giemsa-stained Blood Smears Research Articles

Effectiveness of artemether-lumefantrine for treating uncomplicated malaria in low- and high-transmission areas of Ghana.

Artemisinin-based combination therapy (ACT) has been effective in the supervised treatment of uncomplicated malaria in Ghana. Since ACT usage is primarily unsupervised, this study aimed to determine the effectiveness of artemether-lumefantrine (AL) for treating malaria patients in two transmission settings in Ghana. Eighty-four individuals with uncomplicated Plasmodium falciparum malaria were recruited from Lekma Hospital (LH) in Accra (low-transmission area; N = 28), southern Ghana, and King's Medical Centre (KMC) in Kumbungu (high-transmission area; N = 56), northern Ghana. Participants were followed up for 28days after unsupervised treatment with AL. The presence of asexual parasites was determined by microscopic examination of Giemsa-stained blood smears. Plasmodium species identification was confirmed using species-specific primers targeting the 18S rRNA gene. Parasite recrudescence or reinfection was determined by genotyping the Pfmsp 1 and Pfmsp 2 genes. After AL treatment, 3.6% (2/56) of the patients from KMC were parasitaemic on day 3 compared to none from the LH patients. One patient from KMC with delayed parasite clearance on day 3 remained parasite-positive by microscopy on day 7 but was parasite-free by day 14. While none of the patients from LH experienced parasite recurrence during the 28-day follow-up, three and two patients from KMC had recurrent parasitaemia on days 21 and 28, respectively. Percentage reduction in parasite densities from day 1, 2, and 3 for participants from the KMC was 63.2%, 89.5%, and 84.5%. Parasite densities for participants from the LH reduced from 98.2%, 99.8% on day 1, and 2 to 100% on day 3. The 28-day cumulative incidence rate of treatment failure for KMC was 12.8% (95% confidence interval: 1.9-23.7%), while the per-protocol effectiveness of AL in KMC was 89.47%. All recurrent cases were assigned to recrudescence after parasite genotyping by Pfmsp 1 and Pfmsp 2. While AL is efficacious in treating uncomplicated malaria in Ghana, when taken under unsupervised conditions, it showed an 89.4% PCR-corrected cure rate in northern Ghana, which is slightly below the WHO-defined threshold.

Detection of Hepatozoon spp. in dogs in Shiraz, southern Iran and its effects on the hematological alterations.

Canine hepatozoonosis is a tick-transmitted apicomplexan infection caused by two species of Hepatozoon, H. canis, and H. americanum. The present research aimed at detection of Hepatozoon spp. in dogs and its effects on hematological alterations. Blood samples were taken from 108 dogs to assess Hepatozoon spp. Phylogenetic analysis was performed based on the 18S rDNA marker by PCR assay and Giemsa-stained blood smear examination. Of the 108 blood samples of dogs tested in the present study, eight (7.40%, 95% CI: 3.25-14.07%) were positive by the Hepatozoon-specific PCR assay. However, in the microscopic examination, only one sample (0.93%) was positive. All of the sequenced samples were H. canis. The Hepatozoon sequences obtained from PCR amplicons in the canine-positive cases exhibited 100% similarity to each other and 98.47-100% similarity to other relevant sequences in GenBank. These findings represent the first molecular evidence of H. canis in dog populations in South Iran. Furthermore, according to the hematological analysis, significantly higher average numbers of neutrophils and lymphocytes were found in the infected group compared to the non-infected dogs. In this study, no statistically significant connection (P<0.05) was observed between H. canis infection and the examined risk factors.

Hematological alterations in large Babesia species infection in dogs of Kannur District of Kerala.

Canine babesiosis is a tick-borne disease caused by apicomplexan intraerythrocytic hemoprotozoan parasites. It is caused by the small (Babesia gibsoni, B. conradae, and B. vulpes) and large (B. vogeli, B. canis, and B. rossi) Babesia groups. As per the recent reports, the most prominent Babesia species encountered in the Kerala state are the small Babesia, B. gibsoni followed by the large Babesia, B. vogeli. The latter is regarded as mildly pathogenic, causing subclinical or mild disease; however severe complications like systemic inflammatory response syndrome, multiple organ dysfunction syndrome, etc. have also been reported. The information on the status of hematological alterations in naturally infected dogs with large Babesia is lacking, particularly from the state of Kerala. The present study involves a retrospective study of clinical cases of large Babesia infection in dogs. The complete haematological profile from well-documented laboratory records of 4039 dogs suspected for babesiosis presented to District Veterinary Centre, Kannur during the period from December 2018 to October 2020 was analyzed for the study. Natural infections were recorded in 35 (0.87%) dogs based on the presence of intraerythrocytic piroplasm of large Babesia spp. by light microscopic examination of Giemsa-stained blood smears. The most consistent features observed were mild to moderate regenerative, normocytic and normochromic anemia, lowered to normal neutrophil count and thrombocytopenia. In comparison to hemolytic anemia, thrombocytopenia was the most frequent clinicopathological finding in the study with an increased presence of large activated platelets or macro-platelets.

Development of a novel multiplex PCR assay for the detection and differentiation of Plasmodium caprae from Theileria luwenshuni and Babesia spp. in goats

Intraerythrocytic parasites are traditionally identified by the microscopic examination of Giemsa-stained blood smears. However, this method does not always allow for the identification of individual species in goat's RBCs. Moreover, its unreliability in detecting low levels of parasitemia makes it unsuitable for epidemiological investigations and leaves goat farms vulnerable to potential outbreaks. In the present study, a novel multiplex PCR (mPCR) targeting the cytochrome c oxidase subunit I (COI) gene was developed to detect and subsequently differentiate Plasmodium caprae, Theileria luwenshuni, and Babesia spp. The specificity of each primer set was assessed both in silico and with a panel of DNA samples from the hosts themselves and other goat hemoparasites. Amplicons generated from each pair of primers were 664, 555, and 320-bp for P. caprae, Babesia spp., and T. luwenshuni, respectively. These products were further confirmed by sequencing. Our novel mPCR reactions successfully demonstrated the accurate and simultaneous amplification of the three parasites’ DNA samples. The current mPCR method showed no cross-amplification with unintended targets. The detection limit of the mPCR in this study was 108 parasites’ DNA copies per reaction. The current mPCR was able to detect the minimum parasitemia of approximately 0.001%, 0.000005%, 0.00001% for P. caprae, Babesia spp. and T. luwenshuni, respectively. The diagnostic specificity in the detection of P. caprae and T. luwenshuni ranged from 94.9 to 100 %. The mPCR was further applied to a collection of field blood samples from five provinces in Thailand to validate its reliability and applicability. The results demonstrated the successful detection of P. caprae, Babesia spp. and T. luwenshuni in goat samples with the same sensitivity levels as conventional PCR methods. This study also confirmed the presence of T. luwenshuni and Babesia spp. in Thai goats. The current mPCR method offers an alternative for the diagnosis of P. caprae, T. luwenshuni, and Babesia spp., either single or under co-infection conditions, and for large-scale surveillance.

Cross-sectional survey of cattle haemopathogens in Constantine, Northeast Algeria.

This aim of the present study was to estimate the prevalence of haemopathogens in cattle in Beni Hamidene locality, district of Constantine (Νortheastern Algeria). Between June and October 2014, 169 bovines from 25 farms were included in this survey, 32 (18.9%) among them were suspected of piroplasmosis and/or anaplasmosis. Infection prevalences were estimated by microscopic examination of Giemsa‐stained blood smears and blood samples from all included cattle (n = 169). Animals were infected by Theileria annulata (65/169; 38.46%), Anaplasma marginale (22/169; 13%) and Babesia bovis (5/169; 3%). Two co‐infection patterns were found: Theileria annulata/Anaplasma marginale (7.69%) and Theileria annulata/Babesia bovis (1.18%). Only one farm had no cattle infected by any of the haemopathogens. There was a signification difference of T. annulata infection prevalence according to age category (p =.04). These results emphasised mainly the presence of bovine tropical theileriosis in northeastern, Beni Hamidene locality, province of Constantine, Algeria.

Prevalence and hematological parameters of bovine Trypanosomosis in Nsukka municipal abattoir, Southeastern Nigeria

The prevalence and hematological parameters of trypanosomosis in slaughter cattle in Nsukka, Southeastern Nigeria, were investigated. Blood samples from 200 randomly selected cattle were collected following slaughter into labeled sample bottles with anticoagulant. The sex and breed of the sampled cattle were properly noted. Standard trypanosome detection techniques involving wet blood film examination, buffy coat technique, examination of Giemsa-stained blood smears, and mice inoculation were used to detect and identify the trypanosome species present. The hematological parameters—packed cell volume (PCV), hemoglobin concentration (HbC), total erythrocyte count (TEC), total leucocyte count (TLC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)—were determined following standard procedures. A prevalence rate of 1.5% (95% CI = 0.005–0.043) was obtained for bovine trypanosomosis in Nsukka with 1% (95% CI = 0.003–0.036) and 0.5% (95% CI = 0.001–0.028) prevalence rates in female and male cattle, respectively. Whereas a prevalence rate of 1.5% (95% CI = 0.005–0.043) was recorded in the White Fulani breed, the Sokoto Gudali had 0% prevalence rate. The prevalence recorded in this study was not associated (P ˃ 0.05) with sex or breed. The mean PCV, HbC, TEC, MCV, MCH, MCHC, and TLC values of the infected cattle differed (P < 0.05) from that of the uninfected cattle. In conclusion, trypanosomosis is prevalent in cattle slaughtered in Nsukka, Southeastern Nigeria, with associated hematological changes. Hence, the need for the adoption and implementation of national trypanosomosis integrated control strategies.

Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Diagnosis Cepat Kandidemia Neonatus dengan Pemeriksaan Spesimen Darah Pulasan Giemsa

Candidemia is one of the major problems in neonates with low birth weight (LBW). Neonatal candidemia has a high rateof morbidity and mortality. Early initiation of antifungal therapy could be decreasing the mortality, but this managementoften hampered due to late diagnosis. The gold standard for diagnosing candidemia is blood culture, but it takes a longtime about 5 days. A rapid alternative method is needed to decrease candidemia morbidity and mortality. The methodchosen in this study is Giemsa stain of blood smear which result could be read within one hour. The study wasconducted in the Department of Parasitology, Faculty of Medicine Universitas Indonesia with a total blood samples of170 from 2009-2012. Blood samples are devided in two, first is made thick blood smear than stain with giemsa, than readby microscope by 400×and 1000× magnification. The second is cultured on Sabauraud agar media and incubated inroom temperature for ten days. Thirty four patients (20%) were positive for Candida, 28 (82.4%) positive samples withboth giemsa staining and culture, while 6 (17,6%) positive culture samples but negative giemsa staining. The values ofthe sensitivity and specificity of the Giemsa stained blood smear examination were 82%, and 100%. This result showsthat Giemsa staining has a good diagnostic value for detecting neonatal candidemia.

Anaplasma ovis infection in goat flocks around Gaborone, Botswana

Anaplasma is a genus of gram-negative, intracellular bacteria infecting different blood cells in animals. A. ovis infects erythrocytes of sheep and goats, inducing clinical signs that include haemolytic anaemia, icterus and loss of production. Aim of this study was to evaluate the prevalence of Anaplasma spp. in goats in smallholder flocks around Gaborone, Botswana. Whole blood and serum samples were collected from 100 goats coming from 10 smallholder goat flocks from three villages around Gaborone, Botswana. Body condition and FAMACHA© scores were estimated. Blood was used for packed cell volume (PCV), blood smears, PCR, including species-specific PCR assays for A. marginale and A. ovis, and serum samples were analysed by Anaplasma spp. cELISA. Each farmer was interviewed about management, health and treatment of the goats. Microscopic examination of Giemsa-stained blood smears showed inclusion bodies in 53% of the samples. A seroprevalence of 88% was found on cELISA and 76% of the goats were positive by PCR using general primers for Anaplasma spp. All PCR positive goats were infected with A. ovis and no goats were positive for A. marginale. Positive animals were found in all areas and in all flocks. There was no correlation between infection and clinical signs. In Botswana, A. ovis in goats does not seem to pose a significant problem even though the pathogen is endemic in the goat population, or perhaps, because of being endemic, herd immunity leads to less severe clinical signs.

In situ loop-mediated isothermal amplification (LAMP) for identification of Plasmodium species in wide-range thin blood smears

BackgroundFive species of Plasmodium are known to infect humans. For proper treatment of malaria, accurate identification of the parasite species is crucial. The current gold standard for malaria diagnosis is microscopic examination of Giemsa-stained blood smears. Since the parasite species are identified by microscopists who manually search for the parasite-infected red blood cells (RBCs), misdiagnosis due to human error tends to occur in case of low parasitaemia or mixed infection. Then, molecular methods, such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are required for conclusive identification of the parasite species. However, since molecular methods are highly sensitive, false-positive results tend to occur due to contamination (carry over) or the target gene products may be detected even after clearance of the parasites from the patient’s blood. Therefore, accurate detection of parasites themselves by microscopic examination is essential for the definitive diagnosis. Thus, the method of in situ LAMP for the parasites was developed.ResultsRed blood cell suspensions, including cultured Plasmodium falciparum, strain 3D7, infected-RBCs, were dispersed on cyclic olefin copolymer (COC) plate surfaces rendered hydrophilic by reactive ion-etching treatment using a SAMCO RIE system (hydrophilic-treated), followed by standing for 10 min to allow the RBCs to settle down on the plate surface. By rinsing the plate with RPMI 1640 medium, monolayers of RBCs formed on almost the entire plate surface. The plate was then dried with a hair drier. The RBCs were fixed with formalin, followed by permeabilization with Triton X-100. Then, amplification of the P. falciparum 18S rRNA gene by the LAMP reaction with digoxigenin (DIG)-labelled dUTP and a specific primer set was performed. Infected RBCs as fluorescence-positive cells with anti-DIG antibodies conjugated with fluorescein using fluorescent microscopy could be detected.ConclusionsThe present work shows that the potential of in situ LAMP for the identification of Plasmodium species at the single cell level on hydrophilic-treated COC palates, allowing highly sensitive and accurate malaria diagnosis. The findings will improve the efficacy of the gold standard method for malaria diagnosis.

Comparative evaluation of different diagnostic tests for B. gibsoni in dogs

Babesia gibsoni is the common cause of canine babesiosis in India. Its diagnosis by conventional diagnostic methods result in false negative condition, particularly in those cases in which parasitemia is low. Therefore the present study employed IFAT and PCR along with microscopic examination to overcome this situation. Microscopic examination of Giemsa-stained blood smears revealed presence of small pear-shaped, oval or signet ring shaped B. gibsoni within the erythrocytes of 1.34 % dogs. IFAT and PCR detected a total of 1.80 % and 1.82 % dogs positive for B. gibsoni, respectively. The sensitivity and specificity of IFAT was 93.10% and 88.89%, respectively whereas, sensitivity and specificity of microscopic examination was 71.26% and 95.56%, respectively. The agreement of IFAT and microscopic examination with PCR was 82.30% and 60.73%, respectively. The agreement between IFAT and microscopy was 60.99%. Therefore, IFAT is considered to be highly sensitive test for diagnosing B. gibsoni in subclinically or chronically infected dogs with significantly low level of parasitemia.

Molecular and microscopic detection of Theileria equi and Babesia caballi in horses in Kurdestan Province, Iran

Equine piroplasmosis is a tick-borne disease caused by intra-erythrocyte protozoa, Theileria equi and Babesia caballi. The present study aimed to detect piroplasm infection in horses in Kurdestan Province, Iran, through molecular and microscopic approaches.n this study, 186 blood samples were randomly collected from horses of five regions of Kurdestan Province. The Tbs-S/Tbs-A primer set was used for amplification of Theileria equi and Babesia caballi DNA through polymerase chain reaction. Blood smears of each case were also examined by Giemsa staining method. During microscopic examination of Giemsa-stained blood smears, 3 out of 186 (1.61%) blood samples were positive for piroplasm infection. The product of only one blood sample yielded 426-430 bp-sized fragments on a 1.5% agarose gel electrophoresis, and BLAST analysis of the sequenced sample indicated a 100% similarity with T. equi 18S rRNA gene sequences in GenBank. The results indicated that one out of 186 blood sample was positive (0.54%) for Theileria equi and none of them was positive for Babesia caballi.

A novel quantitative PCR detects Babesia infection in patients not identified by currently available non-nucleic acid amplification tests

BackgroundTicks transmit Babesia microti, the causative agents of babesiosis in North America and Europe. Babesiosis is now endemic in Northeastern USA and affects people of all ages. Babesia species infect erythrocytes and can be transmitted through blood transfusion. Whole blood and blood products, which are not tested for Babesia, can cause transfusion-transmitted babesiosis (TTB) resulting in severe consequences in the immuno-compromised patients. The purpose of this study was epidemiological evaluation of babesiosis in a tick-infested state.ResultsWe examined blood samples from 192 patients who visited clinics during the active tick-borne diseases season, using a newly developed qPCR assay that uses the specific molecular beacon probe. Due to the absence of clear symptomology, clinical laboratories did not test 131 samples by IFA, FISH or microscopic examination of Giemsa-stained blood smears. Babesia infection was detected in all age groups by FISH and microscopy; notably patients >40 years of age represented 64% of tested samples and 13% were younger patients. We tested all samples using qPCR and found that 38% were positive for Babesia. Of 28 samples that were positive by FISH, 27 (96%) were also positive by qPCR indicating high congruency between nucleic acid based tests. Interestingly, of 78 asymptomatic samples not tested by FISH, 22 were positive by our qPCR. Direct detection of Babesia relies upon microscopic examination of patient blood smears, which is labor intensive, difficult to scale up, requires specific expertise and is hence, often not performed. In fact, a clinical laboratory examined only 23 of 86 blood samples obtained from two different counties by microscopy. By considering individuals positive for Babesia infection when results from currently available microscopy, FISH or serological tests were positive, we found that our qPCR is highly sensitive (96.2%) and showed a specificity of 70.5% for Babesia.ConclusionRobust qPCR using specific probes can be highly useful for efficient and appropriate diagnosis of babesiosis in patients in conjunction with conventional diagnostics, or as a stand-alone test, especially for donated blood screening. The use of a nucleic acid amplification test based screening of blood and blood products could prevent TTB.

The first report of Anaplasma phagocytophilum and a novel Theileria spp. co-infection in a South African giraffe

Organisms of the genera Anaplasma and Theileria are important intracellular bacteria and parasites that cause various tick-borne diseases, threatening the health of numerous animals as well as human beings. In the present study, a 12-month-old male wild South African giraffe (Giraffa camelopardalis giraffa) originating from South Africa, and living in Zhengzhou Zoo (located in the urban district of Zhengzhou in the provincial capital of Henan), suddenly developed an unknown fatal disease and died 1day after the onset of the clinical signs. By microscopic examination of Giemsa-stained blood smears combined with nested PCR and DNA sequence analysis, Anaplasma phagocytophilum, Anaplasma bovis and a novel Theileria spp. were found in the blood of this giraffe. The six other Cervidae animals in the zoo and three ruminants living in the same colony house with them were found to be negative for both Anaplasma and Theileria in their blood specimens. We report on the first case of an A. phagocytophilum infection and the occurrence of a novel Theileria spp. in the blood of a giraffe. This is the first reported case of a multi-infection of A. bovis, A. phagocytophilum and Theileria spp. in a giraffe, as revealed by microscopic examination of blood smears and the results of nested PCR and DNA sequencing.

Prevalence of Theileria and Babesia species in Tunisian sheep.

In this study, the prevalence of Theileria and Babesia species in sheep was assessed with Giemsa-stained blood smear examination and polymerase chain reaction to identify the different piroplasms in 270 sheep from three Tunisian bioclimatic zones (north, centre, and south). The overall infection prevalence by Babesia spp. and Theileria spp. in Giemsa-stained blood smears was 2.9% (8/270) and 4.8% (13/270) respectively. The molecular results showed that sheep were more often infected by Theileria ovis than Babesia ovis with an overall prevalence of 16.3% (44/270) and 7.8% (21/270) respectively (p = 0.01). The molecular prevalence by Babesia ovis was significantly higher in females than in males (p < 0.05). According to localities B. ovis was found exclusively in sheep from the centre of Tunisia (Kairouan) whereas Theileria ovis was found in all regions. Infections with T. ovis and B. ovis were confirmed by sequencing. The sequence of T. ovis in this study (accession numbers KM924442) falls into the same clade as T. ovis deposited in GenBank. The T. ovis amplicons (KM924442) showed 99%–100% identities with GenBank sequences. Moreover, comparison of the partial sequences of 18S rRNA gene of B. ovis described in this study (KP670199) revealed 99.4% similarity with B. ovis recently reported in northern Tunisia from sheep and goats. Three nucleotides were different at positions 73 (A/T), 417 (A/T), and 420 (G/T). It also had 99% identity with B. ovis from Spain, Turkey and Iraq. The results suggest a high T. ovis prevalence in Tunisia with a decreasing north-south gradient. This could be correlated to the vector tick distribution.

Accuracy of immunochromatography diagnostic test versus microscopy in the diagnosis of malaria among clinically suspected patients in Jazan area, KSA

Background Malaria has been documented as a major health problem in Saudi Arabia, and it is highly endemic especially in the Southwest (Jazan and Asir). Its control is considered a challenge; hence proper diagnosis is essential for implementation of successful control programs. Accordingly, the diagnostic test used should be easy, rapid, and reliable, besides it must be accurate and cost effective. Objective This work aims to study reliability and diagnostic accuracy of an immunochromatographic test (ICT) using BinaxNOW® malaria test compared to microscopical examination of blood film as gold standard for malaria diagnosis among clinically suspected patients in Jazan area, KSA. Methods A cross sectional prospective designed study was done for 200 patients with prolonged fever attending Jazan general hospitals. Venous blood samples were collected for both microscopic examination of Giemsa-stained thick and thin blood and rapid ICT (BinaxNOW®). Results Microscopic examination of Giemsa-stained blood smears revealed 64 out of 200 cases (32%) positive for Plasmodium spp.; 43 out of 64 (67.2%) were positive for P. falciparum; 12 (18.7%) had mixed infection including P. falciparum; and 9 (14.1%) belonged to other Plasmodium spp. of which 8 (12.5%) were P. vivax and one case (1.6%) was P. ovale. ICT showed 66 out of 200 (33%) cases with positive results for Plasmodium spp. and one sample gave an invalid result. The overall sensitivity and specificity of ICT were 97% and 96%, respectively. While for single P. falciparum infection sensitivity and specificity were 96.7% and 78%, respectively. Regarding Plasmodium spp. other than P. falciparum sensitivity was 91.6% and specificity was 100%. Conclusion The use of ICT complement to microscopy is of great value particularly in Jazan, KSA, where P. falciparum and P. vivax are the most prevalent Plasmodium spp. These methods help in expanding laboratory based diagnosis, and minimize malaria associated morbidity and mortality.

Parasites and vector-borne pathogens in client-owned dogs in Albania. Blood pathogens and seroprevalences of parasitic and other infectious agents.

Knowledge on the epidemiology of parasitic and vector-borne infections is still very limited for Albania, a country located in the Balkan Peninsula in southeast Europe. Recent publications indicated prevalence rates of up to 52% for vector-borne infections in less-cared dogs in Albania. To provide data on the epidemiological situation in dogs under veterinary care, a total of 602 client-owned dogs presented to four small animal clinics between March 2010 and April 2011 in Tirana, Albania, were screened by examination of Giemsa-stained blood smears, PCR, and serological methods for the presence of arthropod-borne infections, as well as Neospora caninum and Toxoplasma gondii. Eight different pathogens, namely Babesia vogeli, Hepatozoon canis, Leishmania infantum, Dirofilaria immitis, Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, and Mycoplasma haemocanis, were detected by direct methods with prevalence rates ranging from 1 to 9%. Seroprevalence for Babesia spp., L. infantum, Anaplasma spp., and E. canis were 6.6, 5.1, 24.1, and 20.8%, respectively. Dogs >1 year of age were positive for vector-borne infections significantly more often than younger dogs (p = 0.003). More than half (51.7%) of the dogs were seroreactive to T. gondii and 18.3% to N. caninum. This is the first report on the detection of A. phagocytophilum, A. platys, E. canis, and M. haemocanis by PCR as well as the serological confirmation of exposure of dogs to N. caninum and T. gondii in Albania. The spectrum of pathogens and the seroprevalences for N. caninum and T. gondii in client-owned dogs from Tirana, Albania, are comparable to that reported in other countries in the Mediterranean Basin. The prevalence rates of vector-borne pathogens are at the lower range of that reported in studies from this geographical region. This is probably due to increased awareness of the owners of pet dogs, including better husbandry conditions and ectoparasiticidal treatment, thus limiting exposure of dogs to vectors.

Sequence-Based Optimization of a Quantitative Real-Time PCR Assay for Detection of Plasmodium ovale and Plasmodium malariae

Although microscopic examination of Giemsa-stained blood smears remains the gold standard for the diagnosis of malaria, molecular detection using PCR is becoming increasingly popular. Due to discrepant PCR and microscopy results, we aimed to optimize our detection assays for Plasmodium malariae and Plasmodium ovale by sequencing the 18S rRNA region and developing a new primer and probe set for real-time quantitative PCR (qPCR). Clinical specimens positive for P. malariae (n = 15) or P. ovale (n = 33) underwent amplification and sequencing of the 18S rRNA region. Based on sequence discrepancies between our current primer/probe and clinical isolates, degenerate P. ovale primer and probe were developed to determine if their performance characteristics improved. The reference (gold) standard was microscopy. No 18S sequence heterogeneity was observed among the P. malariae isolates, and the sensitivity and specificity of our current P. malariae qPCR assay were both 100%. Compared to microscopy, the sensitivity and specificity of our current P. ovale qPCR assay were 72.7% and 100%, respectively. Five single nucleotide polymorphisms (SNPs) were identified in P. ovale. The sensitivity of the new P. ovale assay increased to 100% with 100% specificity. We therefore improved the performance characteristics of our P. ovale molecular detection assay through the development of a degenerate primer and probe set which accommodates 18S SNPs among the 2 subspecies of P. ovale. Given the suboptimal sensitivity of rapid diagnostic tests for non-falciparum malaria and the typically low parasitemia of P. malariae and P. ovale, a well-performing confirmatory molecular assay is imperative for clinical laboratories.

Prevalence of piroplasms in small ruminants in North-West Tunisia and the first genetic characterisation of Babesia ovis in Africa.

In this study, the prevalence of piroplasms in sheep and goats was assessed with Giemsa-stained blood smear examination, PCR and nested PCR-restriction fragment length polymorphism (RFLP) to identify Babesia and Theileria species, respectively, in 338 small ruminants (172 sheep and 166 goats) from three sites in North-West Tunisia during the 2011 summer season. The overall infection prevalence of piroplasms in Giemsa-stained blood smears was 3.2% (11/338), with a parasitaemia ranging from 0.01 to 0.05%. PCR detected two species, namely Babesia ovis (in sheep and goats) and Theileria ovis (in sheep), with an overall prevalence of 16.3%. The molecular prevalence of B. ovis was significantly higher in sheep than in goats (17.4% and 9%, respectively, p = 0.034). The same trend was observed for T. ovis in sheep and goats (5.8% and 0%, respectively, p = 0.004). Comparison of the partial sequences of the 18S ssu rRNA gene revealed 100% similarity amongst Babesia from sheep and goats. The single Theileria sequence in this study showed 100% similarity to T. ovis. A high similarity with all the blasted genotypes was reported for Theileria and Babesia sequences. This is the first molecular detection of B. ovis and genetic characterisation of small ruminants’ piroplasms in Africa.

The Finding and Phylogenetic Evolution Analysis of Bovine Piroplasms in the Rasn Area of North Korea

The objective of this study was to investigate the epidemiology of bovine Piroplasms infections in the Ras▪n area of North Korea. The survey was carried out by light microscopic examination of Giemsa-stained blood smears, PCR, and phylogenetic evolution analysis of 128 blood samples collected from the Ras▪n area. The results showed that the infection rates of the small and large parasites were about 2.5 and 1.5% on average, respectively, in all Theileria sergenti and Babesia ovata positive blood smears by microscopic examination of blood smears. The detection rate of T. sergenti Giemsa-stained smears was 43.75%, while that with PCR was 67.97%. The detection rate of B. ovata Giemsa-stained smears was 49.21%, while that with PCR was 71.88%. The sequence and phylogenetic analysis of DNA showed 98.84% homology between the 18S rRNA gene sequences of T. sergenti isolates from North Korean and that of Yanbian state from China, indicating the closest genetic relationship between both of them. Moreover, 100% homology was shown between the 18S rRNA gene sequence of B. ovata isolates from North Korea and the published sequence AY081192 of GenBank, indicating the closest genetic relationship between both of them. This survey confirmed that Ras▪n is the endemic area of T. sergenti and B. ovata in North Korea.