Detection of Leishmania infantum kinetoplast DNA by Real Time PCR in hair of wild rabbits

The study of potential wild mammal reservoirs is necessary for the surveillance of leishmaniosis, as Leishmania protozoans have been isolated from a wide range of wild and domestic animal species, including Leporidae. Recently, it has been demonstrated that both hares and wild rabbits can act as sylvatic reservoirs of Leishmania. In Spain, most of the research involving wild rabbits has been developed in the central area of Madrid and in the southeastern Mediterranean coast. We studied the presence of Leishmania infantum in 116 wild rabbits (Oryctolagus cuniculus) captured in Santovenia de Pisuerga, Valladolid, Spain. Hair samples were analyzed by real time PCR. L. infantum kinetoplast DNA (kDNA) was detected and quantified in 4 out of 116 analyzed animals. The estimated number of parasites obtained were quite variable, ranging from 2.60 to 276.60. Hair samples can be collected by non-invasive methods, being a proper sample for Leishmania detection in wild Leporidae, which have an important role as reservoirs of Leishmania. Our findings enhance the need for more extensive studies in different geographical areas.

Download Full-text

Serological, molecular and clinicopathological findings associated with Leishmania infantum infection in cats in Northern Italy

Journal of Feline Medicine and Surgery ◽

10.1177/1098612x19895067 ◽

2020 ◽

Vol 22 (10) ◽

pp. 935-943 ◽

Cited By ~ 2

Author(s):

Lorenza Urbani ◽

Alessandro Tirolo ◽

Daniela Salvatore ◽

Michele Tumbarello ◽

Sofia Segatore ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Infantum ◽

Complete Blood Count ◽

Fluorescent Antibody ◽

Leishmania Species ◽

Northern Italy ◽

University Hospital ◽

Hair Samples ◽

Clinicopathological Findings

Objectives The aims of this study were to investigate the prevalence of Leishmania species infection in cats in Northern Italy and to evaluate the associations between infection and signalment and clinicopathological data. Methods The study was carried out in a veterinary university hospital from June to November 2017. Blood, urine, conjunctival swabs and hair were collected from all randomly selected cats. Leishmania species infection was evaluated using the indirect fluorescent antibody test (IFAT), setting a cut-off value of 1:80, and using real-time PCR on blood, conjunctival and hair samples. A complete blood count, serum chemistry profile, serum electrophoresis and urinalysis were also carried out. The cats were grouped on the basis of the results of the diagnostic criteria adopted in positive, negative and unconfirmed Leishmania cases. Non-parametric variables and continuous data were compared among the study groups using the χ2 test and the Mann–Whitney U-test, respectively. Results One hundred and fifty-two cats were included. Nineteen of the 152 (12.5%) cats were positive (18/152 [11.8%] showed an IFAT titre of ⩾1:80 and 1/152 [0.7%] was real-time PCR-positive from a hair sample); 106/152 (69.7%) cats were negative; and 27/152 (17.8%) cats were unconfirmed for Leishmania species. Total proteins, beta2-globulin and gamma-globulin were significantly increased in the positive Leishmania group compared with the negative group. Conclusions and relevance The results of the present study demonstrated the spread of Leishmania infantum infection in cats in Northern Italy. Hyperproteinaemia and hypergammaglobulinaemia appeared to be significant clinicopathological abnormalities in this population of cats with L infantum infection.

Download Full-text

Leishmania-FAST15: A rapid, sensitive and low-cost real-time PCR assay for the detection of Leishmania infantum and Leishmania braziliensis kinetoplast DNA in canine blood samples

Molecular and Cellular Probes ◽

10.1016/j.mcp.2016.08.006 ◽

2017 ◽

Vol 31 ◽

pp. 65-69 ◽

Cited By ~ 19

Author(s):

Filipe Dantas-Torres ◽

Kamila Gaudêncio da Silva Sales ◽

Lidiane Gomes da Silva ◽

Domenico Otranto ◽

Luciana Aguiar Figueredo

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Infantum ◽

Low Cost ◽

Leishmania Braziliensis ◽

Kinetoplast Dna ◽

Pcr Assay ◽

Blood Samples

Download Full-text

Application of a specific quantitative real-time PCR (qPCR) to identify Leishmania infantum DNA in spleen, skin and hair samples of wild Leporidae

Veterinary Parasitology ◽

10.1016/j.vetpar.2017.05.015 ◽

2017 ◽

Vol 243 ◽

pp. 92-99 ◽

Cited By ~ 7

Author(s):

María Victoria Ortega ◽

Inmaculada Moreno ◽

Mercedes Domínguez ◽

María Luisa de la Cruz ◽

Ana Belén Martín ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Infantum ◽

Quantitative Real Time Pcr ◽

Hair Samples

Download Full-text

Real-Time PCR Analysis of Peripheral Blood: A Non-Invasive Technology that can be Used to Assess Tumor Burden in Breast Cancer Patients

10.21236/ada412843 ◽

2002 ◽

Author(s):

Michael Mitas

Keyword(s):

Breast Cancer ◽

Real Time ◽

Cancer Patients ◽

Real Time Pcr ◽

Peripheral Blood ◽

Tumor Burden ◽

Pcr Analysis ◽

Breast Cancer Patients ◽

Non Invasive

Download Full-text

Real-Time PCR Analysis of Peripheral Blood: A Non-Invasive Technology That Can Be Used to Assess Tumor Burden in Breast Cancer Patients

10.21236/ada424096 ◽

2003 ◽

Author(s):

Michael Mitas

Keyword(s):

Breast Cancer ◽

Real Time ◽

Cancer Patients ◽

Real Time Pcr ◽

Peripheral Blood ◽

Tumor Burden ◽

Pcr Analysis ◽

Breast Cancer Patients ◽

Non Invasive

Download Full-text

Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques

Veterinary Parasitology ◽

10.1016/j.vetpar.2011.08.010 ◽

2012 ◽

Vol 184 (1) ◽

pp. 10-17 ◽

Cited By ~ 45

Author(s):

Gabriella Lombardo ◽

Maria Grazia Pennisi ◽

Tiziana Lupo ◽

Antonella Migliazzo ◽

Alessandra Caprì ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Infantum ◽

Diagnostic Techniques

Download Full-text

Reaktive Arthritis beim Hund nach Leishmanien-Infektion?

Tierärztliche Praxis Ausgabe K Kleintiere / Heimtiere ◽

10.1055/s-0038-1622657 ◽

2008 ◽

Vol 36 (01) ◽

pp. 35-41

Author(s):

D. Schaarschmidt ◽

W. Müller ◽

L. Walla ◽

A. Borggräfe

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Infantum ◽

Reaktive Arthritis ◽

Klinische Relevanz

Zusammenfassung: Gegenstand und Ziel: Erstbeschreibung einer möglicherweise leishmaniosebedingten Arthritis. Material und Methode: Fallbericht einer aus Griechenland importierten vierjährigen Mischlingshündin. Ergebnisse: Die Hündin wurde mit einer mittelgradigen Stützbeinlahmheit der rechten Vordergliedmaße vorgestellt. Radiologische, computertomographische Untersuchung sowie Arthroskopie ergaben eine mittelgradige Cubarthrose mit ausgeprägter Entknorpelung der Gelenkflächen. Serologisch wurde eine Leishmanioseinfektion diagnostiziert, wobei offensichtliche klinische Anzeichen einer viszeralen oder kutanen Leishmaniose fehlten. Mittels Real-Time-PCR ließ sich in der Synovia des betroffenen Ellbogengelenks Leishmaniainfantum-DNA nachweisen. Schlussfolgerung und klinische Relevanz: Bei Hunden mit Auslandsvorbericht und Arthropathien unklarer Genese bietet sich der direkte Nachweis von Leishmania-infantum-DNA mittels PCR aus Synovia an.

Download Full-text

Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma

Diagnostics ◽

10.3390/diagnostics10080564 ◽

2020 ◽

Vol 10 (8) ◽

pp. 564

Author(s):

Jana Bohmova ◽

Marek Lubusky ◽

Iva Holuskova ◽

Martina Studnickova ◽

Romana Kratochvilova ◽

...

Keyword(s):

Pregnant Women ◽

Real Time ◽

Real Time Pcr ◽

Dna Analysis ◽

Methodological Approach ◽

Maternal Plasma ◽

Buccal Swab ◽

Non Invasive ◽

Pcr Method ◽

Parallel Reactions

Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.

Download Full-text

Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR

BMC Hematology ◽

10.1186/2052-1839-14-4 ◽

2014 ◽

Vol 14 (1) ◽

Cited By ~ 21

Author(s):

Runa M Grimholt ◽

Petter Urdal ◽

Olav Klingenberg ◽

Armin P Piehler

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Globin Gene ◽

Copy Number Variations ◽

Globin Genes ◽

Quantitative Real Time Pcr ◽

Human Genetic Disease ◽

Large Deletions ◽

Wide Range

Abstract Background Alpha-thalassemia is the most common human genetic disease worldwide. Copy number variations in the form of deletions of α-globin genes lead to α-thalassemia while duplications of α-globin genes can cause a severe phenotype in β-thalassemia carriers due to accentuation of globin chain imbalance. It is important to have simple and reliable methods to identify unknown or rare deletions and duplications in cases in which thalassemia is suspected but cannot be confirmed by multiplex gap-PCR. Here we describe a copy number variation assay to detect deletions and duplications in the α-globin gene cluster (HBA-CNV). Results Quantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the –α3.7 deletion and the HS-40 region. The copy number for each target was determined by the 2-ΔΔCq method. To validate our method, we compared the HBA-CNV method with traditional gap-PCR in 108 samples from patients referred to our laboratory for hemoglobinopathy evaluation. To determine the robustness of the four assays, we analyzed samples with and without deletions diluted to obtain different DNA concentrations. The HBA-CNV method identified the correct copy numbers in all 108 samples. All four assays showed the correct copy number within a wide range of DNA concentrations (3.2-100 ng/μL), showing that it is a robust and reliable method. By using the method in routine diagnostics of hemoglobinopathies we have also identified several deletions and duplications that are not detected with conventional gap-PCR. Conclusions HBA-CNV is able to detect all known large deletions and duplications affecting the α-globin genes, providing a flexible and simple workflow with rapid and reliable results.

Download Full-text