Publicação
Feline hemoplasmas : evaluation of specific antibodies and the molecular and cytological diagnostic
| Resumo: | Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis are three hemoplasmas responsible for feline hemolytic anemias. The diagnosis of the infection can be performed by observation of the agents on blood smears or by their identification and quantification using PCR techniques, presently considered the gold standard method. A recombinant antigen for Mhf (rDnaK) has been identified, characterized, produced and then successfully applied in Western blot and ELISA assays (rELISA) to detect antibodies in samples from experimentally induced infections. To evaluate this application under field conditions, this rELISA was used in samples collected from naturally infected cats, in parallel with quantitative PCR and cytologic examination. Within the scope of the TNR program implemented by the Lisbon City Council, blood samples were collected from 104 cats. After sample collection, blood was immediately used to prepare blood smears which were then stained with Giemsa (Parasitology Lab, FMV/Lisbon University). The remaining blood was split into two aliquots: 1 – Plasma separation, shipped to Vetsuisse Faculty, Zurich University, for antibody testing with rELISA; 2 – Total DNA extraction for identification and quantification of the three species of hemoplasma using qPCR (Virology Lab, FMV/Lisbon University). Out of 22.1% (N=23) samples where the microorganism was identified by qPCR, mycoplasmas were also identified on the blood smears of 15.4% (N=16). 4.8% (N=5) of samples tested as seropositive and 2.9% (N=3) revealed bordeline results. From 77.9% (N=81) of samples that were negative by qPCR, only 44.2% (N=46) were also negative on cytologic examination. 60.6% (N=63) of samples were considered seronegative and 6.7% (N=7) were borderline. Sensitivity for cytology was 69.6% and specificity was 56.8%. Sensibility of rELISA was 25% and its specificity was 85.1%. Cohen’s Kappa (k) was calculated to assess agreement between PCR-Cytology (k=0.1836) and PCR-rELISA (k=0.1093). Given the low agreement, PCR was found to be the most appropriate diagnostic method. Further studies are necessary to characterize the response of the immune system and the role of different antigens in these infections in order to improve the suitability of rELISA in a clinical setting |
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| Autores principais: | Catarino, Patrícia Pacheco Nobre Pina |
| Assunto: | Mycoplasma haemofelis Candidatus Mycoplasma haemominutum Candidatus Mycoplasma turicensis Hemoplasmas quantitative PCR rELISA PCR quantitativo |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis are three hemoplasmas responsible for feline hemolytic anemias. The diagnosis of the infection can be performed by observation of the agents on blood smears or by their identification and quantification using PCR techniques, presently considered the gold standard method. A recombinant antigen for Mhf (rDnaK) has been identified, characterized, produced and then successfully applied in Western blot and ELISA assays (rELISA) to detect antibodies in samples from experimentally induced infections. To evaluate this application under field conditions, this rELISA was used in samples collected from naturally infected cats, in parallel with quantitative PCR and cytologic examination. Within the scope of the TNR program implemented by the Lisbon City Council, blood samples were collected from 104 cats. After sample collection, blood was immediately used to prepare blood smears which were then stained with Giemsa (Parasitology Lab, FMV/Lisbon University). The remaining blood was split into two aliquots: 1 – Plasma separation, shipped to Vetsuisse Faculty, Zurich University, for antibody testing with rELISA; 2 – Total DNA extraction for identification and quantification of the three species of hemoplasma using qPCR (Virology Lab, FMV/Lisbon University). Out of 22.1% (N=23) samples where the microorganism was identified by qPCR, mycoplasmas were also identified on the blood smears of 15.4% (N=16). 4.8% (N=5) of samples tested as seropositive and 2.9% (N=3) revealed bordeline results. From 77.9% (N=81) of samples that were negative by qPCR, only 44.2% (N=46) were also negative on cytologic examination. 60.6% (N=63) of samples were considered seronegative and 6.7% (N=7) were borderline. Sensitivity for cytology was 69.6% and specificity was 56.8%. Sensibility of rELISA was 25% and its specificity was 85.1%. Cohen’s Kappa (k) was calculated to assess agreement between PCR-Cytology (k=0.1836) and PCR-rELISA (k=0.1093). Given the low agreement, PCR was found to be the most appropriate diagnostic method. Further studies are necessary to characterize the response of the immune system and the role of different antigens in these infections in order to improve the suitability of rELISA in a clinical setting |
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