Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Isoanticuerpos/sangre , Trasplante de Riñón/efectos adversos , Linfocitos/metabolismo , Síndrome , Creatinina/sangre , Nefropatías Diabéticas/inmunología , Nefropatías Diabéticas/terapia , Femenino , Hemoglobinas/química , Humanos , Terapia de Inmunosupresión/métodos , Isoanticuerpos/inmunología , Masculino , Persona de Mediana Edad , Globulina Inmune rho(D) , Riesgo , Donantes de TejidosRESUMEN
The aim of this study was to develop quantitative real-time (q)PCR assays to detect all known haemoplasma species, and a human housekeeping gene in order to demonstrate both successful DNA extraction from clinical samples and to test for sample inhibition, and to apply these qPCRs to human blood samples and blood smears. Sensitive and specific generic haemoplasma qPCR assays were developed to amplify haemoplasma species, as well as human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal amplification control. An optimized technique for extracting DNA from stained blood smears was also developed. These methods were applied to anonymized blood samples obtained from 100 human immunodeficiency virus (HIV)-infected South Africans and 920 UK patients undergoing haematological examination, and to 15 blood smears recruited from previous studies describing human haemoplasmosis. Human GAPDH levels were acceptable in all but three of the blood samples and all but two of the blood smears. The latter could have arisen due to DNA degradation due to the old age (over 35 years) of these smears. Haemoplasma infection was found in one HIV-infected South African, but the species could not be characterized due to the very low levels of DNA present. This report describes novel extraction and qPCR methodologies for haemoplasma screening. Previously reported human haemoplasmosis based on cytological diagnosis alone should be viewed with caution.
Asunto(s)
Técnicas Bacteriológicas/métodos , Sangre/microbiología , Infecciones por Mycoplasma/diagnóstico , Mycoplasma/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Animales , Gatos , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Infecciones por VIH/complicaciones , Humanos , Tamizaje Masivo/métodos , Mycoplasma/genética , Reacción en Cadena de la Polimerasa/normas , Estándares de Referencia , Sensibilidad y Especificidad , Sudáfrica , Reino UnidoRESUMEN
The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.