A possible role for hepatitis C virus (HCV) infection in the pathogenesis of idiopathic pulmonary fibrosis (IPF) has recently been suggested on the basis of an unusually high seroprevalence rate of anti-HCV in such patients from Japan. In an attempt to confirm these findings, we tested sera from 62 patients with IPF by two second-generation anti-HCV ELISAs. Only one serum was reactive. Serum from this patient gave an indeterminate result when tested by four-antigen RIBA (c22 band only), and it was negative for the presence of HCV RNA when tested by the reverse transcriptase polymerase chain reaction assay. HCV infection is thus no more prevalent in patients with IPF from the UK than in the general population.
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.
Objective: The aim of this study was to investigate whether the two canine haemoplasma species, Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum,” are commonly associated with immune‐mediated haemolytic anaemia (IMHA) in UK dogs.
Methods: Three groups of dogs were recruited to the study: anaemic dogs with primary IMHA (n=37); anaemic dogs not meeting the inclusion criteria for primary IMHA (n=77) and non‐anaemic dogs (n=113). DNA was extracted from 100 μl of blood and subjected to real‐time quantitative polymerase chain reaction (qPCR) assays for both species of Mycoplasma. Each assay incorporated co‐amplification of canine glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as an endogenous internal control.
Results: Canine GAPDH was successfully amplified by qPCR from all 227 canine blood samples but none contained M. haemocanis or “Candidatus M. haematoparvum” DNA.
Clinical Significance: Haemoplasma infection is uncommon in dogs in the UK and no evidence was found that these organisms act as triggers for IMHA.
An immunogold labelling technique was used to label the pili of the bacterium Bacteroides nodosus. The labelling was distinct and highly specific, and individual pili could be recognised beneath the gold probe. The labelling of somatic antigens could be distinguished from that of pilus antigens. Furthermore, labelling of fragments of cytoplasm released by cell lysis and trapped in the pili could be distinguished from pilus labelling. An antiserum that had been raised against strain 80200 (serotype N) labelled pili of strain 215 (serotype B). Double labelling experiments with this antiserum and the antiserum against strain 215 (serotype B) showed that both antisera label the same pili bundles. The ease of detection of the immunocytochemical reaction should enable this technique to be used as a routine screen for pilus antigens. It also possesses the potential for much wider applications for immunolabelling other antigens, such as viruses, that can be obtained in suspension.
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