Risk exposures for human ornithosis in a poultry processing plant modified by use of personal protective equipment: an analytical outbreak study.

Ornithosis outbreaks in poultry processing plants are well-described, but evidence for preventive measures is currently lacking. This study describes a case-control study into an outbreak of ornithosis at a poultry processing plant in the East of England, identified following three employees being admitted to hospital. Workers at the affected plant were recruited via their employer, with exposures assessed using a self-completed questionnaire. Cases were ascertained using serological methods or direct antigen detection in sputum. 63/225 (28%) staff participated, with 10% of participants showing evidence of recent infection. Exposure to the killing/defeathering and automated evisceration areas, and contact with viscera or blood were the main risk factors for infection. Personal protective equipment (goggles and FFP3 masks) reduced the effect of exposure to risk areas and to self-contamination with potentially infectious material. Our study provides some evidence of effectiveness for respiratory protective equipment in poultry processing plants where there is a known and current risk of ornithosis. Further studies are required to confirm this tentative finding, but in the meantime respiratory protective equipment is recommended as a precautionary measure in plants where outbreaks of ornithosis occur.

Chlamydia trachomatis RNA in the environment: is there potential for false-positive nucleic acid amplification test results?

OBJECTIVES: The ability of molecular methods to detect low levels of nucleic acid has led to the widespread application of techniques based on nucleic acid amplification tests in microbiological diagnosis. This exquisite sensitivity is recognised in the laboratory to require stringent precautions to avoid contamination, but this is not widely appreciated in clinical settings where samples are initially collected, and may be a particular problem in the non-clinical settings used for sampling as part of the National Chlamydia Screening Programme. There is thus the need to characterise the risk of false-positive results caused by environmental contamination in these areas. METHODS: The extent of environmental contamination of Chlamydia trachomatis (CT) nucleic acid in clinical settings was investigated by swabbing surfaces within the vicinity of specimen collection. Laboratory experiments were designed to monitor the persistence of ribosomal RNA under simulated conditions and to investigate whether contamination of patients' specimens is a risk if environmental surfaces are contaminated. The Gen-Probe APTIMA Combo 2 system was used for CT rRNA detection. RESULTS: CT rRNA was detected in swabs taken from examination rooms and toilet areas. Tests showed that this could persist for at least 50 days. The potential for clinical samples to become contaminated as a result of the presence of CT rRNA in the immediate environment was demonstrated in this simulated test. CONCLUSION: This study demonstrated that there is a risk of false-positive nucleic acid amplification test results, when samples are taken in an area that is contaminated with target nucleic acid.

Microbiological profile of community-acquired pneumonia in adults over the last 20 years.

OBJECTIVES: To assess any change in the microbiological profile of community-acquired pneumonia (CAP) in our region over the last 20 years. METHODS: We compared hospital admissions aged between 15 and 74 (n = 61) in Norfolk (UK) for CAP over a 19-month period in 1982-3 (ST1) with all admissions aged over 16 (n = 99) over a 14-month period in 1999-2000 (ST2). Data were collected for ST1 as part of a prospective multicentred research study, in a period of high Mycoplasma pneumoniae activity. ST2 was a prospective study of clinical practice. Chlamydophila species were differentiated in ST2 using whole-cell immunofluorescence. RESULTS: A microbiological diagnosis was made in 38 (62%) in ST1 compared with 48 (48%) in ST2. Streptococcus pneumoniae remained the most common pathogen (26% in ST1, 25% in ST2). The incidence of M. pneumoniae was 18% in ST1 and 4% in ST2. The proportion of viral pathogens identified was similar: nine (15%) in ST1 and 14 (14%) in ST2. No cases of Chlamydophila pneumoniae were diagnosed in ST2. CONCLUSIONS: The microbiological profile of CAP in Norfolk (UK) has not changed over the last 20 years and C. pneumoniae is not a frequent pathogen.

Clinical evaluation of enzyme immunoassay in rapid diagnosis of herpes simplex infections.

AIMS: To evaluate the performance of antigen detection by IDEIA (NovoNordisk Ltd) in the rapid diagnosis of potentially serious herpes simplex (HSV) infections. METHODS: Nine hundred and twelve specimens from a variety of clinical sites, including ocular, mucocutaneous, respiratory and genital material, urines and necropsy tissue, were compared by enzyme immunoassay (EIA) and conventional culture for the presence of HSV. RESULTS: The EIA performed to a high level of sensitivity and specificity using a variety of specimen types. Some problems were encountered using cervical swabs from pregnant women and necropsy brain tissue. Analysis of clinical and contact history data of most patients giving discrepant results supported the evidence of recent HSV infection obtained by EIA. The mean culture time was 2.4 days (range one to eight days). CONCLUSIONS: The HSV EIA test performed to a high level of sensitivity (93.7%) and specificity (96.6%) when compared with culture using a variety of clinical material. These results assumed cell culture was 100% sensitive and specific. The actual performance of the EIA test is probably much higher. This approach to rapid HSV diagnosis should be used more widely, particularly in potentially serious cases.

Detection of Chlamydia pneumoniae and Chlamydia psittaci in sputum samples by PCR.

AIMS: To use the polymerase chain reaction (PCR) to detect Chlamydia pneumoniae and Chlamydia psittaci in sputum samples. METHODS: A nested PCR was developed, the first stage of which amplified DNA from both C pneumoniae and C psittaci while the second stage targeted specifically at C pneumoniae, allowing the two species to be differentiated. The primers were designed not to amplify sequences from C trachomatis. A panel of 26 sputum samples from patients with community acquired pneumonia evaluated previously by enzyme linked immunosorbent assay (ELISA), direct immunofluorescence (DIF), and culture was tested blind by PCR. Most of these specimens also had accompanying serial serum samples which were tested for species specific antibodies using microimmunofluorescence (micro-IF). RESULTS: PCR detected C pneumoniae DNA in 10 of the 26 samples and C psittaci DNA in four. There was good concordance between ELISA, DIF, micro-IF and PCR in the C pneumoniae group. Two of the C psittaci identified by PCR were labelled C pneumoniae by DIF but the PCR results were supported by serology or a history of bird contact. Of the PCR negative group: six were true negative results; two contained C trachomatis. There were four discrepant results. CONCLUSIONS: The data suggest that PCR is effective in the detection of C pneumoniae. The sensitivity for C psittaci is inevitably lower due to the strategy taken but specificity seemed to be good.

Evaluation of Serodia Myco II particle agglutination test for detecting Mycoplasma pneumoniae antibody: comparison with mu-capture ELISA and indirect immunofluorescence.

The Serodia Myco II particle agglutination test, which the manufacturers claim exclusively detects IgM antibody, was compared with two IgM-specific tests, a mu-capture ELISA, and indirect immunofluorescence for their ability to detect recent Mycoplasma pneumoniae infection. In general there was good agreement among the three tests, all three having similar sensitivity. One hundred and nine (78%) of serum samples gave concordant results in all three assays. Several sera gave positive particle agglutination titres, however, while being negative by the two other assays, and the Serodia Myco II test may not be as specific for detecting M pneumoniae IgM as the other two tests. While the Serodia Myco II test may be a good screening assay, it is unlikely to be a definitive test for M pneumoniae IgM, but may be better than the complement fixation test, particularly in younger patients in whom M pneumoniae IgM is found more frequently.

Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples.

AIMS: To develop a multiplex polymerase chain reaction (PCR) for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples. METHODS: Oligonucleotide primers for the amplification of the DNA of these three organisms were optimised for use in combination in the same reaction. PCR products were detected by hybridisation with pooled internal probes using an enzyme linked immunosorbent assay. Those with positive signals were further differentiated using species specific probes. Quality of DNA extraction and PCR inhibition were controlled by amplification of a human mitochondrial gene. A panel of 53 respiratory samples with known results was evaluated blindly. This was followed by a retrospective study on sputa collected from 244 patients with suspected community acquired pneumonia. RESULTS: The multiplex assay had a lower sensitivity than PCR with individual primers by about one log. The resultant sensitivity was considered acceptable for diagnostic use. Of the panel of 53 samples, nine of 11 M pneumoniae, 11 of 11 C pneumoniae, six of seven C psittaci, and 24 of 24 negative samples were correctly identified. Of the 244 patients with pneumonia, seven (2.9%) had detectable M pneumoniae, six (2.5%) had C pneumoniae, and one (0.4%) had C psittaci. The case notes from 11 patients were studied. The PCR finding was of possible significance in at least eight of these patients. CONCLUSIONS: This multiplex PCR assay has the potential to be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical value.

Anticomplementary activity in serum samples from patients with acute parvovirus B19 infection.

Of 65 serum samples submitted for diagnostic purposes which proved to be anti-complementary by complement fixation test, 49 were parvovirus B19 IgM positive. Forty four of the 49 serum samples were from patients with arthropathy. Acute parvovirus B19 infection should be suspected when a patient has symptoms of disease of the joints and the serum is anticomplementary.

The limitations of IgM assays in the serological diagnosis of Mycoplasma pneumoniae infections.

The most useful and reliable serological investigations for the diagnosis of current Mycoplasma pneumoniae infection, including reinfection, were investigated. Paired sera and respiratory specimens from 115 patients with lower respiratory tract symptoms were examined for evidence of current M. pneumoniae infection by serological response, as measured by complement-fixation and indirect immunofluorescence tests for specific IgM, IgA and IgG, and also by culture of M. pneumoniae from respiratory material. Specific IgM was not always detectable in cases where other criteria indicated current or recent infection. On the basis of the present results, it is postulated that primary infection and reinfection may be differentiated by the presence or absence of specific IgM in the presence of elevated specific IgA levels and, therefore, that estimation of both IgM and IgA is necessary for the maximal detection of current M. pneumoniae infection, including reinfections. Specific IgG levels remained elevated for many weeks and were not useful diagnostically.

The rapid serological diagnosis of infectious mononucleosis.

A total of 121 samples of serum collected from 101 patients was tested to determine the sensitivity and specificity of a commercial latex agglutination test for detecting infectious mononucleosis heterophile antibody, a commercial immunofluorescence test for detecting antibody to Epstein-Barr virus capsid antigen and a rapid enzyme immunoassay for detecting antibody to Epstein-Barr virus nuclear antigen. Although the Epstein-Barr virus capsid antigen IgM indirect immunofluorescence test proved to be the most sensitive, false-positive reactions were seen when samples collected from patients with cytomegalovirus, hepatitis A virus, parvovirus and leptospira infection were tested. False-positive reactions were also seen with samples containing rheumatoid factor.

The differentiation of Chlamydia species by antigen detection in sputum specimens from patients with community-acquired acute respiratory infections.

An amplified enzyme immunoassay (IDEIA III: Dako Diagnostics Ltd) for detecting genus-specific chlamydia antigen was evaluated prospectively on 286 respiratory specimens from 275 patients presenting with community-acquired pneumonia or persistent chest infection. Nineteen patients had evidence of recent chlamydial infection, having two or more positive sputum or serological markers. Sputa from two other patients were ELISA-positive in the absence of other positive criteria and were regarded as false-positive results. When compared with a direct immunofluorescence test for chlamydial elementary bodies (EBs) using a genus-specific monoclonal antibody, the ELISA gave a positive predictive value of 91% and a negative predictive value of 99%. Non-specific problems with a wide variety of other micro-organisms isolated from the sputa were not encountered. Attempts to differentiate between Chlamydia psittaci, Chlamydia pneumoniae and Chlamydia trachomatis using genus-specific lipopolysaccharide reactive--and species-specific major outer membrane protein--monoclonal antibodies were encouraging and results were substantiated, in most patients, by the species-specific serological assays of the whole-cell-inclusion immunofluorescence or micro-immunofluorescence assays. The study demonstrated that antigen detection techniques offer scope for routine laboratories to diagnose chlamydial respiratory infections rapidly and reliably and may enable differentiation to species level. Although immunofluorescence offers marginally greater sensitivity and specificity when compared with ELISA, the latter is less subjective and less demanding. Sixty-eight per cent of these infections would have remained undiagnosed despite the general availability of ELISA tests.

Genital mycoplasmas revisited--an evaluation of a new culture medium.

The role of genital mycoplasmas as pathogens causing severe neonatal respiratory and central nervous system disease has been highlighted recently following publication of new data. These organisms are generally neglected by diagnostic laboratories in the United Kingdom, possibly due to the lack of a suitable commercially-available culture medium. We have evaluated the bioMerieux Mycoplasma-Lyo system using qualitative and quantitative studies and have found it to have suitable qualities and practical advantages for detection of Ureaplasma urealyticum and Mycoplasma hominis.

Direct sample polymerase chain reaction for the detection of Mycoplasma pneumoniae: a simple system for clinical application.

A semi-nested polymerase chain reaction system with primers derived from the P1 adhesin gene of Mycoplasma pneumoniae was evaluated for sensitivity and specificity for detection of M. pneumoniae. The method used target DNA within samples of M. pneumoniae broth cultures and clinical material, without a formal extraction process. The sensitivity for detection of DNA was found to be to a level of one copy per sample and was specific to M. pneumoniae only, discriminating from the other human mycoplasma and ureaplasma species tested. The system offers the opportunity for a simple and highly specific laboratory method for diagnosis which may be compared to currently available serological methods, and may provide another method of studying mycoplasma-induced pathology.

Orf (contagious pustular dermatitis) in farmworkers: prevalence and risk factors in three areas of England.

Orf is a zoonotic skin disease which is commonly self-diagnosed by those who tend sheep and goats. This paper reports the prevalence, incidence and risk factors associated with the infection in a cohort of farmworkers from three areas of England, derived from the results of self-reporting and serology. Twenty-three per cent of those employed or living on a sheep farm reported ever having had orf, and the antibody serological profiles indicated a prevalence of 4 per cent and an annual incidence of 2.8 per cen. The main risk factors associated with the infection were contact with sheep, the size of the sheep flock, and contact with dogs.

Seroprevalence of hepatitis E virus in the UK farming population.

Hepatitis E is a zoonosis that can be acquired by the consumption of contaminated food or water, or via person-to-person spread. However, little is known about the transmission of hepatitis E virus (HEV) in the UK. We investigated the epidemiology of indigenous hepatitis E infection using the PHLS Farm Cohort, a sentinel group with a history of close contact with a range of domestic animals. Ten of the 413 subjects tested were positive for hepatitis E IgG antibodies (2.4%). Seroprevalence peaked in those aged 51 to 60 years (relative risk 3.3, 95% CI: 1.0-10.5). Male participants (relative risk 3.6, 95% CI: 0.6-21.2) and those from farms in the Hereford area of the United Kingdom (relative risk 2.7, 95% CI: 0.8-8.4), an area of mixed livestock farming, were more likely to have serological evidence of previous HEVs exposure, although these findings were not statistically significant. Exposure to pigs, or water from a private supply, was not identified as a significant risk factor. The results of this study suggest that UK farming populations are exposed to HEV, but the predominant route of transmission remains elusive.

No evidence of the Chlamydia trachomatis variant in the UK.

OBJECTIVES: The discovery of a variant strain of Chlamydia trachomatis (Ct) in Sweden has raised awareness of its possible undetected spread in the UK. The assays that fail to detect this variant are widely used in this country. This study aimed to determine if this variant is circulating in the UK. METHOD: 1,680 genital specimens tested negative by the Roche assays were retested by Aptima Combo2. Discordant results were sequenced to check for the deletion variant. RESULTS: Of 1,680 specimens tested, 29 were candidates for sequencing: 16 were negative for the variant, 11 failed to amplify, and 2 were lost. DISCUSSION: No Ct deletion variants were found in the UK. If it is circulating, then the prevalence is low (0-0.77%), but even a low level cannot be ignored. The system we describe is simple and suitable for rapid response and phasing of surveillance to match an unknown level of threat if other variants emerge.

A micro-capture ELISA for detecting Mycoplasma pneumoniae IgM: comparison with indirect immunofluorescence and indirect ELISA.

A mu-capture ELISA was developed for detecting Mycoplasma pneumoniae-specific IgM, and compared with an indirect immunofluorescent antibody (IFA) technique and an indirect ELISA. mu-capture ELISA and IFA compared well and were found to be the most sensitive assays. The IFA test can be completed in 2 h whilst the results of the mu-capture ELISA can be available in 24 h. Both tests are amenable to routine diagnostic use and have similar sensitivity. Indirect ELISA was found to be less sensitive and less specific, giving high assay values with several sera having undetectable M. pneumoniae CF antibody or CF antibody in low titre. Serum samples obtained from 11 patients at various times after M. pneumoniae infection showed maximum antibody levels within the first month by all assays, with a gradual fall in amount of IgM with time when assayed by mu-capture ELISA, a more gradual decline by IFA and hardly any decline with indirect ELISA. It was concluded that the indirect ELISA is unsuitable for the investigation of possible M. pneumoniae infection because the sustained high assay values with serum samples taken many months after infection, make interpretation of the test results very difficult.