Preparedness and response to bioterrorism.

As we enter the 21st century the threats of biological warfare and bioterrorism (so called asymmetric threats) appear to be more real than ever before. Historical evidence suggests that biological weapons have been used, with varying degrees of success, for many centuries. Despite the international agreements to ban such weapons, namely the 1925 Geneva Protocol and the 1975 Biological and Toxin Weapons Convention, there is no effective international mechanism for challenging either the development of biological weapons or their use. Advances in technology and the rise of fundamentalist terror groups combine to present a significant threat to western democracies. A timely and definitive response to this threat will require co-operation between governments on a scale never seen before. There is a need for proper planning, good communication between various health, home office, defence and intelligence agencies and sufficient financial support for a realistic state of preparedness. The Department of Health has produced guidelines for responding to real or suspected incidents and the Public Health Laboratory Service (PHLS) has produced detailed protocols to inform the actions required by microbiologists and consultants in communicable disease control. These protocols will be published on the Department of Health and PHLS web sites.

The use of Lenticules for the process control of enumeration techniques in food and environmental microbiology.

AIMS: Lenticules consist of control-dried plano convex discs in which biologically-active materials are contained within a water-soluble matrix. They can be produced to contain stable numbers of bacteria from 10 cfu lenticule-1 to 108 cfu lenticule-1 with a wide variety of organisms. These experiments were carried out to validate their use as a tool for internal quality control in quantitative microbiology. METHODS AND RESULTS: The Lenticules were used routinely in standard quantitative microbiological procedures across five laboratories. Results showed the materials to be stable, homogeneous and capable of identifying systematic errors. CONCLUSIONS: The Lenticules provide suitable, stable control materials. SIGNIFICANCE AND IMPACT OF THE STUDY: Routine internal quality control of quantitative measurements is greatly improved; the materials are easy to use and enable comparisons between laboratories to be made.

Correlations between microbial parameters from water samples: expectations and reality.

Data which are collected in order to estimate the correlation between parameters must be analysed with caution. Classical statistics of correlation are often inappropriate. The "r" statistic is very easily distorted by non-Normal data. Non-parametric statistics can be helpful. The interpretation and usefulness of the estimates of correlation will depend on the study plan. If water samples come from disparate sources (e.g. upstream or downstream from sewage outlets) then parameters A and B may occur in their highest and lowest numbers according to how close the samples were to contamination sources thus correlating closely. However, if all samples come from sources with similar pollution levels then plots of A and B will show considerable scatter and apparently little correlation. So what is the relationship between A and B? An example of "perfect" correlation, as demonstrated by replicate counts of a single parameter from split samples, gave an r value of only 0.63 (p = 0.62) due to random variation in numbers of organisms between the two halves of the sample. Thus large amounts of data are needed for studying true correlation because relationships between parameters are embedded in the natural variation. This also illustrated that Standards for a single parameter can be "passed" or "failed" by two halves of the same sample. Study design is clearly of fundamental importance. Consideration must be given to the appropriate way of asking questions about correlation between different parameters.

An evaluation of a laser scanning device for the detection of Cryptosporidium parvum in treated water samples.

A laser scanning device, the ChemScan RDI (Chemunex, Paris, France), was compared with manual fluorescence microscopy for the detection of oocysts of Cryptosporidium. Pairs of filters were spiked with approximately 100 oocysts. Over 24 h at least 1000 l of treated water was passed through the filters, then concentrated deposits were subjected to an immunomagnetic separation (IMS) protocol described by the manufacturer (Dynal, Oslo, Norway) and examination by fluorescence microscopy, or an IMS protocol (Chemunex) and detection by ChemScan laser scanning. Subsequently a set of five 1-ml samples containing oocysts over a range of concentrations, including a negative control, were examined blind by the two methods (stage two). In stage 1 the average recovery rates were estimated to be 49% (manual fluorescence microscopy) and 73% (ChemScan). The average ratio of ChemScan to manual fluorescence microscopy counts was 1.54 (range 1.08-2.36). In stage 2, statistical comparison of all but one set of results showed there was no significant difference between methods. Differences for the high count sample may possibly have been caused by duplicate counting of oocysts by manual fluorescence microscopy.

Reference laboratory surveillance of syphilis in England and Wales, 1994 to 1996.

Routine surveillance of syphilis of public health importance (infectious, congenital, and neurosyphilis) began in England and Wales in 1994, using reports from six PHLS laboratories that undertook serological and other reference work. One hundred and thirty-one cases were reported in the first two years, including 100 cases of infectious syphilis, with all regions reporting some infectious syphilis. Reports from PHLS laboratories represented one sixth of the number of cases seen in genitourinary clinics (KC60 data), but both systems produced comparable results. Laboratory reports provided more data on risk factors, which were not available elsewhere. This study documents the risk in England and Wales from infections originating in eastern Europe, where sexually transmitted infections including syphilis have reached epidemic proportions. Forty-five per cent of cases of infectious syphilis were reported to have been acquired in the United Kingdom (UK) and 59% of people with infectious syphilis were reported to have been born in the UK. Twenty per cent of the infectious cases were associated directly or indirectly with transmission in Russia or elsewhere in eastern Europe. The majority of infectious cases were from the white ethnic group. Eighty-five per cent of cases of infectious syphilis were reported to have been acquired heterosexually; 26% of male cases were reported to have been acquired homosexually. The PHLS laboratory reporting system is now well established, and could usefully be expanded to include other, non PHLS, laboratories that undertake reference work. It has the capacity to detect changes in the national epidemiology of syphilis, including imported infections.

The detection of enteroviruses in large volume concentrates of recreational waters by the polymerase chain reaction.

A rapid and simple method was developed to detect enteroviruses in large-volume water samples. It relies on the adsorption of the virus capsids to silica particles under acidic conditions, allowing their recovery by relatively gentle centrifugation. Different reagents used in enterovirus concentration and detection were seeded with Coxsackievirus B5 and used to optimise the recovery method, which was then used to detect the enteroviruses from seeded and unseeded 101 seawater samples in one PCR tube rather than in up to 50 sub-sample volumes, demonstrating its use for routine environmental monitoring. Concentrates from 36 recreational water samples from three sites in N.E. England were analysed for enteroviruses by regular and the new method semi-nested PCR, and infectivity in cell culture. Some of the samples were also analysed for faecal indicator bacteria and F-specific bacteriophage. The results showed a marked increase in detection sensitivity when the whole sample concentrate was assayed as compared with a small volume aliquot.

An apparent outbreak of infection with Acinetobacter calcoaceticus reconsidered after investigation by pyrolysis mass spectrometry.

In a previously reported apparent outbreak of infection due to Acinetobacter calcoaceticus on a Regional Burns Unit in which both clinical and environmental isolates were available for study, investigations of the organisms by electrophoretic typing of surface proteins, plasmid profiling and antibiograms were inconclusive and unable to identify a single epidemic strain of the organism. The same collection of isolates has been analysed for inter-strain comparison by pyrolysis mass spectrometry (PyMS). PyMS analysis suggested that a few isolates were very different from the majority but that most of the collection comprised a group of closely similar but nonetheless distinct isolates. These isolates may well be representatives of a limited variety of strains occupying an ecological niche but not yet a single emergent strain. The ability of PyMS to detect a single epidemic strain of A. calcoaceticus when present in such a collection of isolates was demonstrated by analysis of a "constructed outbreak collection", using some of the original isolates. This study illustrates the versatility of PyMS for inter-strain comparison studies of species not yet amenable to conventional typing methods. The application of rapid, highly discriminatory, high-volume inter-strain comparison methods such as PyMS to apparent outbreaks of nosocomial infection is likely to reveal patterns of organism acquisition other than point-source transmission.

Differentiation of Mycobacterium senegalense from related non-chromogenic mycobacteria using pyrolysis mass spectrometry.

Twenty-six representative strains of Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum and Mycobacterium senegalense were compared by Curie point pyrolysis mass spectrometry. The M. chelonae and M. senegalense strains formed distinct groups. A third, relatively diffuse group, contained the M. fortuitum and M. peregrinum strains. These results, together with those from corresponding analyses, suggest that pyrolysis mass spectrometry provides a rapid and reliable way of distinguishing between members of closely related mycobacterial species which are difficult to differentiate using conventional taxonomic procedures.

A highly discriminatory method for the direct comparison of two closely related bacterial populations by pyrolysis mass spectrometry.

The ability of pyrolysis mass spectrometry (Py-MS) to discriminate between paired groups of closely related bacteria has been examined. The technique was challenged with increasing levels of biological difference; from that between different subcultures of the same isolate of Staphylococcus aureus, through that between separate isolates of the same strain and eventually to that between different staphylococcal species and Streptococcus pyogenes. By using an analytical method in which the spectral data from any two groups can be directly compared at a time, it was shown that Py-MS was capable of measuring statistically significant differences between staphylococci and Streptococcus pyogenes, between different Staphylococcus species and between two isolates of the same strain of S. aureus from different sources. Two subcultures of the same isolate were found to be indistinguishable. The differing magnitude of the Py-MS-derived differences corresponded to the "biological differences", being much larger between staphylococci and streptococci than between staphylococcal species and only just statistically significant between isolates of the same strain from different sources. The technique described allows the assessment of the magnitude and significance of Py-MS-derived differences between any two bacterial populations.

Differentiation between bacterial species and sub-species by pyrolysis mass spectrometry of extracted DNA.

Whole cell samples from cultures of Staphylococcus aureus, S. hominis, S. epidermidis and Streptococcus pyogenes were analysed by pyrolysis mass spectrometry and the results were compared with Py-MS-derived analyses of samples of DNA extracted from the same organisms. Py-MS analysis differentiated the four organisms in both circumstances. These results challenge previous assumptions that Py-MS is restricted to detecting phenotypic differences, although the basis for the differentiation of the DNA extracts has yet to be determined.

Note: development of an external quality assurance scheme for the detection and enumeration of Pseudomonas aeruginosa from water and comparison of results using modified King's A broth and a commercial agar.

Two trials of the isolation and enumeration of a given strain of Pseudomonas aeruginosa from water are reported. In each trial participants received concentrated samples from two batches, one with low and one with high counts, to be diluted to 500 ml in sterile distilled or deionized water and examined for Ps. aeruginosa by membrane filtration. Membranes were incubated at 37 degrees C for 48 h on pads soaked in modified King's A broth (MKAB) and Unipath Pseudomonas Agar plus CFC supplement (PCFC). The first trial involved eight Public Health Laboratories (PHL) and the organizers provided media from single batches. The second trial, involving 50 PHL, examined the feasibility of a large scale external quality assessment (EQA) distribution. Participants were invited to use the same two media and their usual medium if different. Average counts were close to expected and the spread of results was comparable to that observed from the EQA scheme for indicator organisms. From the results of the two trials a better isolation of the strain of Ps. aeruginosa under consideration was noted with PCFC compared with MKAB.

Nosocomial enterococci: resistance to heat and sodium hypochlorite.

Six strains each of Enterococcus faecium and E. faecalis were investigated with respect to their resistance to heat and sodium hypochlorite. All enterococci survived the temperatures and holding times specified by the Department of Health (DoH) for the disinfection of 'foul and used' or 'infected' linen (65 degrees C for 10 min or 71 degrees C for 3 min). In addition, three strains (one E. faecium and two E. faecalis) could withstand 150 ppm available chlorine for 5 min, the treatment suggested by the DoH for the disinfection of heat labile materials. Further, our results showed that four strains of E. faecium were able to survive the British Standard for heat disinfection of bedpans (80 degrees C for 1 min). The significance of these findings with particular reference to the potential for enterococci to survive and disseminate in the hospital environment is discussed.

Sporadic isolates of Escherichia coli O157.H7 investigated by pyrolysis mass spectrometry.

Thirty-six encoded isolates of Escherichia coli. 32 of which were of serotype O157, were examined by pyrolysis mass spectrometry (PyMS). Thirty-one of the serotype O157 isolates possessed the flagellar antigen H7 and produced Verocytotoxin (VT), the other isolate serotyped as H45 and was non-toxigenic. Eighteen of the VT-producing E. coli (VTEC) isolates were from sporadic disease in residents of the Northern Region. Standard principal component (PC) and canonical variate (CV) analysis of the data distinguished only the four non-O157 isolates from the remainder which were indistinguishable by this approach. A similarity matrix based on differences between individual CV means distinguished a further ten isolates. The matrix correctly clustered 2 pairs of isolates from siblings and 4 isolates from an affected family. A further 5 clusters of 3 or more isolates and 6 pairs of isolates were defined. These groupings proved to be homogenous for toxin phenotype but occasionally entrained isolates of dissimilar phage type. However, in general, PyMS-derived clustering of apparently sporadic isolates accorded with geographical locations as determined by postcode. PyMS, which is a quick and high volume capacity phenotypic technique, may be a useful addition to existing methods in the investigation of the epidemiology of sporadic VTEC disease.

Direct milk excretion of Campylobacter jejuni in a dairy cow causing cases of human enteritis.

Consumption of milk contaminated with Campylobacter jejuni has been described as a cause of human enteritis. Although faecal contamination of milk with the organism has frequently been described, direct milk excretion of Campylobacter jejuni into milk has rarely been linked with cases of human infection. We describe the investigations undertaken following the isolation of Campylobacter jejuni from samples of unpasteurized milk prior to retail. Results of epidemiological investigations including typing of Campylobacter jejuni isolates using pyrolysis mass spectrometry, Penner and Lior serotyping, biotyping, phage typing and restriction fragment length polymorphism analysis provided convincing evidence implicating direct milk excretion of Campylobacter jejuni by one asymptomatic dairy cow as the source of the milk contamination and the cause of local cases of human enteritis.

Rapid identification of species within the Mycobacterium tuberculosis complex by artificial neural network analysis of pyrolysis mass spectra.

An artificial neural network (ANN) was trained to distinguish between Mycobacterium tuberculosis and M. bovis with averaged pyrolysis mass spectra from duplicate subcultures of four strains of each of these species, each pyrolysed in triplicate. Once trained, the ANN was interrogated with spectrum data from the original organisms (the "training set") and from 26 other mycobacterial isolates (the "challenge set") of the M. tuberculosis complex (MTBC). Eight strains of M. bovis and 13 of M. tuberculosis, whether sensitive or variously resistant to antituberculosis drugs, were identified in agreement with conventional identification. Four strains of "M. africanum" were identified as M. bovis. Of two atypical M. tuberculosis strains from South India, one was identified as M. tuberculosis and the other as M. bovis. Six strains of BCG proved heterogeneous; two gave equivocal identifications, three were identified as M. bovis and one was identified as M. tuberculosis.

External quality assessment in water microbiology: statistical analysis of performance.

A UK-based scheme of water microbiology assessment requires participants to record counts of relevant organisms. Not every sample will contain the target number of organisms because of natural variation and therefore a range of results is acceptable. Results which are tail-end (i.e. at the extreme low or high end of this range) could occasionally be reported by any individual laboratory by chance. Several tail-end results might imply a laboratory problem. Statistical assessment is done in two stages. A non-parametric test of the distribution of tail-end counts amongst laboratories is performed (Cochran's Q) and, if they are not random, then observed and expected frequencies of tail-end counts are compared to identify participants who may have reported excessive numbers of low or high results. Analyses so far have shown that laboratories find high counts no more frequently than would be expected by chance, but that significant clusters of low counts can be detected among participants. These findings have been observed both in short-term and in long-term assessments, thus allowing detection of new episodes of poor performance and intermittent problems. The analysis relies on an objective definition of tail-end results. Working definitions are presented which should identify poor performance in terms of microbiological significance, and which allow fair comparison between membrane-filtration and multiple-tube techniques. Smaller differences between laboratories, which may be statistically significant, will not be detected. Different definitions of poor performance could be incorporated into future assessments.