Changing epidemiology of Clostridium difficile infection following the introduction of a national ribotyping-based surveillance scheme in England.

BACKGROUND: Marked increases in Clostridium difficile infection (CDI) incidence, driven by epidemic strain spread, is a global phenomenon. METHODS: The Clostridium difficile Ribotyping Network (CDRN) was established in 2007 as part of enhanced CDI surveillance in England, to facilitate the recognition and control of epidemic strains. We report on changes in CDI epidemiology in England in the first 3 years of CDRN. RESULTS: CDRN received 12,603 fecal specimens, comprising significantly (P < .05) increasing numbers and proportions of national CDI cases in 2007-2008 (n = 2109, 3.8%), 2008-2009 (n = 4774, 13.2%), and 2009-2010 (n = 5720, 22.3%). The C. difficile recovery rate was 90%, yielding 11,294 isolates for ribotyping. Rates of 9 of the 10 most common ribotypes changed significantly (P < .05) during 2007-2010. Clostridium difficile ribotype 027 predominated, but decreased markedly from 55% to 36% and 21% in 2007-2008, 2008-2009, and 2009-2010, respectively. The largest regional variations in prevalence occurred for ribotypes 027, 002, 015, and 078. Cephalosporin and fluoroquinolone use in CDI cases was reported significantly (P < .05) less frequently during 2007-2010. Mortality data were subject to potential reporting bias, but there was a significant decrease in CDI-associated deaths during 2007-2010, which may have been due to multiple factors, including reduced prevalence of ribotype 027. CONCLUSIONS: Access to C. difficile ribotyping was associated with significant changes in the prevalence of epidemic strains, especially ribotype 027. These changes coincided with markedly reduced CDI incidence and related mortality in England. CDI control programs should include prospective access to C. difficile typing and analysis of risk factors for CDI and outcomes.

Molecular epidemiology of Mycobacterium tuberculosis in East Lancashire 2001-2009.

BACKGROUND: East Lancashire has had high rates of tuberculosis for 40 years. The ethnically diverse population is predominantly of South Asian and white origin. Drug resistance data from 1960 to 1999 indirectly suggest that no significant inter-ethnic transmission has occurred. This study used mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) fingerprinting to assess clustering within and between ethnic groups. METHODS: All isolates of Mycobacterium tuberculosis from January 2001 to July 2009 from East Lancashire postcode areas were MIRU-VNTR fingerprinted. Clusters of strains with indistinguishable profiles were also assessed epidemiologically, and their MIRU-VNTR profiles compared with the UK M tuberculosis Strain Typing Database. RESULTS: 332 strains were typed (63 white patients, and 269 non-white patients). 198 MIRU-VNTR profiles were identified, with 144 profiles occurring only once. The typing clustered 187 strains into 53 clusters indistinguishable at all 12 loci and these were further characterised using the exact tandem repeat loci A, B, and C. The 15 loci clustered 32/63 (50.8%) of white and 110/269 (40.9%) of non-white cases and all but nine clusters were of the same ethnicity. The nine inter-racial clusters were further assessed from an epidemiological and clinical perspective and fingerprinting using nine additional loci. Isolates within two of the clusters were further discriminated using the additional nine loci. However, the additional loci did not further discriminate the isolates in the other seven inter-racial clusters. CONCLUSIONS: MIRU-VNTR fingerprinting indicates that although there is evidence of a high rate of transmission within the South Asian sub-population, the data suggest that there is little inter-ethnic transmission.

Improved differentiation of Mycobacterium tuberculosis isolates in the north of England using additional variable number tandem repeat loci.

Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) genotyping of over 3300 Mycobacterium tuberculosis isolates from the north of England has identified large clusters of strains which share common profiles. However, many apparent clusters identified when typed using the existing 15 loci lack clear epidemiological links. This study seeks to discover whether or not six additional VNTR loci can increasethe discriminatory power of the existing MIRU-VNTR 15-loci technique. Two hundred and six M. tuberculosis isolates were genotyped, including 57 isolates from 20 epidemiologically linked clusters and 149 from unlinked patients belonging to six large MIRU-VNTR-defined clusters. The discriminatory power of the six additional loci was high (Hunter Gaston Discriminatory Index [HGDI]: 0.952). Five of the six loci were highly discriminative (h > 0.6); however, locus 2401 was less discriminative (h = 0.5). The additional VNTR loci were able to subtype all six unlinked common MIRU-VNTR clusters into 56 subclusters, significantly differentiating unrelated strains in a set previously incorrectly clustered using 15 MIRU-VNTR loci. The largest cluster size was 14 (9.3%) when typed using the six additional VNTR loci, compared to 30 (20%) when typed using the original 15 MIRU-VNTR loci. The same loci were also found to be stable as a result of their inability to subdivide any of the epidemiologically linked clusters. This study has demonstrated that expanding the MIRU-VNTR panel beyond the 15 previously used loci significantly increases the discriminatory power of the technique and thus provides a valuable tool in the epidemiological monitoring of this disease.

Mycobacteria and allograft heart valve banking: an international survey.

Since the 1970s many tissue banks have been testing allograft heart valves (HVs) for Mycobacterium tuberculosis (MTB). Donor selection for low risk of tuberculosis (TB) was introduced in the 1980s and appears to have reduced the risk of TB transmission. Regulatory guidance does not specify testing for TB, but does exclude donors with a recent history of TB. This survey of HV international bank practices revealed variations in donor selection, testing and processing of valves. Participant banks (from Europe and the USA) reported that over a period of 15 years, HV tissues from 38,413 donors were banked and 32,289 donors were tested for TB, none being positive. HV-associated tissue from 27,840 donors was stained and underwent microscopy; none of these were positive for acid-fast bacilli (AFB). Non-tuberculosis mycobacteria (NTBM) were detected by culture on 24 HVs. It is recommended that HV banks employ donor selection to exclude donors at risk of TB, to culture material for mycobacteria, and to investigate potential sources when clusters of NTBM are found to facilitate corrective and preventative actions.

Disseminated BCG infection in severe combined immunodeficiency presenting with severe anaemia and associated with gross hypersplenism after bone marrow transplantation.

An infant with severe combined immunodeficiency (SCID) is described, who presented with severe anaemia and hepatosplenomegaly due to disseminated Bacillus Calmette-Guérin (BCG) infection involving the bone marrow, liver and spleen. After BMT, huge splenic enlargement occurred, presumably due to proliferation of engrafted donor lymphocytes, leading to severe hypersplenism. Peripheral blood cell consumption was resolved by splenectomy, but gradual loss of the marrow graft followed.

Rapid differentiation of Mycobacterium xenopi from mycobacteria of the Mycobacterium avium-intracellulare complex by pyrolysis mass spectrometry.

Thirty four cultures of slow growing, Tween-80 negative mycobacteria were analysed by pyrolysis mass spectrometry. The results showed that pyrolysis mass spectrometry could positively distinguish strains of Mycobacterium xenopi from those of the Mycobacterium avium-intracellulare (MAI) complex. Pyrolysis mass spectrometry may be a useful technique for the rapid characterisation of non-tuberculous mycobacteria in such clinical settings as their isolation from immunocompromised patients-for example, those with AIDS.

A rapid polymerase chain reaction technique for detecting M tuberculosis in a variety of clinical specimens.

A rapid in-house polymerase chain reaction (PCR) assay is described for the direct detection of Mycobacterium tuberculosis complex in clinical material. Its performance is compared with two kit based systems. The results of the in-house assay were comparable with the commercial assays, detecting M tuberculosis in 100% of smear positive, culture positive samples. The in-house assay proved to be rapid, easy, and inexpensive to perform, and the inclusion of an internal inhibitor control permitted validation of the PCR results.

Optimisation of acid fast smears for the direct detection of mycobacteria in clinical samples.

AIMS: Despite its long history, the acid fast smear remains unstandardised. Technical variations in both the preparation of clinical material and subsequent staining mean that smear sensitivity relative to culture may vary from 50% to over 80%. This study assessed the sensitivity of acid fast microscopy at each of five stages of sample preparation and by both commonly used staining methods. METHODS: Sputum samples thought for varying reasons to be highly likely to be culture positive were used to prepare a series of smears in which the effects of digestion (liquefaction), concentration (centrifugation), and decontamination (sodium hydroxide) could be assessed, together with a comparison of staining by the auramine/phenol and Ziehl-Neelsen techniques. RESULTS: The most effective method for the demonstration of acid fast organisms in sputum was found to be an auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process. CONCLUSIONS: The auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process is the most effective method for the demonstration of acid fast organisms in sputum.

Epidemiology and molecular typing of an outbreak of tuberculosis in a hostel for homeless men.

AIM: To investigate a possible outbreak of tuberculosis in a hostel for homeless men using IS6110 profiling, a polymerase chain reaction (PCR) based fingerprinting technique. METHODS: Eight cases of tuberculosis were diagnosed in residents of the hostel over a period of 28 months. To provide epidemiological data, a heminested inverse PCR (HIP) assay targeting the insertion sequence IS6110 together with its upstream flanking region was used to fingerprint the eight isolates of M tuberculosis under investigation. RESULTS: The HIP technique gave IS6110 profiles which showed that while three isolates were clearly distinct, the remaining five strains were indistinguishable, suggesting the latter were representatives of a single outbreak strain. CONCLUSIONS: The HIP assay proved discriminatory and facilitated repeated testing for the direct comparison of strains as more patients presented over the protracted course of this outbreak.

A national audit of the laboratory diagnosis of tuberculosis and other mycobacterial diseases within the United Kingdom.

In order to audit United Kingdom laboratory diagnostic and reference services including novel molecular methods for tuberculosis, a questionnaire was sent to laboratories submitting specimens to the PHLS Mycobacterium Reference Unit (MRU) and regional centres and to the Scottish Mycobacteria Reference Laboratory (SMRL) in 1996-7. Nationally, 67.2% of laboratories responded. Most UK laboratories were fully or conditionally CPA accredited and take part in the NEQAS proficiency scheme. On average only 3.3% of primary samples submitted for mycobacterial diagnosis in 1995 produced a mycobacterial culture from approximately half as many patients (that is, a mean of 1488 specimens producing 49 isolates from 23 patients). Potentially over 380,000 specimens are processed for mycobacteria in the UK each year. The majority of laboratories use 4% NaOH +/- NALC for specimen decontamination. Culture on solid media was used by most laboratories and 62.9% also use liquid media. Most laboratories incubated cultures for eight weeks. Few laboratories use molecular diagnostic methods. Laboratories were most likely to use molecular methods for diagnosing tuberculous meningitis and for specimens from immunocompromised patients, although usage was strongly influenced by cost. Within England and Wales 43.9% (47/107) and 56% (61/109) of laboratories wanted a rapid service for rifampicin resistance detection in M tuberculosis from immunocompetent and immunocompromised patients, respectively. In regard to a tuberculous meningitis service, 80.5% (43/112) and 84.3% (102/121) of laboratories wanted this service for immunocompetent and immunocompromised patients, respectively. The quality of reference services was rated as "very good"/"good" by 85.6% of respondents nationally. Rapid molecular amplification diagnostic services were established at the PHLS MRU for rifampicin drug resistance detection nationally and for tuberculous meningitis at the MRU.

Rapid identification of streptomycetes by artificial neural network analysis of pyrolysis mass spectra.

An artificial neural network was trained to distinguish between three putatively novel species of Streptomyces using normalised, scaled prolysis mass spectra from three representative strains of each of the taxa, each sampled in triplicate. Once trained, the artificial neural network was challenged with spectral data from the original organisms, the 'training set', from additional members of the putative novel taxa and from over a hundred strains representing six other actinomycete genera. All of the streptomycetes were correctly identified but many of the other actinomycetes were mis-identified. A modified network topology was developed to recognise the mass spectral patterns of the non-streptomycete strains. The resultant neural network correctly identified the streptomycetes, whereas all of the remaining actinomycetes were recognised as unknown organisms. The improved artificial neural network provides a rapid, reliable and cost-effective method of identifying members of the three target streptomycete taxa.

Enhanced speed and sensitivity in the cultural diagnosis of pulmonary tuberculosis with a continuous automated mycobacterial liquid culture (CAMLiC) system.

A total of 2800 sputum samples referred for mycobacterial investigation was examined by both continuous automated mycobacterial liquid culture (CAMLiC) and conventional Loewenstein-Jensen culture (LJC). The CAMLiC system was more sensitive than LJC, detecting 188 (98.4%) of 191 of all mycobacteria found by one or both methods compared to 150-162 (78.5-84.8%) found by LJC (the range for LJC takes into account all potential 'missed positives' due to contamination). Figures for Mycobacterium tuberculosis complex (MTBC) organisms specifically were 133 (98.4%) of 135 for CAMLiC and 115-122 (85.2-90.4%) for LJC. Detectable growth of MTBC organisms in CAMLiC occurred at a mean of 13.4 days after inoculation (range 3-32; SD 6.49; mode 8 days); 65.4% of such isolates were detected within 14 days and 87.2% within 21 days. In 73 instances the MTBC status of the isolate was defined by gene probe on the day of growth detection. Sufficient biomass for valid gene probe assay was always present. The speed, sensitivity and labour-sparing technology (no manual intervention is necessary before identification or discard) of CAMLiC make it possible for many laboratories to approach the Centers for Disease and Prevention (CDC) standard for culture and identification in mycobacteriology without resort to direct DNA detection techniques and at a much lower cost.

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.

Rapid identification of Mycobacterium bovis BCG by the detection of the RD1 deletion using a multiplex PCR technique.

The BCG vaccine strain cannot, with confidence, be differentiated from other members of the Mycobacterium tuberculosis complex on phenotypic tests alone. Isolates from clinical sites not associated with vaccination may be confused with M. tuberculosis. A characteristic of BCG strains is the deletion of the genomic region RD1; detection of this forms the basis of a multiplex polymerase chain reaction (PCR) assay to distinguish BCG strains. In this study, 28 M. tuberculosis complex strains were analysed by the PCR assay. A DNA sequence displaying the characteristic deletion was detected in all eleven of the BCG strains tested and was not found in representatives of other members of the complex, including M. bovis. Thus, the assay affords a rapid, simple and effective method for the discrimination of the BCG vaccine strain from other members of the M. tuberculosis complex.

A hospital outbreak of antibiotic-resistant Streptococcus pneumoniae.

We report six patients colonised with a multiply resistant strain of Streptococcus pneumoniae.

Tuberculosis transmitted through transplantation.

Tuberculosis in solid organ transplant recipients is associated with relatively high morbidity and mortality and is often extra-pulmonary. Reactivation of dormant infection is the usual mode of acquisition with donor and nosocomial transmission occurring infrequently. We report two cases of probable donor transmitted extra-pulmonary infection where both isolates of Mycobacterium tuberculosis proved to be indistinguishable using hemi-nested inverse PCR of the IS 6110 region.

The use of rpoB sequence analysis in the differentiation of Mycobacterium abscessus and Mycobacterium chelonae: a critical judgement in cystic fibrosis?

Individuals suffering from fibrocystic disease may acquire non-tuberculous mycobacteria as colonizing or infecting organisms. Mycobacterium abscessus is of particular concern because it may be very difficult to eradicate and may mitigate against lung transplantation. However, this species may be difficult to reliably differentiate from the closely related M. chelonae. We have developed a rapid, low-cost, short sequence-based technique to confirm species identity by analysis of a segment of the RNA Polymerase B (rpoB) gene.

The epidemiology of atypical mycobacterial diseases in northern England: a space-time clustering and Generalized Linear Modelling approach.

The incidence of infection by mycobacteria, other than tubercle bacilli (MOTT) is increasing in the United Kingdom, Europe and the United States. These diseases increase morbidity and are an increasing public health concern. However, the epidemiology of disease due to these species is not well characterized. We used space-time clustering approaches and Generalized Linear Modelling to investigate the potential predictors of disease in cases of infection by organisms of the Mycobacterium avium complex (MAC) and M. malmoense recorded in the north of England during 2000-2005. There was significant spatial and temporal clustering in juvenile cases of infection by MAC but not for cases of infection in adults by either species. There were no significant predictors of infection by M. malmoense or juvenile cases of M. avium. Incidence of disease caused by M. avium in adults was significantly related to health deprivation and weakly related to rainfall. We consider possible reasons for the difference in epidemiology in infection by M. avium in adults and juveniles.