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The role of interferon-γ release assays in the diagnosis of active tuberculosis disease is uncertain, and recent guidelines do not support their routine use. We reviewed the clinical records of 415 patients who had a QuantiFERON-TB Gold In-Tube assay between 29 June 2005 and 28 October 2010 to determine its performance in the diagnosis of active tuberculosis disease in a low prevalence setting, specifically in human immunodeficiency virus (HIV) -positive and HIV-negative patients, those of UK and non-UK origin, and those with pulmonary and extrapulmonary disease. For the diagnosis of active tuberculosis disease the overall sensitivity of QuantiFERON-TB Gold In-Tube assay was 71.4% (95% CI 59.3–81.1), specificity was 81.0% (95% CI 75.5–85.6) and negative predictive value was 92.6% (95% CI 88.2–95.5). No significant difference in sensitivity was seen in culture-positive and culture-negative tuberculosis, in pulmonary and extrapulmonary disease, or with HIV infection. Specificity and negative predictive value were significantly higher in patients of UK origin compared with those of non-UK origin (89.3% (95% CI 83.3–93.3) and 97.1% (95% CI 92.7–98.9) versus 66.3% (95% CI 55.6–75.5) and 83.3% (95% CI 72.6–90.4)). Our study suggests that there may be a role for interferon-γ release assays in excluding active tuberculosis disease, particularly extrapulmonary disease, in patients originating from areas of low tuberculosis incidence, with a negative test highly predictive of a lack of active tuberculosis disease in this group. We cannot support the use of these assays in the diagnosis of active tuberculosis infection in patients from areas of higher incidence.

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.

Due to an increase in bovine tuberculosis in cattle in the United Kingdom, we investigated the characteristics of Mycobacterium bovis infection in humans and assessed whether extensive transmission of M. bovis between humans has occurred. A cross-sectional study linking demographic, clinical, and DNA fingerprinting (using 15-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat [MIRU-VNTR] typing) data on cases reported between 2005 and 2008 was undertaken. A total of 129 cases of M. bovis infection in humans were reported over the period, with a decrease in annual incidence from 0.065 to 0.047 cases per 100,000 persons. Most patients were born pre-1960, before widespread pasteurization was introduced (73%), were of white ethnicity (83%), and were born in the United Kingdom (76%). A total of 102 patients (79%) had MIRU-VNTR typing data. A total of 31 of 69 complete MIRU-VNTR profiles formed eight distinct clusters. The overall clustering proportion determined using the n − 1 method was 33%. The largest cluster, comprising 12 cases, was indistinguishable from a previously reported West Midlands outbreak strain cluster and included those cases. This cluster was heterogeneous, having characteristics supporting recent zoonotic and human-to-human transmission as well as reactivation of latent disease. Seven other, smaller clusters identified had demographics supporting recrudescence rather than recent infection. A total of 33 patients had incomplete MIRU-VNTR profiles, of which 11 may have yielded 2 to 6 further small clusters if typed to completion. The incidence of M. bovis in humans in the United Kingdom remains low, and the epidemiology is predominantly that of reactivated disease.

Background: The emergence of multidrug-resistant tuberculosis (MDRTB) and extensively drug-resistant tuberculosis (XDRTB) is a threat to global tuberculosis control. Limited information is, however, available on the outcome of XDRTB cases. This study describes the susceptibility to second- and third-line antituberculosis drugs among MDRTB cases and treatment outcome of identified XDRTB cases. Method: The results of second-line antituberculosis drug susceptibility tests in the UK between January 1995 and December 2007 were retrospectively reviewed and clinicians contacted for treatment outcome of XDRTB cases. Participants included all 678 patients with culture-confirmed MDRTB in the UK. The main outcome measures were the proportion of isolates resistant to second-line antituberculosis drugs and treatment outcome for XDRTB cases. Results: Among MDRTB isolates, levels of resistance to amikacin, capreomycin, ciprofloxacin, cycloserine, ethionamide and p-aminosalicylic acid (PAS) were 5.5, 3.4, 5.6, 5.1, 14.0 and 16.7%, respectively. Six XDRTB cases (0.9% of MDR cases) were identified during this period. Two further cases of XDRTB were reported in 2008. Five individuals with XDRTB died of tuberculosis within 3 years of diagnosis and three are still on treatment. Conclusion: Levels of MDRTB remain low, and those of XDRTB very low, in this high income country. The case fatality ratio among XDRTB cases was high despite low levels of HIV co-infection.

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.

In controlling the spread of tuberculosis, early detection of disease caused by organisms of the Mycobacterium tuberculosis complex (MTBC) is vital. The BD ProbeTec ET system provides a method for the direct detection of MTBC by strand displacement amplification. Two hundred and five respiratory samples from patients with a high probability of tuberculosis were assessed by ProbeTec and by microscopy and culture for mycobacteria. ProbeTec positive results were obtained with 101 of 109 samples from which MTBC organisms were isolated. ProbeTec correctly signalled 78 of 81 samples that gave growths of mycobacteria other than tubercle bacilli (MOTT) as negative. Three samples gave false-positive results, corrected on repeat testing. Positive and negative predictive values (PPV, NPV) were 0.97 and 0.90 and the system showed a sensitivity and specificity of 92.7% and 96.0%, respectively. These values rose to PPV 0.97, NPV 0.96, sensitivity 97.1% and specificity 96.0% when data from the small number of gastric lavage samples tested were removed from the analysis. The BD ProbeTec ET system offers a robust and reliable molecular biological approach to the detection of MTBC organisms in respiratory samples in a semi-automated format.

Background: This study aimed to describe the clinical, microbiological, molecular epidemiology and treatment of multidrug resistant tuberculosis (MDRTB) cases in the UK and to determine factors associated with survival. Methods: Ninety MDRTB cases were identified from 1 January 1996 to 30 June 1997; 69 were DNA fingerprinted. Date of diagnosis was determined and data were collated on key demographic factors, clinical, radiological and treatment details. Variables associated with survival were included in a Cox proportional hazards model. Results: Most of the patients (72.4%) were male, born outside the UK (57.1%), were sputum smear positive (82.2%), and had entered the UK more than 5 years previously (61.9%). Thirty eight of 78 cases (48.7%) had prior TB. Sufficient data on 82 patients were available for survival analysis; 20/27 (74.1%) known to be dead at the end of the observation period had died of tuberculosis. Median survival time overall was 1379 days (95% CI 1336 to 2515) or 3.78 (95% CI 3.66 to 6.89) years (858 days (95% CI 530 to 2515) in immunocompromised individuals (n=32) and 1554 (95% CI 1336 to 2066) days in immunocompetent cases (n=48)). Median survival in patients treated with three drugs to which the bacterium was susceptible on in vitro testing (n=62) was 2066 days (95% CI 1336 to 2515) or 5.66 years, whereas in those not so treated (n=13) survival was 599 days (95% CI 190 to 969) or 1.64 years. Conclusions: Immunocompromised status, failure to culture the bacterium in 30 days or to apply appropriate three drug treatment, and age were significant factors in mortality. An immunocompromised patient was nearly nine times more likely to die, while application of appropriate treatment reduced the risk (risk ratio 0.06). Increasing age was associated with increasing risk of death (risk ratio 2.079; 95% CI 1.269 to 3.402)—that is, for every 10 year increase in age the risk almost doubled. Overall survival was lower than that reported in previous studies.

Aims—To compare clinical information and sputum microscopy as methods for the selection of samples for enhanced mycobacterial detection, identification, and susceptibility systems (EMDISS) to promote the rapid diagnosis of patients with infectious pulmonary tuberculosis. Methods—Two thousand, two hundred and sixty four specimen request forms were examined for clinical details, which were then used to identify specimens likely to yield Mycobacterium tuberculosis on culture. These results were compared with the results of sputum microscopy for acid fast bacilli (AFB). Both methods were assessed against the results of culture using a combination of continuous automated mycobacterial liquid culture (CAMLiC) and conventional solid culture. Results—Classification based on clinical details was an inefficient method of identifying high priority specimens for EMDISS. Although, when given, clinical details were often consistent, a substantial proportion of specimens arrived with no details. This approach would result in the referral of at least 16% of the workload but lead to the detection by culture of only 46% of the M tuberculosis present within it. In contrast, microscopy for AFB defined a much smaller number of specimens (4.8% of the total), which contained 90% of the M tuberculosis isolates. Conclusions—Microscopy for AFB is the most efficient method for defining sputum specimens suitable for referral for enhanced mycobacteriological techniques. However, it is essential that the methods used for smear preparation and microscopy are of the highest possible standard, otherwise some patients with infectious pulmonary tuberculosis will be denied, unnecessarily, the benefits of important advances in mycobacteriology. Key Words: tuberculosis • diagnosis • AFB smear • mycobacterial culture

Two rapid real-time RT-PCR assays, specific for respiratory syncytial virus (RSV) and influenza A and B, were evaluated for the detection of these viruses in clinical respiratory samples. The RSV assay was applied to 100 samples and the Influenza A and B assay applied to 96 samples all of which had been tested previously by an "in-house" multiplex real-time PCR assay. Forty-three samples were negative for RSV by both methods and 56 samples were positive by both methods. One sample was negative by the new RSV assay although it was positive for RSV A by the "in-house" test. Thirty-nine samples were negative for influenza virus by both methods and 55 samples were positive by both assays. Two samples were negative by the new influenza assay however they were positive by the "in-house" influenza assay. The new assays did not cross react with samples containing other viruses including parainfluenza 1, 2, and 4; human metapnuemovirus; coronavirus 229E, NL63, OC43; rhinovirus; adenovirus; bocavirus and had a specificity of 100% and a sensitivity of 98.2% for RSV and 96.5% for influenza respectively. The results of this study demonstrate that the new assays are specific and sensitive for the detection of RSV and influenza viruses in clinical samples.

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.

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.

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.

An artificial neural network was trained to distinguish between three putatively novel species of Streptomyces using normalised, scaled pyrolysis 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.

IS6110, a Mycobacterium tuberculosis complex species-specific insertion element, is targeted primarily for DNA fingerprinting of M. tuberculosis strains. The number and chromosomal positions of copies of this element have been found to be highly variable between unrelated strains and have been exploited for molecular epidemiological purpose but the utility of IS6110 as an informative marker of strain phylogeny has yet to be demonstrated. In the current study, a recently proposed IS6110-targetting PCR based typing methodology, fluorescent amplified fragment length polymorphism (fAFLP) was applied to a global panel of 166 of the clinically more predominant ‘modern’ strains characterised by spoligotype and, where available, Variable Number Tandem Repeat (VNTR) to identify potentially evolutionarily informative common fragments that could define strains as belonging to established genetic lineages. These common fragments are hereby proposed to be ancestral insertion sites present in common ancestors of these strains rather than preferential insertion sites or ‘hot spots’. Results indicate that the exact same spoligotype and VNTR-defined lineages are reflected in the fragment patterns but with greater resolution and are able to clearly define the very distinct Haarlem, LAM, X and also the currently ill-defined T and S and lineages and spoligotypes designated as U, or unknown, without ambiguity. The biogeographical patterns generated reflect the migration of mankind across the globe and indicate that only four successful clones (or individual bacteria) gave rise to virtually all of the tuberculosis in Europe and the Americas. Potential lies in the application of the data to determine IS6110 evolutionary events that have occurred during the evolution of these lineages.

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. Key Words: tuberculosis • epidemiology • molecular typing • IS6110

32 isolates belonging to the Mycobacterium tuberculosis complex were examined by pyrolysis mass spectrometry (PyMS). This technique demonstrated that recent clinical isolates of M. africanum were indistinguishable from those of M. bovis and together formed a homogeneous group distinct from M. tuberculosis. Isolates of BCG were heterogeneous and more closely related to laboratory-adapted strains of M. tuberculosis than to recent isolates of either M. tuberculosis or M. bovis. PyMS is a simple and inexpensive technique which gives interesting information on the relationships between members of the M. tuberculosis complex and can make the clinically important distinction between strains of M. bovis and M. tuberculosis accurately and much more rapidly than conventional techniques.

OBJECTIVE: To identify recent trends in, and factors associated with, resistance to antituberculosis drugs in England, Wales, and Northern Ireland. DESIGN: Cohort of tuberculosis cases reported to the enhanced tuberculosis surveillance system matched to data on drug susceptibility and national strain typing data. SETTING: England, Wales, and Northern Ireland 1998-2005. MAIN OUTCOME MEASURES: Unadjusted and adjusted odds ratios for drug resistance and associated factors. Proportion of multidrug resistant tuberculosis cases clustered. RESULTS: 28 620 culture confirmed cases were available for analysis. The proportion of cases resistant to isoniazid increased from 5% to 7%. Rifampicin resistance increased from 1.0% to 1.2% and multidrug resistance from 0.8% to 0.9%. Ethambutol and pyrazinamide resistance remained stable at around 0.4% and 0.6%, respectively. Regression analyses showed a significant increase in isoniazid resistance outside London (odds ratio 1.04, 95% confidence interval 1.01 to 1.07, a year, associated with changes in age (0.98, 0.98 to 0.99, a year), place of birth (1.49, 1.16 to 1.92), and ethnicity (P

SUMMARYThe 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.

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 strain isolated from a lung abscess in an elephant that died from chronic respiratory disease was found to have properties consistent with its classification in the genus Mycobacterium. An almost complete sequence of the 165 rDNA of the strain was determined following the cloning and sequencing of the amplified gene. The sequence was aligned with those available on mycobacteria and phylogenetic trees inferred by using three tree-making algorithms. The organism, which formed a distinct phyletic line within the evolutionary radiation occupied by rapidly growing mycobacteria, was readily distinguished from members of validly described species of rapidly growing mycobacteria on the basis of its mycolic acid pattern and by a number of other phenotypic features, notably its ability to grow at higher temperatures. The type strain is Mycobacterium elephantis DSM 44368T.

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.

The global increase in tuberculosis which has occurred in the 1980s and 1990s, and the associated re-emergence of resistance to antituberculous drugs, has focused attention on recent trends in resistance in Europe and the United States.1–3 In the United Kingdom overall drug resistance levels have been low.4 A surveillance system, the UK Mycobacterial Resistance Network (MYCOBNET), was established in 1994 by the Public Health Laboratory Service to record drug resistance in laboratory isolates of tuberculosis. We used data from this network to examine resistance among people with newly diagnosed tuberculosis. We analysed the data on initial isolates of Mycobacterium tuberculosis complex referred to United Kingdom reference laboratories5 during 1994 to 1996. Initial isolates were defined as the first positive culture from a person from whom no positive culture had been recorded during the past 12 months. Since M bovis isolates are intrinsically resistant to pyrazinamide …

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.

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.

Mycobacterium (Gr. n. myces a fungus; Gr. neut. dim. n. bakterion a small rod; M.L. neut. n. Mycobacterium a fungus rodlet) is undeniably one of the most clinically important and intensively studied of bacterial taxa. Tuberculosis and leprosy, the most significant diseases caused by mycobacteria, have been recognized throughout recorded times. The taxonomic history of the genus Mycobacterium is intricate and difficult to disentangle from that of related taxa, notably Corynebacterium, Nocardia, and Rhodococcus (1). Comprehensive reviews on the early taxonomic history of these genera are available (2, 3, 4, 5, 6, 7).

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.

The surgical and antimicrobial treatment of non-tuberculous mycobacterial lymphadenopathy in 17 children was reviewed. Node excision was curative, but most nodes were still incised leaving discharging lesions. Standard antituberculous treatment was unhelpful, but a new macrolide/quinolone combination appeared to be effective in three cases.

Sixty representatives of selected Mycobacterium and Nocardia species were examined for their ability to cleave 79 fluorogenic synthetic enzyme substrates based on the fluorophores 7-amino-4-methylcoumarin and 4-methylumbelliferone. The resultant data were analysed using the simple matching coefficient and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. Clusters corresponding to the validly described species Mycobacterium bovis, M. chelonae, M. chitae, M. farcinogenes, M. fortuitum, M. peregrinum, M. senegalense, M. smegmatis, Nocardia asteroides, and N. farcinica were circumscribed at or above the 83% similarity level. Fluorogenic probes prepared from 7-amino-4-methylcoumarin and 4-methylumbelliferone provide a rapid means of detecting taxonomically useful enzymes in small amounts of whole mycobacteria and nocardiae.

A monoclonal antibody-based immunofluorescence test for the detection of Pneumocystis carinii was evaluated in comparison with the conventional direct staining by Grocott's silver methenamine technique. A total of 254 respiratory samples from HIV positive and other immunocompromised patients were examined. Cysts were detected in 30 (12%) of samples using the monoclonal test, but only 15 (6%) positives were found using the Grocott method. There is need for a speedy and efficient test for detection of these organisms, and the monoclonal test was found to be more reliable, quicker and more sensitive than the Grocott technique.

We read with interest the letter by Grobusch et al. (3) on a rapid immunochromatographic assay for the diagnosis of tuberculosis but wish to present evidence that this test may not be as specific as they portrayed it to be. It was long thought that the 38-kDa antigen of Mycobacterium tuberculosis was confined to organisms of the M. tuberculosis complex (MTBC) (1). As suggested in the letter of Grobusch et al., it has therefore been assumed that detection of antibody to the 38-kDa antigen implies infection (although not necessarily disease) with M. tuberculosis, M. africanum, M. bovis, or M. microti. The lack of antibody development after M. bovis BCG vaccination has been ascribed to the lower expression of the 38-kDa antigen in this variant (9). However, Thangaraj and colleagues reported in 1996 (8) the detection of genes encoding the 38-kDa antigen in a strain of M. intracellulare and the expression of the antigen in this species. They speculated that the possession of a homologue of the M. tuberculosis 38-kDa antigen by M. intracellulare might be associated with virulence, since M. intracellulare has the ability to cause disease in immunocompetent hosts, in distinction to M. avium, which does not possess the antigen. Thangaraj et al. did not detect the 38-kDa antigen or the appropriate gene sequence in M. avium, M. paratuberculosis, M. smegmatis, M. fortuitum, M. chelonei, M. leprae, M. kansasii, M. marinum, or M. vaccae. M. malmoense was not included in these investigations. We have produced a recombinant 38-kDa antigen in Escherichia coli as a histidine-tagged fusion protein (7) and used this to raise a panel of 15 mouse monoclonal antibodies (MAbs) by using standard hybridoma technology (6). Selection was based on reactivity with the recombinant antigen by enzyme-linked immunosorbent assay and/or Western blotting of an M. tuberculosis lysate. To further assess the specificity of the MAbs, they were screened against lysates of M. avium, M. intracellulare, M. kansasii, M. malmoense, M. vaccae, and M. xenopi. Upon immunoblotting, 2 of the 15 MAbs (7 and 29) reacted with a 38-kDa protein in M. intracellulare and 2 MAbs (1 and 7) reacted with a 38-kDa protein in M. malmoense (Fig. ​(Fig.1).1). All remaining MAbs were negative against all lysates. Twenty-one of 23 M. malmoense lysates examined by immunoblotting consistently reacted with both MAbs. The two isolates of M. malmoense not expressing the 38-kDa antigen are also atypical when examined by other taxonomic methods, including random amplified polymorphic DNA analysis (5). FIG. 1 Western blot of mycobacterial lysates with murine MAb 7 to recombinant 38-kDa antigen of M. tuberculosis. Lane M, low-molecular-mass standards (in kilodaltons; Bio-Rad); lanes 1 to 6; lysates of M. tuberculosis, M. avium, M. malmoense, M. kansasii, M. ... Our results confirm the observations of Thangaraj et al. in relation to M. intracellulare and strongly suggest that M. malmoense also expresses a homologue of the 38-kDa antigen of M. tuberculosis. M. malmoense is a recognized primary pathogen of immunocompetent children and the elderly, and it has been estimated to cause up to 10% of tuberculosis-like pulmonary disease in the United Kingdom and other northwest European countries (2, 4). We have yet to proceed with studies to detect the gene sequence in M. malmoense which encodes for the antigen. If such a sequence is found it will extend the argument of Thangaraj et al. (8) that possession of the antigen may be associated with virulence, demonstrating the presence of a common immunodominant antigen in the three species of mycobacteria (MTBC organisms, M. intracellulare, and M. malmoense) which cause disease in immunocompetent individuals. These findings may have important implications for vaccine studies. However, it is also clear from our results and those of Thangaraj et al. that the detection of antibodies which react with the 38-kDa antigen of M. tuberculosis cannot be taken to indicate infection or disease due to MTBC organisms alone, since such antibodies may also be evoked by infection or disease with M. intracellulare or M. malmoense.

The rapid technique of pyrosequencing was used to examine 123 samples (in the form of DNA extracts and inactivated sputum) of Mycobacterium spp. Of 99 Mycobacterium tuberculosis samples investigated for single-nucleotide polymorphisms (SNPs), 68% of isoniazid-resistant isolates analysed had an AGC → ACC mutation in katG at codon 315, resulting in the Ser → Thr substitution associated previously with isoniazid resistance. Of the rifampicin-resistant isolates, 92% showed SNPs in rpoB at codons 516, 531 or 526. Inactivated sputum samples and DNA extracts could both be analysed by pyrosequencing, and the method was able to differentiate rapidly between the closely related species of the M. tuberculosis complex (M. tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti and Mycobacterium microti), except between M. tuberculosis, M. canetti and one of two M. africanum strains. This low-cost, high-throughput technique could be used as a rapid screen for drug resistance and as a replacement for some of the time-consuming tests used currently for species identification.

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.

Isolates of Mycobacterium avium-intracellulare complex (MAI) from AIDS and non-AIDS patients were compared by pyrolysis mass spectrometry (PyMS). Those from AIDS patients were more closely related to each other than those from non-AIDS patients which were significantly more disparate.