Investigation of two cases of Mycobacterium chelonae infection in haemato-oncology patients using whole-genome sequencing and a potential link to the hospital water supply.

BACKGROUND: Haemato-oncology patients are at increased risk of infection from atypical mycobacteria such as Mycobacterium chelonae which are commonly found in both domestic and hospital water systems. AIMS: To describe the investigation and control measures following two patient cases of M. chelonae and positive water samples in the study hospital. METHODS: Water testing was undertaken from outlets, storage tanks and mains supply. Whole-genome sequencing (WGS) was used to compare patient and positive water samples. The subsequent infection control measures implemented are described. FINDINGS: The WGS results showed two main populations of M. chelonae within the group of sampled isolates. The results showed that the patient strains were unrelated to each other, but that the isolate from one patient was closely related to environmental samples from water outlets, supporting nosocomial acquisition. CONCLUSIONS: WGS was used to investigate two patient cases of M. chelonae and positive water samples from a hospital water supply. Relevant control measures and the potential for chemical dosing of water systems to enhance proliferation of atypical mycobacteria are discussed.

Characterising transmission of a tuberculosis genotype in Scotland: a qualitative approach to social network enquiry.

SETTING: Contact investigation resulting from specimens sent to the Scottish Mycobacteria Reference Laboratory. OBJECTIVE: To characterise patients and types of exposures associated with transmission of a prevalent Mycobacterium tuberculosis genotype in Scotland. DESIGN: A combined approach using molecular epidemiology and semi-structured patient interviews for social network enquiry. RESULTS: We investigated social connections between 64 patients diagnosed between 1994 and 2004. Fifty-five per cent had > or = 1 identifiable contact. One third (n = 14, 32.6%) of the 43 epidemiological links detected were discerned as a result of patient interviews and were not previously recorded on surveillance reports, nor recognised by nurse specialists (all were non-household contacts). Sixteen putative sites of exposure were identified, 11 were public houses. Rather than a single-source outbreak, eight pockets of transmission were identified, the largest involving UK-born alcohol-misusing males frequenting several public houses. CONCLUSIONS: Using a standardised approach to explore themes around which individuals may have been exposed to TB resulted in the detection of previously unrecognised epidemiological links. Epidemiological data obtained from cluster investigations, e.g., risk and social behaviours that increase the risk of infection and sites of putative exposure, can enhance the development of more appropriate questions for the contact tracing interview.

Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population.

AIMS: If human papillomavirus (HPV) testing is to be included within cervical screening programmes, the importance of multiple HPV infections in cervical neoplasia needs to be determined. This study investigated the diversity of multiple HPV types in a routine cervical screening population, and assessed associations with cervical neoplasia. METHODS: Overall HPV prevalence, type specific prevalence, and extent of multiple infection were assessed in residual material from 3444 liquid based cytology samples, using real time GP5+/GP6+ polymerase chain reaction for screening and linear array assay for genotyping. HPV status was studied in relation to age and concurrent cytological evidence of dyskaryosis. RESULTS: Twenty per cent of samples were HPV positive. HPV type diversity was broad, and multiple HPV infections occurred in half of the HPV positive samples. Younger women were significantly more likely to harbour multiple high risk HPV (HR-HPV) infections. Infections with multiple HR-HPV types were found in 3.4% of samples negative for neoplasia and in 33.3%, 41.8%, and 40.4% of samples with borderline, mild, or high grade dyskaryosis, respectively. Single HR-HPV infections were found in 4.9%, 38.6%, 45.0%, and 51.1% of negative, borderline, mild, or high grade dyskaryosis samples, respectively. CONCLUSIONS: Multiple HR-HPV infections were most prevalent in young women. Multiple HR-HPV infections were not more frequent in high grade than in low grade cervical neoplasia, reflecting common sexual transmission of multiple HR-HPV. Prospective cohort studies linking sequential loss or gain of HPV types with cytological analysis are required to assess the impact of multiple HR-HPV infections on neoplastic progression.

The efficacy of the heat killing of Mycobacterium tuberculosis.

There is concern that current procedures for the heat inactivation of Mycobacterium tuberculosis may not be adequate. This raises serious safety issues for laboratory staff performing molecular investigations such as IS6110 restriction fragment length polymorphism typing. This paper confirms that the protocol of van Embden et al, as performed routinely in this laboratory, is safe and effective for the heat inactivation of M tuberculosis. This procedure involves complete immersion of a tube containing a suspension of one loopfull of growth in a water bath at 80 degrees C for 20 minutes. Seventy four isolates were included in this investigation. Despite prolonged incubation for 20 weeks, none of the heat killed M tuberculosis suspensions produced visible colonies or gave a positive growth signal from liquid culture. This method did not affect the integrity of the DNA for subsequent molecular investigations.

Rapid real time PCR to distinguish between high risk human papillomavirus types 16 and 18.

AIMS: To assess the validity and practicality of real time polymerase chain reaction (PCR) for human papillomavirus (HPV) testing in combination with liquid based cytology samples for cervical screening. METHODS: Real time PCR using consensus (GPS+/6+) and type specific primers was developed to detect genital HPV types. This provides rapid, efficient amplification followed by denaturation of the product and computer analysis of the kinetics data that are generated. Liquid based cytology samples were obtained from patients attending routine cervical screening clinics. DNA was extracted from the residual cellular suspension after cytology using spin columns. RESULTS: Real time PCR successfully distinguished between HPV-16 and HPV-18 on the basis of amplification with consensus primers followed by DNA melting temperature (Tm) analysis. Sensitivities of one to 10 copies of HPV-16 (mean Tm = 79.4 degrees C; 2 SD, 0.8) and four to 40 copies of HPV-18 (mean Tm = 80.4 degrees C; 2 SD, 0.4) were obtained. In a mixed population of SiHa and HeLa cells containing known copy numbers of HPV-16 and HPV-18 genomes, HPV-16 and HPV-18 products were clearly separated by Tm analysis in mixtures varying from equivalence to 111000. Together with detailed melt analysis, type specific primers from the same region of the L1 gene confirmed the differential ability of this system. The method was applied to 100 liquid based cytology samples where HPV status using conventional GP5+/6+ PCR was already known. There was 95% agreement between the methods, with 55 positives detected by conventional PCR and 59 with real time PCR. The method was then tested on 200 routine liquid based cytology samples. Approximately 10% were positive by real time PCR, most of which were classified as HPV-16 by detailed melt analysis. Thirteen (6.8%) HPV positives were identified in 189 samples showing no evidence of cervical cytological abnormality. CONCLUSIONS: Real time PCR is a rapid, efficient method for the detection of HPV with the separation of HPV-16 and HPV-18 on the basis of differential Tm. Preliminary results suggest it could prove

A longitudinal study of HPV detection and cervical pathology in HIV infected women.

OBJECTIVE: To monitor the presence and persistence of high risk (HR) human papillomavirus (HPV) in cervical brushings from HIV infected women. METHODS: Prospective observational cohort study of HIV infected women. Women were enrolled from the cohort of 164 HIV infected women who attend the colposcopy clinic at the Edinburgh Regional Infectious Diseases Unit. A single cervical brush scrape was obtained from 39 women and two or more samples from 63 women who attended regularly at approximately 6 monthly intervals. HPV typing was carried out using a commercial hybrid capture assay (HCA). Details of antiretroviral therapy, cytological assessment, and histological evaluation were made available and the interrelation with HR-HPV detection analysed. RESULTS: Abnormal cervical cytology, particularly of low grade, was common in these HIV infected women. HR-HPV types were detected in 25% of the women with normal cytology, while over 80% of those with abnormal cytology of any grade were HR-HPV positive. Persistent HR-HPV, as defined by two or more consecutive HPV positive results, was common and found in 27/63 women from whom multiple samples were obtained. HR-HPV was detected at high levels whether or not patients were receiving antiretroviral therapy. Profound immunosuppression was not necessarily associated with progression of cervical disease and no cases of invasive cervical disease were seen. CONCLUSION: While mild dyskaryosis (low grade squamous intraepithelial lesion (LSIL)) and persistence of HR-HPV are common in HIV infected women in Edinburgh, regular cytological and colposcopic evaluation with appropriate intervention and treatment appears to limit the progression of cervical disease.

Detection of high-risk human papillomavirus nucleic acid in archival cervical smears.

OBJECTIVE: To test the usefulness of a commercial DNA hybridization assay for the detection of high-risk (HR) human papillomavirus (HPV) types in archival cervical smears and to compare the sensitivity with that of polymerase chain reaction (PCR) using consensus primers. STUDY DESIGN: Stained material was scraped from archival slides and the pellet volume noted. DNA was extracted using silica/guanidinium isothiocyanate and the quality checked by amplification of the beta-globin gene. HR-HPV DNA was detected using a commercial hybrid capture assay (HCA) and the results compared with an in-house amplification system with consensus primers. RESULTS: Of 156 archival smears stored for 12-13 years, 20 were positive by HCA using an HR probe cocktail. Ninety-eight were also tested by PCR, and 35 were positive. The percentage of HPV-positive samples increased with the increasing size of the pellet. HR-HCA detected more positives in samples with high grade squamous intraepithelial lesion (moderate/severe dyskaryosis). CONCLUSION: Both hybridization by HCA and amplification by PCR could be used to detect genital HPV in archival smears. The general primers PCR detected more positives than HR-HCA but included HPV 6/11. While variation in sample size and prolonged storage may reduce the quality of DNA, the use of archival material for longitudinal studies of HPV presence is potentially worthwhile.