An evaluation of three processing methods and the effect of reduced culture times for faster direct identification of pathogens from BacT/ALERT blood cultures by MALDI-TOF MS

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker’s MALDI Sepsityper kit, the commercially available Molzym’s MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification

Benign anal lesions, inflammatory bowel disease and risk for high-risk human papillomavirus-positive and -negative anal carcinoma.

A central role in anal carcinogenesis of high-risk types of human papillomaviruses (hrHPV) was recently established, but the possible role of benign anal lesions has not been addressed in hrHPV-positive and -negative anal cancers. As part of a population-based case-control study in Denmark and Sweden, we interviewed 417 case patients (93 men and 324 women) diagnosed during the period 1991-94 with invasive or in situ anal cancer, 534 patients with adenocarcinoma of the rectum and 554 population controls. Anal cancer specimens (n = 388) were tested for HPV by the polymerase chain reaction. Excluding the 5 years immediately before diagnosis, men, but not women, with anal cancer reported a history of haemorrhoids [multivariate odds ratio (OR) 1.8; 95% confidence interval (CI) 1.04-3.2] and unspecific anal irritation (OR 4.5; CI 2.3-8.7) significantly more often than controls. Women with anal cancer did not report a history of benign anal lesions other than anal abscess to any greater extent than controls, but they had used anal suppositories more often (OR 1.5; CI 1.1-2.0). Patients with hrHPV in anal cancer tissue (84%) and those without (16%) reported similar histories of most benign anal lesions, but anal fissure or fistula was more common among hrHPV-positive cases. Ulcerative colitis and Crohn's disease, reported by <1% of study participants, were not associated with anal cancer risk. The higher proportion of hrHPV-positive anal cancers among case patients with anal fissure or fistula suggests that such mucosal lesions may provide direct viral access to basal epithelial layers. Since risk associations with benign anal lesions in men may be confounded by unreported sexual behaviour, and since risk associations in women were generally negative, it seems unlikely that benign anal lesions act as promoters in hrHPV-associated anal carcinogenesis. Moreover, benign anal lesions appear not to be linked to an alternative, hrHPV-unassociated causal pathway to anal cancer. Ulcerative colitis and Crohn's disease were not supported as causal factors for anal cancer

Analysis of MHC class I and II expression in relation to presence of HPV genotypes in premalignant and malignant cervical lesions.

Cervical intraepithelial neoplasia (CIN) grades I to III lesions (n = 94) and squamous cell carcinomas of the uterine cervix (n = 27) were analysed for MHC class I and II expression and presence of HPV genotypes. MHC class I and II expression was studied by immunohistochemistry and HPV typing was performed by general primer- and type-specific primer mediated PCR (GP/TS PCR). Both techniques were performed on paraffin embedded tissue sections. Results show disturbed MHC class I heavy chain expression in CIN I to CIN III, as well as in cervical carcinomas. Upregulated MHC class II expression on dysplastic epithelial cells was also found in the different CIN groups and carcinomas. Prevalence of HPV genotypes increased with the severity of the lesion, mainly due to the contribution of the HPV types 16 and 18. No correlation could be established between the presence of specific HPV genotypes and any MHC expression pattern in the different CIN groups or cervical carcinomas. In some cases these data were confirmed by RNA in situ hybridisation showing HPV 16 E7 transcripts in the same dysplastic/neoplastic cells from which MHC status was determined. The results indicate that local differences may exist in the type of cellular immune response to HPV induced lesions

Low grade squamous intra-epithelial lesions and human papillomavirus infection in Colombian women

Low grade squamous intra-epithelial lesions could be considered as a manifestation of human papillomavirus exposition, however the discrepancy between rates of infection with human papillomavirus and development of low grade squamous intra-epithelial lesions is notable. Here we report a cross-sectional three-armed case–control study in the Colombian population, to compare the risk factors of women with low grade squamous intra-epithelial lesions with that of human papillomavirus DNA-negative and positive women with normal cytology

Introduction of primary screening using high-risk HPV DNA detection in the Dutch cervical cancer screening programme:a population-based cohort study

Background: In January 2017, the Dutch cervical cancer screening programme transitioned from cytomorphological to primary high-risk HPV (hrHPV) DNA screening, including the introduction of self-sampling, for women aged between 30 and 60 years. The Netherlands was the first country to switch to hrHPV screening at the national level. We investigated the health impact of this transition by comparing performance indicators from the new hrHPV-based programme with the previous cytology-based programme. Methods: We obtained data from the Dutch nationwide network and registry of histo- and cytopathology (PALGA) for 454,573 women eligible for screening in 2017 who participated in the hrHPV-based programme between 1 January 2017 and 30 June 2018 (maximum follow-up of almost 21 months) and for 483,146 women eligible for screening in 2015 who participated in the cytology-based programme between 1 January 2015 and 31 March 2016 (maximum follow-up of 40 months). We compared indicators of participation (participation rate), referral (screen positivity; referral rate) and detection (cervical intraepithelial neoplasia (CIN) detection; number of referrals per detected CIN lesion). Results: Participation in the hrHPV-based programme was significantly lower than that in the cytology-based programme (61% vs 64%). Screen positivity and direct referral rates were significantly higher in the hrHPV-based programme (positivity rate: 5% vs 9%; referral rate: 1% vs 3%). CIN2+ detection increased from 11 to 14 per 1000 women screened. Overall, approximately 2.2 times more clinical irrelevant findings (i.e. ≤CIN1) were found in the hrHPV-based programme, compared with approximately 1·3 times more clinically relevant findings (i.e. CIN2+); this difference was mostly due to a national policy change recommending colposcopy, rather than observation, of hrHPV-positive, ASC-US/LSIL results in the hrHPV-based programme. Conclusions: This is the first time that comprehensive results of nationwide implementation of hrHPV-based screening have been reported using high-quality data with a long follow-up. We have shown that both benefits and potential harms are higher in one screening round of a well-implemented hrHPV-based screening programme than in an established cytology-based programme. Lower participation in the new hrHPV programme may be due to factors such as invitation policy changes and the phased roll-out of the new programme. Our findings add further to evidence from trials and modelling studies on the effectiveness of hrHPV-based screening

Prevalence and determinants of HPV infection among Colombian women with normal cytology

Human papillomavirus is the principal risk factor associated with cervical cancer, the most common malignancy among women in Colombia. We conducted a survey, aiming to report type specific prevalence and determinants of human papillomavirus infection in women with normal cytology. A total of 1859 women from Bogota, Colombia were interviewed and tested for human papillomavirus using a general primer GP5+/GP6+ mediated PCR–EIA. The overall HPV DNA prevalence was 14.8%; 9% of the women were infected by high risk types, 3.1% by low risk types, 2.3% by both high risk/low risk types and 0.4% by uncharacterized types (human papillomavirus X). Thirty-two different human papillomavirus types were detected, being human papillomavirus 16, 58, 56, 81(CP8304) and 18 the most common types. The human papillomavirus prevalence was 26.1% among women younger than 20 years, 2.3% in women aged 45–54 years, and 13.2% in women aged 55 years or more. For low risk types the highest peak of prevalence was observed in women aged 55 years or more. Compared to women aged 35–44 years, women aged less than 20 years had a 10-fold increased risk of having multiple infections. Besides age, there was a positive association between the risk of human papillomavirus infection and number of regular sexual partners and oral contraceptive use. In women aged below 25 years, high educational level and having had casual sexual partners predicted infection risk. In conclusion, there was a broad diversity of human papillomavirus infections with high risk types being the most common types detected. In this population multiplicity of sexual partners and, among young women, high educational level and casual sexual partners seem to determine risk

Papillomavirus infection in rural women in southern India

To investigate the prevalence of, and the risk factors for, cervical infection with 44 types of human papillomavirus (HPV) in a rural area in the Dindigul District, Tamil Nadu, India, we interviewed and obtained cervical cell samples from 1891 married women aged 16–59 years. HPV prevalence was 16.9% overall and 14.0% among women without cervical abnormalities, or 17.7 and 15.2%, respectively, age-standardised to the world standard population. In all, 21.9% of infections involved more than one HPV type. High-risk HPV types predominated, particularly HPV 16 (22.5% of women infected), followed by HPV 56, HPV 31, HPV 33 and HPV 18. Unlike most populations studied in developed countries, HPV prevalence was constant across the age groups. HPV positivity was inversely associated with education level (odds ratio (OR) among women with high school vs no education=0.6) and positively associated with widowhood and divorce (OR=1.7), nulligravidity (OR=2.3), and condom use (OR=2.6). It is unclear how much low clearance of, or frequent reinfection with HPV accounted for the study prevalence of infection in different age groups

The contribution of HPV18 to cervical cancer is underestimated using high-grade CIN as a measure of screening efficiency

In one geographical area, 14 high-risk human papillomavirus types in cervical intraepithelial neoplasia (CIN2/3; n=139) and cervical squamous cell carcinoma (SCC; n=84) were analysed. HPV18 was more prevalent in SCC than CIN2/3 (OR 9.8; 95% confidence interval: 2.5–39). Other high-risk types prevalences corresponded in CIN2/3 and SCC. Evaluations using CIN2/3 as a measure of efficiency underestimate the contribution of HPV18 to SCC

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay