Identification of a methylation panel as an alternative triage to detect CIN3+ in hrHPV-positive self-samples from the population-based cervical cancer screening programme.

BACKGROUND: The Dutch population-based cervical cancer screening programme (PBS) consists of primary high-risk human papilloma virus (hrHPV) testing with cytology as triage test. In addition to cervical scraping by a general practitioner (GP), women are offered self-sampling to increase participation. Because cytological examination on self-sampled material is not feasible, collection of cervical samples from hrHPV-positive women by a GP is required. This study aims to design a methylation marker panel to detect CIN3 or worse (CIN3+) in hrHPV-positive self-samples from the Dutch PBS as an alternative triage test for cytology. METHODS: Fifteen individual host DNA methylation markers with high sensitivity and specificity for CIN3+ were selected from literature and analysed using quantitative methylation-specific PCR (QMSP) on DNA from hrHPV-positive self-samples from 208 women with CIN2 or less (< CIN2) and 96 women with CIN3+. Diagnostic performance was determined by area under the curve (AUC) of receiver operating characteristic (ROC) analysis. Self-samples were divided into a train and test set. Hierarchical clustering analysis to identify input methylation markers, followed by model-based recursive partitioning and robustness analysis to construct a predictive model, was applied to design the best marker panel. RESULTS: QMSP analysis of the 15 individual methylation markers showed discriminative DNA methylation levels between < CIN2 and CIN3+ for all markers (p < 0.05). The diagnostic performance analysis for CIN3+ showed an AUC of ≥ 0.7 (p < 0.001) for nine markers. Hierarchical clustering analysis resulted in seven clusters with methylation markers with similar methylation patterns (Spearman correlation> 0.5). Decision tree modeling revealed the best and most robust panel to contain ANKRD18CP, LHX8 and EPB41L3 with an AUC of 0.83 in the training set and 0.84 in the test set. Sensitivity to detect CIN3+ was 82% in the training set and 84% in the test set, with a specificity of 74% and 71%, respectively. Furthermore, all cancer cases (n = 5) were identified. CONCLUSION: The combination of ANKRD18CP, LHX8 and EPB41L3 revealed good diagnostic performance in real-life self-sampled material. This panel shows clinical applicability to replace cytology in women using self-sampling in the Dutch PBS programme and avoids the extra GP visit after a hrHPV-positive self-sampling test.

Pectin limits epithelial barrier disruption by Citrobacter rodentium through anti-microbial effects.

SCOPE: C. rodentium is the murine equivalent of Enteropathogenic Escherichia. coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) which induce damage to the intestinal epithelial barrier that results in diarrhea and intestinal inflammation. Dietary fibre intake can be an effective approach to limit epithelial damage by these enteric pathogens. Therefore, the protective effect of dietary fibre pectin against dysfunction of epithelial barrier integrity upon C. rodentium infection was investigated. METHODS AND RESULTS: Pectins that structurally differed in the degree and distribution of methylesters were tested on barrier protective effects on epithelial cells against C. rodentium by measuring transepithelial electrical resistance and lucifer yellow fluxes. All three pectins protected the epithelial barrier from C. rodentium induced damage in a structure-independent manner. These barrier protective effects were also independent of pectin-induced TLR2 activation. Furthermore, the pectins induced anti-adhesive effects on C. rodentium by interacting with C. rodentium and not with epithelial cells. This may be explained by antimicrobial effects of pectins on C. rodentium and not on other enteric bacteria including Lactobacillus plantarum and E. coli. A competition ELISA for binding of C. rodentium to pectin supported this finding as it showed that pectin interacts strongly with C. rodentium, whereas it interacts weakly or not with L. plantarum or E. coli. CONCLUSION: These findings demonstrate that pectin protects the epithelial barrier from C. rodentium induced damage by inducing anti-microbial effects.

The value of a dedicated multidisciplinary expert panel to assess treatment strategy in patients suffering from colorectal cancer liver metastases.

BACKGROUND: For patients with colorectal cancer liver metastases (CRLM), local treatment is the only treatment with curative intent. The majority of patients with CRLM are however evaluated in multidisciplinary teams of colorectal cancer specialists often lacking expertise in local treatment of liver tumors. The aim of this study was therefore to assess the value of a dedicated multidisciplinary panel consisting of hepatobiliary surgeons and interventional radiologists for patients suffering from liver-only CRLM. METHODS: Patients diagnosed with liver-only CRLM in 2016 were identified in a tertiary referral hospital, and two of the referring hospitals in the Netherlands. Diagnostic imaging was independently reviewed by a panel of four hepatobiliary surgeons and two interventional radiologists to re-evaluate treatment strategy retrospectively. If two or more panelists assessed all lesions eligible for resection and/or ablation, patients were deemed eligible for local treatment with curative intent. Interrater reliability between hepatobiliary surgeons was assessed through intraclass correlation coefficient (ICC) and weighted Cohen's kappa. RESULTS: Diagnostic imaging of 61 patients with liver-only metastases were reviewed. Local treatment strategies appeared feasible in 40/61 (65.6%) patients. Five out of 25 patients (20.0%) initially assigned to systemic therapy were deemed eligible for upfront local treatment with curative intent (p = 0.015). In this subgroup, interrater reliability between hepatobiliary surgeons was substantial (ICC: 0.704, 95% CI: 0.536-0.838, n = 25). CONCLUSION: Assessment of treatment strategy by a dedicated multidisciplinary panel including liver experts may result in an increased number of patients eligible for potentially curative treatment and reduce undertreatment of patients suffering from liver-only CRLM.

Source investigation of two outbreaks of skin and soft tissue infection by Mycobacterium abscessus subsp. abscessus in Venezuela.

Outbreaks of soft tissue or skin infection due to non-tuberculous mycobacteria are reported frequently in scientific journals but in general the infection source in these outbreaks remains unknown. In Venezuela, in two distinct outbreaks, one after breast augmentation surgery and another after hydrolipoclasy therapy, 16 patients contracted a soft tissue infection due to Mycobacterium abscessus subsp. abscessus. Searching for the possible environmental infection sources in these outbreaks, initially the tap water (in the hydrolipoclasy therapy outbreak) and a surgical skin marker (in the breast implant surgery outbreak), were identified as the infection sources. Molecular typing of the strains with a variable number tandem repeat typing assay confirmed the tap water as the infection source but the molecular typing technique excluded the skin marker. We discuss the results and make a call for the implementation of stringent hygiene and disinfection guidelines for cosmetic procedures in Venezuela.

Understanding the relationship between Mycobacterium bovis spoligotypes from cattle in Latin American countries.

Spoligotyping is the most frequently used method for genotyping isolates of Mycobacterium bovis worldwide. In the current work, we compared spoligotypes from 1684 M. bovis isolates from Argentina (816), Brazil (412), Chile (66), Mexico (274) and Venezuela (116), obtained from cattle, humans, pigs, wild boars, farmed deer, goats, buffaloes, cats, and wild animals. A total of 269 different spoligotypes were found: 142 (8.4%) isolates presented orphan spoligotypes, whereas 1542 (91.6%) formed 113 different clusters. In cattle, SB0140 was the most representative spoligotype with 355 (24.6%) isolates, followed by SB0121 with 149 (10.3%) isolates. Clustering of spoligotypes ranged from 95.2% in Argentina to 85.3% in Mexico. Orphan spoligotypes were also variable, ranging from 23.7% in Mexico to 4.1% in Brazil. A large proportion of spoligotypes were common to the neighboring countries Argentina, Brazil and Chile. In conclusion, despite the diversity of spoligotypes found in the five countries studied, there are major patterns that predominate in these neighboring countries. These clusters may reflect a long-lasting active transmission of bovine tuberculosis or common historical origins of infection.

Pharmacokinetics of anti-tuberculosis drugs in Venezuelan children younger than 16 years of age: supportive evidence for the implementation of revised WHO dosing recommendations.

OBJECTIVES: The World Health Organization (WHO) recently issued revised first-line antituberculosis (anti-TB) drug dose recommendations for children, with dose increases proposed for each drug. No pharmacokinetic data are available from South American children. We examined the need for implementation of these revised guidelines in Venezuela. METHODS: Plasma isoniazid, rifampicin, pyrazinamide and ethambutol concentrations were assessed prior to and at 2, 4 and 8 h after intake of TB drugs by 30 TB patients aged 1-15 years. The effects of dose in mg/kg, age, sex, body weight, malnutrition and acetylator phenotype on maximum plasma drug concentrations (Cmax) and exposure (AUC0-24) were determined. RESULTS: 25 patients (83%) had an isoniazid Cmax below 3 mg/l and 23 patients (77%) had a rifampicin Cmax below 8 mg/l. One patient (3%) had a pyrazinamide Cmax below 20 mg/l. The low number of patients on ethambutol (n = 5) precluded firm conclusions. Cmax and AUC0-24 of all four drugs were significantly and positively correlated with age and body weight. Patients aged 1-4 years had significantly lower Cmax and AUC0-24 values for isoniazid and rifampicin and a trend to lower values for pyrazinamide compared to those aged 5-15 years. The geometric mean AUC0-24 for isoniazid was much lower in fast acetylators than in slow acetylators (5.2 vs. 12.0, P < 0.01). CONCLUSION: We provide supportive evidence for the implementation of the revised WHO pediatric TB drug dose recommendations in Venezuela. Follow-up studies are needed to describe the corresponding plasma levels that are achieved by the recommended increased doses of TB drugs.

Helminths and skewed cytokine profiles increase tuberculin skin test positivity in Warao Amerindians.

The immune regulatory mechanisms involved in the acquisition of Mycobacterium tuberculosis infection in children are largely unknown. We investigated the influence of parasitic infections, malnutrition and plasma cytokine profiles on tuberculin skin test (TST) positivity in Warao Amerindians in Venezuela. Pediatric household contacts of sputum smear-positive tuberculosis (TB) cases were enrolled for TST, chest radiograph, plasma cytokine analyses, QuantiFERON-TB Gold In-Tube (QFT-GIT) testing and stool examinations. Factors associated with TST positivity were studied using generalized estimation equations logistic regression models. Of the 141 asymptomatic contacts, 39% was TST-positive. After adjusting for age, gender and nutritional status, TST positivity was associated with Trichuris trichiura infections (OR 3.5, 95% CI 1.1-11.6) and low circulating levels of T helper 1 (Th1) cytokines (OR 0.51, 95% CI 0.33-0.79). Ascaris lumbricoides infections in interaction with Th2- and interleukin (IL)-10-dominated cytokine profiles were positively associated with TST positivity (OR 3.1, 95% CI 1.1-8.9 and OR 2.4, 95% CI 1.04-5.7, respectively). A negative correlation of QFT-GIT mitogen responses with Th1 and Th2 levels and a positive correlation with age were observed (all p < 0.01). We conclude that helminth infections and low Th1 cytokine plasma levels are significantly associated with TST positivity in indigenous Venezuelan pediatric TB contacts.

Carriage and invasive isolates of Streptococcus pneumoniae in Caracas, Venezuela: the relative invasiveness of serotypes and vaccine coverage.

The introduction of a pneumococcal conjugate vaccine in Venezuela needs previous studies to assess vaccine efficiency. We conducted a survey of nasopharyngeal pneumococcal carriage in urban children in Caracas and studied the distribution of serotypes. We compared these data with survey data available for invasive strains isolated in the same area and in the same time period. An overall pneumococcal carriage rate of 27% was observed. The most predominant capsular serotypes among carriage isolates were 6B (29%), 19A (13.8%), 23F (10%), 14 (8.3%), 6A (8.3%) and 15B/C (3.3%) and among invasive isolates 6B (25%), 14 (15%), and 19A, 6A, 7F, and 18 (7.5% each). The serotypes/groups 1, 5, 7F and 18, jointly covering 30% of the invasive strains, represented less than 0.7% of the carrier strains. The theoretical coverage of the pneumococcal conjugate vaccine PCV13 for carriage and invasive strains was calculated to be 74% and 90%, respectively. Our study demonstrates important differences for the serotype distribution in disease and carriage isolates and provides a key baseline for future studies addressing the prevalence and replacement of invasive and carriage serotypes after the introduction of the PCV 13 vaccine in Venezuela in the year 2010.

[Pneumococcal carriage in mothers and children of the Panare Amerindians from the State of Bolivar, Venezuela]. / Estado de portador nasofaríngeo de Streptococcus pneumoniae en madres e hijos de la población indígena Panare del estado Bolívar, Venezuela.

In North America, the indigenous groups have been identified as a population with increased risk of pneumococcal colonization and pneumococcal invasive disease. However, little information is available from South American natives. In the present study we evaluated the nasopharyngeal carriage and serotype distribution of Streptococcus pneumoniae in mothers and children of the Panare people from Venezuela. In May 2008, in 8 distinct geographically isolated communities, 148 nasopharyngeal samples were obtained from 64 healthy mothers and 84 healthy Panare children under 5 years of age. S. pneumoniae was isolated and identified by standard techniques. Strains were typified by multiplex PCR and resistance patterns were determined by the disk diffusion method. A total of 65 strains were isolated; 11% of the mothers and 69% of the children carried S. pneumoniae. Serotypes 6B (48%), 33F (21,5%), 6A (6%), 19A (3,1%) and 23F (1,5%) were the most predominant. Of the 6 colonized mother-child pairs, 3 pairs (2 with 6B), were colonized with the same serotype. All strains were sensitive to penicillin and 13,7% were resistant to macrolides. The high colonization rates in the Panare people suggest that the children are at increased risk of pneumococcal invasive disease and could benefit from vaccination. Four conjugate vaccine serotypes (6B, 6A, 19A and 23F) representing 58 % of all strains were present in the population at the moment of sampling. Resistance to antibiotics is (still) not a problem.

99mTc Nanocoll: a radiopharmaceutical for sentinel node localisation in breast cancer--in vitro and in vivo results.

This study evaluated labelling efficiency and radiochemical purity of (99m)Tc colloid albumin to identify an optimal labelling protocol for sentinel node detection. Results indicate that a 72 h eluate is not recommended for high specific labelling of (99m)Tc colloid albumin. Ex vivo, significantly higher count rates were reached using a 2h eluate in vacuum or nitrogen. Labelling 26 MBq/microg (99m)Tc colloid albumin with a 2 h eluate under nitrogen is recommended because of the ease of labelling.

Characterization of IS6110 insertions in the dnaA-dnaN intergenic region of Mycobacterium tuberculosis clinical isolates.

Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to be clonally related. The presence of IS6110 in the dnaA-dnaN intergenic region, one preferential locus for the integration of IS6110, was evaluated in 125 M. tuberculosis isolates. Five isolates had IS6110 inserted in this region, and two consisted of a mix of isogenic strains that putatively have evolved during a single infection. Strains from the same isolate had identical spoligo and mycobacterial interspersed repetitive unit-variable-number tandem repeat profiles, but had slight variations in IS6110 RFLP patterns, due to the presence of IS6110 in the dnaA-dnaN intergenic region. Duplication of the dnaA-dnaN intergenic region was found in one isogenic strain.

(99M)Technetium-sestamibi scintimammography in non-palpable breast lesions found on screening X-ray mammography.

OBJECTIVE: The sensitivity and specificity of (99m)Tc-sestamibi scintimammography in patients with non-palpable breast lesions diagnosed by screening mammography and the value of (99m)Tc-sestamibi to detect axillary lymph node metastases was determined. METHODS: Between September 2000 and December 2003, 103 females with non-palpable breast lesions were included for further evaluation. X-ray mammography was repeated and 99mTc-sestamibi scintimammography performed within one-week. Anterior, and left and right lateral images were obtained. The scintimammography was analysed by 2 experienced observers who were blinded to the clinical, pathological, and radiological results. The sensitivity and specificity of scintimammography to diagnose non-palpable lesion(s), including the axillary regions, was compared with histopathology, clinical, and radiological follow up. RESULTS: Two patients (one non-small lung cancer and one non-Hodgkin's disease) were excluded. Both showed (99m)Tc-sestamibi avid lesions in the breast and axillary region. In the remaining 101 patients, 37 true positive (TP), 4 false positive (FP), 52 true negative (TN), and 8 false negative (FN) breast carcinomas were found. The specificity was 92.8%, sensitivity 82.2%, positive predictive value (PPV) 90.2%, and negative predictive value (NPV) 86.6%. (99m)Tc-sestamibi scintimammography showed axillary lesions in 5/15 (33%) patients with axillary lymph node metastasis. CONCLUSION: In patients with non-palpable lesions diagnosed by screening- X-ray-mammography, (99m)Tc-sestamibi scintimammography provided high specificity and PPV. Furthermore, (99m)Tc-sestamibi scintimammography detected 33% of patients with axillary lymph node metastases. Therefore, (99m)Tc-sestamibi scintimammography could be of incremental value in the surgical work-up of these patients.

Effect of mechanical bowel preparation with polyethyleneglycol on bacterial contamination and wound infection in patients undergoing elective open colon surgery.

Mechanical bowel preparation is common practice in elective colon surgery. In order to verify the effect of mechanical bowel preparation on the colonic flora, this study followed 185 patients undergoing elective open colon surgery, 90 of whom were assigned randomly to receive mechanical bowel preparation with polyethyleneglycol. Swabs of the anastomosis and the subcutis were taken during surgery. Further swabs were taken of any subsequent wound infections. Mechanical bowel preparation did not reduce contamination of the peritoneal cavity or the subcutis during surgery, and there appeared to be more sterile subcutaneous swabs in the control group.

Effect of increased (99m)Tc/(99)Tc ratios on count rates in sentinel node procedures: a randomised study.

The aim of this study was to evaluate the count rates of sentinel lymph nodes (SLNs) in patients with breast cancer in the operating theatre, using (99m)Tc-Nanocoll with different ratios of technetium-99m to technetium-99. After written informed consent had been obtained, we tested different ratios of (99m)Tc/(99)Tc-Nanocoll in a double-blinded randomised study performed in 161 patients. Twenty-five MBq/ microg (99m)Tc-colloid albumin was prepared in vacuum. In 87 patients (group A) a 2-h elution was used and in 74 patients (group B) a 24-h elution was used. Patients were subcategorised into subgroups 1 and 3, in which an SLN procedure for breast carcinoma was performed simultaneously with lumpectomy, and subgroups 2 and 4, in which an SLN procedure was performed 2-3 weeks after prior excision biopsy. All patients were injected along the lateral border of the areola (two injections: 50 MBq/0.3 ml intradermally and 50 MBq/2 ml intraparenchymally). Ex vivo measurement of count rates was performed with a gamma probe. Comparing groups A and B in respect of registered counts per second (cps) of excised SLNs, a significant difference was found ( P<0.004). When comparisons were made between subgroups 1 and 2 (2-h elution) and between subgroups 3 and 4 (24-h elution) in respect of registered cps of excised SLNs, no significant difference was found (subgroup 1 vs 2, P=0.825; subgroup 3 vs 4, P=0.915). Use of a 2-h elution in vacuum yielded a significantly higher count rate of maximum specific activity of (99m)Tc-colloid albumin in SLNs than was achieved using a 24-h elution in vacuum. SLN procedures performed 2-3 weeks after prior excision biopsy proved reliable as compared to SLN procedures performed simultaneously with lumpectomy.

[Radionuclide-guided minimally invasive rib biopsy for metastases]. / Radionuclidegeleide minimaal invasieve biopsie van voor metastase verdachte ribafwijkingen.

OBJECTIVE: To determine the feasibility of radionuclide-guided surgical rib biopsy in patients in whom a local metastasis is suspected. DESIGN: Descriptive. METHOD: In eight patients (three men and five women) with a known primary carcinoma, radionuclide-guided surgical rib biopsy was performed because a local metastasis was suspected. The skin was marked on the basis of a technetium-99m-(99mTc)-medronate scintigram. During surgery, a gamma probe was used to look for the hot spot in the rib in the vicinity of the skin marking; this hot spot was then minimally excised and submitted for pathological examination. RESULTS: In all patients, the radionuclide-guided rib biopsy resulted in an unequivocal histopathologic diagnosis: tumour metastasis (n = 2), necrosis (n = 1), old fracture (n = 5). CONCLUSION: Radionuclide-guided surgical bone biopsy has been shown to be a specific, simple, minimally invasive technique for obtaining a representative histological specimen with little stress on the patient. This technique can be used in every hospital that is equipped to carry out the sentinal node procedure in case of breast cancer or melanoma.

[Request for labia correction: sometimes more than a simple question]. / Verzoek om labiumcorrectie: soms meer dan een simpele vraag.

Three women aged 19, 50 and 33 years, requested surgical correction of their labia minora because of subjective complaints attributed to the size of their labia minora. However, during consultation, for one of the patients it transpired that she did not know anything about the normal physiological changes of the external genitals during puberty and the enormous variety and diversity of the length of labia minora between women. It turned out that the other woman felt uncertain about her genitals following a recent divorce. For the third woman a vulvar pain syndrome and a sexual abuse history became clear. Two of the patients decided not to undergo surgery and the third sought a cure elsewhere. The request for a surgical correction of the labia minora seems quite simple and the operation does not seem to be complicated either. However, the question remains as to whether an operation is the solution for the psychological and behavioural consequences the woman experiences. A conservative approach is recommended, with attention for other possible problems that can be hidden behind the request for labia minora correction.

Further optimisation of 99mTc-Nanocoll sentinel node localisation in carcinoma of the breast by improved labelling.

The sentinel node (SLN) procedure has gained widespread acceptance for the axillary staging of patients who present with carcinoma of the breast. A first series of 21 patients were imaged 2 and 4 h after the injection of technetium-99m colloidal albumin. Preoperatively, axillary foci were located scintigraphically and with a hand-held gamma probe that was also used to detect the radiolabelled nodes 22 h later in the operating theatre. In a high percentage of our first series it was difficult to localise the sentinel node owing to a low count rate ex vivo. The aim of this study was to facilitate the detection of sentinel nodes by improving the count rate by the use of higher specific concentrations of 99mTc-colloidal albumin, with increased radiochemical labelling efficiency and stability. In vitro tests were performed to establish the radiochemical labelling efficiency and stability of different concentrations of 99-mTc-colloidal albumin. Concentrations of 2.5, 10, 20, 25, 30, 37 and 50 MBq 99mTc/microg colloidal albumin (Nanocoll) were prepared under nitrogen and in vacuum. The quality of the solutions was assessed by testing the radiochemical labelling efficiency and stability after 0.5, 2, 5, 8 and 24 h. The particle size of colloidal albumin was tested by dynamic light scattering at 30.2 degrees, 62.6 degrees and 90.0 degrees angles. Following the results of the in vitro studies, higher concentrations of 99mTc-colloidal albumin were used in vivo in a further series of 98 patients. For labelling under nitrogen, a maximum acceptable concentration of 10 MBq/microg was found, which complies with the specifications of the manufacturer. By preparing the labelling in vacuum vials, a 2.5 times greater radiochemical labelling efficiency over the entire period was achieved, and a significant improvement (P<0.002) in the in vivo series was found. It is concluded that although the rate of successful visualisation of the SLN was high in all studies, a better count rate (nine times higher) was achieved with the highest concentration of 99mTc-colloidal albumin, which facilitated the detection of the SLN by the gamma probe during surgery.