Development and validation of multiplex real-time PCR assay for noninvasive prenatal assessment of fetal RhD status and fetal sex in maternal plasma.

OBJECTIVE: Noninvasive prenatal detection of RhD status and fetal sex is becoming part of daily practice in clinical laboratories. We evaluated a high throughput procedure for automated DNA extraction and developed a multiplex real-time PCR (rt-PCR) for the simultaneous detection of three fetal loci in a single reaction to assess fetal sex and RhD status in maternal plasma. METHODS: An automated DNA extraction method was evaluated together with a new multiplex rt-PCR assay for the simultaneous detection of exons 5 and 7 of the RHD gene together with the Y chromosome marker DYS14 in maternal plasma. The test was evaluated on 60 samples of known fetal genotype obtained from RhD-negative pregnant women before being applied prospectively on 158 consecutive clinical cases. Results were compared with newborn phenotypes. RESULTS: Automated DNA extraction allowed successful analysis of all samples. DYS14 was detected in 118 cases (male fetuses) and both RHD exon 5 and 7 were detected in 148 samples. In 70 samples neither RHD exon 5 nor RHD exon 7 were detected (RhD-negative fetuses). Absence of all three sequences (female RhD-negative fetuses) was assessed in 33 samples. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. CONCLUSION: The automated DNA extraction procedure coupled with a novel multiplex rt-PCR assay proved accurate, efficient and reliable allowing rapid and high throughput noninvasive determination of fetal sex and RhD status in clinical samples.

Condylomata, cytological abnormalities and human papillomavirus infection in the anal canal in HIV-infected men.

BACKGROUND: Genital infections with low-risk (LR) and high-risk (HR) human papillomavirus (HPV) genotypes are associated with ano-genital condylomata and anal squamous cell cancer. HPV-related pathologies in HIV-infected men are a serious concern. In this study, the prevalence of anal condylomata and their association with cytological abnormalities and HPV infection in the anal canal in HIV-infected men [men who have sex with men (MSM) and heterosexuals] were estimated. METHODS: This was a cross-sectional study based on the first visits of patients in the Can Ruti HIV-positive Men (CARH·MEN) cohort. Anal condylomata were assessed by clinical and proctological examination. Samples from the anal canal were collected for HPV genotyping and cytological diagnoses. RESULTS: A total of 640 HIV-infected men (473 MSM and 167 heterosexuals) were included in the study. The overall prevalence of anal condylomata was 25% [157 of 640; 95% confidence interval (CI) 21-28%]; in MSM it was 28% and in heterosexuals it was 15% [odds ratio (OR) 2.2; 95% CI 1.4-3.5]. In patients with anal condylomata, HPV infection in the anal canal was more prevalent (92% vs. 67% in those without anal condylomata; OR 8.5; 95% CI 3.2-22). This higher HPV prevalence involved at least two HPV genotypes (OR 4.0; 95% CI 2.2-7.1), mainly HR genotypes (OR 3.3; 95% CI 1.7-6.4). Similarly, the cumulative prevalence of HPV-6 and HPV-11 was higher in patients with anal condylomata (63% vs. 19% in those without anal condylomata). Having anal condylomata was associated with higher prevalences of cytological abnormalities (83% vs. 32% in those without anal condylomata; OR 6.9; 95% CI 3.8-12.7) and high-grade squamous intraepithelial lesions (HSILs) (9% vs. 3% in those without anal condylomata; OR 9.0; 95% CI 2.9-28.4) in the anal canal. CONCLUSIONS: HIV-infected men with anal condylomata were at risk of presenting HSILs and harbouring multiple HR HPV infections in the anal canal. Although MSM presented the highest prevalence of anal condylomata, heterosexual men also had a clinically important prevalence. Our findings emphasize the importance of screening and follow-up for condylomata in the anal canal in HIV-infected men.

Short-term anti-HIV activity of the combination of didanosine and hydroxyurea.

The synergistic action of hydroxyurea with some other antiretroviral drugs led us to evaluate the effect of therapy with the combination of didanosine and hydroxyurea in HIV-1-infected patients. We aimed to assess the anti-HIV activity of therapy with this combination by measuring variations in viral load and in CD4 cell counts. We also evaluated the potential side effects of this drug combination in HIV-1-positive patients with advanced disease. A total of 15 HIV-1-seropositive homosexual men with a mean baseline CD4 cell count of 149 cells/mm3 (range: 1-430 cells/mm3) were recruited to the study, and received didanosine (200 mg) plus hydroxyurea (500 mg) twice daily for 12 weeks. Ten patients were didanosine naive and five had previously received didanosine (for > 3 months). The combination therapy was well tolerated, although grade 2-3 alopecia appeared in four patients who had very low CD4 cell counts (< 50 cells/mm3). No significant variation in renal, hepatic and pancreatic functions occurred. A significant reduction in the plasma HIV-1 RNA (> 0.5 logs) was observed in seven of ten patients naive to didanosine after weeks 4 and 12 of the study; five of these patients had a decrease in plasma HIV-1 RNA of > 1.5 logs, with two having a decrease of > 2.0 logs. The viral load became undetectable (below 200 copies/ml) in three patients. The patients whose plasma HIV-1 RNA levels were not significantly reduced by the combination therapy had a higher baseline viral load. CD4 cell counts did not increase significantly in most patients. We observed a better response in those patients who had virus of the non-syncytium-inducing phenotype. In conclusion, hydroxyurea in combination with didanosine was well tolerated and led to a reduction in viral load mainly in patients who were initially naive to didanosine.

Uhl's anomaly associated with pulmonary atresia.

We present a case of Uhl's anomaly associated with atresia of the pulmonary valve. The rarity of this association is noteworthy, only three cases having previously been published. Our case presents the peculiarity of a patient with a normal tricuspid valve.

Histiocytoid hemangioma of the innominate vein.

Primary malignancies originating in large veins are infrequent. Leiomyosarcomas and malignant vascular tumors have been occasionally reported. The recently described histiocytoid hemangioma has a predilection for medium-sized and large vessels. A case is reported of histiocytoid hemangioma arising in the innominate vein and presenting clinically as a mediastinal mass.

Estrogen and progesterone receptors, c-erbB-2, p53, and Bcl-2 in thirty-three invasive micropapillary breast carcinomas.

Tests for estrogen (ER) and progesterone (PR) receptors, c-erbB-2, p53 and Bcl-2 were made on paraffin sections of thirty-three cases of invasive micropapillary carcinoma (MPCa) of the breast. The relations between these proteins and general parameters and the patients' evolution, were analyzed and their statistical significance determined by Fisher's exact test. Follow up was available on twenty one patients of whom thirteen were alive after a mean of sixty months. Tumor size, metastatic nodes, c-erbB-2 and Bcl-2 all showed higher values in the dead patient group, but only nuclear grade and extensive lymphatic vessel invasion (LVI) were statistically significant prognostic factors. Hormone receptors and oncogenes were positive in quite similar figures to those of common breast cancers (NOSCa) and offered supplementary information about differentiation and cell atypia of individual cancers. Accordingly, ER (72.7%), PR (45.4%) and Bcl-2 (69.6%) were directly interrelated and inversely related with nuclear grade, mitotic grade, c-erbB-2 (36.3%) and p53 (12.1%). In conclusion, MPCa is a lymphotropic cancer phenotype whose prognosis can be influenced by known prognostic factors, including molecular. The lack of discriminative power between MPCa and NOSCa of ER, PR, c-erbB-2, p53, and Bcl-2 reinforces the importance of recognizing this particular type of cancer.

[Parachute tricuspid valve associated with Fallot's tetralogy]. / Valvule tricuspide en parachute associée à une tétralogie de Fallot.

Parachute deformity of the tricuspid valve was observed with a severe form of Fallot's Tetralogy with pulmonary atresia in a newborn. Signs of tricuspid stenosis (peripheral oedema, right atrial dilatation, hepatomegaly and conduction defects) were associated with the clinical signs of the principal malformation (severe hypoxaemia). To our knowledge, this is the first reported case of parachute deformity of the tricuspid valve.

[Clinicopathological correlations in the common atrioventricular canal]. / Corrélations clinico-pathologiques dans le canal auriculo-ventriculaire complet.

20 patients with endocardial cushion defects were studied. Partial forms of common atrioventricular canal and those associated with complex congenital malformations were not considered. The right and left ventricular inflow and outflow tracts were measured and average values of inflow tract/outflow tract were calculated for each anatomical form of the disease. We found this ratio to be significantly smaller in Group B2 compared to Group B1 of Enriquez de Salamanca's classification, the first group being considered to be a more severe congenital malformation than the second. The inflow tract/outflow tract ratio of the right ventricle ranged between 80% and 50%, with an average of 62.3% in Group B1 compared to 62% and 24% with an average of 49% in Group B2. Using Studert's test, this difference was shown to be statistically significant (p = 0.02). On the other hand, there was no correlation between the other parameters or between the severity of the anatomical lesions and the clinical condition. The reduction of the right ventricular inflow tract may give an indication as to the type of atrioventricular valve which may be present.

[Right ventricle with double outlet and intact interventricular septum. Report of 1 case and review of the literature]. / Ventricule droit à double issue avec septum interventriculaire intact. Présentation d'un cas et révision de la littérature.

Double outlet right ventricle with an intact interventricular septum is an exceedingly rare malformation. Only six other cases have been found in the world literature. The clinical, electrocardiographic, radiological and anatomical features of this condition were reviewed on the occasion of the diagnosis of a new case. Cardiac catheterisation and angiography performed in only one other case is not definitive in the presence of mitral atresia.

[Human papillomavirus and human immunodeficiency virus infections as risk factors for cervix cancer in women prisoners]. / Infección por los virus del papiloma humano y de la inmunodeficiencia humana como factores de riesgo para el cáncer de cuello uterino en mujeres reclusas.

BACKGROUND: The identification of high risk groups for genital human papillomavirus (HPV) infection may contribute to cervical cancer prevention. The study was designed to estimate the prevalence of HPV infection and the related risk of cervical cancer among imprisoned women. PATIENTS AND METHODS: 157 women were visited at the Medical Office of a prison in Barcelona, Spain. Women underwent a structured interview, determination of HIV serostatus and detection of HPV cervical infection by means of PCR. RESULTS: The prevalence of HPV infection was 46%. Prostitution was reported by 38.2% and intravenous drug use by 64.3%. HIV infection was detected in 56.1%. Cervical cytology revealed 19 women with ASCUS and 28 with squamous intraepithelial lesions (SIL) (all grades). HPV infection was associated with an increased risk of intravenous drug use for more than 10 years (prevalence odds ratio [POR] = 2.9) and seropositivity to HIV (POR = 4.7). The increase in risk for SIL related to HIV was explained by the presence of HPV. HIV positive women with low CD4 counts may increase the risk for SIL independently of HPV. CONCLUSION: HIV positive women are at high risk for HPV infection and as a consequence, for developing SIL. HIV positive women should be closely monitored for cervical cancer.

Oral human papillomavirus type-specific infection in HIV-infected men: a prospective cohort study among men who have sex with men and heterosexual men.

The natural history of type-specific oral infection of human papillomavirus (HPV) was assessed in a cohort of HIV-infected men (538 men who have sex with men (MSM); 195 heterosexuals). Risk factors associated with oral HPV infections were examined. The overall prevalence of HPV was 16%: HPV-16 was the most prevalent type (3.7% MSM; 7.8% heterosexuals). The prevalence of HPV-16 in heterosexuals was associated with CD4 nadir counts <200 cells/µL (ORadjusted = 3.0, 95% CI, 1.4-6.3). The overall incidence of HPV was similar between groups (11%), but the incidence of HPV-16 was higher in heterosexuals (ORadjusted = 3.2, 95% CI, 1.1-9.5). Not only MSM but also HIV-infected heterosexual men are at risk of HPV infection. Regular and careful oral inspection is needed.

Evaluation of sample stability and automated DNA extraction for fetal sex determination using cell-free fetal DNA in maternal plasma.

OBJECTIVE: The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. METHODS: A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. RESULTS: Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. CONCLUSION: We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

Circumcision and penile human papillomavirus prevalence in human immunodeficiency virus-infected men: heterosexual and men who have sex with men.

Male circumcision is associated with a lower risk of penile human papillomavirus (HPV) infection in human immunodeficiency virus (HIV) uninfected men. Few studies have evaluated the role of male circumcision in penile HPV infection in HIV-infected men. The aim of this cross-sectional study was to examine the association between male circumcision and the prevalence of penile HPV infection among HIV-infected men-both men who have sex with men (MSM) and heterosexual men. Samples from 706 consecutive men included in the CARH-MEN cohort (overall 24% circumcised: 26% of MSM, 18% of heterosexual men) were examined by Multiplex-PCR. In the overall group (all HIV-infected men included), the prevalence of any penile HPV infection was 22% in circumcised men and 27% in uncircumcised men (OR = 1.0, 95% CI 0.6-1.6, adjusted analysis). In the circumcised group the overall prevalence of HPV infection was 22% in MSM and 24% in the heterosexual men, whereas in the uncircumcised group the prevalence was 26% and 28%, respectively. The prevalence of high-risk HPV types tended to be lower in the circumcised MSM (14% vs 21%, OR = 0.6, 95% CI 0.3-1.1, p 0.088), but it was similar in the heterosexual men (18% in circumcised vs 20% in uncircumcised). These results suggest that male circumcision may be associated with a lower prevalence of oncogenic high-risk penile HPV infection in HIV-infected MSM.

Pixelated CdTe detectors to overcome intrinsic limitations of crystal based positron emission mammographs.

A positron emission mammograph (PEM) is an organ dedicated positron emission tomography (PET) scanner for breast cancer detection. State-of-the-art PEMs employing scintillating crystals as detection medium can provide metabolic images of the breast with significantly higher sensitivity and specificity with respect to standard whole body PET scanners. Over the past few years, crystal PEMs have dramatically increased their importance in the diagnosis and treatment of early stage breast cancer. Nevertheless, designs based on scintillators are characterized by an intrinsic deficiency of the depth of interaction (DOI) information from relatively thick crystals constraining the size of the smallest detectable tumor. This work shows how to overcome such intrinsic limitation by substituting scintillating crystals with pixelated CdTe detectors. The proposed novel design is developed within the Voxel Imaging PET (VIP) Pathfinder project and evaluated via Monte Carlo simulation. The volumetric spatial resolution of the VIP-PEM is expected to be up to 6 times better than standard commercial devices with a point spread function of 1 mm full width at half maximum (FWHM) in all directions. Pixelated CdTe detectors can also provide an energy resolution as low as 1.5% FWHM at 511 keV for a virtually pure signal with negligible contribution from scattered events.

Validation of a small-animal PET simulation using GAMOS: a GEANT4-based framework.

Monte Carlo-based modelling is a powerful tool to help in the design and optimization of positron emission tomography (PET) systems. The performance of these systems depends on several parameters, such as detector physical characteristics, shielding or electronics, whose effects can be studied on the basis of realistic simulated data. The aim of this paper is to validate a comprehensive study of the Raytest ClearPET small-animal PET scanner using a new Monte Carlo simulation platform which has been developed at CIEMAT (Madrid, Spain), called GAMOS (GEANT4-based Architecture for Medicine-Oriented Simulations). This toolkit, based on the GEANT4 code, was originally designed to cover multiple applications in the field of medical physics from radiotherapy to nuclear medicine, but has since been applied by some of its users in other fields of physics, such as neutron shielding, space physics, high energy physics, etc. Our simulation model includes the relevant characteristics of the ClearPET system, namely, the double layer of scintillator crystals in phoswich configuration, the rotating gantry, the presence of intrinsic radioactivity in the crystals or the storage of single events for an off-line coincidence sorting. Simulated results are contrasted with experimental acquisitions including studies of spatial resolution, sensitivity, scatter fraction and count rates in accordance with the National Electrical Manufacturers Association (NEMA) NU 4-2008 protocol. Spatial resolution results showed a discrepancy between simulated and measured values equal to 8.4% (with a maximum FWHM difference over all measurement directions of 0.5 mm). Sensitivity results differ less than 1% for a 250-750 keV energy window. Simulated and measured count rates agree well within a wide range of activities, including under electronic saturation of the system (the measured peak of total coincidences, for the mouse-sized phantom, was 250.8 kcps reached at 0.95 MBq mL(-1) and the simulated peak was 247.1 kcps at 0.87 MBq mL(-1)). Agreement better than 3% was obtained in the scatter fraction comparison study. We also measured and simulated a mini-Derenzo phantom obtaining images with similar quality using iterative reconstruction methods. We concluded that the overall performance of the simulation showed good agreement with the measured results and validates the GAMOS package for PET applications. Furthermore, its ease of use and flexibility recommends it as an excellent tool to optimize design features or image reconstruction techniques.

New molecular method for the detection of human papillomavirus type 16 integration.

Human papillomavirus (HPV) infection is the cause of cervical cancer. Integration of HPV-16 DNA in cervical cells is considered to be a key event in the progression towards invasive cancer, but little is known about this event in anal carcinogenesis. The integration could be a useful biomarker for cancer progression. Optimized assays are needed to determine the value of real-time detection of HPV integration in longitudinal studies, and this approach is only possible with a high-throughput assay. The aim of this study was to develop a new multiplex real-time PCR assay based on simultaneous amplification of the E2 and E6 HPV open reading frames (ORFs) in order to assess the physical status (episomal and/or integrated) of HPV-16 in anal cells of HIV-positive men. The comparative threshold (Ct) cycle values for E2 and E6 obtained for SiHA cells and artificial mixtures of episomal and integrated DNA were as expected: similar Ct for episomal forms and absence of E2 amplification for integrated forms. The multiplex real-time PCR was tested in 77 consecutive samples from individual HIV-infected patients with HPV-16 anal infection. The integration of HPV-16 was detected in 25 (32%) patients: 23 as mixed (episomal and integrated) and two as completed integrated forms. The integration occurs in the early stage of anal lesions and was associated with the severity of the lesions (p 0.004). The multiplex real-time PCR assay developed in the course of this study was shown to be a simple, sensitive, specific and inexpensive technique which may be applied routinely to detect HPV-16 integration.

"European Commission Initiative on Breast Cancer": recomendaciones seleccionadas de cribado de cáncer de mama de las guías europeas / "European Commission Initiative on Breast Cancer": selected breast cancer screening recommendations from the European guidelines

OBJETIVO: La European Commission Initiative on Breast Cancer (ECIBC) ha desarrollado nuevas recomendaciones sobre cribado y diagnóstico de cáncer de mama. El objetivo de este trabajo fue adaptar dichas recomendaciones al castellano con el fin de implementar y garantizar la calidad y el éxito de los programas de cribado de cáncer de mama (PCCM) en todo el territorio español. MÉTODOS: Las nuevas Guías Europeas sobre Cribado y Diagnóstico de Cáncer de Mama han sido elaboradas por un panel multidisciplinar de expertos y pacientes (Guidelines Development Group, GDG). Las recomendaciones que se incluyen se apoyan en revisiones sistemáticas de la evidencia realizadas por un equipo de investigadores del Centro Cochrane Iberoamericano. Para su elaboración se aplicaron las reglas de manejo de conflicto de intereses de la Comisión Europea y se utilizó la metodología GRADE (Grading of Recommendations Assessment, Development and Evaluation). Se utilizaron los marcos de la evidencia a la decisión (EtD) de GRADE para minimizar posibles influencias de intereses en las recomendaciones. RESULTADOS: Como resultado de las revisiones sistemáticas realizadas, el GDG publicó en la web de la ECIBC una lista de recomendaciones como parte de las guías para el cribado y diagnóstico del cáncer de mama, que fueron traducidas al castellano en este trabajo. CONCLUSIONES: La adaptación al castellano de las nuevas recomendaciones ayuda a su implementación y a crear un PCCM uniforme en todo el territorio español. Todo ello mejora la toma de decisiones informadas y el éxito de los PCCM

OBJECTIVE: The European Commission Initiative on Breast Cancer (ECIBC) has developed new recommendations on breast cancer screening and diagnosis. The objective of this work was to adapt these recommendations to Spanish in order to implement and guarantee the quality and success of breast cancer screening programmes (PCCM) throughout the Spanish territory. METHODS: The new European Guidelines on Screening and Diagnosis of Breast Cancer have been prepared by a multidisciplinary panel of experts and patients (Guidelines Development Group, GDG). The recommendations inclu-ded are supported by systematic reviews of the evidence conducted by a team of researchers from the Ibero-American Cochrane Center. For its preparation, the European Commission's conflict of interest management rules were applied and the GRADE (Grading of Recommendations Assessment, Development and Evaluation) methodology was used. The GRADE evidence-to-decision (EtD) frameworks were used to minimize potential influence of interests on the recommendations. RESULTS: As a result of the systematic reviews carried out, the GDG published on the ECIBC website a list of recommendations as part of the guidelines for the screening and diagnosis of breast cancer, which were translated into Spanish in this work. CONCLUSIONS: The adaptation to Spanish of the new recommendations helps their implementation and the creation of a uniform PCCM throughout the Spanish territory. All of this improves informed decision making and the success of PCCM

Human papillomavirus HPV-16, 18, 52 and 58 integration in cervical cells of HIV-1-infected women.

BACKGROUND: Genomic integration of high-risk human papillomavirus into the cellular genome is considered an important event in the pathogenesis of cervical cancer related to the progression from premalignant cervical lesions to invasive cervical carcinoma. OBJECTIVE: This cross-sectional study was aimed to characterize the viral integration of HPV-16, HPV-18, HPV-52 and HPV-58 in cervical cells. STUDY DESIGN: HPV genotypes were determined by PCR and HPV integration by multiplex PCR in HIV-1-infected women without a background of HPV-related pathology. RESULTS: This study included 251 cervical cells samples of consecutive HIV-positive women who were visited between 1999 and 2003. The overall prevalence of HPV infection was 53% (133/251, 95%CI: 47-59%). The most prevalent genotypes were HPV-16 (27%), HPV-33 (15%), HPV-52 (8%) and HPV-58 (8%). The prevalence of abnormal cervical cytology was 33% (83/251, 95%CI: 27-39%). The overall prevalence of HPV integration was 11% (27/251, 95%CI: 7-15%), and the prevalence of HPV-16 integration was 33% (22/67, 95%CI: 22-45%), HPV-18 integration was 30% (3/10, 95%CI: 7-65%) and HPV-52 integration was 10% (2/19, 95%CI: 1-32%). No HPV-58 integration was detected. The percentage of HPV-16 and HPV-18 integration increased with the severity of the cervical lesions, HPV-16 integration was almost 70% and HPV-18 integration was 50% in high-grade squamous intraepithelial lesions. Integration was the most important risk factor associated with cervical dysplasia (OR=30.6, 95%CI: 3.5-270.6). CONCLUSION: HPV integration might represent a good biomarker of the evolution from HPV infection to cervical cancer. Further prospective studies are required to validate our findings.