Raw milk cheeses from Beira Baixa, Portugal-A contributive study for the microbiological hygiene and safety assessment.

Due to specific bacterial microbiota, raw milk cheeses have appreciated sensory properties. However, they may pose a threat to consumer safety due to potential pathogens presence. This study evaluated the microbiological contamination of 98 raw milk cheeses from Beira Baixa, Portugal. Presence and enumeration of Coagulase Positive Staphylococci (CPS), Listeria monocytogenes, Salmonella spp., pathogenic Escherichia coli, and indicator microorganisms (non-pathogenic E. coli and Listeria spp.) was attained. E. coli antimicrobial resistance (AMR) was also evaluated. PCR and/or Whole genome sequencing (WGS) was used to characterize E. coli, Salmonella spp. and L. monocytogenes isolates. Sixteen cheeses (16.3%) were classified as Satisfactory, 59 (60.2%) as Borderline and 23 (23.5%) as Unsatisfactory/Potential Injurious to Health. L. monocytogenes, CPS > 104 cfu g-1, Extraintestinal pathogenic E. coli (ExPEC) and Salmonella spp. were detected in 4.1%, 6.1%, 3.1% and 1.0% of the samples, respectively. Listeria innocua (4.1%) and E. coli > 104 cfu g-1 (16.3%) were also detected. AMR E. coli was detected in 23/98 (23.5%) of the cheese samples, of which two were multidrug resistant. WGS identified genotypes already associated to human disease and Listeria spp. cluster analysis indicated that cheese contamination might be related with noncompliance with Good Hygiene Practices during cheese production.

Unveiling a Listeria monocytogenes Outbreak in a Rabbit Farm: Clinical Manifestation, Antimicrobial Resistance, Genomic Insights and Environmental Investigation.

Listeria monocytogenes poses a threat to both human and animal health. This work describes an L. monocytogenes outbreak in a Portuguese rabbit farm, detailing the isolates' clinical manifestations, necropsy findings, and phenotypic and genomic profiles. Clinical signs, exclusively observed in does, included lethargy and reproductive signs. Post-mortem examination of does revealed splenomegaly, hepatomegaly with a reticular pattern, pulmonary congestion, and haemorrhagic lesions in the uterus, with thickening of the uterine wall and purulent greyish exudates. Positive L. monocytogenes samples were identified in fattening and maternity units across different samples, encompassing does and environmental samples. Core-genome Multi Locus Sequence Typing (cgMLST) analysis confirmed the outbreak, with the 16 sequenced isolates (lineage II, CC31, and ST325) clustering within a ≤2 allelic difference (AD) threshold. Antimicrobial susceptibility testing for five antibiotics revealed that 15 out of 19 outbreak isolates were resistant to sulfamethoxazole-trimethoprim (SXT). Concordantly, all SXT-resistant sequenced isolates were found to exclusively harbour a plasmid containing a trimethoprim-resistance gene (dfrD), along with loci linked to resistance to lincosamides (lnuG), macrolides (mphB), and polyether ionophores (NarAB operon). All sequenced outbreak isolates carried the antibiotic resistance-related genes tetM, fosX, lin, norB, lmrB, sul, and mprF. The outbreak cluster comprises isolates from does and the environment, which underscores the ubiquitous presence of L. monocytogenes and emphasizes the importance of biosecurity measures. Despite limited data on listeriosis in rabbit farming, this outbreak reveals its significant impact on animal welfare and production.

Genetic Diversity of Salmonella enterica subsp. enterica Serovar Enteritidis from Human and Non-Human Sources in Portugal.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the leading causes of foodborne infections associated with broilers and laying hens. Portugal has had the lowest notification rates of salmonellosis in recent years, due to the vaccinations of layer and breeder flocks and strict compliance with biosecurity measures. However, data about the genetic diversity of S. Enteritidis in Portugal are scarce. In this study, 102 S. Enteritidis isolates selected from human (n = 63) and non-human sources (n = 39) were characterized by serotyping, antimicrobial susceptibility, and whole genome sequencing. The S. Enteritidis population was mainly resistant to fluoroquinolones, and a sole isolate showed resistance to extended-spectrum cephalosporins. ST11 was the most frequent sequence type, and three novel STs from human isolates (ST9236, ST4457, and ST9995) were assigned. Several Salmonella pathogenic islands (SPI) and Putative SPI were present in the genomes, namely SPI-1, 2, 3, 4, 5, 9, 10, 12, 13, and 14, C63PI, CS54_island, and 170 virulence genes were identified. The phylogenetic analysis revealed that strains from Portugal are genetically heterogeneous regarding sample type, collection date, and genetic content. This study increases the available data, essential to a better characterization of strains in a global context.

Occurrence of Multidrug-Resistant Bacteria Resulting from the Selective Pressure of Antibiotics: A Comprehensive Analysis of ESBL K. pneumoniae and MRSP Isolated in a Dog with Rhinorrhea.

Because of public health concerns, much greater scrutiny is now placed on antibiotic use in pets, especially for antimicrobial agents that have human analogs. Therefore, this study aimed to characterize the phenotypic and genotypic profiles of multidrug-resistant bacteria isolated from nasal swabs samples taken from a one-year-old male Serra da Estrela dog with rhinorrhea that was treated with amikacin. An extended-spectrum ß-lactamases (ESBL) Klebsiella pneumoniae was isolated in the first sample taken from the left nasal cavity of the dog. Seven days later, methicillin-resistant (MRSP) Staphylococcus pseudintermedius was also isolated. Nevertheless, no alterations to the therapeutic protocol were performed. Once the inhibitory action of the antibiotic disappeared, the competitive advantage of the amikacin-resistant MRSP was lost, and only commensal flora was observed on both nasal cavities. The genotypic profile of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae revealed the same characteristics and close relation to other strains, mainly from Estonia, Slovakia and Romania. Regarding MRSP isolates, although resistance to aminoglycosides was present in the first MRSP, the second isolate carried aac(6')-aph(2″), which enhanced its resistance to amikacin. However, the veterinary action was focused on the treatment of the primary agent (ESBL K. pneumoniae), and the antibiotic applied was according to its phenotypic profile, which may have led to the resolution of the infectious process. Therefore, this study highlights the importance of targeted therapy, proper clinical practice and laboratory-hospital communication to safeguard animal, human and environmental health.

Building an International One Health Strain Level Database to Characterise the Epidemiology of AMR Threats: ESBL-AmpC Producing E. coli as An Example-Challenges and Perspectives.

Antimicrobial resistance (AMR) is one of the top public health threats nowadays. Among the most important AMR pathogens, Escherichia coli resistant to extended spectrum cephalosporins (ESC-EC) is a perfect example of the One Health problem due to its global distribution in animal, human, and environmental sources and its resistant phenotype, derived from the carriage of plasmid-borne extended-spectrum and AmpC ß-lactamases, which limits the choice of effective antimicrobial therapies. The epidemiology of ESC-EC infection is complex as a result of the multiple possible sources involved in its transmission, and its study would require databases ideally comprising information from animal (livestock, companion, wildlife), human, and environmental sources. Here, we present the steps taken to assemble a database with phenotypic and genetic information on 10,763 ESC-EC isolates retrieved from multiple sources provided by 13 partners located in eight European countries, in the frame of the DiSCoVeR Joint Research project funded by the One Health European Joint Programme (OH-EJP), along with its strengths and limitations. This database represents a first step to help in the assessment of different geographical and temporal trends and transmission dynamics in animals and humans. The work performed highlights aspects that should be considered in future international efforts, such as the one presented here.

Listeria monocytogenes, Escherichia coli and Coagulase Positive Staphylococci in Cured Raw Milk Cheese from Alentejo Region, Portugal.

Traditional cheeses are part of the Portuguese gastronomic identity, and raw milk of autochthonous species is a common primary ingredient. Here, we investigated the presence of Listeria monocytogenes, Coagulase Positive Staphylococci (CPS) and pathogenic Escherichia coli, as well as of indicator microorganisms (E. coli and other Listeria spp.) in 96 cured raw milk cheeses from the Alentejo region. Whole genome sequencing (WGS) of pathogenic E. coli and Listeria spp. as well as antimicrobial resistance (AMR) screening of E. coli isolates was also performed. L. monocytogenes, CPS > 104 cfu/g and Extraintestinal E. coli were detected in 15.6%, 16.9% and 10.1% of the samples, respectively. Moreover, L. monocytogenes > 102 cfu/g and Staphylococcal enterotoxins were detected in 4.2% and 2.2% of the samples, respectively. AMR was observed in 27.3% of the E. coli isolates, six of which were multidrug resistant. WGS analysis unveiled clusters of high closely related isolates for both L. monocytogenes and L. innocua (often correlating with the cheese producer). This study can indicate poor hygiene practices during milk collection/preservation or during cheese-making procedures and handling, and highlights the need of more effective prevention and control measures and of multi-sectoral WGS data integration, in order to prevent and detect foodborne bacterial outbreaks.

Gulls in Porto Coastline as Reservoirs for Salmonella spp.: Findings from 2008 and 2023.

Gulls act as intermediaries in the exchange of microorganisms between the environment and human settlements, including Salmonella spp. This study assessed the antimicrobial resistance and molecular profiles of Salmonella spp. isolates obtained from fecal samples of gulls in the city of Porto, Portugal, in 2008 and 2023 and from water samples in 2023. Antimicrobial susceptibility profiling revealed an improvement in the prevalence (71% to 17%) and antimicrobial resistance between the two collection dates. Two isolate collections from both 2008 and 2023 underwent serotyping and whole-genome sequencing, revealing genotypic changes, including an increased frequency in the monophasic variant of S. Typhimurium. qacE was identified in 2008 and 2023 in both water and fecal samples, with most isolates exhibiting an MDR profile. The most frequently observed plasmid types were IncF in 2008 (23%), while IncQ1 predominated in 2023 (43%). Findings suggest that Salmonella spp. circulate between humans, animals, and the environment. However, the genetic heterogeneity among the isolates from the gulls' feces and the surface water may indicate a complex ecological and evolutionary dynamic shaped by changing conditions. The observed improvements are likely due to measures to reduce biological contamination and antimicrobial resistance. Nevertheless, additional strategies must be implemented to reduce the public health risk modeled by the dissemination of pathogens by gulls.

Pathogenic Escherichia coli, Salmonella spp. and Campylobacter spp. in Two Natural Conservation Centers of Wildlife in Portugal: Genotypic and Phenotypic Characterization.

Human-wildlife coexistence may increase the potential risk of direct transmission of emergent or re-emergent zoonotic pathogens to humans. Intending to assess the occurrence of three important foodborne pathogens in wild animals of two wildlife conservation centers in Portugal, we investigated 132 fecal samples for the presence of Escherichia coli (Shiga toxin-producing E. coli (STEC) and non-STEC), Salmonella spp. and Campylobacter spp. A genotypic search for genes having virulence and antimicrobial resistance (AMR) was performed by means of PCR and Whole-Genome Sequencing (WGS) and phenotypic (serotyping and AMR profiles) characterization. Overall, 62 samples tested positive for at least one of these species: 27.3% for STEC, 11.4% for non-STEC, 3.0% for Salmonella spp. and 6.8% for Campylobacter spp. AMR was detected in four E. coli isolates and the only Campylobacter coli isolated in this study. WGS analysis revealed that 57.7% (30/52) of pathogenic E. coli integrated genetic clusters of highly closely related isolates (often involving different animal species), supporting the circulation and transmission of different pathogenic E. coli strains in the studied areas. These results support the idea that the health of humans, animals and ecosystems are interconnected, reinforcing the importance of a One Health approach to better monitor and control public health threats.

Hygiene Indicators and Salmonellae on Surfaces of Swine Carcasses from Two Slaughterhouses in Northern Portugal.

ABSTRACT: The monitoring of carcass surface contamination along the slaughter line enables verification of slaughter operation hygiene and the use of good manufacturing practices. Pork meat is a common source of human nontyphoidal salmonellosis, one of the most frequently reported foodborne illnesses worldwide. This study was conducted to gather data on microbial loads before and after evisceration on the surfaces of swine carcasses in two slaughterhouses. The presence of Salmonella enterica was evaluated only after evisceration on carcass surfaces and in livers and floor drains (environmental samples) because pigs are common carriers of this pathogen. The contamination of carcass surfaces was evaluated by delimitation of surface area with sterilized templates (100 cm2), and surface samples were collected with gauze swabs. Total aerobic mesophilic bacteria, Enterobacteriaceae, and Escherichia coli were enumerated. Significant differences (P < 0.05) in counts of mesophilic bacteria, Enterobacteriaceae, and E. coli were found on the external carcass surfaces, with higher counts after evisceration. The neck and abdominal areas had higher levels of mesophilic bacteria, Enterobacteriaceae, and E. coli and a high prevalence of Salmonella. Salmonella was detected in only one of the studied slaughterhouses; 19 (7.3%) of 259 analyzed carcass samples were positive for Salmonella, and Salmonella was detected in two livers and two floor drains. A total of 52 Salmonella isolates (44 from carcasses, 5 from livers, and 3 from drains) were recovered. Three Salmonella serovars (Typhimurium 4,5:i- , Wernigerone, and Derby) were identified, and 53.8% of the 52 isolates were multidrug resistant. The results reveal the need for continuous improvement of slaughtering operations and implementation of good manufacturing practices to ensure the safety of pork produced in Portugal.

Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland Portugal.

Birds are potential carriers of pathogens affecting humans and agriculture. Aiming to evaluate the occurrence of the top three most important foodborne pathogens in free-living birds in Portugal, we investigated 108 individual fecal samples from free-living birds and one pooled sample of gull feces (n = 50) for the presence of Escherichia coli (pathogenic and non-pathogenic), Salmonella spp. and Campylobacter spp. Virulence- and antimicrobial resistance- (AMR) associated genes were detected by PCR and Whole-Genome Sequencing (WGS), and phenotypic (serotyping and AMR profiles) characterization was performed. Overall, 8.9% of samples tested positive for pathogenic E. coli, 2.8% for Salmonella spp., and 9.9% for Campylobacter spp. AMR was performed on all pathogenic isolates and in a fraction of non-pathogenic E. coli, being detected in 25.9% of them. Ten of the tested E. coli isolates were multidrug-resistant (MDR), and seven of them were Extended-spectrum ß-lactamase (ESBL) producers. Among Salmonella (n = 3) and Campylobacter (n = 9), only one strain of C. jejuni was identified as MDR. Most of the identified serotypes/sequence types had already been found to be associated with human disease. These results show that free-living birds in Portugal may act as carriers of foodborne pathogens linked to human disease, some of them resistant to critically important antimicrobials.

Characterization of Multidrug-Resistant Isolates of Salmonella enterica Serovars Heidelberg and Minnesota from Fresh Poultry Meat Imported to Portugal.

Salmonella enterica serovars Heidelberg and Minnesota frequently display several genetic mobile elements making them potential spreaders of resistance genes. Here, we phenotypically determined the antibiotic resistance profile and subsequently performed whole-genome sequencing on 36 isolates recovered from samples of fresh poultry meat, within the Portuguese Official Inspection Plan for Imported Foodstuffs. Several isolates of both serovars showed high genetic relatedness either with isolates from raw poultry meat imported to the Netherlands from Brazil or with isolates from samples from the broiler production chain in Brazil. The multidrug-resistant (MDR) character was common to the vast majority (94.4%) of isolates from both serovars, and several isolates carried the plasmid IncA/C2 containing the ß-lactamase gene blaCMY-2 and IncX1 containing a type IV secretion system. These results somehow mirror the scenario observed in the Netherlands, showing the introduction, through fresh imported poultry meat in compliance with European legislation, of MDR Salmonella enterica serovars Heidelberg and Minnesota in Europe, with the potential spread of resistance markers. These data suggest the need to revise the hygiene criteria for foodstuffs monitoring before its placement on the market, with the determination of the resistome being an invaluable contribute to limit the dissemination of resistance markers.

High Prevalence of Human Papillomavirus on Anal and Oral Samples from Men and Women with External Anogenital Warts: The HERCOLES Study.

Human papillomavirus (HPV) infection is highly prevalent in the sexually active population. This study estimates the prevalence of HPV DNA in anal and oral samples from a cohort of men and women with incident anogenital warts. Anal and/or oral samples from 541 patients with anogenital warts were tested for 35 HPV genotypes using a PCR assay. The overall prevalence of anal HPV and oral HPV DNA was 59.9% (n = 305/509; 95% confidence interval (CI) 55.6-64.1%) and 14.5% (n = 78/538; 95% CI 11.8-17.7%), respectively. Among patients with perianal warts, the anal HPV DNA prevalence was 92.3% (95% CI 87.0-95.5%). Anal HPV DNA prevalence in patients with genital warts but no perianal warts was 55.7% (95% CI 50.6-60.7%). Both anal and oral HPV infections were more common in men who have sex with men than in heterosexual men (90.4% versus 38.5% and 20.8% versus 11.8%, respectively). Anal high risk-HPV infection was more common in women (58.8%) and in men who have sex with men (67.7%). We found that anogenital warts represent a clinical marker for both anal and oral HPV infections, including anal high risk-HPV infections, particularly among women and men who have sex with men.

Budget impact analysis of cervical cancer screening in Portugal: comparison of cytology and primary HPV screening strategies.

BACKGROUND: Primary Human Papilloma Virus (HPV) testing is the currently recommended cervical cancer (CxCa) screening strategy by the Portuguese Society of Gynecology (SPG) clinical consensus. However, primary HPV testing has not yet been adopted by the Portuguese organized screening programs. This modelling study compares clinical benefits and costs of replacing the current practice, namely cytology with ASCUS HPV triage, with 2 comparative strategies: 1) HPV (pooled) test with cytology triage, or 2) HPV test with 16/18 genotyping and cytology triage, in organized CxCa screenings in Portugal. METHODS: A budget impact model compares screening performance, clinical outcomes and budget impact of the 3 screening strategies. A hypothetical cohort of 2,078,039 Portuguese women aged 25-64 years old women is followed for two screening cycles. Screening intervals are 3 years for cytology and 5 years for the HPV strategies. Model inputs include epidemiological, test performance and medical cost data. Clinical impacts are assessed with the numbers of CIN2-3 and CxCa detected. Annual costs, budget impact and cost of detecting one CIN2+ were calculated from a public healthcare payer's perspective. RESULTS: HPV testing with HPV16/18 genotyping and cytology triage (comparator 2) shows the best clinical outcomes at the same cost as comparator 1 and is the most cost-effective CxCa screening strategy in the Portuguese context. Compared to screening with cytology, it would reduce annual CxCa incidence from 9.3 to 5.3 per 100,000, and CxCa mortality from 2.7 to 1.1 per 100,000. Further, it generates substantial cost savings by reducing the annual costs by €9.16 million (- 24%). The cost of detecting CIN2+ decreases from the current €15,845 to €12,795. On the other hand, HPV (pooled) test with cytology triage (comparator 1) reduces annual incidence of CxCa to 6.9 per 100,000 and CxCa mortality to 1.6 per 100,000, with a cost of €13,227 per CIN2+ detected with annual savings of €9.36 million (- 24%). The savings are mainly caused by increasing the length of routine screening intervals from three to five years. CONCLUSION: The results support current clinical recommendations to replace cytology with HPV with 16/18 genotyping with cytology triage as screening algorithm.

Multidrug-Resistant Salmonella enterica Serovar Rissen Clusters Detected in Azores Archipelago, Portugal.

Gastrointestinal infections caused by nontyphoidal Salmonella (NTS) remain one of the main causes of foodborne illness worldwide. Within the multiple existing Salmonella enterica serovars, the serovar Rissen is rarely reported, particularly as a cause of human salmonellosis. Between 2015 and 2017, the Portuguese National Reference Laboratory of Gastrointestinal Infections observed an increase in the number of clinical cases caused by multidrug-resistant (MDR) S. enterica serovar Rissen, particularly from the Azores archipelago. In the present study, we analyzed by whole genome sequencing (WGS) all clinical, animal, food, and environmental isolates received up to 2017 in the Portuguese Reference Laboratories. As such, through a wgMLST-based gene-by-gene analysis, we aimed to identify potential epidemiological clusters linking clinical and samples from multiple sources, while gaining insight into the genetic diversity of S. enterica serovar Rissen. We also investigated the genetic basis driving the observed multidrug resistance. By integrating 60 novel genomes with all publicly available serovar Rissen genomes, we observed a low degree of genetic diversity within this serovar. Nevertheless, the majority of Portuguese isolates showed high degree of genetic relatedness and a potential link to pork production. An in-depth analysis of these isolates revealed the existence of two major clusters from the Azores archipelago composed of MDR isolates, most of which were resistant to at least five antimicrobials. Considering the well-known spread of MDR between gastrointestinal bacteria, the identification of MDR circulating clones should constitute an alert to public health authorities. Finally, this study constitutes the starting point for the implementation of the "One Health" approach for Salmonella surveillance in Portugal.

Understanding differences in cervical cancer incidence in Western Europe: comparing Portugal and England.

Background: Cervical cancer incidence has decreased over time in England particularly after the introduction of organized screening. In Portugal, where opportunistic screening has been widely available with only slightly lower coverage than that of the organized programme in England, rates of cervical cancer have been higher than in England. We compared the burden of cervical cancer, risk factors and preventive interventions over time in both countries, to identify elements hindering the further decline in incidence and mortality in Portugal. Methods: We used joinpoint regression to identify significant changes in rate time-trends. We also analyzed individual-level Portuguese data on sexual behaviour and human papillomavirus prevalence, and recent aggregate data on organized and opportunistic screening coverage. We compared published estimates of survival, risk factors and historical screening coverage for both countries. Results: Despite stable incidence, cervical cancer mortality has declined in both countries in the last decade. The burden has been 4 cases and 1 death per 100 000 women annually higher in Portugal than in England. Differences in human papillomavirus prevalence and risk factors for infection and disease progression do not explain the difference found in cervical cancer incidence. Significant mortality declines in both countries followed the introduction of different screening policies, although England showed a greater decline than Portugal over nearly 2 decades after centralizing organized screening. Conclusion: The higher rates of cervical cancer in Portugal compared to England can be explained by differences in screening quality and coverage.

Potential impact of nonavalent HPV vaccine in the prevention of high-grade cervical lesions and cervical cancer in Portugal.

OBJECTIVE: To estimate the potential impact of the nonavalent HPV vaccine for high-grade cervical lesions and invasive cervical cancer (ICC) in Portugal. METHODS: The present secondary analysis used data collected in the CLEOPATRE II study on the prevalence of HPV 6/11/16/18/31/33/45/52/58 among female patients aged 20-88 years. The prevalence of HPV types in patients with cervical intraepithelial neoplasia (CIN) grades 2/3 and ICC was examined. RESULTS: Data were included from 582 patients. There were 177, 341, and 64 patients with CIN2, CIN3, and ICC, respectively, and 169 (95.5%), 339 (99.4%), and 62 (96.9) of them had HPV infections. Of patients with HPV infections, HPV 16, 18, 31, 33, 45, 52, and 58 infections were identified in 150 (88.8%), 329 (97.1%), and 60 (96.8%) patients with CIN2, CIN3, and ICC, respectively. HPV genotypes 6, 11, 16, 18, 31, 33, 45, 52, and 58 were identified in 540 (94.7%) of the patients with HPV infections. CONCLUSION: The addition of the five HPV genotypes included in the nonavalent HPV vaccine (HPV 31/33/45/52/58) could result in the new HPV vaccine preventing 94.7% of CIN2/3 and ICC occurrences.

Use of the NucliSENS EasyQ HPV assay in the management of cervical intraepithelial neoplasia.

Persistent infection by high-risk human papillomavirus is a necessary cause for cervical cancer. DNA-based human papillomavirus (HPV) assays show high sensitivity but poor specificity in detecting high-grade cervical lesions. Assays detecting mRNA of the oncoproteins E6 and E7 show higher specificity but lack either detection of all high-risk genotypes or the ability to specify the detected genotypes. The aim of this study was to evaluate the clinical performance of the NucliSENS EasyQ HPV assay in comparison with the Hybrid Capture 2 test (HC2) and the CLART Human Papillomavirus 2 assay (CLART), using a clinical cut-off of cervical intraepithelial neoplasia grade 2 or worse. In the 554 studied women, the lowest HPV positivity rate was detected for NucliSENS EasyQ HPV assay (55.1%), while HC2 and CLART showed similar results (HC2: 77.4%; CLART: 78.0%). In comparison with the other tests, the NucliSENS EasyQ HPV assay showed a lower clinical sensitivity (79.3% vs. 96.4% for HC2 and 95.9% for CLART) but a higher clinical specificity (72.6% vs. 42.8% for HC2 and 42.5% for CLART). Detection of E6/E7 mRNA transcripts may provide a higher specificity for cervical intraepithelial neoplasia grade 2 lesions or worse, since the oncogenic potential of HPV infection depends on the over-expression of these two oncoproteins.

[Biomarkers of cervical carcinogenesis associated with genital human papillomavirus infection]. / Indicadores de Prognóstico da Carcinogénese do Colo do Útero Associada à Infeção por Vírus do Papiloma Humano.

INTRODUCTION/OBJECTIVE: Persistent infection with high-risk human papillomavirus (HPV) types is a necessary cause for cervical cancer development. The aim of this study was to evaluate the significance of different molecular markers for cervical carcinogenesis, and to assess their association with cervical intraepithelial neoplasia. MATERIALS AND METHODS: 378 cervical samples from women attending to primary Health Clinics of the National Health Service and Gynaecological Outpatient Clinics and referred for HPV testing were analyzed between between January 2007 and December 2010. According to cytological diagnosis, five groups were defined: normal, ASCUS, LSIL, HSIL, and ICC. For the determination of viral DNA physical status was performed by using a real-time PCR methodology, over expression of E6/E7 mRNA NASBA amplification was performed with the NucliSENS EasyQ HPV assay and viral load was determined by a real-time PCR. HPV status was studied in relation to lesion severity. Statistical analysis was performed with SPSS software 16.0 and Chi-Square test. RESULTS: No significant statistical differences were found between the physical status of HPV 16 or 18 and lesion severity. Overexpression of E6/E7 mRNA increased with lesion severity. Viral load was significantly associated with the development of cervical intraepithelial lesion. CONCLUSIONS: Data suggests that viral integration for HPV 16 seems to be an early event on cervical carcinogenesis, not being suitable as a molecular marker. E6/E7 mRNA and viral load can be more valuable approaches to use as biomarkers in the prevention of cervical cancer development.

Introdução/Objetivos: A infeção persistente pelo Vírus do Papiloma Humano de alto risco (HPVar) é considerada como a causa necessária, embora não suficiente, para o desenvolvimento do cancro do colo do útero, sugerindo que outros fatores estarão envolvidos no processo de carcinogénese. Este estudo pretendeu avaliar indicadores de prognóstico da persistência da infeção por HPV, nomeadamente o estado físico e a carga viral dos HPV 16 e 18 e a superexpressão dos transcritos do RNAm dos HPV 16, 18, 31, 33 e 45, num grupo de mulheres com ou sem sintomatologia clínica e citopatológica. Material e Métodos: Foram estudadas 378 alíquotas de células epiteliais congeladas pertencentes a utentes dos centros de saúde do Serviço Nacional de Saúde e de clínicas privadas, referenciadas para teste HPV, entre Janeiro de 2007 e Dezembro de 2010. De acordo com o diagnóstico citopatológico, foram definidos cinco grupos: normal, ASCUS, LSIL, HSIL e carcinoma invasivo do colo do útero. Para a determinação do estado físico do DNA e da carga viral dos HPV 16 e 18 foi utilizada metodologia de PCR em tempo real, e para a superexpressão dos transcritos dos oncogenes E6 e E7 o sistema comercial NucliSENS EasyQ HPV®. Os indicadores foram analisados em associação com os tipos de lesão do colo do útero. Para a análise estatística foi utilizado o o programa informático SPSS versão 16.0 e o teste de Chi-Quadrado. Resultados: Os resultados mostraram ausência de associação estatisticamente significativa entre a gravidade da lesão e o estado físico do DNA dos HPV 16 e 18. A superexpressão dos transcritos do RNAm E6/E7 e a carga viral dos HPV 16 e 18 aumentaram significativamente em função do grau da lesão. Conclusões: Os resultados obtidos sugerem que a determinação do estado físico do DNA dos HPV 16 e 18, isoladamente, não constitui um indicador de prognóstico para o desenvolvimento e progressão das lesões. A superexpressão dos transcritos dos oncogenes E6 e E7 está associada à progressão das lesões do colo do útero e apresenta maior especificidade no diagnóstico precoce das lesões pré-malignas. A quantificação do DNA dos HPVar pode ser um indicador promissor de prognóstico das lesões pré-neoplásicas do colo do útero.