Cervix cytology samples revealed increased methylation of the human markers FAM19A4/miR124-2 up to 8 years before adenocarcinoma.

INTRODUCTION: Methylation analysis of the promoter region of tumor-suppressor genes has previously shown high sensitivity for detection of high-grade cervical intraepithelial neoplasia (CIN) and cancer. HPV-testing has a high sensitivity to identify women at risk to develop cancer, and has been implemented in cervical screening programs in several countries. But in most HPV-positive women the infection will clear and they will not develop cancer. Testing for methylation could help to identify women who have potentially progressive cervical disease and need closer follow-up. The goal of the present study was to investigate the potential use of methylation as a triage test of HPV-positive women in the screening program. MATERIAL AND METHODS: A collection of liquid-based cytology (LBC) samples from 106 women, collected between 4 months and 8 years before histologically confirmed cervical cancer or CIN3, was analyzed for hypermethylation of the human genes FAM19A4 and miR124-2. RESULTS: Methylation was detected in 45% (33/73) of normal LBC samples from women who later developed CIN3+, compared with 10% (3/31) of normal LBC samples from women without subsequent dysplasia (P = 0.0006). Overall, methylation was detected in 39% (14/36), 51% (19/37), 61% (14/23) and 70% (7/10) of LBC samples from women who later developed CIN3, adenocarcinoma in situ (AIS), squamous cell carcinoma (SCC) and adenocarcinoma (ADC), respectively. Positive methylation analysis was not significantly more frequent than abnormal cytology of atypical squamous cells of unclear significance or worse (ASCUS+) in LBC samples collected 4 months to 8 years before SCC or AIS; however, prior to the development of ADC, methylation was observed in 7/10 LBC samples, despite normal cytology. Overall, LBC samples collected before invasive cancer (ADC and SCC) were more frequently positive in the methylation analysis than in cytological analysis of ASCUS+ (P = 0.048). For LBC samples collected more than 2 years before the development of AIS, SCC or ADC, methylation analysis showed a higher positivity rate than cytology did. CONCLUSIONS: Testing for methylation of FAM19A4/miR124-2 as a triage for HPV-positive women would be useful to identify women at risk of cancer development, especially adenocarcinoma. Further studies are needed to estimate the cost-effectiveness before introducing methylation testing in the screening program.

Seroprevalence of tick-borne encephalitis virus and vaccination coverage of tick-borne encephalitis, Sweden, 2018 to 2019.

BackgroundIn Sweden, information on seroprevalence of tick-borne encephalitis virus (TBEV) in the population, including vaccination coverage and infection, is scattered. This is largely due to the absence of a national tick-borne encephalitis (TBE) vaccination registry, scarcity of previous serological studies and use of serological methods not distinguishing between antibodies induced by vaccination and infection. Furthermore, the number of notified TBE cases in Sweden has continued to increase in recent years despite increased vaccination.AimThe aim was to estimate the TBEV seroprevalence in Sweden.MethodsIn 2018 and 2019, 2,700 serum samples from blood donors in nine Swedish regions were analysed using a serological method that can distinguish antibodies induced by vaccination from antibodies elicited by infection. The regions were chosen to reflect differences in notified TBE incidence.ResultsThe overall seroprevalence varied from 9.7% (95% confidence interval (CI): 6.6-13.6%) to 64.0% (95% CI: 58.3-69.4%) between regions. The proportion of vaccinated individuals ranged from 8.7% (95% CI: 5.8-12.6) to 57.0% (95% CI: 51.2-62.6) and of infected from 1.0% (95% CI: 0.2-3.0) to 7.0% (95% CI: 4.5-10.7). Thus, more than 160,000 and 1,600,000 individuals could have been infected by TBEV and vaccinated against TBE, respectively. The mean manifestation index was 3.1%.ConclusionA difference was observed between low- and high-incidence TBE regions, on the overall TBEV seroprevalence and when separated into vaccinated and infected individuals. The estimated incidence and manifestation index argue that a large proportion of TBEV infections are not diagnosed.

The risk for celiac disease after Covid-19 infection.

BACKGROUND: Celiac disease (CD) is an autoimmune disease leading to gastrointestinal symptoms and mineral deficiencies. The pathogenetic mechanisms, besides the clear HLA association, are elusive. Among environmental factors infections have been proposed. Covid-19 infection results in a systemic inflammatory response that often also involves the gastrointestinal tract. The aim of the present study was to investigate whether Covid-19 infection could increase the risk for CD. PATIENTS AND METHODS: All patients, both children and adults, in the county Skåne (1.4 million citizens) in southern Sweden with newly diagnosed biopsy- or serology-verified CD or a positive tissue transglutaminase antibody test (tTG-ab) during 2016-2021 were identified from registries at the Departments of Pathology and Immunology, respectively. Patients with a positive Covid-19 PCR or antigen test in 2020 and 2021 were identified from the Public Health Agency of Sweden. RESULTS: During the Covid-19 pandemic (March 2020 - December 2021), there were 201 050 cases of Covid-19 and 568 patients with biopsy- or serology-verified CD or a first-time positive tTG-ab tests, of which 35 patients had been infected with Covid-19 before CD. The incidence of verified CD and tTG-ab positivity was lower in comparison to before the pandemic (May 2018 - February 2020; 22.5 vs. 25.5 cases per 100 000 person-years, respectively, incidence rate difference (IRD) -3.0, 95% CI -5.7 - -0.3, p = 0.028). The incidence of verified CD and tTG-ab positivity in patients with and without prior Covid-19 infection was 21.1 and 22.4 cases per 100 000 person-years, respectively (IRD - 1.3, 95% CI -8.5-5.9, p = 0.75). CONCLUSIONS: Our results indicate that Covid-19 is not a risk factor for CD development. While gastrointestinal infections seem to be an important part of the CD pathogenesis, respiratory infections probably are of less relevance.

Rapid diagnostic testing for SARS-CoV-2: Validation and comparison of three point-of-care antibody tests.

With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a need for diagnostic tests has surfaced. Point-of-care (POC) antibody tests can detect immunoglobulin (Ig) G and M against SARS-CoV-2 in serum, plasma, or whole blood and give results within 15 min. Validation of the performance of such tests is needed if they are to be used in clinical practice. In this study, we evaluated three POC antibody tests. Convalescent serum samples from 47 reverse transcription-polymerase chain reaction (RT-PCR) verified patients with coronavirus disease 2019 (COVID-19) collected at least 28 days post RT-PCR diagnosis as well as 50 negative pre-COVID-19 controls were tested. The three tests (denoted the J-, N-, and Z-tests) displayed the sensitivities of 87%, 96%, and 85%, respectively, for the detection of IgG. All tests had the same specificity for IgG (98%). The tests did not differ significantly for the detection of IgG. The sensitivities for IgM were lower (15%, 67%, and 70%) and the specificities were 90%, 98%, and 90%, respectively. The positive and negative predictive values were similar among the tests. Our results indicate that these POC antibody tests might be accurate enough to use in routine clinical practice.

Population-based primary HPV mRNA cervical screening compared with cytology screening.

Primary HPV screening for cervical cancer by HPV mRNA testing (Aptima) was implemented in January 2017, for women ≥30 through 70 years, in the Region of Skåne, Sweden. HPV positive samples underwent cytology assessment, and women with any degree of abnormal cytology were referred for colposcopy. The aim was to audit the primary HPV screening program, by comparing the cytology results to those of corresponding women (aged ≥30 through 65 years) screened with conventional cytology during 2016. Overall, HPV was detected among 7.0% (4433/63,055) of the women ≥30-70 years in the primary HPV screening program. Among a co-tested (cytology and HPV) subgroup aged 40-42 years (N = 5039), HPV was detected in 100% (28/28) of high-grade squamous intraepithelial lesions (HSIL) and atypical squamous cells of undetermined significance (ASCUS) where HSIL could not be excluded (ASCH) (9/9), and in 80% (4/5) of cases of atypical glandular cells (AGC). Among women ≥30-65 years, the proportion ASCUS or worse (ASCUS+) was similar with cytology (3.52% [2016]) and primary HPV screening (3.70% [2017]). Only the proportion of ASC-H changed by the use of primary HPV screening, from 0.13% (2016) to 0.23% (2017) (p < 0.001). The colposcopy referral rate increased by 54% (3.70 vs 2.41%), when primary HPV screening was introduced. In conclusion, the implemented primary HPV screening approach demonstrated similar prevalence of ASCUS+ cytology as conventional screening. In addition, primary HPV screening decreased cytology assessments by 86% in our screening population of women 30 through 70 years taken into account the co-tested women.

Finnish new variant of Chlamydia trachomatis escaping detection in the Aptima Combo 2 assay also present in Örebro County, Sweden, May 2019.

We identified the first two cases of the Finnish new variant of Chlamydia trachomatis (F-nvCT) beyond Finland in two clinical urogenital specimens in Örebro County, Sweden. These Aptima Combo 2 assay-negative specimens were Aptima Chlamydia trachomatis (CT) assay positive and had the characteristic C1515T mutation in the 23S rRNA gene. From 22 March to 31 May 2019, 1.3% (2/158) of the CT-positive cases in Örebro County were missed because of the F-nvCT. International awareness, investigations and actions are essential.

Changes and sex differences in patient reported outcomes in rheumatoid factor positive RA-results from a community based study.

BACKGROUND: Patient reported outcomes (PROs) are important measures in rheumatoid arthritis (RA). A register of patients with RA from all rheumatology care providers in Malmö, Sweden, was established in 1997 and has been continually updated. This register includes virtually all the RA patients in the area. The aim of this study was to analyse PROs in surveys of this population conducted between 1997 and 2009, and to assess differences in treatment and outcome in male and female patients. METHOD: In 1997, 2002, 2005 and 2009, questionnaires were sent to the RA patients in the register (n = 1016 in 1997; n = 916 in 2002; n = 1625 in 2005; n = 1700 in 2009). Response rates varied between 62 % and 74 %, and 72-74 % was women. Questionnaire data included medication and measures of disability and health related quality of life. Data on rheumatoid factor (RF) tests were retrieved from the databases of the two clinical immunology laboratories in the area. In order to limit the impact of changes in the case mix over time, the study was restricted to RF positive patients. The analyses were stratified by sex. RESULTS: Patients reported less severe outcomes for all measures in the later surveys compared to 1997, and patients' global disease activity assessment and self-reported pain were further improved in 2009 compared to 2005. Treatment with biologics increased over time from 1997 (none) to 2009 (29%), with no difference between men and women. Visual analogue scales (0-100) for patients' global assessment of disease activity [mean 45 (95 % CI (45-47) vs. 38 (35-40)] and pain [mean 46 (44-49) vs. 38 (36-40)] decreased from 1997 to 2009, with numerically greater improvement in male patients. The mean SF-36 physical component scores also improved, and were higher in men than in women in all surveys. CONCLUSION: Pharmacologic treatment of RA became more extensive over time, and there was improvement in all PROs. Despite similar treatment, male patients reported better outcomes, in particular for pain and physical function, compared to female patients. We suggest that patient reported outcomes should be reported separately in male and female patients with RA.

First National Genomic Epidemiological Study of Neisseria gonorrhoeae Strains Spreading Across Sweden in 2016.

The increasing transmission and antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global health concern with worrying trends of decreasing susceptibility to also the last-line extended-spectrum cephalosporin (ESC) ceftriaxone. A dramatic increase of reported gonorrhea cases has been observed in Sweden from 2016 and onward. The aim of the present study was to comprehensively investigate the genomic epidemiology of all cultured N. gonorrhoeae isolates in Sweden during 2016, in conjunction with phenotypic AMR and clinical and epidemiological data of patients. In total, 1279 isolates were examined. Etest and whole-genome sequencing (WGS) were performed, and epidemiological data obtained from the Public Health Agency of Sweden. Overall, 51.1%, 1.7%, and 1.3% resistance to ciprofloxacin, cefixime, and azithromycin, respectively, was found. No isolates were resistant to ceftriaxone, however, 9.3% of isolates showed a decreased susceptibility to ceftriaxone and 10.5% to cefixime. In total, 44 penA alleles were found of which six were mosaic (n = 92). Using the typing schemes of MLST, NG-MAST, and NG-STAR; 133, 422, and 280 sequence types, respectively, and 93 NG-STAR clonal complexes were found. The phylogenomic analysis revealed two main lineages (A and B) with lineage A divided into two main sublineages (A1 and A2). Resistance and decreased susceptibility to ESCs and azithromycin and associated AMR determinants, such as mosaic penA and mosaic mtrD, were predominantly found in sublineage A2. Resistance to cefixime and azithromycin was more prevalent among heterosexuals and MSM, respectively, and both were predominantly spread through domestic transmission. Continuous surveillance of the spread and evolution of N. gonorrhoeae, including phenotypic AMR testing and WGS, is essential for enhanced knowledge regarding the dynamic evolution of N. gonorrhoeae and gonorrhea epidemiology.

Systematic evaluation of SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.

OBJECTIVE: The COVID-19 pandemic poses an immense need for accurate, sensitive and high-throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high-throughput multiplex bead-based serological assay. METHODS: More than 100 representations of SARS-CoV-2 proteins were included for initial evaluation, including antigens produced in bacterial and mammalian hosts as well as synthetic peptides. The five best-performing antigens, three representing the spike glycoprotein and two representing the nucleocapsid protein, were further evaluated for detection of IgG antibodies in samples from 331 COVID-19 patients and convalescents, and in 2090 negative controls sampled before 2020. RESULTS: Three antigens were finally selected, represented by a soluble trimeric form and the S1-domain of the spike glycoprotein as well as by the C-terminal domain of the nucleocapsid. The sensitivity for these three antigens individually was found to be 99.7%, 99.1% and 99.7%, and the specificity was found to be 98.1%, 98.7% and 95.7%. The best assay performance was although achieved when utilising two antigens in combination, enabling a sensitivity of up to 99.7% combined with a specificity of 100%. Requiring any two of the three antigens resulted in a sensitivity of 99.7% and a specificity of 99.4%. CONCLUSION: These observations demonstrate that a serological test based on a combination of several SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.

Spectrum of HPV types before and after treatment of cervical intraepithelial neoplasia grade 2 and 3.

BACKGROUND: To monitor residual disease after treatment of high grade cervical intraepithelial neoplasia (CIN), cytology together with human papillomavirus (HPV) testing are commonly performed. OBJECTIVES: To analyse the spectrum of HPV types before and after treatment. STUDY DESIGN: This register-based study included 446 women treated for CIN2 or 3, where cytology samples had been HPV-tested before and after treatment by the use of the MGP-PCR Luminex HPV L1-DNA-assay identifying 39 HPV types, including 12 high risk (HR) HPV types. RESULTS: Before and after treatment, 706 and 248 HPV isolates were detected of 36 and 34 different HPV types, respectively. Among all the HR HPV isolates, type-specific persistency was observed among 14% (76/542) after treatment, compared to 34% (31/92) of low-risk (LR) HPV isolates (p<0.001). Among the potential high risk (PHR) HPV isolates, 8.3% (6/72) persisted. Totally, 99% (440/446) and 40% (179/446) of the women were HPV-positive before and after treatment, respectively. At least one of the 12 HR HPV types was present in 91% (404/446) and 24% (109/446) of the women before and after treatment, respectively (p<0.0001). HR HPV types were present both before and after treatment among 23% (102/446) of the women, and 16% (71/446) manifested at least one persistent HR HPV type. The sensitivity, specificity and negative predictive value of HR HPV testing for detection of residual high grade squamous intraepithelial lesion (HSIL) was based on the first cytology after treatment, and was 91.7% (95% CI: 61.5%-99.8%), 84.1% (95% CI: 80.0%-87.7%) and 99.7% (95% CI: 98.2%-100.0%), respectively. CONCLUSIONS: About one out six treated women (16%) manifested at least one persistent HR HPV type, that was associated with recurrent or residual HSIL disease (odds ratio 58.1, 95% CI 7.4-457) (p=0.0001). Testing for HR HPV demonstrated high sensitivity (92%) for residual HSIL. The higher persistency rate of LR HPV types suggests that they are more likely to be outside the treated area.

Non-germ-line encoded residues are critical for effective antibody recognition of a poorly immunogenic neutralization epitope on glycoprotein B of human cytomegalovirus.

The capability of the antibody (Ab) repertoire to mount a response to appropriate epitopes on infectious agents will strongly affect the ability of the immune system to provide protection against disease. Certain epitopes may be poor inducers of immunity but are nevertheless able to promote biologically important protection when recognized by the immune repertoire. We have investigated the recognition by Ab of one such poorly immunogenic target, antigenic domain 2 (AD-2) on human cytomegalovirus glycoprotein B. To date, only two well-characterized human monoclonal Ab reactive with this epitope are known. To define parameters important for establishment of a human paratope recognizing this epitope, we created variants of the variable genes utilized by one of these Ab and used phage display technology to select Ab fragments with retained antigen specificity. We show here that residues in the first complementarity determining region of both the heavy and the light chain are involved in determining the AD-2 specificity and, in addition, that key mutations in the germ-line sequence are required for effective interaction with this epitope. Thus, the products of the human germ-line IGHV3-30 and IGKV3-11 genes, the only V genes that have been demonstrated to participate in an AD-2 specific Ab response, do not have the intrinsic features required for high-affinity recognition of this epitope. We propose that the inability of the human germ-line gene-encoded Ab repertoire to directly recognize this and possibly other antigenic determinants results in their poor immunogenicity in vivo. This may favor responses to other epitopes, which have a high-affinity imprint in the human germ-line Ab repertoire.