Molecular epidemiology of HIV among people who inject drugs after the HIV-outbreak in Athens, Greece: Evidence for a 'slow burn' outbreak.

BACKGROUND: New diagnoses of HIV-1 infection among people who inject drugs (PWID) in Athens, Greece, saw a significant increase in 2011 and a subsequent decline after 2013. Despite this, ongoing HIV-1 transmission persisted from 2014 to 2020 within this population. Our objective was to estimate the time of infection for PWID in Athens following the HIV-1 outbreak, explore the patterns of HIV-1 dispersal over time, and determine the duration from infection to diagnosis. METHODS: Time from HIV-1 infection to diagnosis was estimated for 844 individuals infected within 4 PWID-specific clusters and for 8 PWID infected with sub-subtype A6 diagnosed during 2010-2019. Phylogeny reconstruction was performed using the maximum-likelihood method. HIV-1 infection dates were based on molecular clock calculations. RESULTS: In total 86 of 92 (93.5%) sequences from PWID diagnosed during 2016-2019 were either related to the previously identified PWID-specific clusters (n = 81) or belonged to a new A6 cluster (n = 5). The median time between infection and diagnosis was 0.42 years during the outbreak period and 0.70 years during 2016-2019 (p < 0.001). The proportion of clustered sequences from PWID was very low at 5.3% during the pre-outbreak period (1998-2009), saw an increase to 41.7% one year before the outbreak in 2010, and consistently remained high during the whole period after 2011, spanning the post-outbreak period (2016-2019) with a range from 92.9% to 100%. CONCLUSIONS: The substantial proportion of clustered infections (93.5%) during 2016-2019 implies a persistent 'slow burn' HIV outbreak among PWID in Athens, suggesting that the outbreak was not successfully eliminated. The consistently high proportion of clustered sequences since the onset of the outbreak suggests the persistence of ongoing HIV-1 transmission attributed to injection practices. Our findings underscore the importance of targeted interventions among PWID, considering the ongoing transmission rate and prolonged time from infection to diagnosis.

Standardized Cumulative Metrics of Excess Mortality to Monitor Health System Resilience Throughout COVID-19 and Other Respiratory Virus Resurgences.

Monitoring morbidity and mortality in resurgences of respiratory infections poses significant challenges, as shown by coronavirus disease 2019 (COVID-19). For example, case fatality rates and deaths attributed to specific respiratory pathogens are known to suffer from significant biases undermining their comparability through time and space. As a result, it is difficult to evaluate the protective effect of public health interventions or quantify the impact of a resurgence on the general population through direct recording of COVID-19 related deaths. To overcome these limitations, more robust, less-biased metrics, such as all-cause deaths, have been proposed for monitoring the effect of an epidemic over a population and over time. More specifically, metrics of excess mortality over time, which have been used for influenza surveillance in the past, are increasingly considered important for COVID-19 surveillance. Here, we discuss excess mortality surveillance focusing on standardized single-point and standardized cumulative metrics that allow comparability of excess mortality through space and time. We explain why z score allows for comparison of excess mortality between countries and different periods, while cumulative z score allows assessment of excess mortality over long periods. Our commentary reiterates the importance of standardized statistics of excess mortality for COVID-19 surveillance as we move toward a coexistence with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that will allow drawing conclusions from best practices in different health systems and different periods.

On the impact of mass screening for SARS-CoV-2 through self-testing in Greece.

Background: Screening programs that pre-emptively and routinely test population groups for disease at a massive scale were first implemented during the COVID-19 pandemic in a handful of countries. One of these countries was Greece, which implemented a mass self-testing program during 2021. In contrast to most other non-pharmaceutical interventions (NPIs), mass self-testing programs are particularly attractive for their relatively small financial and social burden, and it is therefore important to understand their effectiveness to inform policy makers and public health officials responding to future pandemics. This study aimed to estimate the number of deaths and hospitalizations averted by the program implemented in Greece and evaluate the impact of several operational decisions. Methods: Granular data from the mass self-testing program deployed by the Greek government between April and December 2021 were obtained. The data were used to fit a novel compartmental model that was developed to describe the dynamics of the COVID-19 pandemic in Greece in the presence of self-testing. The fitted model provided estimates on the effectiveness of the program in averting deaths and hospitalizations. Sensitivity analyses were used to evaluate the impact of operational decisions, including the scale of the program, targeting of sub-populations, and sensitivity (i.e., true positive rate) of tests. Results: Conservative estimates show that the program reduced the reproduction number by 4%, hospitalizations by 25%, and deaths by 20%, translating into approximately 20,000 averted hospitalizations and 2,000 averted deaths in Greece between April and December 2021. Conclusion: Mass self-testing programs are efficient NPIs with minimal social and financial burden; therefore, they are invaluable tools to be considered in pandemic preparedness and response.

HERVs: Expression Control Mechanisms and Interactions in Diseases and Human Immunodeficiency Virus Infection.

Human endogenous retroviruses (HERVs) are the result of retroviral infections acquired millions of years ago; nowadays, they compose around 8% of human DNA. Multiple mechanisms have been employed for endogenous retroviral deactivation, rendering replication and retrotransposition defective, while some of them have been co-opted to serve host evolutionary advantages. A pleiad of mechanisms retains the delicate balance of HERV expression in modern humans. Thus, epigenetic modifications, such as DNA and histone methylation, acetylation, deamination, chromatin remodeling, and even post-transcriptional control are recruited. In this review, we aim to summarize the main HERV silencing pathways, revisit paradigms of human disease with a HERV component, and emphasize the human immunodeficiency virus (HIV) and HERV interactions during HIV infection.

Performance of Different Risk Scores for the Detection of Atrial Fibrillation Among Patients With Cryptogenic Stroke.

BACKGROUND: Atrial fibrillation (AF) is a frequent underlying cause of cryptogenic stroke (CS) and its detection can be increased using implantable cardiac monitoring (ICM). We sought to evaluate different risk scores and assess their diagnostic ability in identifying patients with CS with underlying AF on ICM. METHODS: Patients with CS, being admitted to a single tertiary stroke center between 2017 and 2022 and receiving ICM, were prospectively evaluated. The CHA2DS2-VASc, HAVOC, Brown ESUS-AF, and C2HEST scores were calculated at baseline. The primary outcome of interest was the detection of AF, which was defined as at least 1 AF episode on ICM lasting for 2 consecutive minutes or more. The diagnostic accuracy measures and the net reclassification improvement were calculated for the 4 risk scores. Stroke recurrence was evaluated as a secondary outcome. RESULTS: A total of 250 patients with CS were included, and AF was detected by ICM in 20.4% (n=51) during a median monitoring period of 16 months. Patients with CS with AF detection were older compared with the rest (P=0.045). The median HAVOC, Brown ESUS-AF, and C2HEST scores were higher among the patients with AF compared with the patients without AF (all P<0.05), while the median CHA2DS2-VASc score was similar between the 2 groups. The corresponding C statistics for CHA2DS2-VASc, HAVOC, Brown ESUS-AF, and C2HEST for AF prediction were 0.576 (95% CI, 0.482-0.670), 0.612 (95% CI, 0.523-0.700), 0.666 (95% CI, 0.587-0.746), and 0.770 (95% CI, 0.699-0.839). The C2HEST score presented the highest diagnostic performance based on C statistics (P<0.05 after correction for multiple comparisons) and provided significant improvement in net reclassification for AF detection (>70%) compared with the other risk scores. Finally, stroke recurrence was documented in 5.6% of the study population, with no difference regarding the 4 risk scores between patients with and without recurrent stroke. CONCLUSIONS: The C2HEST score was superior to the CHA2DS2-VASc, HAVOC, and Brown ESUS-AF scores for discriminating patients with CS with underlying AF using ICM.

Standardized Cumulative Metrics of Excess Mortality to Monitor Health System Resilience Throughout COVID-19 and Other Respiratory Virus Resurgences.

Monitoring morbidity and mortality in resurgences of respiratory infections poses significant challenges, as shown by coronavirus disease 2019 (COVID-19). For example, case fatality rates and deaths attributed to specific respiratory pathogens are known to suffer from significant biases undermining their comparability through time and space. As a result, it is difficult to evaluate the protective effect of public health interventions or quantify the impact of a resurgence on the general population through direct recording of COVID-19 related deaths. To overcome these limitations, more robust, less-biased metrics, such as all-cause deaths, have been proposed for monitoring the effect of an epidemic over a population and over time. More specifically, metrics of excess mortality over time, which have been used for influenza surveillance in the past, are increasingly considered important for COVID-19 surveillance. Here, we discuss excess mortality surveillance focusing on standardized single-point and standardized cumulative metrics that allow comparability of excess mortality through space and time. We explain why z score allows for comparison of excess mortality between countries and different periods, while cumulative z score allows assessment of excess mortality over long periods. Our commentary reiterates the importance of standardized statistics of excess mortality for COVID-19 surveillance as we move toward a coexistence with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that will allow drawing conclusions from best practices in different health systems and different periods.

The interaction of human immunodeficiency virus-1 and human endogenous retroviruses in patients (primary cell cultures) and cell line models.

IMPORTANCE: In this work, we demonstrated that human immunodeficiency virus (HIV) infection leads to the modification of the human endogenous retrovirus (HERV) expression. Differential expression of multiple HERVs was found in peripheral blood mononuclear cells derived from HIV-infected patients compared to healthy donors and HIV-infected T cell cultures compared to non-infected. The effect of HIV presence on HERV expression appears to be more restricted in cells of monocytic origin, as only deregulation of HERV-W and HERV-K (HML-6) was found in these cell cultures after their infection with HIV. Multiple factors contribute to this aberrant HERV expression, and its levels appear to be modified in a time-dependent manner. Further studies and the development of optimized in vitro protocols are warranted to elucidate the interactions between HIV and HERVs in detail.

Interplay between endogenous and exogenous human retroviruses.

In humans, retroviruses thrive more as symbionts than as parasites. Apart from the only two modern exogenous human retroviruses (human T-cell lymphotropic and immunodeficiency viruses; HTLV and HIV, respectively), ~8% of the human genome is occupied by ancient retroviral DNA [human endogenous retroviruses (HERVs)]. Here, we review the recent discoveries about the interactions between the two groups, the impact of infection by exogenous retroviruses on the expression of HERVs, the effect of HERVs on the pathogenicity of HIV and HTLV and on the severity of the diseases caused by them, and the antiviral protection that HERVs can allegedly provide to the host. Tracing the crosstalk between contemporary retroviruses and their endogenized ancestors will provide better understanding of the retroviral world.

Prolonged cardiac monitoring for stroke prevention: A systematic review and meta-analysis of randomized-controlled clinical trials.

Introduction: Prolonged cardiac monitoring (PCM) substantially improves the detection of subclinical atrial fibrillation (AF) among patients with history of ischemic stroke (IS), leading to prompt initiation of anticoagulants. However, whether PCM may lead to IS prevention remains equivocal. Patients and methods: In this systematic review and meta-analysis, randomized-controlled clinical trials (RCTs) reporting IS rates among patients with known cardiovascular risk factors, including but not limited to history of IS, who received PCM for more than 7 days versus more conservative cardiac rhythm monitoring methods were pooled. Results: Seven RCTs were included comprising a total of 9048 patients with at least one known cardiovascular risk factor that underwent cardiac rhythm monitoring. PCM was associated with reduction of IS occurrence compared to conventional monitoring (Risk Ratio: 0.76; 95% CI: 0.59-0.96; I 2 = 0%). This association was also significant in the subgroup of RCTs investigating implantable cardiac monitoring (Risk Ratio: 0.75; 95% CI: 0.58-0.97; I 2 = 0%). However, when RCTs assessing PCM in both primary and secondary prevention settings were excluded or when RCTs investigating PCM with a duration of 7 days or less were included, the association between PCM and reduction of IS did not retain its statistical significance. Regarding the secondary outcomes, PCM was related to higher likelihood for AF detection and anticoagulant initiation. No association was documented between PCM and IS/transient ischemic attack occurrence, all-cause mortality, intracranial hemorrhage, or major bleeding. Conclusion: PCM may represent an effective stroke prevention strategy in selected patients. Additional RCTs are warranted to validate the robustness of the reported associations.

Human endogenous retroviruses in cancer: Oncogenesis mechanisms and clinical implications.

Human Endogenous Retroviruses (HERVs) are viral sequences integrated into the human genome, resulting from the infection of human germ-line cells by ancient exogenous retroviruses. Despite losing their replication and retrotransposition abilities, HERVs appear to have been co-opted in human physiological functions while their aberrant expression is linked to human disease. The role of HERVs in multiple malignancies has been demonstrated, however, the extent to which HERV activation and expression participate in the development of cancer is not yet fully comprehended. In this review article, we discuss the presumed role of HERVs in carcinogenesis and their promising diagnostic and prognostic implications. Additionally, we explore recent data on the HERVs in cancer therapeutics, either through the manipulation of their expression, to induce antitumor innate immunity responses or as cancer immunotherapy targets. Finally, more precise and higher resolution high-throughput sequencing approaches will further elucidate HERV participation in human physiological and pathological processes.

On the evolution of SARS-CoV-2 and the emergence of variants of concern.

The epidemiology of COVID-19 has dramatically changed since the beginning of the pandemic as we witnessed significant changes in transmissibility and pathogenicity with the emergence of SARS-CoV-2 variants of concern (VoCs). Here I present and comment on working hypotheses about the evolutionary dynamics of the emergence of VoCs.

Postgenomic Bioinformatic Analysis of Nucleic Acid Sequences Expressed in the Salivary Gland Epithelial Cells of Primary Sjögren's Syndrome Patients in Search of Microorganisms and Endogenous Retroviruses.

Several previous studies from our laboratory have indicated that the salivary gland epithelia of primary Sjögren's syndrome (SS) patients are not only the target of autoimmune immune responses, but also key instigators of the chronic salivary gland inflammatory infiltrates of patients. In particular, the comparative analysis of salivary gland tissue specimens and of in-vitro cultured non-neoplastic salivary gland epithelial cell lines (SGEC, of ductal type) from SS-patients and non-SS disease-controls, have unequivocally highlighted the presence of intrinsic activation in the ductal epithelia of SS-patients and of aberrant expression of inflammagenic molecules thereof, that correlate with the severity of local histopathologic changes, as well as of systemic manifestations of the disease. In the same context, we have recently shown that the ductal epithelia of SS-patients manifest cell-autonomous activation of the AIM2 inflammasome owing to the presence of aberrant cytoplasmic accumulations of damaged DNA. These findings not only provide a mechanistic explanation for the intrinsic activation and inflammatory status of SS ductal epithelia, but may also point towards the putative instigating role of an exogenous or endogenous agent (i.e., a micro-organism or an endogenous retrovirus, respectively). On this basis and to further explore the nature of epithelial cell-intrinsic activation in SS, the present proposal aims to investigate the expression of endogenous retroviral and/or non-human nucleic acid sequences of microbial origin in the ductal salivary gland epithelia of SS-patients, using metagenomic analysis of high throughput DNA and RNA genome sequencing data, which will be obtained from SGEC lines derived from SS-patients and disease-controls.

Estimation of the determinants for HIV late presentation using the traditional definition and molecular clock-inferred dates: Evidence that older age, heterosexual risk group and more recent diagnosis are prognostic factors.

OBJECTIVES: HIV late presentation (LP) has been increasing in recent years in Europe. Our aim was to investigate the characteristics of LP in Greece using in addition to the traditional definition for LP, the time interval between HIV infection and diagnosis. METHODS: Our nationwide sample included HIV-1 sequences generated from 6166 people living with HIV (PLWH) in Greece during the period 1999-2015. Our analysis was based on the molecularly inferred HIV-1 infection dates for PLWH infected within local molecular transmission clusters of subtypes A1 and B. RESULTS: Analysis of the determinants of LP was conducted using either CD4 counts or AIDS-defining condition at diagnosis or the time from infection to diagnosis. Older age, heterosexual transmission risk group and more recent diagnosis were associated with increased risk for LP. In contrast to previous studies, people who inject drugs (PWID) had a shorter median time to diagnosis (0.63 years) compared to men who have sex with men (MSM) (1.72 years) and heterosexuals (2.43 years). Using HIV infection dates that provide an unbiased marker for LP compared to CD4 counts at diagnosis, which are age-dependent, we estimated that the time to diagnosis increased gradually with age. Migrants infected regionally do not differ with respect to LP status compared to native Greeks. CONCLUSIONS: We demonstrate that older people and heterosexuals are among those at higher risk for LP; and given the growing number of older people among newly diagnosed cases, tailored interventions are needed in these populations.

Development and Analytical Validation of a One-Step Five-Plex RT-ddPCR Assay for the Quantification of SARS-CoV-2 Transcripts in Clinical Samples.

Highly sensitive methodologies for SARS-CoV-2 detection are essential for the control of COVID-19 pandemic. We developed and analytically validated a highly sensitive and specific five-plex one-step RT-ddPCR assay for SARS-CoV-2. We first designed in-silico novel primers and probes for the simultaneous absolute quantification of three different regions of the nucleoprotein (N) gene of SARS-CoV-2 (N1, N2, N3), a synthetic RNA as an external control (RNA-EC), and Beta-2-Microglobulin (B2M) as an endogenous RNA internal control (RNA-IC). The developed assay was analytically validated using synthetic DNA and RNA calibrator standards and then was applied to 100 clinical specimens previously analyzed with a commercially available CE-IVD RT-qPCR assay. The analytical validation of the developed assay resulted in very good performance characteristics in terms of analytical sensitivity, linearity, analytical specificity, and reproducibility and recovery rates even at very low viral concentrations. The simultaneous absolute quantification of the RNA-EC and RNA-IC provides the necessary metrics for quality control assessment. Direct comparison of the developed one-step five-plex RT-ddPCR assay with a CE-IVD RT-qPCR kit revealed a very high concordance and a higher sensitivity [concordance: 99/100 (99.0%, Spearman's correlation coefficient: -0.850, p < 0.001)]. The developed assay is highly sensitive, specific, and reproducible and has a broad linear dynamic range, providing absolute quantification of SARS-COV-2 transcripts. The inclusion of two RNA quality controls, an external and an internal, is highly important for standardization of SARS-COV-2 molecular testing in clinical and wastewater samples.

Immunogenicity and Safety of the BNT162b2 mRNA COVID-19 Vaccine in Patients with Melanoma Treated with Immunotherapy.

The BNT162b2 vaccine against SARS-CoV-2 has a proven efficacy and a favorable safety profile. In cancer patients under immunotherapy in the form of immune-checkpoint inhibitors (ICIs), the efficacy of the vaccine has not been thoroughly studied, while a theoretical concern has also been raised about triggering immune-related adverse events (irAEs) by the vaccine. We conducted a prospective, non-interventional study on the immunogenicity and safety of the BNT162b2 vaccine in patients with advanced or metastatic melanoma treated with ICIs. Blood samples were obtained 0-4 days before the first dose and 12-21 days after the second dose of the vaccine for the quantification of the SARS-CoV-2 anti-spike antibody using an ELISA and immunophenotyping of the T and myeloid cell subpopulations. The active recording of AEs for a two-month period was conducted. Forty patients were included in the study. All but one (97.3%) achieved seroconversion after two doses of the vaccine and no correlations of the antibody titers with any of the studied parameters (age, gender, stage and duration of the disease, type of ICI, previous treatment, etc.) were found. Moreover, no differences in the subpopulations of the T cells (including the T-regulatory cells) or the myeloid cells were found pre- and post-vaccination. All AEs were low-grade, while one case of arthritis exacerbation was noted. The seroconversion rate in the studied population was high and was comparable to that of healthy subjects, while no major safety issues were raised during the safety follow-up. Finally, no derangements in the subpopulations of T cells or myeloid cells were noted. This is the first study focusing on the immunogenicity, safety, and effect of anti-SARS-CoV-2 vaccines on the blood-cell immunophenotype status of patients with melanoma treated with ICIs.

Significance of serum HBV RNA in non-cirrhotic HBeAg-negative chronic hepatitis B patients who discontinue effective antiviral therapy.

HBV RNA is considered as a promising predictor in patients who discontinue nucleos(t)ide analogues (NAs). We determined HBV RNA levels in non-cirrhotic HBeAg-negative patients who discontinued NAs and assessed their predictability for 12-month outcomes. Fifty-seven patients of DARING-B study were included. HBV RNA levels were determined in stored monthly serum samples drawn at 0-3 months after end of therapy (EOT). Other markers previously determined in the same cohort including hepatitis B core-related antigen (HBcrAg) were also assessed. HBV RNA at EOT was detectable in 7% of patients, who developed virological/clinical relapse and required retreatment at month 2; in patients with undetectable EOT HBV RNA, 12-month cumulative rates of virological relapse, clinical relapse and retreatment were 68%, 28% and 21%, respectively (p ≤ 0.008). HBV RNA at month-1 after EOT was detectable in 19% of patients being associated with higher probability only of virological relapse (p = 0.001). HBV RNA levels correlated significantly to HBV DNA, HBcrAg, ALT and interferon-induced protein-10, but not HBsAg levels. Combined EOT HBV RNA and HBcrAg detection and/or HBsAg >1000 IU/ml was associated only with higher probability of retreatment having higher sensitivity and lower specificity than HBV RNA alone. In conclusion, serum HBV RNA is detectable in a minority of non-cirrhotic HBeAg-negative patients under effective long-term NAs therapy offering low sensitivity but 100% specificity for early retreatment due to severe clinical relapses after NA discontinuation. The combinations of EOT HBV RNA with HBcrAg and/or high HBsAg levels increase sensitivity but decrease specificity for prediction of retreatment after NAs withdrawal.

Cold-Season Epidemic Dynamics of COVID-19 in Two Major Metropolitan Areas in Greece: Hypotheses and Implications for Public Health Interventions.

Many respiratory viruses, including coronaviruses, follow seasonal transmission dynamics. Analyzing the social and environmental mechanics of the emergence of SARS-CoV-2 over the first cold season provides insight into designing targeted interventions. We analyzed all fully anonymized SARS-CoV-2 case data in two metropolitan areas, Attika and Thessaloniki, diagnosed between September 1st and December 31st, 2020. The emergence of the second wave in Greece occurred in October-November. SARS-CoV-2 diagnoses in Thessaloniki increased quasi-exponentially in mid-October, coinciding with the increase in the proportion of diagnoses in young people aged 18-39. The same pattern was observed in Attika with an almost 2-week delay, even though Attika had a higher prevalence of cases throughout summer until the second wave. Crucially, the nighttime temperature in Thessaloniki dropped below 18°C 3 weeks earlier than that in Attika. Epidemic growth was independently associated with the proportion of cases attributed to the 18-39 age group as well as with the drop in nighttime temperature below 18°C in both metropolitan areas but with a time difference. This pattern can be explained by a shift of nighttime entertainment activities from open-air to closed spaces, which occurs as nighttime temperature drops. Vaccination of young individuals can be crucial in decelerating the cold-season dynamics of SARS-CoV-2.