Diagnosing Viral Infections Through T-Cell Receptor Sequencing of Activated CD8+ T Cells.

T-cell-based diagnostic tools identify pathogen exposure but lack differentiation between recent and historical exposures in acute infectious diseases. Here, T-cell receptor (TCR) RNA sequencing was performed on HLA-DR+/CD38+CD8+ T-cell subsets of hospitalized coronavirus disease 2019 (COVID-19) patients (n = 30) and healthy controls (n = 30; 10 of whom had previously been exposed to severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]). CDR3α and CDR3ß TCR regions were clustered separately before epitope specificity annotation using a database of SARS-CoV-2-associated CDR3α and CDR3ß sequences corresponding to >1000 SARS-CoV-2 epitopes. The depth of the SARS-CoV-2-associated CDR3α/ß sequences differentiated COVID-19 patients from the healthy controls with a receiver operating characteristic area under the curve of 0.84 ± 0.10. Hence, annotating TCR sequences of activated CD8+ T cells can be used to diagnose an acute viral infection and discriminate it from historical exposure. In essence, this work presents a new paradigm for applying the T-cell repertoire to accomplish TCR-based diagnostics.

COVID-19 in three waves in a tertiary referral hospital in Belgium: a comparison of patient characteristics, management, and outcome.

PURPOSE: Few studies have compared patient characteristics, clinical management, and outcome of patients with COVID-19 between the different epidemic waves. In this study, we describe patient characteristics, treatment, and outcome of patients admitted for COVID-19 in the Antwerp University Hospital over the first three epidemic waves of 2020-2021. METHODS: Retrospective observational study of COVID-19 patients in a Belgian tertiary referral hospital. All adult patients with COVID-19, hospitalized between February 29, 2020, and June 30, 2021, were included. Standardized routine medical data was collected from patient records. Risk factors were assessed with multivariable logistic regression. RESULTS: We included 722 patients, during the first (n = 179), second (n = 347) and third (n = 194) wave. We observed the lowest disease severity at admission during the first wave, and more elderly and comorbid patients during the second wave. Throughout the subsequent waves we observed an increasing use of corticosteroids and high-flow oxygen therapy. In spite of increasing number of complications throughout the subsequent waves, mortality decreased each wave (16.6%,15.6% 11.9% in 1st, 2nd and 3rd wave respectively). C-reactive protein above 150 mg/L was predictive for the need for intensive care unit admission (odds ratio (OR) 3.77, 95% confidence interval (CI) 2.32-6.15). A Charlson comorbidity index ≥ 5 (OR 5.68, 95% CI 2.54-12.70) and interhospital transfers (OR 3.78, 95% CI 2.05-6.98) were associated with a higher mortality. CONCLUSIONS: We observed a reduction in mortality each wave, despite increasing comorbidity. Evolutions in patient management such as high-flow oxygen therapy on regular wards and corticosteroid use may explain this favorable evolution.

Presymptomatic viral shedding in high-risk mpox contacts: A prospective cohort study.

The risk of infection after exposure to clade IIb mpox virus (MPXV) is unknown, and potential presymptomatic shedding of MPXV remains to be demonstrated. High-risk contacts of mpox patients were followed-up in a prospective longitudinal cohort study. Individuals reporting sexual contact, >15 min skin-to-skin contact, or living in the same household with an mpox patient were recruited in a sexual health clinic in Antwerp, Belgium. Participants kept a symptom diary, performed daily self-sampling (anorectal, genital, and saliva), and presented for weekly clinic visits for physical examination and sampling (blood and oropharyngeal). Samples were tested for MPXV by PCR. Between June 24 and July 31, 2022, 25 contacts were included, of which 12/18 (66.0%) sexual and 1/7 (14.0%) nonsexual contacts showed evidence of infection by MPXV-PCR. Six cases had typical mpox symptoms. Viral DNA was detected as early as 4 days before symptom onset in 5 of them. In 3 of these cases, replication-competent virus was demonstrated in the presymptomatic phase. These findings confirm the existence of presymptomatic shedding of replication-competent MPXV and emphasize the high risk of transmission during sexual contact. Sexual contacts of mpox cases should abstain from sex during the incubation period, irrespective of symptoms.

Understanding sexual transmission dynamics and transmission contexts of monkeypox virus: a mixed-methods study of the early outbreak in Belgium (May-June 2022).

OBJECTIVE: The available epidemiological and clinical evidence from the currently ongoing monkeypox (MPX) outbreak in non-endemic areas suggests an important factor of sexual transmission. However, limited information on the behaviour and experiences of individuals with an MPX infection has to date been provided. We aimed to describe the initial phase of the MPX outbreak in Belgium, and to provide a more in-depth description of sexual behaviour and transmission contexts. METHODS: We used routine national surveillance data of 139 confirmed MPX cases with date of symptom onset until 19 June 2022, complemented with 12 semistructured interviews conducted with a subsample of these cases. RESULTS: Sexualised environments, including large festivals and cruising venues for gay men, were the suspected exposure setting for the majority of the cases in the early outbreak phase. In-depth narratives of sexual behaviour support the hypothesis of MPX transmission through close physical contact during sex. Despite awareness of the ongoing MPX outbreak, low self-perceived risk of MPX acquisition and confusing initial signs and symptoms for other STIs or skin conditions delayed early detection of an MPX infection. In addition, we describe relevant contextual factors beyond individual behaviour, related to sexual networks, interpersonal interactions and health systems. Some of these factors may complicate early MPX detection and control efforts. CONCLUSION: Our results highlight the role of sexual contact and networks in the transmission of MPX during the early phase of the outbreak in Belgium. Risk communication messages should consistently and transparently state the predominant sexual transmission potential of MPX virus, and prevention and control measures must be adapted to reflect multilevel factors contributing to MPX transmission risk.

Persistent morbidity in Clade IIb mpox patients: interim results of a long-term follow-up study, Belgium, June to November 2022.

While mpox was well characterised during the 2022 global Clade IIb outbreak, little is known about persistent morbidity. We present interim results of a prospective cohort study of 95 mpox patients assessed 3-20 weeks post-symptom onset. Two-thirds of participants had residual morbidity, including 25 with persistent anorectal and 18 with genital symptoms. Loss of physical fitness, new-onset/worsened fatigue and mental health problems were reported in 36, 19 and 11 patients, respectively. These findings require attention by healthcare providers.

Human Filariasis in Travelers and Migrants: A Retrospective 25-year Analysis at the Institute of Tropical Medicine, Antwerp, Belgium.

BACKGROUND: Information on human filariasis in international travelers is scarce. We describe the epidemiology, clinical presentation, and outcome of these infections in a reference travel clinic over the past decades. METHODS: We reviewed all cases of filariasis diagnosed at the Institute of Tropical Medicine, Antwerp, Belgium, from 1994 to 2018. Diagnosis was obtained by either parasitological methods (confirmed) or strict clinical case definitions (probable). We assessed the characteristics of cases at diagnosis and response to therapy within 3-12 months. RESULTS: A total of 320 patients (median age: 41 years; 71% males) were diagnosed with 327 filarial infections (Wuchereria bancrofti = 6, Onchocerca volvulus = 33, Loa loa = 150, Mansonella perstans = 130, unspecified species = 8). Diagnosis was confirmed in 213/320 (67%) patients. European long-term travelers accounted for 166 patients (52%) and visitors/migrants from tropical countries for another 110 (34%). Central Africa was the likely region of acquisition for 294 (92%) patients. The number of filariasis cases decreased from 21.5/year on average in the 1990s to 6.3/year in the past decade, when loiasis became predominant. Cases reported symptoms in >80% of all filarial infections but mansonellosis (45/123 single infections; 37%). Lymphatic filariasis and onchocerciasis cases responded well to conventional therapy. However, 30% of patients with loiasis and mansonellosis experienced treatment failure (with diethylcarbamazine and levamisole-mebendazole, respectively). CONCLUSIONS: The burden and species distribution of filariasis in travelers evolved in the past decades. Most presentations were symptomatic. Case management would benefit from more effective therapies for loiasis and mansonellosis.

Severe mpox (formerly monkeypox) disease in five patients after recent vaccination with MVA-BN vaccine, Belgium, July to October 2022.

Vaccination is important in containing the 2022 mpox (formerly monkeypox) epidemic. We describe five Belgian patients with localised severe symptoms of proctitis and penile oedema, occurring between 4 and 35 days after post-exposure preventive vaccination or after one- or two-dose off-label pre-exposure preventive vaccination with MVA-BN vaccine. Genome sequencing did not reveal evidence for immune escape variants. Healthcare workers and those at risk should be aware of possible infections occurring shortly after vaccination and the need for other preventive measures.

Severe steatosis induces portal hypertension by systemic arterial hyporeactivity and hepatic vasoconstrictor hyperreactivity in rats.

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease. The presence of portal hypertension has been demonstrated in NAFLD prior to development of inflammation or fibrosis, and is a result of extrahepatic and intrahepatic factors, principally driven by vascular dysfunction. An increased intrahepatic vascular resistance potentially contributes to progression of NAFLD via intralobular hypoxia. However, the exact mechanisms underlying vascular dysfunction in NAFLD remain unknown. This study investigates systemic hemodynamics and both aortic and intrahepatic vascular reactivity in a rat model of severe steatosis. Wistar rats were fed a methionine-choline-deficient diet, inducing steatosis, or control diet for 4 weeks. In vivo hemodynamic measurements, aortic contractility studies, and in situ liver perfusion experiments were performed. The mean arterial blood pressure was lower and portal blood pressure was higher in steatosis compared to controls. The maximal contraction force in aortic rings from steatotic rats was markedly reduced compared to controls. While blockade of nitric oxide (NO) production did not reveal any differences, cyclooxygenase (COX) blockade reduced aortic reactivity in both controls and steatosis, whereas effects were more pronounced in controls. Effects could be attributed to COX-2 iso-enzyme activity. In in situ liver perfusion experiments, exogenous NO donation or endogenous NO stimulation reduced the transhepatic pressure gradient (THPG), whereas NO synthase blockade increased the THPG only in steatosis, but not in controls. Alpha-1-adrenergic stimulation and endothelin-1 induced a significantly more pronounced increase in THPG in steatosis compared to controls. Our results demonstrate that severe steatosis, without inflammation or fibrosis, induces portal hypertension and signs of a hyperdynamic circulation, accompanied by extrahepatic arterial hyporeactivity and intrahepatic vascular hyperreactivity. The arterial hyporeactivity seems to be NO-independent, but appears to be mediated by specific COX-2-related mechanisms. Besides, the increased intrahepatic vascular resistance in steatosis appears not to be NO-related but rather to vasoconstrictor hyperreactivity.

Rabies post-exposure prophylaxis: A retrospective analysis of timing of initiation and antibody responses in a Belgian cohort.

BACKGROUND: We aimed to determine the timeliness of rabies post-exposure prophylaxis (PEP) and the proportion of individuals with an adequate antibody response post-PEP among those attending the Belgian national reference center. METHODS: Retrospective analysis of patient records who attended our center from 2018 to 2023. Delay was defined as rabies immunoglobulin (RIG) and vaccine initiation beyond 2 calendar days after exposure. Antibodies were measured by rapid fluorescent focus inhibition test (RFFIT) after PEP in high-risk exposures. A titer ≥0.5 IU/ml was considered adequate. RESULTS: We reviewed 317 patient records. Among individuals with inland exposure (n = 103), 85 % timely received PEP. Among travelers exposed abroad (n = 214), administration of RIG and vaccine initiation were timely in 30 % and 50 % of cases, respectively. An adequate antibody response was detected in 99.5 % (195/196) individuals. CONCLUSION: Substantial PEP delays among travelers were observed. The robust antibody responses suggest that routine serological follow-up is not necessary for all patients.

Antimalarial artesunate-mefloquine versus praziquantel in African children with schistosomiasis: an open-label, randomized controlled trial.

Schistosomiasis treatment entirely relies on a single drug, praziquantel, prompting research into alternative therapeutics. Here we evaluated the efficacy and safety of the antimalarial combination artesunate-mefloquine for the treatment of schistosomiasis in a proof-of-concept, pragmatic, open-label, randomized controlled trial in primary schools of six villages endemic for schistosomiasis in northern Senegal. Children (6-14 years) were eligible if Schistosoma eggs were detected by microscopy in urine and/or stool. In total, 726 children were randomized 1:1 to praziquantel (standard care: 40 mg kg-1 single dose; n = 364) or to artesunate-mefloquine (antimalarial dosage: artesunate 4 mg kg-1 and mefloquine 8 mg kg-1 daily for three consecutive days; n = 362). Eight children not meeting the inclusion criteria were excluded from efficacy analysis. Median age of the remaining 718 participants was 9 years; 399 (55.6%) were male, and 319 (44.4%) female; 99.3% were infected with Schistosoma haematobium and 15.2% with S. mansoni. Primary outcomes were cure rate, assessed by microscopy, and frequency of drug-related adverse effects of artesunate-mefloquine versus praziquantel at 4 weeks after treatment. Cure rate was 59.6% (208/349) in the artesunate-mefloquine arm versus 62.1% (211/340) in the praziquantel arm. The difference of -2.5% (95% confidence interval (CI) -9.8 to 4.8) met the predefined criteria of noninferiority (margin set at 10%). All drug-related adverse events were mild or moderate, and reported in 28/361 children receiving artesunate-mefloquine (7.8%; 95% CI 5.4 to 11.0) versus 8/363 (2.2%; 95% CI 1.1 to 4.3) receiving praziquantel (P < 0.001). Artesunate-mefloquine at antimalarial dosage was moderately safe and noninferior to standard-care praziquantel for the treatment of schistosomiasis, predominantly due to S. haematobium. Multicentric trials in different populations and epidemiological settings are needed to confirm these findings. ClinicalTrials.gov identifier: NCT03893097 .

Potential determinants of the decline in mpox cases in Belgium: A behavioral, epidemiological and seroprevalence study.

OBJECTIVES: The 2022 mpox epidemic reached a peak in Belgium and the rest of Europe in July 2022, after which it unexpectedly subsided. This study investigates epidemiological, behavioral, and immunological factors behind the waning of the epidemic in Belgium. METHODS: We investigated temporal evolutions in the characteristics and behavior of mpox patients using national surveillance data and data from a prospective registry of mpox patients in the Institute of Tropical Medicine (Antwerp). We studied behavioral changes in the population at risk using a survey among HIV-preexposure prophylaxis (PrEP) users. We determined the seroprevalence of anti-orthopoxvirus antibodies among HIV-PrEP users across four-time points in 2022. RESULTS: Mpox patients diagnosed at the end of the epidemic had less sexual risk behavior compared to those diagnosed earlier: they engaged less in sex at mass events, had fewer sexual partners, and were less likely to belong to the sexual network's central group. Among HIV-PrEP users there were no notable changes in sexual behavior. Anti-orthopoxvirus seroprevalence did not notably increase before the start of national vaccination campaigns. CONCLUSION: The observed changes in group immunity and behavior in the population at greater risk of exposure to mpox seem unable to explain the waning of the mpox epidemic. A change in the profile of mpox patients might have contributed to the decline in cases.

Sustained human outbreak of a new MPXV clade I lineage in eastern Democratic Republic of the Congo.

Outbreaks of monkeypox (mpox) have historically resulted from zoonotic spillover of clade I monkeypox virus (MPXV) in Central Africa and clade II MPXV in West Africa. In 2022, subclade IIb caused a global epidemic linked to transmission through sexual contact. Here we describe the epidemiological and genomic features of an mpox outbreak in a mining region in eastern Democratic Republic of the Congo, caused by clade I MPXV. Surveillance data collected between September 2023 and January 2024 identified 241 suspected cases. Genomic analysis demonstrates a distinct clade I lineage divergent from previously circulating strains in the Democratic Republic of the Congo. Of the 108 polymerase chain reaction-confirmed mpox cases, the median age of individuals was 22 years, 51.9% were female and 29% were sex workers, suggesting a potential role for sexual transmission. The predominance of APOBEC3-type mutations and the estimated emergence time around mid-September 2023 imply recent sustained human-to-human transmission.

Alternative sampling specimens for the molecular detection of mpox (formerly monkeypox) virus.

BACKGROUND: Mpox (formerly monkeypox) is a viral disease caused by the mpox virus (MPXV), endemic in Central and West Africa and currently causing a global outbreak of international concern. Much remains unknown about sample types most suited for mpox laboratory diagnosis. While it is established that high viral loads can be found in active skin lesions (currently the recommended mpox laboratory confirmation specimen type), WHO mpox testing guidelines encourage the use of oropharyngeal swabs as an additional sample type for mpox diagnosis and suggest investigating the value of other specimens like blood samples. OBJECTIVE: In this study, we verified the value of select alternative specimen types for mpox laboratory confirmation. METHODS: We included 25 patients with MPXV-confirmed skin lesions to compare diagnostic sensitivity of MPXV PCR testing on EDTA plasma and two upper respiratory specimens: oropharyngeal swabs and saliva. RESULTS: In our patient cohort with MPXV-confirmed skin lesions, diagnostic sensitivity of MPXV PCR was 80% in EDTA plasma, 64% in oropharyngeal swabs, and 88% in saliva. MPXV viral loads were significantly higher in saliva compared to oropharyngeal swabs and EDTA plasma. DISCUSSION: The WHO recommendation to collect oropharyngeal swabs as an additional specimen for mpox diagnosis might need to be revised to include saliva wherever feasible. We suggest investigating saliva as a diagnostic specimen in the absence of active skin lesions or during the phase preceding skin manifestations. Moreover, the relatively high MPXV DNA content of saliva warrants elucidating its potential role in disease transmission.

Characteristics of confirmed mpox cases among clinical suspects: A prospective single-centre study in Belgium during the 2022 outbreak.

Background: The presentation of mpox clade IIb during the 2022 outbreak overlaps with a range of other diseases. Understanding the factors associated with mpox is important for clinical decision making. Methods: We described the characteristics of mpox patients who sought care at Belgian sexual health clinic. Furthermore we compared their characteristics to those of patients with a clinical suspicion of mpox but who tested negative on polymerase chain reaction. Results: Between May 23 and September 20, 2022, 155 patients were diagnosed with mpox, and 51 patients with suspected symptoms tested negative. All mpox patients self-identified as men and 148/155 (95.5%) as gay or bisexual MSM. Systemic symptoms were present in 116/155 (74.8%) patients. All but 10 patients (145/155, 93.5%) presented with skin lesions. Other manifestations were lymphadenopathy (72/155, 46.5%), proctitis (50/155, 32.3%), urethritis (12/155, 7.7%), tonsillitis (2/155, 1.3%). Complications involved bacterial skin infection (13/155, 8.4%) and penile oedema with or without paraphimosis (4/155, 2.6%). In multivariable logistic regression models, the presence of lymphadenopathy (OR 3.79 95% CI 1.44-11.49), skin lesions (OR 4.35 95% CI 1.15-17.57) and proctitis (OR 9.41 95% CI 2.72-47.07) were associated with the diagnosis of mpox. There were no associations with age, HIV status, childhood smallpox vaccination, number of sexual partners and international travel. Conclusions: The presence of proctitis, lymphadenopathies and skin lesions should increase clinical suspicion of mpox in patients with compatible symptoms.

Leveraging T-cell receptor - epitope recognition models to disentangle unique and cross-reactive T-cell response to SARS-CoV-2 during COVID-19 progression/resolution.

Despite the general agreement on the significance of T cells during SARS-CoV-2 infection, the clinical impact of specific and cross-reactive T-cell responses remains uncertain. Understanding this aspect could provide insights for adjusting vaccines and maintaining robust long-term protection against continuously emerging variants. To characterize CD8+ T-cell response to SARS-CoV-2 epitopes unique to the virus (SC2-unique) or shared with other coronaviruses (CoV-common), we trained a large number of T-cell receptor (TCR) - epitope recognition models for MHC-I-presented SARS-CoV-2 epitopes from publicly available data. These models were then applied to longitudinal CD8+ TCR repertoires from critical and non-critical COVID-19 patients. In spite of comparable initial CoV-common TCR repertoire depth and CD8+ T-cell depletion, the temporal dynamics of SC2-unique TCRs differed depending on the disease severity. Specifically, while non-critical patients demonstrated a large and diverse SC2-unique TCR repertoire by the second week of the disease, critical patients did not. Furthermore, only non-critical patients exhibited redundancy in the CD8+ T-cell response to both groups of epitopes, SC2-unique and CoV-common. These findings indicate a valuable contribution of the SC2-unique CD8+ TCR repertoires. Therefore, a combination of specific and cross-reactive CD8+ T-cell responses may offer a stronger clinical advantage. Besides tracking the specific and cross-reactive SARS-CoV-2 CD8+ T cells in any TCR repertoire, our analytical framework can be expanded to more epitopes and assist in the assessment and monitoring of CD8+ T-cell response to other infections.

Enhanced surveillance of monkeypox in Bas-Uélé, Democratic Republic of Congo: the limitations of symptom-based case definitions.

BACKGROUND: Following an outbreak of cases of vesicular-pustular rash with fever, evocative of human monkeypox, in Bas-Uélé province, Democratic Republic of Congo, surveillance was strengthened. METHODS: Households with at least one active generalized vesicular-pustular rash case were visited, and contact and clinical history information were collected from all household members. Whenever possible, skin lesions were screened by polymerase chain reaction for the monkeypox virus, followed by the varicella-zoster virus, when negative for the former. RESULTS: Polymerase chain reaction results were obtained for 77 suspected cases, distributed in 138 households, of which 27.3% were positive for monkeypox, 58.4% positive for chickenpox, and 14.3% negative for both. Confirmed monkeypox cases presented more often with monomorphic skin lesions on the palms of the hands and on the soles of the feet. Integrating these three features into the case definition raised the specificity to 85% but would miss 50% of true monkeypox cases. A predictive model fit on patient demographics and symptoms had 97% specificity and 80% sensitivity but only 80% and 33% in predicting out-of-sample cases. CONCLUSION: Few discriminating features were identified and the performance of clinical case definitions was suboptimal. Rapid field diagnostics are needed to optimize worldwide early detection and surveillance of monkeypox.

Retrospective detection of asymptomatic monkeypox virus infections among male sexual health clinic attendees in Belgium.

The magnitude of the 2022 multi-country monkeypox virus (MPXV) outbreak has surpassed any preceding outbreak. It is unclear whether asymptomatic or otherwise undiagnosed infections are fuelling this epidemic. In this study, we aimed to assess whether undiagnosed infections occurred among men attending a Belgian sexual health clinic in May 2022. We retrospectively screened 224 samples collected for gonorrhea and chlamydia testing using an MPXV PCR assay and identified MPXV-DNA-positive samples from four men. At the time of sampling, one man had a painful rash, and three men had reported no symptoms. Upon clinical examination 21-37 days later, these three men were free of clinical signs, and they reported not having experienced any symptoms. Serology confirmed MPXV exposure in all three men, and MPXV was cultured from two cases. These findings show that certain cases of monkeypox remain undiagnosed and suggest that testing and quarantining of individuals reporting symptoms may not suffice to contain the outbreak.

Proline-specific peptidase activities (DPP4, PRCP, FAP and PREP) in plasma of hospitalized COVID-19 patients.

BACKGROUND: COVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospitalized COVID-19 patients. METHODS: Fifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longitudinal follow-up samples. RESULTS: Compared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 patients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge. CONCLUSION: The changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel.