Antibody-drug conjugates in lung and breast cancer: current evidence and future directions-a position statement from the ETOP IBCSG Partners Foundation.

Following the approval of the first antibody-drug conjugates (ADCs) in the early 2000s, development has increased dramatically, with 14 ADCs now approved and >100 in clinical development. In lung cancer, trastuzumab deruxtecan (T-DXd) is approved in human epidermal growth factor receptor 2 (HER2)-mutated, unresectable or metastatic non-small-cell lung cancer, with ADCs targeting HER3 (patritumab deruxtecan), trophoblast cell-surface antigen 2 [datopotamab deruxtecan and sacituzumab govitecan (SG)] and mesenchymal-epithelial transition factor (telisotuzumab vedotin) in late-stage clinical development. In breast cancer, several agents are already approved and widely used, including trastuzumab emtansine, T-DXd and SG, and multiple late-stage trials are ongoing. Thus, in the coming years, we are likely to see significant changes to treatment algorithms. As the number of available ADCs increases, biomarkers (of response and resistance) to better select patients are urgently needed. Biopsy sample collection at the time of treatment selection and incorporation of translational research into clinical trial designs are therefore critical. Biopsy samples taken peri- and post-ADC treatment combined with functional genomics screens could provide insights into response/resistance mechanisms as well as the impact of ADCs on tumour biology and the tumour microenvironment, which could improve understanding of the mechanisms underlying these complex molecules. Many ADCs are undergoing evaluation as combination therapy, but a high bar should be set to progress clinical evaluation of any ADC-based combination, particularly considering the high cost and potential toxicity implications. Efforts to optimise ADC dosing/duration, sequencing and the potential for ADC rechallenge are also important, especially considering sustainability aspects. The ETOP IBCSG Partners Foundation are driving strong collaborations in this field and promoting the generation/sharing of databases, repositories and registries to enable greater access to data. This will allow the most important research questions to be identified and prioritised, which will ultimately accelerate progress and help to improve patient outcomes.

Amivantamab plus chemotherapy with and without lazertinib in EGFR-mutant advanced NSCLC after disease progression on osimertinib: primary results from the phase III MARIPOSA-2 study.

BACKGROUND: Amivantamab plus carboplatin-pemetrexed (chemotherapy) with and without lazertinib demonstrated antitumor activity in patients with refractory epidermal growth factor receptor (EGFR)-mutated advanced non-small-cell lung cancer (NSCLC) in phase I studies. These combinations were evaluated in a global phase III trial. PATIENTS AND METHODS: A total of 657 patients with EGFR-mutated (exon 19 deletions or L858R) locally advanced or metastatic NSCLC after disease progression on osimertinib were randomized 2 : 2 : 1 to receive amivantamab-lazertinib-chemotherapy, chemotherapy, or amivantamab-chemotherapy. The dual primary endpoints were progression-free survival (PFS) of amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy. During the study, hematologic toxicities observed in the amivantamab-lazertinib-chemotherapy arm necessitated a regimen change to start lazertinib after carboplatin completion. RESULTS: All baseline characteristics were well balanced across the three arms, including by history of brain metastases and prior brain radiation. PFS was significantly longer for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy [hazard ratio (HR) for disease progression or death 0.48 and 0.44, respectively; P < 0.001 for both; median of 6.3 and 8.3 versus 4.2 months, respectively]. Consistent PFS results were seen by investigator assessment (HR for disease progression or death 0.41 and 0.38 for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy, respectively; P < 0.001 for both; median of 8.2 and 8.3 versus 4.2 months, respectively). Objective response rate was significantly higher for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy (64% and 63% versus 36%, respectively; P < 0.001 for both). Median intracranial PFS was 12.5 and 12.8 versus 8.3 months for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy (HR for intracranial disease progression or death 0.55 and 0.58, respectively). Predominant adverse events (AEs) in the amivantamab-containing regimens were hematologic, EGFR-, and MET-related toxicities. Amivantamab-chemotherapy had lower rates of hematologic AEs than amivantamab-lazertinib-chemotherapy. CONCLUSIONS: Amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy improved PFS and intracranial PFS versus chemotherapy in a population with limited options after disease progression on osimertinib. Longer follow-up is needed for the modified amivantamab-lazertinib-chemotherapy regimen.

From intensive care to rehabilitation: survey on the satisfaction with care received during prolonged hospitalization for COVID-19 at a northern Italian university hospital.

OBJECTIVE: Investigating the experiences perceived by COVID-19 inpatients is a fundamental research area that is starting to be explored. For this reason, our objective was to provide the first Italian survey on COVID-19 inpatients' satisfaction, obtained through a self-completed questionnaire previously used in a reference study in a UK cohort of COVID-19 patients. SUBJECTS AND METHODS: Hospitalized COVID-19 patients (>20 days) admitted to Ferrara University Hospital who underwent rehabilitation during their hospital stay were invited to complete an anonymous questionnaire. The survey's questions explored the patients' satisfaction with the health services received, and their completion took place approximately one year after hospitalization. Information on sex, number of wards, ICU stays, and hospital discharge dates was collected. RESULTS: Sixty-two completed questionnaires were analyzed. The average overall satisfaction score obtained from the answers indicated by the participants in the tenth question was 4.7 out of 5.0. Very positive responses were observed for information about discharge plans, privacy, management of pain, sleep quality, and feeling of safety. The possibility of being consulted about medications and side effects received a very low satisfaction score. Considering overall satisfaction, no significant differences were noted for sex or ICU stay. The obtained results were almost superimposable to those reported in the cohort of COVID-19 patients of the reference study. CONCLUSIONS: This survey suggested that COVID-19 patients' healthcare satisfaction was high. Nevertheless, some areas must be improved, such as the communication and involvement of the patients in the decision-making of care and the discussion about medications or possible side effects.

Effect of the association of continuous shortwave diathermy and Pilates-based exercises on pain, depression, and anxiety in chronic non-specific low back pain: a randomized clinical trial.

Chronic nonspecific low back pain (CNLBP) is the most common musculoskeletal condition, which can be influenced by nociceptive, psychosocial, cognitive, and affective aspects, causing vulnerabilities and impairing the individual's ability to manage pain. The association of continuous shortwave diathermy (CSWD) with Pilates-based exercises may contribute to reduce pain, depression, and anxiety in patients with CNLBP. A single-blind randomized clinical trial was performed in which 36 patients with CNLBP were divided into a control group that received placebo CSWD and an intervention group that received active CSWD. Both groups received 12 sessions of Pilates-based exercises. Pain, depression, and anxiety variables were evaluated using the McGill questionnaire, the Beck Depression Inventory, and the Visual Analog Anxiety Scale. Assessments were performed at baseline, after three and six weeks of treatment, and at the three-month follow-up. The Shapiro-Wilk test, Student's t-test, Mann-Whitney U test, chi-squared test, and repeated measures ANOVA, with α=0.05, were used to compare the outcomes, and indicated that active CSWD did not present additional improvement in the assessed variables in CNLBP patients compared to the placebo group. Both groups improved pain and depression at follow-up and reduced anxiety only during Pilates-based exercises. Therefore, only Pilates-based exercises seemed sufficient to manage patients with CNLBP.

Safety of mRNA-COVID-19 Vaccines in Patients With Thoracic Cancers.

BACKGROUND: Pivotal trials of COVID-19 vaccines did not include cancer patients with questions remaining in this population. Particularly in patients with thoracic malignancies receiving anticancer treatments, the safety of these vaccines has so far been little investigated. METHODS: This is a prospective trial of patients with thoracic cancer receiving anticancer treatments and COVID-19 vaccines at the Division of Thoracic Oncology of European Institute of Oncology between February and September 2021. RESULTS: A total 207 patients affected by thoracic cancers (199 lung cancers and 8 mesotheliomas) had received Covid-19 vaccines (206 mRNA vaccines and 1 virus-vectored vaccine). The majority of patients had at least one comorbidity (76.3%). They were concomitantly treating with targeted therapy (TT) (45.9%), immunotherapy (IO) (22.7%), and chemotherapy (CT) (14%). A total of 64 AEs (15.6%) were observed after administration of Sars-Cov-2 vaccine. The majority of AEs were grade 1 [G1] (6.3%) and G2 (8.8%), only two events were G3 (0.5%). The median follow-up was 9 months (range 1-22 months), during this follow-up 21 patients (10.1%) had a positive nasal swab, most of the patients were asymptomatic (67%) and the symptomatic ones (33%) had mild symptoms and fewer complications and hospitalizations. CONCLUSIONS: COVID-19 m-RNA vaccines appear to be safe in the cohort of patients with thoracic malignances in active treatment, including those receiving immunotherapy. Considering the high morbidity and mortality associated with COVID-19 in patients with lung cancer receiving active treatments, our study supports the current vaccine prioritization, third and/or fourth dose, of all cancer patients with active treatment.

Effect of the association of continuous shortwave diathermy and Pilates-based exercises on pain, depression, and anxiety in chronic non-specific low back pain: a randomized clinical trial

Chronic nonspecific low back pain (CNLBP) is the most common musculoskeletal condition, which can be influenced by nociceptive, psychosocial, cognitive, and affective aspects, causing vulnerabilities and impairing the individual's ability to manage pain. The association of continuous shortwave diathermy (CSWD) with Pilates-based exercises may contribute to reduce pain, depression, and anxiety in patients with CNLBP. A single-blind randomized clinical trial was performed in which 36 patients with CNLBP were divided into a control group that received placebo CSWD and an intervention group that received active CSWD. Both groups received 12 sessions of Pilates-based exercises. Pain, depression, and anxiety variables were evaluated using the McGill questionnaire, the Beck Depression Inventory, and the Visual Analog Anxiety Scale. Assessments were performed at baseline, after three and six weeks of treatment, and at the three-month follow-up. The Shapiro-Wilk test, Student's t-test, Mann-Whitney U test, chi-squared test, and repeated measures ANOVA, with α=0.05, were used to compare the outcomes, and indicated that active CSWD did not present additional improvement in the assessed variables in CNLBP patients compared to the placebo group. Both groups improved pain and depression at follow-up and reduced anxiety only during Pilates-based exercises. Therefore, only Pilates-based exercises seemed sufficient to manage patients with CNLBP.

Age, Sex, Hypertension and HDL-C Alter Serum BACE1 Activity in Cognitively Normal Subjects: Implications for Alzheimer's Disease.

BACKGROUND: Increasing evidence indicates that ß-secretase 1 (BACE1) activity and concentration in blood are candidate biomarkers for Alzheimer's disease (AD). Investigating potential demographic, biological, and clinical determinants of BACE1 in the blood matrix is the critical step to validate and qualify BACE1 bio-indicators for different contexts-of-use (CoU), such as risk assessment, early detection, diagnosis, prognosis, management of AD, and outcome of amyloid pathway targeted drugs. OBJECTIVES: To evaluate the influence of age, sex, HDL-cholesterol and comorbidities (cardiovascular diseases, hypertension, diabetes) on circulating BACE-1 activity. DESIGN: prospective analysis of serum samples, clinical, biological, and demographic variables. SETTING: Three cohorts: 1) Memory Clinic of the Department of Internal Medicine, S. Anna University Hospital, Ferrara (Italy); 2) outpatients attending the Menopause and Osteoporosis Centre (MOC) of the University of Ferrara (Ferrara, Italy); 3) Prevention Center of the University of Ferrara. PARTICIPANTS: 504 cognitively healthy individuals (median age: 62 years, interquartile range: 51-73) and 175 patients with AD (78 years, 74-82). MEASUREMENTS: serum BACE1 (sBACE1), age, sex, HDL-cholesterol, major comorbidities. RESULTS: Age was the strongest independent predictor of sBACE1 variance (ß=0.425, p<0.0001), followed by sex (ß=0.180, p<0.0001), high density lipoprotein-cholesterol (HDL-C) (ß=-0.168, p<0.0001) and hypertension (ß=0.111, p<0.05) (overall model, R2: 0.232). The probability of having elevated sBACE1 activity increased after 70 years of age, with women being more susceptible to higher sBACE1 activity than men. CONCLUSIONS: We provide evidence about potential clinical and biological determinants of sBACE1 activity with a strong association among biomarker, female sex, and older age.

COVID-19: identifying the main outcome predictors. A retrospective cohort study in Northern Italy.

OBJECTIVE: The need for efficient drugs and early treatment of patients with SARS-CoV-2 infection developing COVID-19 symptoms is of primary importance in daily clinical practice and it is certainly among the most difficult medical challenges in the current century. Recognizing those patients who will need stronger clinical efforts could effectively help doctors anticipate the eventual need for intensification of care (IoC) and choose the best treatment in order to avoid worse outcomes. PATIENTS AND METHODS: We enrolled 501 patients, consecutively admitted to our two COVID hospitals, and collected their clinical, anamnestic and laboratory data on admission. The aim of this retrospective study was to identify those data that are strictly associated with COVID-19 outcomes (IoC and in-hospital death) and that could somehow be intended as predictors of these outcomes. This allowed us to provide a "sketch" of the patient who undergoes, more often than others, an intensification of care and/or in-hospital death. RESULTS: Males were found to have a double risk of needing an IoC (OR=2.11) and a significant role was played by both the PaO2/FiO2 ratio on admission (OR=0.99) and serum LDH (OR=1.01). The main predictors of in-hospital death were age (OR=1.08) and the PaO2/FiO2 ratio on admission (OR=0.99). CONCLUSIONS: Male patients with high serum LDH on admission are those who undergo more often an intensification of care among COVID-19 inpatients. Both age and respiratory performances on admission modify the prognosis within the hospitalization period.

ESMO expert consensus statements on the management of EGFR mutant non-small-cell lung cancer.

The European Society for Medical Oncology (ESMO) held a virtual consensus-building process on epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer in 2021. The consensus included a multidisciplinary panel of 34 leading experts in the management of lung cancer. The aim of the consensus was to develop recommendations on topics that are not covered in detail in the current ESMO Clinical Practice Guideline and where the available evidence is either limited or conflicting. The main topics identified for discussion were: (i) tissue and biomarkers analyses; (ii) early and locally advanced disease; (iii) metastatic disease and (iv) clinical trial design, patient's perspective and miscellaneous. The expert panel was divided into four working groups to address questions relating to one of the four topics outlined above. Relevant scientific literature was reviewed in advance. Recommendations were developed by the working groups and then presented to the entire panel for further discussion and amendment before voting. This manuscript presents the recommendations developed, including findings from the expert panel discussions, consensus recommendations and a summary of evidence supporting each recommendation.

Giomer composite compared to glass ionomer in occlusoproximal ART restorations of primary molars: 24-month RCT.

BACKGROUND: Occlusoproximal restorations of primary molars usually fail, so it is necessary to investigate new materials that may overcome this challenge. Thus, this trial aimed to evaluate the longevity of occlusoproximal ART restorations in primary molars using a glass ionomer cement - GIC (Equia Forte® - GC Corp) and a Giomer resin composite - GCR (Beautifil Bulk Restorative® - Shofu Inc) after 24 months. METHODS: One hundred and eighty-two (182) children aged from 4 to 8 years were selected and randomly assigned to GIC or GCR. A paediatric dentist treated them in the school setting in Cerquilho, Brazil, and the restorations were assessed after 3, 6, 12, 18 and 24 months. The primary outcome was the restoration survival, evaluated using the Kaplan-Meier and superiority Cox regression analyses. Intention to treat (ITT) was performed as a sensitivity analysis using superiority test P value and confidence interval (CI = 95%). Independent variables included gender, age, molar, jaw, cavity volume and caries experience. RESULTS: The restoration survival after 24 months was GIC = 58.1% and GCR = 49.1% (HR = 1.24; CI = 0.97-1.59). ITT analysis showed a success of GIC = 61.1% and GCR = 52.2% (RR = 1.17; CI = 0.91-1.52). The superiority hypothesis was not proved in both analyses (P > 0.05). CONCLUSION: GCR does not have superior longevity than GIC in occlusoproximal ART restorations of primary molars.

SARS-CoV-2 infection and H1N1 vaccination: does a relationship between the two factors really exist? A retrospective analysis of a territorial cohort in Ferrara, Italy.

OBJECTIVE: SARS-CoV-2 has been compared with other strains of coronaviruses, SARS-CoV and MERS-CoV, and with the flu viruses: all of them manifest themselves with respiratory symptoms and, although their genetic patterns are similar, the spread of SARS-CoV-2 infection has quickly reached global dimensions, demonstrating that SARS-CoV-2 is a virus with greater spreading capacity, albeit less lethal. Compared with influenza viruses, coronaviruses have a longer incubation period and the patients with coronaviruses' syndromes develop more severe diseases requiring frequent hospitalizations and intensive care admissions. The aim was to explore the relationships between seasonal influenza vaccination and coronavirus infection and to understand whether this hypothetic role by the flu vaccines modifies SARS-CoV-2 infection's outcomes. PATIENTS AND METHODS: In this retrospective, multicenter study, we enrolled 952 patients diagnosed with SARS-CoV-2 infection; 448 were admitted to our two main hospitals in Ferrara territory, while the remaining 504 were isolated at home. We compared the group of patients who had been vaccinated for influenza in the previous 12 months to that of unvaccinated patients. RESULTS: Significant differences were found for both the need for hospitalization and 30-day mortality between vaccinated and unvaccinated patients. We found age to be the only independent risk factor for a worse 30-day prognosis, while gender, influenza vaccinations and age itself were independent risk factors for undergoing hospitalization. CONCLUSIONS: In our groups of patients, we found a relationship between seasonal influenza vaccinations and SARS-CoV-2 infection. Age seems to be the main risk factor for short-term mortality in COVID-19 inpatients, while the influenza vaccination is, together with gender and age itself, a determining factor in predicting the need for hospitalization.

Biases in study design, implementation, and data analysis that distort the appraisal of clinical benefit and ESMO-Magnitude of Clinical Benefit Scale (ESMO-MCBS) scoring.

BACKGROUND: The European Society for Medical Oncology-Magnitude of Clinical Benefit Scale (ESMO-MCBS) is a validated, widely used tool developed to score the clinical benefit from cancer medicines reported in clinical trials. ESMO-MCBS scores assume valid research methodologies and quality trial implementation. Studies incorporating flawed design, implementation, or data analysis may generate outcomes that exaggerate true benefit and are not generalisable. Failure to either indicate or penalise studies with bias undermines the intention and diminishes the integrity of ESMO-MCBS scores. This review aimed to evaluate the adequacy of the ESMO-MCBS to address bias generated by flawed design, implementation, or data analysis and identify shortcomings in need of amendment. METHODS: As part of a refinement of the ESMO-MCBS, we reviewed trial design, implementation, and data analysis issues that could bias the results. For each issue of concern, we reviewed the ESMO-MCBS v1.1 approach against standards derived from Helsinki guidelines for ethical human research and guidelines from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, the Food and Drugs Administration, the European Medicines Agency, and European Network for Health Technology Assessment. RESULTS: Six design, two implementation, and two data analysis and interpretation issues were evaluated and in three, the ESMO-MCBS provided adequate protections. Seven shortcomings in the ability of the ESMO-MCBS to identify and address bias were identified. These related to (i) evaluation of the control arm, (ii) crossover issues, (iii) criteria for non-inferiority, (iv) substandard post-progression treatment, (v) post hoc subgroup findings based on biomarkers, (vi) informative censoring, and (vii) publication bias against quality-of-life data. CONCLUSION: Interpretation of the ESMO-MCBS scores requires critical appraisal of trials to understand caveats in trial design, implementation, and data analysis that may have biased results and conclusions. These will be addressed in future iterations of the ESMO-MCBS.

SARS-CoV-2 and finding of vein thrombosis: can IMPROVE and IMPROVEDD scores predict COVID-19 outcomes?

OBJECTIVE: Diffuse thrombosis represents one of the most predominant causes of death by COVID-19 and SARS-CoV-2 infection seems to increase the risk of developing venous thromboembolic diseases (VTE). Aim of this study is to analyze the relationship between validated predictive scores for VTE such as IMPROVE and IMPROVEDD and: (1) Intensification of Care (IoC, admission to Pulmonology Department or Intensive Care Unit) (2) in-hospital mortality rate 3) 30-days mortality rate. PATIENTS AND METHODS: We retrospectively evaluated 51 adult patients with laboratory diagnosis of SARS-CoV-2 infection and calculated IMPROVE and IMPROVEDD scores. All patients underwent venous color-Doppler ultrasound of the lower limbs to assess the presence of superficial vein thrombosis (SVT) and/or deep vein thrombosis (DVT). Patients with normal values of D-dimer did not receive heparin therapy (LMWH); patients with ≥ 4 ULN values of D-dimer or with a diagnosis of DVT were treated with therapeutic LMWH dosage, while the remaining patients were treated with prophylactic LMWH dosages. RESULTS: We found strong relations between IMPROVE score and the need for IoC and with the in-hospital mortality rate and between the IMPROVEDD score and the need for IoC. We defined that an IMPROVE score greater than 4 points was significantly associated to in-hospital mortality rate (p = 0.05), while an IMPROVEDD score greater than 3 points was associated with the need for IoC (p = 0.04). Multivariate logistic analysis showed how IMPROVE score was significantly associated to in-hospital and 30-days mortality rates. CONCLUSIONS: IMPROVE score can be considered an independent predictor of in-hospital and 30-days mortality.

Prediction of in-hospital mortality of patients with SARS-CoV-2 infection by comorbidity indexes: an Italian internal medicine single center study.

OBJECTIVE: Clinical outcomes in patients hospitalized for severe acute respiratory syndrome due to coronavirus (SARS-CoV-2) infection seems to be closely related with burden of comorbidities. A comorbidity score could help in clinical stratification of patients admitted to internal medicine units. Our aim was to assess a novel modified Elixhauser index (mEi) and the Charlson Comorbidity Index (CCI) for predicting in-hospital mortality (IHM) in internal medicine patients with SARS-CoV-2 infection. PATIENTS AND METHODS: This single-center retrospective study enrolled all consecutive patients discharged from internal medicine unit with confirmed SARS-CoV-2 infection. Both the mEi and CCI were easily calculated from administrative data. Comorbidity scores were tested using receiver operating characteristic (ROC) analysis, and the respective area under the curve (AUC). RESULTS: The total sample consisted of 151 individuals, and 30 (19.9%) died during their hospital stay. Deceased subjects were older (82.8±10.8 vs. 63.3±18.1 years; p<0.001) and had a higher burden of comorbidities: the mEi and CCI were 29.9±11 vs. 8.8±9.2 and 4.6±2.6 vs. 1.2±2 (p<0.001), respectively. Only the mEi was independently associated with IHM (OR 1.173), and ROC curves analysis showed that the AUCs were 0.863 and 0.918 for the CCI and for mEi, respectively. CONCLUSIONS: In patients admitted to internal medicine wards with SARS-CoV-2 infection, the mEi showed a better performance in predicting IHM than CCI.

SARS-CoV-2 RNA contamination on surfaces of a COVID-19 ward in a hospital of Northern Italy: what risk of transmission?

OBJECTIVE: SARS-CoV-2 can reportedly exist on inanimate surfaces for a long duration, but there is limited data available from Italian COVID-19 hospital wards, especially for non-intensive care units hosting patients that do not require mechanical ventilation. Identification of the extent of environmental contamination can help in understanding possible virus transmission routes, limit hospital infections and protect healthcare workers. Thus, we investigated virus contamination on surfaces of the acute COVID-19 ward of an Italian hospital. MATERIALS AND METHODS: Ward surfaces, including four points inside and six points outside the patients' rooms were sampled by swabs, seven hours after routine sanitation. To minimize the risk of underestimation of virus detection, two different sensitive molecular methods were used comparatively, and specific internal controls were added to enhance the efficiency of all the analysis steps. RESULTS: SARS-CoV-2 contamination was detected in only three out of all the collected samples, i.e., on two floors and one-bathroom sink, likely reflecting aerosol and saliva contamination, respectively. The overall level of contamination was low, and the floors exhibited a very low level of SARS-CoV-2 presence, evidenced by only one of the two methods used. CONCLUSIONS: The existence of SARS-CoV-2 on hospital surfaces may be limited, although it was reported to persist for a longer duration on surfaces under controlled laboratory conditions. Thus, effective transmission of SARS-CoV-2 by surfaces/fomites within the hospital ward may be a rare event. However, the results highlight the importance of assessing method sensitivity and including controls when investigating low-level virus contamination so as to avoid the risk of underestimation of virus presence.