The biological variation of insulin resistance markers: data from the European Biological Variation Study (EuBIVAS).

OBJECTIVES: An insulin resistant state is characteristic of patients with type 2 diabetes, polycystic ovary syndrome, and metabolic syndrome. Identification of insulin resistance (IR) is most readily achievable using formulae combining plasma insulin and glucose results. In this study, we have used data from the European Biological Variation Study (EuBIVAS) to examine the biological variability (BV) of IR using the Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) and the Quantitative Insulin sensitivity Check Index (QUICKI). METHODS: Ninety EuBIVAS non-diabetic subjects (52F, 38M) from five countries had fasting HOMA-IR and QUICKI calculated from plasma glucose and insulin samples collected concurrently on 10 weekly occasions. The within-subject (CVI) and between-subject (CVG) BV estimates with 95 % CIs were obtained by CV-ANOVA after analysis of trends, variance homogeneity and outlier removal. RESULTS: The CVI of HOMA-IR was 26.7 % (95 % CI 25.5-28.3), driven largely by variability in plasma insulin and the CVI for QUICKI was 4.1 % (95 % CI 3.9-4.3), reflecting this formula's logarithmic transformation of glucose and insulin values. No differences in values or BV components were observed between subgroups of men or women below and above 50 years. CONCLUSIONS: The EuBIVAS, by utilising a rigorous experimental protocol, has produced robust BV estimates for two of the most commonly used markers of insulin resistance in non-diabetic subjects. This has shown that HOMA-IR, in particular, is highly variable in the same individual which limits the value of single measurements.

Non-O1 and non-O139 Vibrio cholerae Septicemia and Cellulitis: a Case Report.

Non-O1 and non-O139 Vibrio cholerae (NOVC) are serogroups that do not produce cholera toxin and are not responsible for epidemics. Even though rarely encountered in clinical practice, they can cause a spectrum of different conditions ranging from mild gastrointestinal syndrome to extraintestinal diseases, of which bacteremia and wound infections are the most severe. Risk factors for severe disease are cirrhosis, neoplasms, and diabetes mellitus. The mortality rate of NOVC bacteremia in hospitalized patients ranges from 24 to 61.5%. Incidence of NOVC infections is still rare, and consensus recommendations on treatment are not available. We report a case of NOVC bacteremia associated with severe cellulitis in an immunocompetent 75-year-old man who had eaten raw seafood in a location by the northern Adriatic Sea (Italy). Twenty-four hours after intake, he developed a high fever and vomiting. Afterwards, he started noticing the appearance of cellulitis in his right leg, which worsened in a matter of hours. The patient had a history of compensated type 2 diabetes mellitus. NOVC was isolated from both blood cultures and the leg ulcer. The non-O1, non-O139 serogroup was confirmed, and the detection of the cholera toxin gene was negative. Both tests were performed by the Reference National Laboratory of Istituto Superiore di Sanità (ISS). Multiple antimicrobial regimens were administered, with complete recovery. In conclusion, considering the severity of NOVC-associated manifestations, it is of pivotal importance to reach etiological diagnosis for a target antimicrobial therapy and to consider V. cholerae infection in the differential diagnosis in the presence of risk factors and potential exposure.

Smallpox vaccination-elicited antibodies cross-neutralize 2022-Monkeypox virus Clade II.

Since early May 2022, some monkeypox virus (MPXV) infections have been reported from countries where the disease is not endemic. Within 2 months, the number of patients has increased extensively, becoming the most considerable MPXV outbreak described. Smallpox vaccines demonstrated high efficacy against MPXVs in the past and are considered a crucial outbreak control measure. However, viruses isolated during the current outbreak carry distinct genetic variations, and the cross-neutralizing capability of antibodies remains to be assessed. Here we report that serum antibodies elicited by first-generation smallpox vaccines can neutralize the current MPXV more than 40 years after vaccine administration.

Review on adherence of the literature to official recommendations on albuminuria harmonization and standardization.

Albuminuria standardization is a key issue to produce reliable and equivalent results between laboratories. We investigated whether official recommendations on albuminuria harmonization are followed in the literature. The PubMed database was searched from June 1 to September 26, 2021. The search terms included urine albumin, urine albumin-to-creatinine ratio (uACR), and albuminuria. A total of 159 articles were considered eligible; 50.9 % reported the type of urine collection. Specifically, 58.1 % collected a random spot urine specimen, 21 % collected a first morning void, and 6.2 % collected a 24-h specimen. Overall, 15 % of articles reported data on sample shipping, storage, and centrifugation and 13.3 % mentioned the preanalytical phase without any data on albuminuria. The method for albuminuria was properly described in 31.4 % of articles; of these, 54.9 % used immunological methods, and 8.9 % contained errors or missing data. Most articles (76.7 %) expressed test results as albuminuria-to-creatininuria ratio. Different decision levels were utilized in 130 articles; of these, 36 % used a decision level of ≤30 mg/g creatininuria and 23.7 % used three decision levels (≤30, 30-300, and ≥300 mg/g). The failure to follow guidelines on albuminuria harmonization was mainly found in the preanalytical phase. The poor awareness of the importance of preanalytical steps on test result may be a possible explanation.

European Biological Variation Study (EuBIVAS): within- and between-subject biological variation estimates for serum thyroid biomarkers based on weekly samplings from 91 healthy participants.

OBJECTIVES: Thyroid biomarkers are fundamental for the diagnosis of thyroid disorders and for the monitoring and treatment of patients with these diseases. The knowledge of biological variation (BV) is important to define analytical performance specifications (APS) and reference change values (RCV). The aim of this study was to deliver BV estimates for thyroid stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), thyroglobulin (TG), and calcitonin (CT). METHODS: Analyses were performed on serum samples obtained from the European Biological Variation Study population (91 healthy individuals from six European laboratories; 21-69 years) on the Roche Cobas e801 at the San Raffaele Hospital (Milan, Italy). All samples from each individual were evaluated in duplicate within a single run. The BV estimates with 95% CIs were obtained by CV-ANOVA, after analysis of variance homogeneity and outliers. RESULTS: The within-subject (CV I ) BV estimates were for TSH 17.7%, FT3 5.0%, FT4 4.8%, TG 10.3, and CT 13.0%, all significantly lower than those reported in the literature. No significant differences were observed for BV estimates between men and women. CONCLUSIONS: The availability of updated, in the case of CT not previously published, BV estimates for thyroid markers based on the large scale EuBIVAS study allows for refined APS and associated RCV applicable in the diagnosis and management of thyroid and related diseases.

The European Biological Variation Study (EuBIVAS): a summary report.

Biological variation (BV) data have many important applications in laboratory medicine. Concerns about quality of published BV data led the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) 1st Strategic Conference to indicate need for new studies to generate BV estimates of required quality. In response, the EFLM Working Group on BV delivered the multicenter European Biological Variation Study (EuBIVAS). This review summarises the EuBIVAS and its outcomes. Serum/plasma samples were taken from 91 ostensibly healthy individuals for 10 consecutive weeks at 6 European centres. Analysis was performed by Siemens ADVIA 2400 (clinical chemistry), Cobas Roche 8000, c702 and e801 (proteins and tumor markers/hormones respectively), ACL Top 750 (coagulation parameters), and IDS iSYS or DiaSorin Liaison (bone biomarkers). A strict preanalytical and analytical protocol was applied. To determine BV estimates with 95% CI, CV-ANOVA after analysis of outliers, homogeneity and trend analysis or a Bayesian model was applied. EuBIVAS has so far delivered BV estimates for 80 different measurands. Estimates for 10 measurands (non-HDL cholesterol, S100-ß protein, neuron-specific enolase, soluble transferrin receptor, intact fibroblast growth-factor-23, uncarboxylated-unphosphorylated matrix-Gla protein, human epididymis protein-4, free, conjugated and %free prostate-specific antigen), prior to EuBIVAS, have not been available. BV data for creatinine and troponin I were obtained using two analytical methods in each case. The EuBIVAS has delivered high-quality BV data for a wide range of measurands. The BV estimates are for many measurands lower than those previously reported, having an impact on the derived analytical performance specifications and reference change values.

Within- and between-subject biological variation data for tumor markers based on the European Biological Variation Study.

OBJECTIVES: Reliable biological variation (BV) data are required for the clinical use of tumor markers in the diagnosis and monitoring of treatment effects in cancer. The European Biological Variation Study (EuBIVAS) was established by the EFLM Biological Variation Working Group to deliver BV data for clinically important measurands. In this study, EuBIVAS-based BV estimates are provided for cancer antigen (CA) 125, CA 15-3, CA 19-9, carcinoembryonic antigen, cytokeratin-19 fragment, alpha-fetoprotein and human epididymis protein 4. METHODS: Subjects from five European countries were enrolled in the study, and weekly samples were collected from 91 healthy individuals (53 females and 38 males; 21-69 years old) for 10 consecutive weeks. All samples were analyzed in duplicate within a single run. After excluding outliers and homogeneity analysis, the BVs of tumor markers were determined by CV-ANOVA on trend-corrected data, when relevant (Røraas method). RESULTS: Marked individuality was found for all tumor markers. CYFRA 21-1 was the measurand with the highest index of individuality (II) at 0.67, whereas CA 19-9 had the lowest II at 0.07. The CV I s of HE4, CYFRA 21-1, CA 19-9, CA 125 and CA 15-3 of pre- and postmenopausal females were significantly different from each other. CONCLUSIONS: This study provides updated BV estimates for several tumor markers, and the findings indicate that marked individuality is characteristic. The use of reference change values should be considered when monitoring treatment of patients by means of tumor markers.

Serum Irisin May Predict Cardiovascular Events in Elderly Patients With Chronic Kidney Disease Stage 3-5.

OBJECTIVE: Irisin is a circulating myokine released from skeletal muscles after physical exercise. Irisin production decreases during the course of chronic kidney disease (CKD) as a potential consequence of sarcopenia and physical inactivity. METHODS: This observational study explored the relationship of serum irisin with cardiovascular outcome in 79 patients with stage 3-5 CKD. RESULTS: Serum irisin was significantly higher in healthy subjects (n = 20) than that in CKD patients (7 ± 2 vs. 3.1 ± 0.9 µg/mL; P = .0001) and was higher in patients with CKD stage 3 (3.2 ± 1 µg/mL) than in patients at stage 4 and 5 taken together (n = 36, 2.8 ± 0.7 µg/mL, P = .05). Patients in the lowest serum irisin tertile had lower serum 1,25(OH)2D levels (21 ± 11 pg/mL) than patients in the middle (30 ± 13 pg/mL; P = .005) and the highest tertile (27 ± 14 pg/mL; P = .047). Patients in the highest tertile had lower Kauppila score (10.6 ± 6.9) than patients in the middle (11.8 ± 5.5; P = .007) and the lowest tertile (6.9 ± 6.8; P = .043). Twenty patients suffered from cardiovascular events during a 3-year follow-up. A Cox regression model using age, body weight, presence of diabetes mellitus, gender, Kauppila calcification score, serum values of FGF23 (as logarithm), phosphate, sclerostin, albumin and cholesterol, estimated glomerular filtration rate, and serum irisin tertiles as covariates showed that patients in the highest tertile of serum irisin had a lower cardiovascular risk than patients in the middle tertile (B, 2.38; odds ratio, 10.8; 95% confidence interval, 1.65-58.13; P = .013) or in the lowest tertile (B, 1.61; odds ratio, 5; 95% confidence interval, 1.09-22.83; P = .038). CONCLUSIONS: These findings suggest that serum irisin may be a marker of cardiovascular outcome in patients with CKD.

Smart Immunosensors for Point-of-Care Serological Tests Aimed at Assessing Natural or Vaccine-Induced SARS-CoV-2 Immunity.

Innovative and highly performing smart voltammetric immunosensors for rapid and effective serological tests aimed at the determination of SARS-CoV-2 antibodies were developed and validated in human serum matrix. Two immunosensors were developed for the determination of immunoglobulins directed against either the nucleocapsid or the spike viral antigen proteins. The immunosensors were realized using disposable screen-printed electrodes modified with nanostructured materials for the immobilization of the antigens. Fast quantitative detection was achieved, with analysis duration being around 1 h. Signal readout was carried out through a smart, compact and battery-powered potentiostat, based on a Wi-Fi protocol and devised for the Internet of Things (IoT) paradigm. This device is used for the acquisition, storage and sharing of clinical data. Outstanding immunosensors' sensitivity, specificity and accuracy (100%) were assessed, according to the diagnostic guidelines for epidemiological data. The overall performance of the sensing devices, combined with the portability of the IoT-based device, enables their suitability as a high-throughput diagnostic tool. Both of the immunosensors were validated using clinical human serum specimens from SARS-CoV-2 infected patients, provided by IRCCS Ospedale San Raffaele.

MYD88 L265P mutation and interleukin-10 detection in cerebrospinal fluid are highly specific discriminating markers in patients with primary central nervous system lymphoma: results from a prospective study.

Reliable biomarkers are needed to avoid diagnostic delay and its devastating effects in patients with primary central nervous system (CNS) lymphoma (PCNSL). We analysed the discriminating sensitivity and specificity of myeloid differentiation primary response (88) (MYD88) L265P mutation (mut-MYD88) and interleukin-10 (IL-10) in cerebrospinal fluid (CSF) of both patients with newly diagnosed (n = 36) and relapsed (n = 27) PCNSL and 162 controls (118 CNS disorders and 44 extra-CNS lymphomas). The concordance of MYD88 mutational status between tumour tissue and CSF sample and the source of ILs in PCNSL tissues were also investigated. Mut-MYD88 was assessed by TaqMan-based polymerase chain reaction. IL-6 and IL-10 messenger RNA (mRNA) was assessed on PCNSL biopsies using RNAscope technology. IL levels in CSF were assessed by enzyme-linked immunosorbent assay. Mut-MYD88 was detected in 15/17 (88%) PCNSL biopsies, with an 82% concordance in paired tissue-CSF samples. IL-10 mRNA was detected in lymphomatous B cells in most PCNSL; expression of IL-6 transcripts was negligible. In CSF samples, mut-MYD88 and high IL-10 levels were detected, respectively, in 72% and 88% of patients with newly diagnosed PCNSL and in 1% of controls; conversely, IL-6 showed a low discriminating sensitivity and specificity. Combined analysis of MYD88 and IL-10 exhibits a sensitivity and specificity to distinguish PCNSL of 94% and 98% respectively. Similar figures were recorded in patients with relapsed PCNSL. In conclusion, high detection rates of mut-MYD88 and IL-10 in CSF reflect, respectively, the MYD88 mutational status and synthesis of this IL in PCNSL tissue. These biomarkers exhibit a very high sensitivity and specificity in detecting PCNSL both at initial diagnosis and relapse. Implications of these findings in patients with lesions unsuitable for biopsy deserve to be investigated.

Weak correlation between antibody titers and neutralizing activity in sera from SARS-CoV-2 infected subjects.

Plenty of serologic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed so far, thus documenting the importance of evaluating the relevant features of the immune response to this viral agent. The performance of these assays is currently under investigation. Amongst them, LIAISON® SARS-CoV-2 S1/S2 IgG by DiaSorin and Elecsys Anti-SARS-CoV-2 cobas® by Roche are currently used by laboratory medicine hospital departments in Italy and many other countries. In the present study, we firstly compared two serologic tests on serum samples collected at two different time points from 46 laboratory-confirmed coronavirus disease-2019 (COVID-19) subjects. Secondly, 85 negative serum samples collected before the SARS-CoV-2 pandemic were analyzed. Thirdly, possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS-CoV-2 were evaluated. Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased to 97.8% and 100% for samples collected after 15 days for DiaSorin and Roche tests, respectively. The specificity evaluated for the two tests ranges from 96.5% to 100%, respectively. Importantly, a poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study. These data further shed light on both potentials and possible limitations related to SARS-CoV-2 serology. In this context, great efforts are still necessary for investigating antibody kinetics to develop novel diagnostic algorithms. Moreover, further investigations on the role of neutralizing antibodies and their correlate of protection will be of paramount importance for the development of effective vaccines.

Quantitative serological evaluation as a valuable tool in the COVID-19 vaccination campaign.

OBJECTIVES: After exceptional research efforts, several vaccines were developed against SARS-CoV-2 which sustains the pandemic COVID-19. The Comirnaty vaccine showed high efficacy in clinical trials and was the first to be approved for its distribution to the general population. We evaluated the immune response induced by the first vaccine dose in different sex/age groups and subjects with or without naturally present anti-SARS-CoV-2 antibodies. METHODS: As part of an Italian multicenter project (Covidiagnostix), serum samples from 4,290 health-professionals were serologically tested the day of the first vaccination dose, and 21 days later, using two different instrumentations (Siemens-Healthineers and Roche). RESULTS: In total, 97% of samples showed the presence of specific antibodies 21 days after the vaccination dose; the percentage of non-responders increased with age in both genders. Remarkably, naturally seropositive individuals showed antibody persistence up to 11 months and an exceptionally higher vaccination response compared to subjects never infected by SARS-CoV-2. CONCLUSIONS: This study highlighted the importance of the serological test i) to identify naturally SARS-CoV-2 seropositive individuals and ii) to evaluate the antibody level elicited by the first vaccination dose. Both tests, highlighted differences in the immune response, when subjects were stratified by sex and age, and between naturally seropositive and seronegative subjects. The data obtained show how serological tests could play a crucial role in the triage of the population subjected to the vaccination campaign for COVID-19. The definition of suitable instrumentation-specific thresholds is needed to correctly follow eventually acquired post-vaccination immunity in the general population.

Is there a link between vitamin D status, SARS-CoV-2 infection risk and COVID-19 severity?

The outbreak of COVID-19 emerged in December 2019 rapidly spread across the globe and has become pandemic. Little is known about the protective factors of this infection, which is equally distributed between genders and different ages while severe and poor prognosis cases are strongly associated to old males and the presence of comorbidities. Thus, preventive measures aiming at reducing the number of infection and/or their severity are strongly needed. Vitamin D has got great attention and has been claimed as potentially protective against the infection since it may be associated with immunocompetence, inflammation, aging, and those diseases involved in determining the outcomes of COVID-19. This narrative review aims at collecting the literature available on the involvement of the vitamin D status in the pathogenesis of COVID-19 and the putative utility of vitamin D supplementation in the therapeutics. It emerges that a poor vitamin D status seems to associate with an increased risk of infection whereas age, gender and comorbidities seem to play a more important role in COVID-19 severity and mortality. While randomized control trials are needed to better inquire into this topic, vitamin D supplementation may be useful beside its potential effects on SARS-CoV-2 infection and COVID-19.

COVIDIAGNOSTIX: health technology assessment of serological tests for SARS-CoV-2 infection.

OBJECTIVE: In vitro diagnostic tests for SARS-COV-2, also known as serological tests, have rapidly spread. However, to date, mostly single-center technical and diagnostic performance's assessments have been carried out without an intralaboratory validation process and a health technology assessment (HTA) systematic approach. Therefore, the rapid HTA for evaluating antibody tests for SARS-COV-2 was applied. METHODS: The use of rapid HTA is an opportunity to test innovative technology. Unlike traditional HTA (which evaluates the benefits of new technologies after being tested in clinical trials or have been applied in practice for some time), the rapid HTA is performed during the early stages of developing new technology. A multidisciplinary team conducted the rapid HTA following the HTA Core Model® (version 3.0) developed by the European Network for Health Technology Assessment. RESULTS: The three methodological and analytical steps used in the HTA applied to the evaluation of antibody tests for SARS-COV-2 are reported: the selection of the tests to be evaluated; the research and collection of information to support the adoption and appropriateness of the technology; and the preparation of the final reports and their dissemination. Finally, the rapid HTA of serological tests for SARS-CoV-2 is summarized in a report that allows its dissemination and communication. CONCLUSIONS: The rapid-HTA evaluation method, in addition to highlighting the characteristics that differentiate the tests from each other, guarantees a timely and appropriate evaluation, becoming a tool to create a direct link between science and health management.

No significant association between vitamin D and COVID-19: A retrospective study from a northern Italian hospital.

The world is currently overwhelmed by a novel coronavirus disease (COVID-19). Clinically, COVID-19 shows a broad range of manifestations: from asymptomatic to severe and possibly lethal interstitial pneumonia. Several studies suggested the involvement of Vitamin-D (VitD) in reducing the risk of COVID-19 infections/severity. However, most of them are based on circumstantial evidences, like the association between latitude-related sunlight exposure and mortality rate, while studies based on patients' VitD measurements are still scarce. Therefore, we retrospectively analyzed the VitD levels (measured as 25-hydroxyvitamin-D) from a cohort of 347 patients admitted to a northern Italian Hospital as suspected COVID-19s. Of them, 128 were positive (83 males, aged 62.7 ± 14.2 and 45 females, aged 69.3 ± 15.6) and 219 were negative (107 males, aged 62.8 ± 19.5 and 112 females, aged 54.3 ± 20.1). The averaged VitD levels were similar in the two groups: 21.8 ± 16.1 ng/mL and 22.8 ± 14.0 ng/mL for the COVID-19 positive and negative group respectively (p-value: 0.39), as well as the percentage of individuals having VitD levels below 30 ng/mL: 78.9% and 73.5% for the COVID-19 positive and negative group respectively. Because a large portion of patients were below the suggested 30 ng/mL threshold, we can't exclude that VitD supplementation, restoring normal levels, might be beneficial in reducing the risk of infection.

Early predictors of clinical outcomes of COVID-19 outbreak in Milan, Italy.

BACKGROUND: National health-system hospitals of Lombardy faced a heavy burden of admissions for acute respiratory distress syndromes associated with coronavirus disease (COVID-19). Data on patients of European origin affected by COVID-19 are limited. METHODS: All consecutive patients aged ≥18 years, coming from North-East of Milan's province and admitted at San Raffaele Hospital with COVID-19, between February 25th and March 24th, were reported, all patients were followed for at least one month. Clinical and radiological features at admission and predictors of clinical outcomes were evaluated. RESULTS: Of the 500 patients admitted to the Emergency Unit, 410 patients were hospitalized and analyzed: median age was 65 (IQR 56-75) years, and the majority of patients were males (72.9%). Median (IQR) days from COVID-19 symptoms onset was 8 (5-11) days. At hospital admission, fever (≥ 37.5 °C) was present in 67.5% of patients. Median oxygen saturation (SpO2) was 93% (range 60-99), with median PaO2/FiO2 ratio, 267 (IQR 184-314). Median Radiographic Assessment of Lung Edema (RALE) score was 9 (IQR 4-16). More than half of the patients (56.3%) had comorbidities, with hypertension, coronary heart disease, diabetes and chronic kidney failure being the most common. The probability of overall survival at day 28 was 66%. Multivariable analysis showed older age, coronary artery disease, cancer, low lymphocyte count and high RALE score as factors independently associated with an increased risk of mortality. CONCLUSION: In a large cohort of COVID-19 patients of European origin, main risk factors for mortality were older age, comorbidities, low lymphocyte count and high RALE.

Routine blood tests as a potential diagnostic tool for COVID-19.

Objectives The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to date, the epidemic has gradually spread to 209 countries worldwide with more than 1.5 million infected people and 100,000 deaths. Amplification of viral RNA by rRT-PCR serves as the gold standard for confirmation of infection, yet it needs a long turnaround time (3-4 h to generate results) and shows false-negative rates as large as 15%-20%. In addition, the need of certified laboratories, expensive equipment and trained personnel led many countries to limit the rRT-PCR tests only to individuals with pronounced respiratory syndrome symptoms. Thus, there is a need for alternative, less expensive and more accessible tests. Methods We analyzed the plasma levels of white blood cells (WBCs), platelets, C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), alkaline phosphatase and lactate dehydrogenase (LDH) of 207 patients who, after being admitted to the emergency room of the San Raffaele Hospital (Milan, Italy) with COVID-19 symptoms, were rRT-PCR tested. Of them, 105 tested positive, whereas 102 tested negative. Results Statistically significant differences were observed for WBC, CRP, AST, ALT and LDH. Empirical thresholds for AST and LDH allowed the identification of 70% of either COVID-19-positive or -negative patients on the basis of routine blood test results. Conclusions Combining appropriate cutoffs for certain hematological parameters could help in identifying false-positive/negative rRT-PCR tests. Blood test analysis might be used as an alternative to rRT-PCR for identifying COVID-19-positive patients in those countries which suffer from a large shortage of rRT-PCR reagents and/or specialized laboratory.

Lower nasopharyngeal viral load during the latest phase of COVID-19 pandemic in a Northern Italy University Hospital.

Objectives: A milder clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been anecdotally reported over the latest phase of COVID-19 pandemic in Italy. Several factors may contribute to this observation, including the effect of lockdown, social distancing, lower humidity, lower air pollution, and potential changes in the intrinsic pathogenicity of the virus. In this regard, the clinical severity of COVID-19 could be attenuated by mutations in SARS-CoV-2 genome that decrease its virulence, as well as by lower virus inocula. Methods: In this pilot study, we compared the reverse transcription polymerase chain reaction (RT-PCR) amplification profile of 100 nasopharyngeal swabs consecutively collected in April, during the peak of SARS-CoV-2 epidemic, to that of 100 swabs collected using the same procedure in May. Results: The mean Ct value of positive samples collected in May was significantly higher than that of samples collected in the previous period (ORF 1a/b gene: 31.85 ± 0.32 vs. 28.37 ± 0.5, p<0.001; E gene: 33.76 ± 0.38 vs. 29.79 ± 0.63, p<0.001), suggesting a lower viral load at the time of sampling. No significant differences were observed between male and females in the two periods, whilst higher viral loads were found in (i) patients over 60-years old, and (ii) patients that experienced severe COVID-19 during the early stages of the pandemic. Conclusions: This pilot study prompts further investigation on the correlation between SARS-CoV-2 load and different clinical manifestation of COVID-19 during different phases of the pandemic. Laboratories should consider reporting quantitative viral load data in the molecular diagnosis of SARS-CoV-2 infection.

Development, evaluation, and validation of machine learning models for COVID-19 detection based on routine blood tests.

Objectives: The rRT-PCR test, the current gold standard for the detection of coronavirus disease (COVID-19), presents with known shortcomings, such as long turnaround time, potential shortage of reagents, false-negative rates around 15-20%, and expensive equipment. The hematochemical values of routine blood exams could represent a faster and less expensive alternative. Methods: Three different training data set of hematochemical values from 1,624 patients (52% COVID-19 positive), admitted at San Raphael Hospital (OSR) from February to May 2020, were used for developing machine learning (ML) models: the complete OSR dataset (72 features: complete blood count (CBC), biochemical, coagulation, hemogasanalysis and CO-Oxymetry values, age, sex and specific symptoms at triage) and two sub-datasets (COVID-specific and CBC dataset, 32 and 21 features respectively). 58 cases (50% COVID-19 positive) from another hospital, and 54 negative patients collected in 2018 at OSR, were used for internal-external and external validation. Results: We developed five ML models: for the complete OSR dataset, the area under the receiver operating characteristic curve (AUC) for the algorithms ranged from 0.83 to 0.90; for the COVID-specific dataset from 0.83 to 0.87; and for the CBC dataset from 0.74 to 0.86. The validations also achieved good results: respectively, AUC from 0.75 to 0.78; and specificity from 0.92 to 0.96. Conclusions: ML can be applied to blood tests as both an adjunct and alternative method to rRT-PCR for the fast and cost-effective identification of COVID-19-positive patients. This is especially useful in developing countries, or in countries facing an increase in contagions.

Detection of COVID-19 Infection from Routine Blood Exams with Machine Learning: A Feasibility Study.

The COVID-19 pandemia due to the SARS-CoV-2 coronavirus, in its first 4 months since its outbreak, has to date reached more than 200 countries worldwide with more than 2 million confirmed cases (probably a much higher number of infected), and almost 200,000 deaths. Amplification of viral RNA by (real time) reverse transcription polymerase chain reaction (rRT-PCR) is the current gold standard test for confirmation of infection, although it presents known shortcomings: long turnaround times (3-4 hours to generate results), potential shortage of reagents, false-negative rates as large as 15-20%, the need for certified laboratories, expensive equipment and trained personnel. Thus there is a need for alternative, faster, less expensive and more accessible tests. We developed two machine learning classification models using hematochemical values from routine blood exams (namely: white blood cells counts, and the platelets, CRP, AST, ALT, GGT, ALP, LDH plasma levels) drawn from 279 patients who, after being admitted to the San Raffaele Hospital (Milan, Italy) emergency-room with COVID-19 symptoms, were screened with the rRT-PCR test performed on respiratory tract specimens. Of these patients, 177 resulted positive, whereas 102 received a negative response. We have developed two machine learning models, to discriminate between patients who are either positive or negative to the SARS-CoV-2: their accuracy ranges between 82% and 86%, and sensitivity between 92% e 95%, so comparably well with respect to the gold standard. We also developed an interpretable Decision Tree model as a simple decision aid for clinician interpreting blood tests (even off-line) for COVID-19 suspect cases. This study demonstrated the feasibility and clinical soundness of using blood tests analysis and machine learning as an alternative to rRT-PCR for identifying COVID-19 positive patients. This is especially useful in those countries, like developing ones, suffering from shortages of rRT-PCR reagents and specialized laboratories. We made available a Web-based tool for clinical reference and evaluation (This tool is available at https://covid19-blood-ml.herokuapp.com/ ).