ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections.

Sequencing the SARS-CoV-2 genome from clinical samples can be challenging, especially in specimens with low viral titer. Here we report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. The protocol was optimized to improve flexibility and the combination of technical replicates was established as the central strategy to achieve accurate analysis of low-titer/suboptimal samples. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.

Assessment of real-time PCR for Helicobacter pylori DNA detection in stool with co-infection of intestinal parasites: a comparative study of DNA extraction methods.

BACKGROUND: Many studies reported high prevalence of H. pylori infection among patients co-infected with intestinal parasites. Molecular approach for the DNA detection of those microbes in stool have been proposed. However there are a few reports that evaluated the effect of bead-beating in relation to the H. pylori outcome. Therefore, we developed and evaluated two TaqMan-based real-time PCR (rt-PCR) qualitative assays for the detection of ureC (glmM) and cagA of Helicobacter pylori on DNA extracted by three procedures. RESULTS: The two PCRs were analysed on 100 stool samples from patients who were screened for intestinal parasites. Three DNA extraction procedures were used: 1) automation with bead beating, 2) automation without bead beating and 3) hand column. The specificity of the new assays was confirmed by sequencing the PCR products and by the lack of cross-reactivity with other bacteria or pathogens DNA. Rt-PCR assays showed a detection limit of 10^4 bacteria/200 mg stool. The ureC_PCR with bead beating process was compared to conventional stool antigen test (SAT), with 94.12 and 93.75% of respectively sensitivity and specificity. However, the discordant samples were confirmed by DNA sequencing suggesting a potential higher sensitivity and specificity of PCR. CONCLUSIONS: Our findings showed that the automation with bead-beating -suggested procedure for intestinal parasitic infections- can reach highly sensitive results in H. pylori detection on stool compared also with SAT. Thus, this work can provide new insights into the practice of a clinical microbiology laboratory in order to optimize detection of gastro-intestinal infections. Further studies are needed to better define the clinical value of this technique.

Dynamics of anti-malarial antibodies in non-immune patients during and after a first and unique Plasmodium falciparum malaria episode.

BACKGROUND: Malaria is a major travel medicine issue. Retrospective confirmation of a malaria episode diagnosed in an endemic area can have relevant implications in transfusional medicine in Europe, where blood donors are excluded from donation on the basis of positive malaria serology. However, there is scarce evidence on the dynamics of anti-malarial antibodies after a first malaria episode in non-immune individuals. The first aim of this study was to describe the dynamics of anti-malarial antibodies in a first malaria episode in non-immune travellers. Secondary objectives were to assess the sensitivity of serology for a retrospective diagnosis in non-immune travellers diagnosed while abroad and to discuss the implications in transfusional medicine. METHODS: Retrospective analysis of the results of an indirect fluorescence antibody test (IFAT) for malaria available for patients with a first malaria episode by Plasmodium falciparum and admitted at the IRCCS Sacro Cuore Don Calabria hospital in a 14-year period. The antibody titres were collected at baseline and during further follow up visits. Epidemiological, demographic and laboratory test results (including full blood count and malaria parasite density) were anonymously recorded in a study specific electronic Case Report Form created with OpenClinica software. Statistical analysis was performed with SAS software version 9.4. RESULTS: Thirty-six patients were included. Among them, all but two were Europeans (one African and one American). Median length of fever before diagnosis was 2 days (IQR 1-3). Thirty-five patients had seroconversion between day 1 and day 4 from admission, and the titre showed a sharply rising titre, often to a very high level in a few days. Only a single patient remained negative in the first 5 days from admission, after which he was no more tested. Six patients were followed up for at least 2 months, and they all showed a decline in IFAT titre, tending to seroreversion (confirmed in one patient with the longest follow up, almost 4 years). CONCLUSIONS: Serology demonstrated reliable for retrospective diagnosis in non-immune travellers. The decline in the anti-malarial titre might be included in the screening algorithms of blood donors, but further studies are needed.

Improved detection of DNA Schistosoma haematobium from eggs extracted by bead beating in urine.

Diagnosis of Schistosoma haematobium relies primarily on microscopical analysis of urine. The method is time consuming and requires some expertise. Genus-specific real-time PCRs have been developed, but we still observed low sensitivity. In the present study, in order to achieve a more sensitive DNA detection of eggs of S. haematobium in urine samples, we wanted to develop a novel protocol of DNA extraction using mechanic disruption of eggs by bead beating as supplementary step. We tested Schistosoma spp. internal transcribed spacer 2 real-time PCR after both methods with and without bead beating. First, we preliminary assessed the DNA detection after bead beating using dilution of 2, 10, 50, and 90 eggs/10 mL, and the Ct value analysis showed significant improved DNA detection per each point of egg concentration using the novel supplementary step. Twenty microscopy positive and five microscopy negative urine samples were used to validate the procedure. All urines came from imported cases and admitted at center for tropical medicine, and were examined by microscopy. PCR results after novel method with bead beating showed 100% to be positive for S. haematobium, compared with 85% positive by our standard extraction procedure. Results confirmed mechanic disruption of eggs by bead beating before DNA extraction to be highly effective method for the detection of S. haematobium DNA in urine.

Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.

Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17ß-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24-48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin.

Effects of Two Different Rhodiola rosea Extracts on Primary Human Visceral Adipocytes.

Rhodiola rosea (Rro) has been reported to have various pharmacological properties, including anti-fatigue, anti-stress and anti-inflammatory activity. It is also known to improve glucose and lipid metabolism, but the effects of Rhodiola rosea on adipocyte differentiation and metabolism are not still elucidated. In this study the anti-adipogenic and lipolytic activity of two extracts of Rhodiola rosea, containing 3% salidroside (RS) or 1% salidroside and 3% rosavines (RR) on primary human visceral adipocytes was investigated. Pre-adipocytes were analyzed after 10 and 20 days of treatment during differentiation and after 7 days of treatment when they reached mature shape. The RS extract significantly induced higher apoptosis and lipolysis in comparison to control cells and to RR extract. In contrast, RR extract significantly reduced triglyceride incorporation during maturation. Differentiation of pre-adipocytes in the presence of RS and RR extracts showed a significant decrease in expression of genes involved in adipocyte function such as SLC2A4 and the adipogenic factor FGF2 and significant increase in expression of genes involved in inhibition of adipogenesis, such as GATA3, WNT3A, WNT10B. Furthermore RR extract, in contrast to RS, significantly down-regulates PPARG, the master regulator of adipogenesis and FABP4. These data support the lipolytic and anti-adipogenetic activity of two different commercial extracts of Rhodiola rosea in primary human visceral pre-adipocytes during differentiation.

Urine: A Pitfall for Molecular Detection of Toscana Virus? An Analytical Proof-of-Concept Study.

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID50 vs. 1 × 104 TCID50 limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches.

Exploring Viral Genome Profile in Mpox Patients during the 2022 Outbreak, in a North-Eastern Centre of Italy.

In 2022, an unprecedented outbreak of mpox raged in several nations. Sequences from the 2022 outbreak reveal a higher nucleotide substitution if compared with the estimated rate for orthopoxviruses. Recently, intra-lesion SNVs (single nucleotide variants) have been described, and these have been suggested as possible sources of genetic variation. Until now, it has not been clear if the presence of several SNVs could represents the result of local mutagenesis or a possible co-infection. We investigated the significance of SNVs through whole-genome sequencing analysis of four unrelated mpox cases. In addition to the known mutations harboured by the circulating strains of virus (MPXV), 7 novel mutations were identified, including SNVs located in genes that are involved in immune evasion mechanisms and/or viral fitness, six of these appeared to be APOBEC3-driven. Interestingly, three patients exhibited the coexistence of mutated and wild-type alleles for five non-synonymous variants. In addition, two patients, apparently unrelated, showed an analogous pattern for two novel mutations, albeit with divergent frequencies. The coexistence of mixed viral populations, harbouring non-synonymous mutations in patients, supports the hypothesis of possible co-infection. Additional investigations of larger clinical cohorts are essential to validating intra-patient viral genome heterogeneity and determining the possibility of co-presence events of slightly divergent MPXV strains.

Wide Real-Life Data Support Reduced Sensitivity of Antigen Tests for Omicron SARS-CoV-2 Infections.

With the continuous spread of new SARS-CoV-2 variants of concern (VOCs), the monitoring of diagnostic test performances is mandatory. We evaluated the changes in antigen diagnostic tests' (ADTs) accuracy along the Delta to Omicron VOCs transition, exploring the N protein mutations possibly affecting ADT sensitivity and assessing the best sampling site for the diagnosis of Omicron infections. In total, 5175 subjects were enrolled from 1 October 2021 to 15 July 2022. The inclusion criteria were SARS-CoV-2 ADT combined with a same-day RT-PCR swab test. For the sampling site analysis, 61 patients were prospectively recruited during the Omicron period for nasal and oral swab analyses by RT-PCR. Next-Generation Sequencing data were obtained to evaluate the different sublineages. Using RT-PCR as a reference, 387 subjects resulted in becoming infected and the overall sensitivity of the ADT decreased from 63% in the Delta period to 33% in the Omicron period. This decrease was highly statistically significant (p < 0.001), and no decrease in viral load was detected at the RNA level. The nasal site presented a significantly higher viral load than the oral site during the Omicron wave. The reduced detection rate of Omicron infections by ADT should be considered in the global testing strategy to preserve accurate diagnoses across the changing SARS-CoV-2 variants.

Strongyloides questions-a research agenda for the future.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

sgRNAs: A SARS-CoV-2 emerging issue.

Like for other coronaviruses, SARS-CoV-2 gene expression strategy is based on the synthesis of a nested set of subgenomic mRNA species (sgRNAs). These sgRNA are synthesized using a "discontinuous transcription" mechanism that relies on template switching at Transcription Regulatory Sequences (TRS). Both canonical (c-sgRNA) and non-canonical (nc-sgRNA, less numerous) subgenomic RNA species can be produced. Currently, sgRNAs are investigated on the basis of sequence data obtained through next generation sequencing (NGS), and bioinformatic tools are crucial for their identification, characterization and quantification. To date, few software have been developed to this aim, whose reliability and applicability to all the available NGS platforms need to be established, to build confidence on the information resulting from such tools. In fact, these information may be crucial for the in depth elucidation of viral expression strategy, particularly in respect of the significance of nc-sgRNAs, and for the possible use of sgRNAs as potential markers of virus replicative activity in infected patients.

Evaluation of a ddPCR Commercial Assay for the Absolute Quantification of the Monkeypox Virus West Africa in Clinical Samples.

BACKGROUND: Monkeypox virus (MPXV) is a double-stranded DNA virus belonging to the orthopoxvirus genus in the family Poxviridae. Distinct clades are identified: the clade I belonging to the Central African (or Congo Basin) clade and the subclades IIa and IIb belonging to the West African clade. Here, a commercial droplet digital PCR (ddPCR) assay was evaluated for the quantification of the MPXV West Africa clade in clinical samples. METHODS: The ddPCR reaction was assessed as a duplex assay using RPP30 as an internal amplification control. A total of 60 clinical specimens were tested, 40 positives (skin lesions, n=10; rectal swabs, n = 10; pharyngeal swabs, n = 10; and whole blood, n = 10), and 20 negatives (n = 5 for each biological matrix) were found at the routine molecular diagnostics (orthopoxvirus qPCR followed by confirmation with Sanger sequencing). To evaluate the analytical sensitivity, the ddPCR reaction was first analyzed on serial dilutions of synthetic DNA spiked in water and in negative biological matrices, achieving a limit of detection of 3.5 copy/µL. RESULTS: Regarding the clinical samples, compared to routine molecular diagnostics, the ddPCR duplex assay showed 100% of specificity for all biological matrices and 100% sensitivity (10/10) for lesions, 100% (10/10) for rectal swabs, 90% (9/10) for pharyngeal swabs, and 60% (6/10) for whole blood. CONCLUSION: Overall, our data showed that the commercial ddPCR assay allowed the DNA detection of MPXV in 87.5% (35/40) of our cohort, highlighting useful technical indications for the different specimens with a potential greatest performance for skin lesions and rectal swabs.

Leprosy in an Adopted Woman Diagnosed by Molecular Tools: A Case Report from a Non-Endemic Area.

Coupled with its rarity in non-endemic areas, the clinical heterogeneity of leprosy makes diagnosis very challenging. We report a diagnosis of multibacillary leprosy in a 22-year-old Indian woman, adopted at the age of 10 and living in Italy. The patient presented with painful skin lesions on the face, trunk, and lower and upper extremities, associated with dysesthesia and a motor deficit in her left leg following corticosteroid therapy interruption. Histopathology results from the skin lesions suggested leprosy, but no acid-fast bacilli were identified. Molecular biology in a center specializing in tropical diseases confirmed the diagnosis, allowing prompt and adequate treatment. Genotype analysis allowed the identification of a genotype 1D of M. leprae, facilitating the epidemiological investigation of the plausible infection origin. No resistances to rifampicin, dapsone, or ofloxacin were detected. Leprosy will continue to exist in high-income nations, and the incidence may rise over time due to increasing migration and globalization. CARE guidelines were followed.

Assessing the prevalence of Female Genital Schistosomiasis and comparing the acceptability and performance of health worker-collected and self-collected cervical-vaginal swabs using PCR testing among women in North-Western Tanzania: The ShWAB study.

BACKGROUND: Female Genital Schistosomiasis (FGS) is a neglected disease of the genital tract due to the inflammatory response to the presence of Schistosoma haematobium eggs in the genital tract. The WHO has prioritized the improvement of diagnostics for FGS and previous studies have explored the PCR-based detection of Schistosoma DNA on genital specimens, with encouraging results. This study aimed to determine the prevalence of FGS among women living in an endemic district in North-western Tanzania, using PCR on samples collected though cervical-vaginal swabs, and to compare the performance of self-collected and healthcare worker-collected (operator-collected) samples, and the acceptability of the different sampling methods. METHODS/PRINCIPAL FINDINGS: A cross-sectional study was conducted involving 211 women living in 2 villages in the Maswa district of North-western Tanzania. Urine, self-collected and operator-collected cervical-vaginal swabs were obtained from participants. A questionnaire was administered, focusing on the comfortability in undergoing different diagnostic procedures. Prevalence of urinary schistosomiasis, as assessed by eggs in urine, was 8.5% (95%CI 5.1-13.1). DNA was pre-isolated from genital swabs and transported at room temperature to Italy for molecular analysis. Prevalence of active schistosomiasis, urinary schistosomiasis, and FGS were 10.0% (95% CI 6.3-14.8), 8.5% (95%CI 5.1-13.1), and 4.7% (95%CI 2.3-8.5), respectively. When real-time PCR was performed after a pre-amplification step, the prevalence of active schistosomiasis increased to 10.4% (95%CI 6.7-15.4), and FGS to 5.2% (95%CI 2.6-9.1). Of note, more cases were detected by self-collected than operator-collected swabs. The vast majority of participants (95.3%) declared that they were comfortable/very comfortable about genital self-sampling, which was indicated as the preferred sampling method by 40.3% of participants. CONCLUSIONS/SIGNIFICANCE: The results of this study show that genital self-sampling followed by pre-amplified PCR on room temperature-stored DNA is a useful method from both technical and acceptability point of views. This encourages further studies to optimize samples processing, and identify the best operational flow to allow integration of FGS screening into women health programmes, such as HPV screening.

Comparative analysis of bioinformatics tools to characterize SARS-CoV-2 subgenomic RNAs.

During the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), positive-sense genomic RNA and subgenomic RNAs (sgRNAs) are synthesized by a discontinuous process of transcription characterized by a template switch, regulated by transcription-regulating sequences (TRS). Although poorly known about makeup and dynamics of sgRNAs population and function of its constituents, next-generation sequencing approaches with the help of bioinformatics tools have made a significant contribution to expand the knowledge of sgRNAs in SARS-CoV-2. For this scope to date, Periscope, LeTRS, sgDI-tector, and CORONATATOR have been developed. However, limited number of studies are available to compare the performance of such tools. To this purpose, we compared Periscope, LeTRS, and sgDI-tector in the identification of canonical (c-) and noncanonical (nc-) sgRNA species in the data obtained with the Illumina ARTIC sequencing protocol applied to SARS-CoV-2-infected Caco-2 cells, sampled at different time points. The three software showed a high concordance rate in the identification and in the quantification of c-sgRNA, whereas more differences were observed in nc-sgRNA. Overall, LeTRS and sgDI-tector result to be adequate alternatives to Periscope to analyze Fastq data from sequencing platforms other than Nanopore.

Tropheryma whipplei, Helicobacter pylori, and Intestinal Protozoal Co-Infections in Italian and Immigrant Populations: A Cross-Sectional Study.

Tropheryma whipplei (TW), Helicobacter pylori (HP), and intestinal protozoa (IP) are widespread pathogens with similar routes of transmission and epidemiological risk factors. Epidemiological data on co-infection between TW, HP, and IP are scarce. We aim to more deeply investigate the co-infection rate for these pathogens, evaluating the risk factors and symptoms. Methods: This is a cross-sectional study conducted at the IRCCS Sacro Cuore Don Calabria Hospital in Northern Italy, a referral center for tropical and Whipple's disease (WD). Stored stool samples from 143 subjects previously tested for TW DNA by real-time PCR were explored for HP and IP DNA detection. The virulence factor cagA was investigated in HP-positive patients. Results: A history of migration was reported significantly more in TW-positive than in negative subjects (34.1% vs. 9.1%, p = 0.001) and in HP-infected than in those non-infected (59.1% vs. 9.1%, p < 0.001). The HP infection rate differed significantly between TW-infected and uninfected groups (31.8% vs. 8.1%, p = 0.001), while no difference was observed for IP infection. Significantly higher TW intestinal colonization was found in HP-infected patients than in non-infected (63.6% vs. 24.8%, p < 0.001). In addition, the proportion of Blastocysts positive finding was also significantly higher in HP-infected than in non-infected (40.9% vs. 17.4%, p = 0.018). Conclusions: The present study is the first to report a high TW and HP co-infection rate. To reduce the risk of morbidity from a chronic infection of either pathogen, clinicians may consider TW-HP molecular screening on the same stool sample for patients with suspected HP disease or WD, particularly in case of travel history.

Identification of miRNAs of Strongyloides stercoralis L1 and iL3 larvae isolated from human stool.

Strongyloidiasis is a neglected tropical disease caused by the soil-transmitted nematode by Strongyloides stercoralis, that affects approximately 600 million people worldwide. In immunosuppressed individuals disseminated strongyloidiasis can rapidly lead to fatal outcomes. There is no gold standard for diagnosing strongyloidiasis, and infections are frequently misdiagnosed. A better understanding of the molecular biology of this parasite can be useful for example for the discovery of potential new biomarkers. Interestingly, recent evidence showed the presence of small RNAs in Strongyloididae, but no data was provided for S. stercoralis. In this study, we present the first identification of miRNAs of both L1 and iL3 larval stages of S. stercoralis. For our purpose, the aims were: (i) to analyse the miRNome of L1 and iL3 S. stercoralis and to identify potential miRNAs of this nematode, (ii) to obtain the mRNAs profiles in these two larval stages and (iii) to predict potential miRNA target sites in mRNA sequences. Total RNA was isolated from L1 and iL3 collected from the stool of 5 infected individuals. For the miRNAs analysis, we used miRDeep2 software and a pipeline of bio-informatic tools to construct a catalog of a total of 385 sequences. Among these, 53% were common to S. ratti, 19% to S. papillosus, 1% to Caenorhabditis elegans and 44% were novel. Using a differential analysis between the larval stages, we observed 6 suggestive modulated miRNAs (STR-MIR-34A-3P, STR-MIR-8397-3P, STR-MIR-34B-3P and STR-MIR-34C-3P expressed more in iL3, and STR-MIR-7880H-5P and STR-MIR-7880M-5P expressed more in L1). Along with this analysis, we obtained also the mRNAs profiles in the same samples of larvae. Multiple testing found 81 statistically significant mRNAs of the total 1553 obtained (FDR < 0.05; 32 genes expressed more in L1 than iL3; 49 genes expressed more in L3 than iL1). Finally, we found 33 predicted mRNA targets of the modulated miRNAs, providing relevant data for a further validation to better understand the role of these small molecules in the larval stages and their valuein clinical diagnostics.

High-dose ivermectin for early treatment of COVID-19 (COVER study): a randomised, double-blind, multicentre, phase II, dose-finding, proof-of-concept clinical trial.

High concentrations of ivermectin demonstrated antiviral activity against SARS-CoV-2 in vitro. The aim of this study was to assess the safety and efficacy of high-dose ivermectin in reducing viral load in individuals with early SARS-CoV-2 infection. This was a randomised, double-blind, multicentre, phase II, dose-finding, proof-of-concept clinical trial. Participants were adults recently diagnosed with asymptomatic/oligosymptomatic SARS-CoV-2 infection. Exclusion criteria were: pregnant or lactating women; CNS disease; dialysis; severe medical condition with prognosis <6 months; warfarin treatment; and antiviral/chloroquine phosphate/hydroxychloroquine treatment. Participants were assigned (ratio 1:1:1) according to a randomised permuted block procedure to one of the following arms: placebo (arm A); single-dose ivermectin 600 µg/kg plus placebo for 5 days (arm B); and single-dose ivermectin 1200 µg/kg for 5 days (arm C). Primary outcomes were serious adverse drug reactions (SADRs) and change in viral load at Day 7. From 31 July 2020 to 26 May 2021, 32 participants were randomised to arm A, 29 to arm B and 32 to arm C. Recruitment was stopped on 10 June because of a dramatic drop in cases. The safety analysis included 89 participants and the change in viral load was calculated in 87 participants. No SADRs were registered. Mean (S.D.) log10 viral load reduction was 2.9 (1.6) in arm C, 2.5 (2.2) in arm B and 2.0 (2.1) in arm A, with no significant differences (P = 0.099 and 0.122 for C vs. A and B vs. A, respectively). High-dose ivermectin was safe but did not show efficacy to reduce viral load.

Molecular techniques for the genomic viral RNA detection of West Nile, Dengue, Zika and Chikungunya arboviruses: a narrative review.

Introduction: Molecular technology has played an important role in arboviruses diagnostics. PCR-based methods stand out in terms of sensitivity, specificity, cost, robustness, and accessibility, and especially the isothermal amplification (IA) method is ideal for field-adaptable diagnostics in resource-limited settings (RLS).Areas covered: In this review, we provide an overview of the various molecular methods for West Nile, Zika, Dengue and Chikungunya. We summarize literature works reporting the assessment and use of in house and commercial assays. We describe limitations and challenges in the usage of methods and opportunities for novel approaches such as NNext-GenerationSequencing (NGS).Expert opinion: The rapidity and accuracy of differential diagnosis is essential for a successful clinical management, particularly in co-circulation area of arboviruses. Several commercial diagnostic molecular assays are available, but many are not affordable by RLS and not usable as Point-of-care/Point-of-need (POC/PON) such as RReal-TimeRT-PCR, Array-based methods and NGS. In contrast, the IA-based system fits better for POC/PON but it is still not ideal for the multiplexing detection system. Improvement in the characterization and validation of current molecular assays is needed to optimize their translation to the point of care.

Overall decrease in SARS-CoV-2 viral load and reduction in clinical burden: the experience of a hospital in northern Italy.

OBJECTIVES: In Italy the burden of patients with coronavirus disease 2019 (COVID-19) gradually decreased from March to the end of May. In this work we aimed to evaluate a possible association between the severity of clinical manifestations and viral load over time during the epidemiological transition from high-to low-transmission settings. METHODS: We reviewed the cases of COVID-19 diagnosed at the emergency room of our hospital, retrieving the proportion of patients admitted to the intensive care unit. A raw estimation of the viral load was done evaluating the Ct (cycle threshold) trend obtained from our diagnostic reverse transcriptase real-time PCR test. RESULTS: The proportion of patients requiring intensive care significantly decreased from 6.7% (19/281) in March to 1.1% (1/86) in April, and to none in May (Fisher's test p 0.0067). As for viral load, we observed a trend of Ct increasing from a median value of 24 (IQR 19-29) to 34 (IQR 29-37) between March and May, with a statistically significant difference between March and April (pairwise Wilcoxon test with stepdown Bonferroni adjustment for multiple testing, p 0.0003). CONCLUSIONS: We observed a reduction over time in the proportion of patients with COVID-19 requiring intensive care, along with decreasing median values of viral load. As the epidemiological context changes from high-to low-transmission settings, people are presumably exposed to a lower viral load which has been previously associated with less severe clinical manifestations.