ddPCR for the Detection and Absolute Quantification of Oropouche Virus.

BACKGROUND: Oropouche virus (OROV) is a segmented RNA virus belonging to the genus Orthobunyavirus in the family Peribunyaviridae. Herein, an in-house droplet digital PCR (ddPCR) assay was used for the detection and quantification of OROV. METHODS: The ddPCR reaction was assessed as duplex assay using the human housekeeping gene RPP30. Limit of detection (LoD) analysis was performed in whole blood, serum, and urine. The assay was executed on a total of 28 clinical samples (whole blood n = 9, serum n = 11, and urine n = 8), of which 16 specimens were tested positive at the routine molecular diagnostics (endpoint and real-time PCRs). RESULTS: The LoD of the ddPCR performed using 10-fold serial dilution of OROV detected up to 1 cp/µL in all the biological matrices. Compared to the routine molecular diagnostics, the ddPCR assay showed 100% sensitivity for whole blood and serum and 75% for urine, highlighting higher positive rate of ddPCR. CONCLUSION: We have established a quantitative RNA detection method of OROV with high sensitivity and specificity based on ddPCR. This test is capable of quantitatively monitoring the viral load of OROV and can contribute, in addition to laboratory diagnosis, to shed light on the pathogenesis, filling in the knowledge gaps of this neglected disease and to the vector control programs.

A Pilot Study for the Characterization of Bacillus spp. and Analysis of Possible B. thuringiensis/Strongyloides stercoralis Correlation.

Differentiating between Bacillus species is relevant in human medicine. Bacillus thuringiensis toxins might be effective against Strongyloides stercoralis, a nematode causing relevant human morbidity. Our first objective was to evaluate genomic and MALDI-TOF identification methods for B. thuringiensis. Our secondary objective was to evaluate a possible negative selection pressure of B. thuringiensis against S. stercoralis. PCR and Sanger were compared to MALDI-TOF on a collection of 44 B. cereus group strains. B. thuringiensis toxin genes were searched on 17 stool samples from S. stercoralis-infected and uninfected dogs. Metagenomic 16S rRNA was used for microbiome composition. The inter-rate agreement between PCR, Sanger, and MALDI-TOF was 0.631 k (p-value = 6.4 × 10-10). B. thuringiensis toxins were not found in dogs' stool. Bacteroidota and Bacillota were the major phyla in the dogs' microbiome (both represented >20% of the total bacterial community). Prevotella was underrepresented in all Strongyloides-positive dogs. However, the general composition of bacterial communities was not significantly linked with S. stercoralis infection. The genomic methods allowed accurate differentiation between B. thuringiensis and B. cereus. There was no association between B. thuringiensis and S. stercoralis infection, but further studies are needed to confirm this finding. We provide the first descriptive results about bacterial fecal composition in dogs with S. stercoralis infection.

Tropheryma whipplei Colonization in Adults and Children: A Prospective Study.

We conducted a prospective cohort study at the IRCCS Sacro Cuore Don Calabria Hospital in Negrar di Valpolicella from 2019 to 2021 to investigate the duration of T. whipplei colonization. In addition, the correlation between persistent colonization and the continent of origin, current treatment regimen, clinical manifestations, and parasite coinfection was evaluated. The cohort included subjects who were tested in a previous study (years 2014-2016) and found to be positive for T. whipplei DNA in fecal samples. Thirty-three subjects were enrolled in a prospective study between 2019 and 2021. Feces, saliva, urine, and blood were collected at baseline and after 12 months. Medical history, current treatment, and symptoms were recorded. Among them, 25% showed persistent intestinal or oral colonization, 50% had no colonization at both visits, and 25% had intermittent colonization. No association was found between persistent T. whipplei colonization and subjects' continent of origin, current treatment regimen, initial clinical manifestations, and parasite coinfection. The longest duration of persistent T. whipplei intestinal colonization exceeded six years, with 11 subjects presenting persistent positivity for more than three years, including 1 minor. Our research was limited by the lack of a strain-specific identification of T. whipplei that made it impossible to distinguish between persistence of the same T. whipplei strain, reinfection from household exposure, or infection by a new strain. Larger prospective studies are needed to further explore the implications of this persistence and determine the key factors influencing the duration of colonization and its potential health impacts.

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.

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.

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.

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'.

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.

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.

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.

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.

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.

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.

Impact of Full Vaccination with mRNA BNT162b2 on SARS-CoV-2 Infection: Genomic and Subgenomic Viral RNAs Detection in Nasopharyngeal Swab and Saliva of Health Care Workers.

SARS-CoV-2 infection was monitored in 1898 health care workers (HCWs) after receiving full vaccination with BNT162b2. Untill 30 June 2021, 10 HCWs tested positive for SARS-CoV-2 using real time RT-PCR, resulting in a 4-month cumulative incidence of 0.005%. The infection was mildly symptomatic in six (60%) and asymptomatic in four (40%) individuals. Among the infected HCWs, eight consenting individuals provided paired NPS and saliva during the course of infection, for the purpose of the analysis performed in the present study. Genomic and subgenomic viral RNAs were investigated using real-time RT-PCR in both biological specimens. The temporal profile of viral load was measured using ddPCR. Viral mutations were also analysed. Subgenomic viral RNA was detected in 8/8 (100%) NPS and in 6/8 (75%) saliva specimens at the baseline. The expression of subgenomic RNA was observed for up to 7 days in 3/8 (38%) symptomatic cases. Moreover, concordance was observed between NPS and saliva in the detection of viral mutations, and both N501Y and 69/70del (associated with the B.1.1.7 variant) were detected in the majority 6/8 (75%) of subjects, while the K417T mutation (associated with the P.1-type variants) was detected in 2/8 (25%) individuals. Overall, our findings report a low frequency of infected HCWs after full vaccination. It is, therefore, important to monitor the vaccinees in order to identify asymptomatic infected individuals. Saliva can be a surrogate for SARS-CoV-2 surveillance, particularly in social settings such as hospitals.

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.