Discovery and Genomic Characterization of a Novel Henipavirus, Angavokely Virus, from Fruit Bats in Madagascar.

The genus Henipavirus (family Paramyxoviridae) currently comprises seven viruses, four of which have demonstrated prior evidence of zoonotic capacity. These include the biosafety level 4 agents Hendra (HeV) and Nipah (NiV) viruses, which circulate naturally in pteropodid fruit bats. Here, we describe and characterize Angavokely virus (AngV), a divergent henipavirus identified in urine samples from wild, Madagascar fruit bats. We report the nearly complete 16,740-nucleotide genome of AngV, which encodes the six major henipavirus structural proteins (nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, and L polymerase). Within the phosphoprotein (P) gene, we identify an alternative start codon encoding the AngV C protein and a putative mRNA editing site where the insertion of one or two guanine residues encodes, respectively, additional V and W proteins. In other paramyxovirus systems, C, V, and W are accessory proteins involved in antagonism of host immune responses during infection. Phylogenetic analysis suggests that AngV is ancestral to all four previously described bat henipaviruses-HeV, NiV, Cedar virus (CedV), and Ghanaian bat virus (GhV)-but evolved more recently than rodent- and shrew-derived henipaviruses, Mojiang (MojV), Gamak (GAKV), and Daeryong (DARV) viruses. Predictive structure-based alignments suggest that AngV is unlikely to bind ephrin receptors, which mediate cell entry for all other known bat henipaviruses. Identification of the AngV receptor is needed to clarify the virus's potential host range. The presence of V and W proteins in the AngV genome suggest that the virus could be pathogenic following zoonotic spillover. IMPORTANCE Henipaviruses include highly pathogenic emerging zoonotic viruses, derived from bat, rodent, and shrew reservoirs. Bat-borne Hendra (HeV) and Nipah (NiV) are the most well-known henipaviruses, for which no effective antivirals or vaccines for humans have been described. Here, we report the discovery and characterization of a novel henipavirus, Angavokely virus (AngV), isolated from wild fruit bats in Madagascar. Genomic characterization of AngV reveals all major features associated with pathogenicity in other henipaviruses, suggesting that AngV could be pathogenic following spillover to human hosts. Our work suggests that AngV is an ancestral bat henipavirus that likely uses viral entry pathways distinct from those previously described for HeV and NiV. In Madagascar, bats are consumed as a source of human food, presenting opportunities for cross-species transmission. Characterization of novel henipaviruses and documentation of their pathogenic and zoonotic potential are essential to predicting and preventing the emergence of future zoonoses that cause pandemics.

BK virus associated haemorrhagic cystitis in related donor haematopoietic stem cell transplant recipients: A developing country experience.

A retrospective cross sectional study was conducted at the Virology Department, Armed Forces Institute of Pathology (AFIP) and Armed Forces Bone Marrow Transplant Centre (AFBMTC), Rawalpindi, from January 2016 to July 2018. Medical records of 193 patients were examined to determine the number of patients developing Haemorrhagic Cystitis associated with BK virus (BKV). BKV PCR testing was done on the patients' urine samples. Cytomegalovirus reactivation was also assessed weekly from day 30 to day 100, by CMV quantitative PCR testing on blood samples. Out of 193 patients, 11 (5.6%) developed haemorrhagic cystitis and all these patients were positive for BKV on urine samples. The maximum number of positive cases, i.e. 5 (2.6%) was in the age group three months to 10 years. Primary disease in seven out of 11 cases was Beta-Thalassemia Major.

Urinary genome detection and tracking of Hantaan virus from hemorrhagic fever with renal syndrome patients using multiplex PCR-based next-generation sequencing.

BACKGROUND: Hantavirus infection occurs through the inhalation of aerosolized excreta, including urine, feces, and saliva of infected rodents. The presence of Hantaan virus (HTNV) RNA or infectious particles in urine specimens of patient with hemorrhagic fever with renal syndrome (HFRS) remains to be investigated. METHODOLOGY/PRINCIPAL FINDINGS: We collected four urine and serum specimens of Republic of Korea Army (ROKA) patients with HFRS. We performed multiplex PCR-based next-generation sequencing (NGS) to obtain the genome sequences of clinical HTNV in urine specimens containing ultra-low amounts of viral genomes. The epidemiological and phylogenetic analyses of HTNV demonstrated geographically homogenous clustering with those in Apodemus agrarius captured in highly endemic areas, indicating that phylogeographic tracing of HTNV genomes reveals the potential infection sites of patients with HFRS. Genetic exchange analyses showed a genetic configuration compatible with HTNV L segment exchange in nature. CONCLUSION/SIGNIFICANCE: Our results suggest that whole or partial genome sequences of HTNV from the urine enabled to track the putative infection sites of patients with HFRS by phylogeographically linking to the zoonotic HTNV from the reservoir host captured at endemic regions. This report raises awareness among physicians for the presence of HTNV in the urine of patients with HFRS.

JEV-nanobarcode and colorimetric reverse transcription loop-mediated isothermal amplification (cRT-LAMP).

Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/µL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2-4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of  rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A530) and 650 (A650), which have a limit of detection of 23.3 ng/µL. The AuNP:polyA10-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs. The JEV RT-LAMP nanobarcode assay targeting the envelope (E) gene and MgSO4 induced AuNP aggregation, indicated by an instant pink-to-violet colorimetric read-out.

Utility of Nested Reverse-Transcriptase Polymerase Chain Reaction of Clinical Specimens for Early Diagnosis of Hemorrhagic Fever with Renal Syndrome.

Hemorrhagic fever with renal syndrome (HFRS) is confirmed by the isolation of hantavirus from serum, detection of virus-specific IgM, or a four-fold change in IgG titers during the acute and convalescent periods measured using an immunofluorescence assay (IFA). However, these tests are inefficient for early diagnosis. Therefore, this study investigated the usefulness of reverse-transcriptase nested polymerase chain reaction (RT-nPCR) for early diagnosis of HFRS using clinical samples such as urine and serum. Electronic medical records of eight patients with confirmed HFRS using IFA and RT-nPCR between May 2016 and May 2020 at Chosun University Hospital were reviewed. The virus was detected in all patients using RT-nPCR targeting the large (L) segment of hantavirus during the early phase in urine and serum. Importantly, the virus was identified in urine at a time when it was not identified in serum. Additionally, the virus was detected in urine and serum for up to 1 month after initial presentation with illness, but not in saliva, using RT-nPCR. We report eight HFRS cases diagnosed using urine and serum, but not using saliva, with RT-nPCR targeting the L-segment. Hantavirus RNA detection by RT-nPCR in urine and serum may aid the rapid diagnosis of HFRS during the early phase of the disease. In particular, HFRS should not be ruled out based on negative RT-PCR results in serum, and RT-PCR should be performed using urine as well as serum during the early phase of symptoms.

Longitudinal Secretion of Paramyxovirus RNA in the Urine of Straw-Coloured Fruit Bats (Eidolon helvum).

The straw-coloured fruit bat (Eidolon helvum) is widespread in sub-Saharan Africa and is widely hunted for bushmeat. It is known to harbour a range of paramyxoviruses, including rubuloviruses and henipaviruses, but the zoonotic potential of these is unknown. We previously found a diversity of paramyxoviruses within a small, captive colony of E. helvum after it had been closed to contact with other bats for 5 years. In this study, we used under-roost urine collection to further investigate the paramyxovirus diversity and ecology in this colony, which had been closed to the outside for 10 years at the time of sampling. By sampling urine weekly throughout an entire year, we investigated possible seasonal patterns of shedding of virus or viral RNA. Using a generic paramyxovirus L-gene PCR, we detected eight distinct paramyxovirus RNA sequences. Six distinct sequences were detected using a Henipavirus-specific PCR that targeted a different region of the L-gene. Sequence detection had a bi-annual pattern, with the greatest peak in July, although different RNA sequences appeared to have different shedding patterns. No significant associations were detected between sequence detection and birthing season, environmental temperature or humidity, and no signs of illness were detected in any of the bats in the colony during the period of sample collection.

Persistence of Multiple Paramyxoviruses in a Closed Captive Colony of Fruit Bats (Eidolon helvum).

Bats have been identified as the natural hosts of several emerging zoonotic viruses, including paramyxoviruses, such as Hendra and Nipah viruses, that can cause fatal disease in humans. Recently, African fruit bats with populations that roost in or near urban areas have been shown to harbour a great diversity of paramyxoviruses, posing potential spillover risks to public health. Understanding the circulation of these viruses in their reservoir populations is essential to predict and prevent future emerging diseases. Here, we identify a high incidence of multiple paramyxoviruses in urine samples collected from a closed captive colony of circa 115 straw-coloured fruit bats (Eidolon helvum). The sequences detected have high nucleotide identities with those derived from free ranging African fruit bats and form phylogenetic clusters with the Henipavirus genus, Pararubulavirus genus and other unclassified paramyxoviruses. As this colony had been closed for 5 years prior to this study, these results indicate that within-host paramyxoviral persistence underlies the role of bats as reservoirs of these viruses.

Clinical and analytical evaluation of the RealTime High Risk HPV assay in Colli-Pee collected first-void urine using the VALHUDES protocol.

OBJECTIVE: Urine self-sampling has gained increasing interest for cervical cancer screening. In contrast to analytical performance, little information is available regarding the clinical accuracy for high-risk Human Papillomavirus (hrHPV) testing on urine. METHODS: VALHUDES is a diagnostic test accuracy study comparing clinical accuracy to detect high-grade cervical precancer (CIN2+) of HPV testing on self-collected compared to clinician-collected samples (NCT03064087). Disease outcome was assessed by colposcopy and histology. The Abbott RealTime High Risk HPV assay performance was evaluated on Colli-Pee collected first-void urine with cervical outcomes as comparator. RESULTS: As no assay cut-off for urine has been clinically validated, we used the predefined cut-off for cervical samples (CN ≤ 32). Using this cut-off, hrHPV testing was similarly sensitive (relative sensitivity 0.95; 95% CI: 0.88-1.01) and specific (relative specificity 1.03; 95% CI: 0.95-1.13) for detection of CIN2+ compared to testing cervical samples. In the subgroup of women of 30 years and older, similar relative sensitivity (0.97; 95% CI: 0.89-1.05) and specificity (1.02; 95% CI: 0.93-1.12) was found. Additionally, an exploratory cut-off (CN ≤ 33.86) was defined which further improved sensitivity and analytical test performance. CONCLUSION: HrHPV-DNA based PCR testing on home-collected first-void urine has similar accuracy for detecting CIN2+ compared to cervical samples taken by a clinician.

High SARS-CoV-2 Viral Load in Urine Sediment Correlates with Acute Kidney Injury and Poor COVID-19 Outcome.

BACKGROUND: AKI is a complication of coronavirus disease 2019 (COVID-19) that is associated with high mortality. Despite documented kidney tropism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are no consistent reports of viral detection in urine or correlation with AKI or COVID-19 severity. Here, we hypothesize that quantification of the viral load of SARS-CoV-2 in urine sediment from patients with COVID-19 correlates with occurrence of AKI and mortality. METHODS: The viral load of SARS-CoV-2 in urine sediments (U-viral load) was quantified by qRT-PCR in 52 patients with PCR-confirmed COVID-19 diagnosis, who were hospitalized between March 15 and June 8, 2020. Immunolabeling of SARS-CoV-2 proteins Spike and Nucleocapsid was performed in two COVID-19 kidney biopsy specimens and urine sediments. Viral infectivity assays were performed from 32 urine sediments. RESULTS: A total of 20 patients with COVID-19 (39%) had detectable SARS-CoV-2 U-viral load, of which 17 (85%) developed AKI with an average U-viral load four-times higher than patients with COVID-19 who did not have AKI. U-viral load was highest (7.7-fold) within 2 weeks after AKI diagnosis. A higher U-viral load correlated with mortality but not with albuminuria or AKI stage. SARS-CoV-2 proteins partially colocalized with the viral receptor ACE2 in kidney biopsy specimens in tubules and parietal cells, and in urine sediment cells. Infective SARS-CoV-2 was not detected in urine sediments. CONCLUSION: Our results further support SARS-CoV-2 kidney tropism. A higher SARS-CoV-2 viral load in urine sediments from patients with COVID-19 correlated with increased incidence of AKI and mortality. Urinary viral detection could inform the medical care of patients with COVID-19 and kidney injury to improve prognosis.

Ticking on Pandora's box: a prospective case-control study into 'other' tick-borne diseases.

BACKGROUND: Tick-borne pathogens other than Borrelia burgdorferi sensu lato - the causative agent of Lyme borreliosis - are common in Ixodes ricinus ticks. How often these pathogens cause human disease is unknown. In addition, diagnostic tools to identify such diseases are lacking or reserved to research laboratories. To elucidate their prevalence and disease burden, the study 'Ticking on Pandora's Box' has been initiated, a collaborative effort between Amsterdam University Medical Center and the National Institute for Public Health and the Environment. METHODS: The study investigates how often the tick-borne pathogens Anaplasma phagocytophilum, Babesia species, Borrelia miyamotoi, Neoehrlichia mikurensis, spotted fever group Rickettsia species and/or tick-borne encephalitis virus cause an acute febrile illness after tick-bite. We aim to determine the impact and severity of these tick-borne diseases in the Netherlands by measuring their prevalence and describing their clinical picture and course of disease. The study is designed as a prospective case-control study. We aim to include 150 cases - individuals clinically suspected of a tick-borne disease - and 3 matched healthy control groups of 200 persons each. The controls consist respectively of a group of individuals with either a tick-bite without complaints, the general population and of healthy blood donors. During a one-year follow-up we will acquire blood, urine and skin biopsy samples and ticks at baseline, 4 and 12 weeks. Additionally, participants answer modified versions of validated questionnaires to assess self-reported symptoms, among which the SF-36, on a 3 monthly basis. DISCUSSION: This article describes the background and design of the study protocol of 'Ticking on Pandora's Box'. With our study we hope to provide insight into the prevalence, clinical presentation and disease burden of the tick-borne diseases anaplasmosis, babesiosis, B. miyamotoi disease, neoehrlichiosis, rickettsiosis and tick-borne encephalitis and to assist in test development as well as provide recommendations for national guidelines. TRIAL REGISTRATION: NL9258 (retrospectively registered at Netherlands Trial Register, trialregister.nl in in February 2021).

Elimination of human cytomegalovirus DNA degradation in urine.

Congenital cytomegalovirus infection (cCMVi) is an important cause of sensorineural hearing loss in newborns. Detection of human cytomegalovirus (HCMV) DNA in urine has been used to screen for cCMVi in newborns. However, the matrix effect of urine on HCMV DNA detection is unclear. To evaluate the matrix effect of urine on HCMV DNA detection and optimize the sample process strategy to eliminate or minimize the impact of urine on HCMV DNA detection, DNA in spiked samples was extracted using different DNA extraction methods, and urine samples that could inhibit HCMV DNA detection were mixed to evaluate the inhibitory substances, inhibitory mechanism, and elimination of the inhibitory effect. The optimal urine sample process strategy was evaluated using 42 adult female urine samples and 42 newborn urine samples spiked with HCMV. Some urine samples were found to inhibit HCMV DNA detection due to DNA degradation. The addition of ≥5 mM EDTA to the urine before extraction eliminated the inhibitory effect of urine and did not affect the detection results of urine exhibiting no inhibition. Of the 42 adult female and 42 newborn urine samples, four and two samples, respectively, could inhibit HCMV DNA detection. However, the inhibitory effects of these six urine samples were eliminated after the addition of EDTA. The collective results indicate that the addition of EDTA can completely eliminate the impact of inhibitors present in urine on HCMV DNA extraction and improve the detection of HCMV in urine.

In Vivo Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads.

Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory digital droplet PCR (ddPCR) of urine samples taken from immunosuppressed individuals, we show that clinical BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were derived from BKPyV genotypes Ia, Ib-1, and Ic. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology.IMPORTANCE Defective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens; no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes, with the majority affecting genes required for viral replication. Longitudinal analysis showed that these DVGs can persist during an infection but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests that these parasitic sequences exist across the many classes of viruses capable of causing human disease.

Zika virus RNA excretion in sweat with concomitant detection in other body fluid specimens.

We evaluated sweat, blood and urine specimens obtained from an ongoing cohort study in Brazil. Samples were collected at pre-established intervals after the initial rash presentation and tested for Zika virus (ZIKV) RNA presence by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). From 254 participants with confirmed infection, ZIKV RNA was detected in the sweat of 46 individuals (18.1%). Sweat presented a median cycle threshold (Ct) of 34.74 [interquartile range (IQR) 33.44-36.04], comparable to plasma (Ct 35.96 - IQR 33.29-36.69) and higher than urine (Ct 30.78 - IQR 28.72-33.22). Concomitant detection with other specimens was observed in 33 (72%) of 46 participants who had a positive result in sweat. These findings represent an unusual and not yet investigated virus shedding through eccrine glands.

Analysis of viral load in different specimen types and serum antibody levels of COVID-19 patients.

BACKGROUND: COVID-19 has caused a global pandemic and the death toll is increasing. However, there is no definitive information regarding the type of clinical specimens that is the best for SARS-CoV-2 detection, the antibody levels in patients with different duration of disease, and the relationship between antibody level and viral load. METHODS: Nasopharyngeal swabs, anal swabs, saliva, blood, and urine specimens were collected from patients with a course of disease ranging from 7 to 69 days. Viral load in different specimen types was measured using droplet digital PCR (ddPCR). Meanwhile, anti-nucleocapsid protein (anti-N) IgM and IgG antibodies and anti-spike protein receptor-binding domain (anti-S-RBD) IgG antibody in all serum samples were tested using ELISA. RESULTS: The positive detection rate in nasopharyngeal swab was the highest (54.05%), followed by anal swab (24.32%), and the positive detection rate in saliva, blood, and urine was 16.22%, 10.81%, and 5.41%, respectively. However, some patients with negative nasopharyngeal swabs had other specimens tested positive. There was no significant correlation between antibody level and days after symptoms onset or viral load. CONCLUSIONS: Other specimens could be positive in patients with negative nasopharyngeal swabs, suggesting that for patients in the recovery period, specimens other than nasopharyngeal swabs should also be tested to avoid false negative results, and anal swabs are recommended. The antibody level had no correlation with days after symptoms onset or the viral load of nasopharyngeal swabs, suggesting that the antibody level may also be affected by other factors.

Gestation and COVID-19: clinical and microbiological observational study (Gesta-COVID19).

BACKGROUND: The Coronavirus Disease 2019 (COVID-19) is a novel disease which has been having a worldwide affect since December 2019. Evidence regarding the effects of SARS-CoV-2 during pregnancy is conflicting. The presence of SARS-CoV-2 has been demonstrated in biological samples during pregnancy (placenta, umbilical cord or amniotic fluid); however, maternal and fetal effects of the virus are not well known. METHODS: Descriptive, multicentre, longitudinal, observational study in eight tertiary care hospitals throughout Spain, that are referral centres for pregnant women with COVID-19. All pregnant women with positive SARS-CoV-2 real-time reverse transcriptase polymerase chain reaction during their pregnancy or 14 days preconception and newborns born to mothers infected with SARS-CoV-2 will be included. They will continue to be followed up until 4 weeks after delivery. The aim of the study is to investigate both the effect of COVID-19 on the pregnancy, and the effect of the pregnancy status with the evolution of the SARS-CoV-2 disease. Other samples (faeces, urine, serum, amniotic fluid, cord and peripheral blood, placenta and breastmilk) will be collected in order to analyse whether or not there is a risk of vertical transmission and to describe the behaviour of the virus in other fluids. Neonates will be followed until 6 months after delivery to establish the rate of neonatal transmission. We aim to include 150 pregnant women and their babies. Ethics approval will be obtained from all the participating centres. DISCUSSION: There is little information known about COVID-19 and its unknown effects on pregnancy. This study will collect a large number of samples in pregnant women which will allow us to demonstrate the behaviour of the virus in pregnancy and postpartum in a representative cohort of the Spanish population.

A Randomized Comparison of Different Vaginal Self-sampling Devices and Urine for Human Papillomavirus Testing-Predictors 5.1.

BACKGROUND: Human papillomavirus (HPV)-based screening is rapidly replacing cytology as the cervical screening modality of choice. In addition to being more sensitive than cytology, it can be done on self-collected vaginal or urine samples. This study will compare the high-risk HPV positivity rates and sensitivity of self-collected vaginal samples using four different collection devices and a urine sample. METHODS: A total of 620 women referred for colposcopy were invited to provide an initial stream urine sample collected with the Colli-Pee device and take two vaginal self-samples, using either a dry flocked swab (DF) and a wet dacron swab (WD), or a HerSwab (HS) and Qvintip (QT) device. HPV testing was performed by the BD Onclarity HPV Assay. RESULTS: A total of 600 vaginal sample pairs were suitable for analysis, and 505 were accompanied by a urine sample. Similar positivity rates and sensitivities for CIN2+ and CIN3+ were seen for DF, WD, and urine, but lower values were seen for QT and HS. No clear user preferences were seen between devices, but women found urine easiest to collect, and were more confident they had taken the sample correctly. The lowest confidence in collection was reported for HS. CONCLUSIONS: Urine, a DF swab, and WD swab all performed well and were well received by the women, whereas the Qvintip and HerSwab devices were less satisfactory. IMPACT: This is the first study to compare five self-sampling methods in the same women taken at the same time. It supports wider use of urine or vaginal self-sampling for cervical screening.

No evidence of HEV genotype 1 infections harming the male reproductive system.

Extrahepatic disorders are recorded with hepatitis E virus (HEV) infection. The impact of HEV infection on the male reproductive system is a query. In this study, we retrospectively analyzed semen from infertile men and prospectively examined the semen from acute hepatitis E patients (AHE) for HEV markers. HEV RNA and HEV Ag were not detectable in the semen of infertile men nor the semen of AHE patients. Although HEV markers were detectable in the urine of patients infected with HEV-1, these markers were absent in their semen. There is no significant difference in the level of reproductive hormones between AHE patients and healthy controls. Semen analysis of AHE patients did not show a notable abnormality and there was no significant difference in the semen quality and sperm characteristics between AHE and healthy controls.

No detection of SARS-CoV-2 from urine, expressed prostatic secretions, and semen in 74 recovered COVID-19 male patients: A perspective and urogenital evaluation.

BACKGROUND: The coronavirus disease 2019 (COVID-19) has been spreading all over the world since December 2019. However, medical information regarding the urogenital involvement in recovered COVID-19 patients is limited or unknown. OBJECTIVES: To comprehensively evaluate urogenital involvement in recovered COVID-19 patients. MATERIALS AND METHODS: Men aged between 20 years and 50 years who were diagnosed with SARS-CoV-2 infection and recovered when the study was conducted were enrolled in our study. Demographic and clinical characteristics, and history of hospitalization were collected and analyzed. Urine, expressed prostatic secretions (EPSs), and semen samples were collected for SARS-CoV-2 RNA detection. Semen quality and hormonal profiles were analyzed. RESULTS: Among 74 male recovered COVID-19 patients, 11 (14.9%) were asymptomatic, classified into mild type, and 31 (41.9%) were classified into moderate type. The remaining patients (32/74, 43.2%) had severe pneumonia. No critically ill recovered COVID-19 patient was recruited in our cohort. The median interval between last positive pharyngeal swab RT-PCR test and semen samples collection was 80 days (IQR, 64-93). The median age was 31 years (IQR, 27-36; range, 21-49), and the median body mass index (BMI) was 24.40 (IQR, 22.55-27.30). Forty-five (61.6%) men were married, and 28 (38.4%) were unmarried. Fifty-three (72.6%) patients denied cigarette smoking, 18 (24.7%) were active smokers, and 2 of them were past smokers. The majority of our participants (53/74, 72.6%) did not consume alcohol. Fever occurred in most of the patients (75.3%), and 63 of them had abnormal chest CT images. Only one patient complained of scrotal discomfort during the course of COVID-19, which was ruled out orchitis by MRI (data not shown). A total of 205 samples were collected for SARS-CoV-2 detection (74 urine samples, 70 semen samples, and 61 EPS samples). However, viral nucleic acid was not detected in body fluids from the urogenital system. In terms of hormonal profiles, the levels of FSH, LH, testosterone, and estradiol were 5.20 [4.23] mIU/mL, 3.95 [1.63] mIU/mL, 3.65 [1.19] ng/mL, and 39.48 [12.51] pg/mL, respectively. And these values were within the normal limits. The overall semen quality of recovered COVID-19 patients was above the lower reference limit released by the WHO. While compared with healthy control, sperm concentration, total sperm count, and total motility were significantly declined. In addition, different clinical types of COVID-19 have no significant difference in semen parameters, but total sperm count showed a descending trend. Interestingly, subjects with a longer recovery time showed worse data for sperm quality. Small sample size and lacking semen parameters before the infection are the major limitations of our study. DISCUSSION AND CONCLUSIONS: To the best of our knowledge, it is the largest cohort study with longest follow-up for urogenital evaluation comprehensively so far. Direct urogenital involvement was not found in the recovered COVID-19 male patients. SARS-CoV-2 RNA was undetectable in the urogenital secretions, and semen quality declined slightly, while hormonal profiles remained normal. Moreover, patients with a long time (≥90 days) since recovery had lower total sperm count. Great attention and further study should be conducted and follow-up on the reproductive function in the following months.

Pilot Study: Long-Term Shedding of SARS-CoV-2 in Urine: A Threat for Dispersal in Wastewater.

Only 4 months after the beginning of SARS-CoV-2 epidemic, the world is facing a global pandemic due to a complex and insidious virus that today constantly poses new challenges. In this study, we highlight a persistent shedding of SARS-CoV-2 RNA into the urine, even in patients with a negative nasopharyngeal swab and in patients considered recovered. What does it mean? Besides the fact that the kidney is a probable site of viral replication, the prolonged viral excretion is a matter of great concern for our drainage system contamination.