First Detection and Characterization of Smacovirus in the Human Vagina in Two Sequential Samples over a Twelve-Day Interval.

BACKGROUND: Smacovirus is a CRESS-DNA virus identified almost exclusively in transient fecal samples from various vertebrate species. OBJECTIVE: We evaluated human vaginal samples for the presence and maintenance of Smacovirus. METHODS: Viral metagenomics analysis was performed on vaginal samples collected from 28 apparently healthy women in New York City, USA. Twenty-one of the women provided duplicate samples over a 12-21-day interval. RESULTS: Phylogenetic analysis identified two samples from the same individual, collected over a twelve-day interval, that were positive for the complete Smacovirus genome. All detected sequence contigs belonged to a single variant of CRESS-DNA. CONCLUSIONS: The continuous presence of Smacovirus in the human vagina over a twelve-day period was identified.

Characterization of multiple human papillomavirus types in the human vagina following ovarian hormonal stimulation.

The objective of study was to characterize HPV in vaginal samples from women being seen at the Center for Reproductive Medicine and Infertility at Weill Cornell Medicine before and following ovarian stimulation. A total of 29 women made samples available for analysis by viral metagenomics. Eighteen women were HPV-positive, six (33.3%) at their initial visit and 15 (83.3%) following hormone stimulation (p = 0.0059). Pairwise comparison of nucleotide sequences and phylogenetic analysis showed the classification sequences into two genera: Alphapapillomavirus and Gammapapillomavirus. Sequences were from 8 HPV types: HPV 51 (n = 2), HPV 68 (n = 1), HPV 83 (n = 9), HPV 84 (n = 2), HPV 121 (n = 6), HPV 175 (n = 1) and HPV 190 (n = 1). Additionally, C16b and C30 likely represent new types. In summary, multiple HPV types are present in the vagina of reproductive age women and are induced by hormone used to stimulate ovulation.

Characterization of CRESS-DNA viruses in human vaginal secretions: An exploratory metagenomic investigation.

The Phylum Cressdnaviricota consists of a large number of circular Rep-encoding single-stranded (CRESS)-DNA viruses. Recently, metagenomic analyzes revealed their ubiquitous distribution in a diverse range of eukaryotes. Data relating to CRESS-DNA viruses in humans remains scarce. Our study investigated the presence and genetic diversity of CRESS-DNA viruses in human vaginal secretions. Vaginal swabs were collected from 28 women between 29 and 43 years old attending a fertility clinic in New York City. An exploratory metagenomic analysis was performed and detection of CRESS-DNA viruses was confirmed through analysis of near full-length sequences of the viral isolates. A phylogenetic tree was based on the REP open reading frame sequences of the CRESS-DNA virus genome. Eleven nearly complete CRESS-DNA viral genomes were identified in 16 (57.1%) women. There were no associations between the presence of these viruses and any demographic or clinical parameters. Phylogenetic analysis indicated that one of the sequences belonged to the genus Gemycircularvirus within the Genomoviridae family, while ten sequences represented previously unclassified species of CRESS-DNA viruses. Novel species of CRESS-DNA viruses are present in the vaginal tract of adult women. Although they be transient commensal agents, the potential clinical implications for their presence at this site cannot be dismissed.

Changes in cytokine and sequestosome-1 levels during twin pregnancy progression: Association with outcome.

BACKGROUND: Twin pregnancies are associated with complications and adverse outcomes. The number of twin pregnancies has increased in the last decades, due to the use of assisted reproductive techniques and delayed childbearing. Analysis of changes that occur during twin pregnancy progression and their association with outcome will lead to improved clinical interventions. OBJECTIVE: We evaluated if the plasma concentration of select cytokines and the level of sequestosome-1 (p62) in peripheral blood mononuclear cells (PBMCs) during each trimester of twin gestations was predictive of pregnancy outcome. STUDY DESIGN: This prospective, observational study was conducted at Careggi University Hospital, Florence, Italy. Plasma from 82 women with twin pregnancies was collected in each trimester for measurement of interleukin (IL)-1ß, IL-6, IL-10, IL-12 and tumor necrosis factor (TNF)-α. The intracellular PBMC concentration of p62, a protein involved in autophagy, kinase activity and cell differentiation, was also determined. RESULTS: IL-1ß (p < 0.001), IL-6 (p < 0.001), TNF-α (p < 0.001) and p62 (p < 0.05) increased from the 1st to the 2nd to the 3rd trimester. The TNF-α level was correlated with the IL-1ß concentration in the 1st and 3rd trimesters p < 0.01) and with the IL-6 concentration in each of the three trimesters (p < 0.01). The intracellular p62 level in PBMCs was negatively correlated with the concentration of IL-1ß in the 2nd trimester (p < 0.05) and negatively correlated with the IL-6 level in the 3rd trimester (p < 0.05). The TNF-α level was significantly higher in the 2nd (p < 0.05) and 3rd (p < 0.001) trimester in women with a spontaneous preterm delivery. The TNF-α concentrations in the 2nd (p < 0.05) and 3rd (p < 0.01) trimester, respectively, and 3rd trimester IL-6 (p < 0.01), were negatively associated with gestational age at delivery. The concentration of IL-6 was highest in the 2nd (p < 0.05) and 3rd (p < 0.05) trimesters in women who utilized assisted reproductive technologies. An elevated IL-1ß level in the 3rd trimester was associated with gestational diabetes mellitus (p < 0.05). CONCLUSION: Variations in cytokine levels between individual women during the three trimesters of twin gestations are predictive of spontaneous preterm delivery and the onset of gestational diabetes.

Ocular manifestations of Monkeypox virus (MPXV) infection with viral persistence in ocular samples: A case series.

OBJECTIVE: We describe the clinical presentation and ocular viral dynamics in patients with Monkeypox virus-related ophthalmic disease (MPXROD). METHODS: In this case series, we investigated five consecutive patients with confirmed mpox, diagnosed through a positive Monkeypox virus (MPXV) Polymerase Chain Reaction (PCR) test and presenting with ocular symptoms. They were referred from the Reference Center for Sexually Transmitted Infections in São Paulo (CRT) to the Uveitis Sector at the Federal University of São Paulo, between August and December 2022. We performed PCR testing on ocular samples and culture supernatants for MPXV in all patients. Viral sequencing was conducted in one of the cases. RESULTS: Replicating MPXV was identified in at least one ocular sample of all patients, between day 31 and day 145 after the onset of skin lesions. All patients presented with keratitis, 3 with uveitis (60%) and two exhibited hypopyon (40%). The onset of ocular symptoms occurred at a mean of 21.2 days after the appearance of the first skin lesion and persisted, on average, for 61,.6 days, with a worsening trend observed until the initiation of tecovirimat treatment. Tecovirimat treatment was administered to all patients, with initiation occurring between 31 and 145 days after the onset of skin lesions. MPXV genome sequencing of an isolate from one patient classified it as belonging to lineage B1 in clade IIb. CONCLUSION: This study reveals a late onset and persistence of sight threatening ocular disease, along with potential viral infectivity even after systemic resolution in mpox cases. These findings highlight the risk of ongoing transmission from individuals with prolonged ocular manifestations, particularly through ocular discharge.

Evaluation of enterovirus concentration, species identification, and cerebrospinal fluid parameters in patients of different ages with aseptic meningitis in São Paulo, Brazil.

Human enteroviruses (EV) are the most common cause of aseptic meningitis worldwide. Data on EV viral load in cerebrospinal fluid (CSF) and related epidemiological studies are scarce in Brazil. This study investigated the influence of EV viral load on CSF parameters, as well as identifying the involved species. CSF samples were collected in 2018-2019 from 140 individuals at The Hospital das Clínicas, São Paulo. The EV viral load was determined using real-time quantitative polymerase chain reaction, while EV species were identified by 5'UTR region sequencing. Median viral load was 5.72 log10 copies/mL and did not differ by subjects' age and EV species. Pleocytosis was observed in 94.3% of cases, with the highest white blood cell (WBC) counts in younger individuals. Viral load and WBC count were correlated in children (p = 0.0172). Elevated lactate levels were observed in 60% of cases and correlated with the viral load in preteen-teenagers (p = 0.0120) and adults (p = 0.0184). Most individuals had normal total protein levels (70.7%), with higher in preteen-teenagers and adults (p < 0.0001). By sequencing, 8.2% were identified as EV species A and 91.8% as species B. Age-specific variations in CSF characteristics suggest distinct inflammatory responses in each group.

Association between torquetenovirus in vaginal secretions and infertility: An exploratory metagenomic analysis.

PROBLEM: The association of viruses with infertility remains incompletely evaluated. METHOD OF STUDY: Vaginal secretions from 46 women seeking treatment in the Center for Reproductive Medicine and Infertility at Weill Cornell Medicine were tested for viruses by metagenomic analysis by lab personnel blinded to all clinical data. RESULTS: Torquetenovirus (TTV) was identified in 16 women, alphapapillomavirus in seven women and most were positive for bacteriophages. Twelve of the subjects were fertile and sought to freeze their oocytes for future implantation. These women were all negative for TTV. In contrast, 16 of the 34 women (47.1%) being treated for infertility were TTV-positive (p = .0035). Evaluating the women by cause of infertility, five of nine women (55.6%) whose male partner had inadequate sperm parameters and six of 14 women (42.9%) with defective ovulation were TTV positive (p = .0062 and p = .0171, respectively, vs. the fertile women). Alphapapillomavirus was identified in one (8.3%) fertile woman, five (35.7%) women with ovulation deficiency, and one (11.1%) woman with male factor infertility. These differences were not statistically significant. There were no differences in bacteriophage families or the presence of Lactobacillus phages between fertile or infertile women or between different causes of infertility. There was a negative association between TTV detection and Lactobacillus crispatus dominance in the vaginal microbiota (p = .0184), but no association between TTV detection and the presence of alphapapillomavirus or Candida species. CONCLUSION: Detection of TTV in the vagina might be a biomarker for specific causes of infertility.

Encephalopathy Caused by Human Parvovirus B19 Genotype 1 Associated with Haemophilus influenzae Meningitis in a Newborn.

Parvovirus B19 infection is associated with a wide range of clinical manifestations, from asymptomatic to severe neurological disorders. Its major clinical symptoms, fever and rash, are common to multiple viruses, and laboratory tests to detect B19 are frequently not available. Thus, the impact of B19 on public health remains unclear. We report the case of a 38-day old girl admitted to São Paulo Clinical Hospital, Brazil, with an initial diagnosis of bacterial meningitis, seizures, and acute hydrocephalus. Antibiotic therapy was maintained for one week after admission and discontinued after negative laboratory results were obtained. Nine days after symptoms onset, a cerebral spinal fluid (CSF) sample revealed persistent pleocytosis. The complete B19 complete genome was subsequently identified in her CSF by a metagenomic next-generation sequencing approach. This report highlights the possible involvement of B19 in the occurrence of acute neurological manifestations and emphasizes that its possible involvement might be better revealed by the use of metagenomic technology to detect viral agents in clinical situations of unknown or uncertain etiology.

Detailed characterization of Redondovirus in saliva of SARS-CoV-2-infected individuals in Sao Paulo, Brazil.

BACKGROUND: Redondovirus (ReDoV) is a DNA virus present in the respiratory tract of many healthy individuals. Since SARS-CoV-2, the virus responsible for COVID-19, also primarily infects the same site, we evaluated whether ReDoV was present at increased frequency in patients with COVID-19 and influenced infection parameters. METHODS: Saliva samples were collected weekly from 59 individuals with COVID-19 and from 132 controls. ReDoV was detected by polymerase chain reaction and the genotypes were identified by metagenomics. Torque Teno Virus (TTV) in these samples were previously reported. RESULTS: ReDoV was detected in saliva more frequently from COVID-19 patients (72.9%) than from controls (50.0%) (p = 0.0015). There were no associations between ReDoV detection and either continuous or intermittent SARS-CoV-2 shedding, the duration of SARS-CoV-2 detection in saliva, patients' sex or if infection was by the B1 or Gamma strain. The two ReDoV strains, Brisavirus and Vientovirus, were present in equivalent frequencies in ReDoV-positive COVID-19 patients and controls. Phylogenetic analysis suggested that the two ReDoV strains in Brazil were similar to strains previously detected on other continents. CONCLUSION: ReDoV expression in saliva is increased in males and females in Brazil with mild COVID-19 but its presence does not appear to influence properties of the SARS-CoV-2 infection.

Characterization of Torquetenovirus in amniotic fluid at the time of in utero fetal surgery: correlation with early premature delivery and respiratory distress.

Torquetenovirus (TTV) is a commensal virus present in many healthy individuals. Although considered to be non-pathogenic, its presence and titer have been shown to be indicative of altered immune status in individuals with chronic infections or following allogeneic transplantations. We evaluated if TTV was present in amniotic fluid (AF) at the time of in utero surgery to correct a fetal neurological defect, and whether its detection was predictive of adverse post-surgical parameters. AF was collected from 27 women by needle aspiration prior to a uterine incision. TTV titer in the AF was measured by isolation of viral DNA followed by gene amplification and analysis. The TTV genomes were further characterized and sequenced by metagenomics. Pregnancy outcome parameters were subsequently obtained by chart review. Three of the AFs (11.1%) were positive for TTV at 3.36, 4.16, and 4.19 log10 copies/mL. Analysis of their genomes revealed DNA sequences similar to previously identified TTV isolates. Mean gestational age at delivery was >2 weeks earlier (32.5 vs. 34.6 weeks) and the prevalence of respiratory distress was greater (100% vs. 20.8%) in the TTV-positive pregnancies. TTV detection in AF prior to intrauterine surgery may indicate elevated post-surgical risk for earlier delivery and newborn respiratory distress.

Association between development of severe COVID-19 and a polymorphism in the CIAS1 gene that codes for an inflammasome component.

An elevated pro-inflammatory cytokine response is associated with severe life-threatening symptoms in individuals with Coronavirus Disease-2019 (COVID). The inflammasome is an intracellular structure responsible for generation of interleukin (IL)-1ß and IL-18. NALP3, a product of the CIAS1 gene, is the rate-limiting component for inflammasome activity. We evaluated if a CIAS1 42 base pair length polymorphism (rs74163773) was associated with severe COVID. DNA from 93 individuals with severe COVID, 38 with mild COVID, and 98 controls were analyzed for this polymorphism. The 12 unit repeat allele is associated with the highest inflammasome activity. Five alleles, corresponding to 6, 7, 9, 12 or 13 repeat units, divided into 12 genotypes were identified. The frequency of the 12 unit repeat allele was 45.3% in those with severe disease as opposed to 30.0% in those with mild disease and 26.0% in controls (p < 0.0001, severe vs. controls). In contrast, the 7 unit repeat allele frequency was 30.1% in controls as opposed to 14.0% and 12.5% in those with severe or mild disease, respectively (p ≤ 0.0017). We conclude that individuals positive for the CIAS1 12 allele may be at elevated risk for development of severe COVID due to an increased level of induced pro-inflammatory cytokine production.

SARS-CoV-2 Detection and Culture in Different Biological Specimens from Immunocompetent and Immunosuppressed COVID-19 Patients Infected with Two Different Viral Strains.

Introduction-The dynamics of SARS-CoV-2 shedding and replication in humans remain incompletely understood. Methods-We analyzed SARS-CoV-2 shedding from multiple sites in individuals with an acute COVID-19 infection by weekly sampling for five weeks in 98 immunocompetent and 25 immunosuppressed individuals. Samples and culture supernatants were tested via RT-PCR for SARS-CoV-2 to determine viral clearance rates and in vitro replication. Results-A total of 2447 clinical specimens were evaluated, including 557 nasopharyngeal swabs, 527 saliva samples, 464 urine specimens, 437 anal swabs and 462 blood samples. The SARS-CoV-2 genome sequences at each site were classified as belonging to the B.1.128 (ancestral strain) or Gamma lineage. SARS-CoV-2 detection was highest in nasopharyngeal swabs regardless of the virus strain involved or the immune status of infected individuals. The duration of viral shedding varied between clinical specimens and individual patients. Prolonged shedding of potentially infectious virus varied from 10 days up to 191 days, and primarily occurred in immunosuppressed individuals. Virus was isolated in culture from 18 nasal swab or saliva samples collected 10 or more days after onset of disease. Conclusions-Our findings indicate that persistent SARS-CoV-2 shedding may occur in both competent or immunosuppressed individuals, at multiple clinical sites and in a minority of subjects is capable of in vitro replication.

Does the addition of electrical stimulation or kinesiotherapy improve outcomes of amitriptyline treatment for women with vulvodynia? A randomized clinical trial.

INTRODUCTION AND HYPOTHESIS: Women diagnosed with provoked vulvodynia frequently report a great deal of frustration in achieving symptomatic relief. Physical therapy and drug treatment are among the interventions most indicated by guidelines; however, whether those modalities are effective when combined remains unclear. The objective was to evaluate the effectiveness of adding a physical therapy modality compared with amitriptyline alone for the treatment of vulvodynia. METHODS: Eighty-six women with vulvodynia were randomized to (G1) 25 mg amitriptyline, once a day (n=27), (G2) amitriptyline + electrical stimulation therapy (n=29) or (G3) amitriptyline + kinesiotherapy (n=30). All treatment modalities were administered for 8 weeks. The primary endpoint was the reduction in vestibular pain. Secondary measurements focused on sexual pain, frequency of vaginal intercourse, Friedrich score, and overall sexual function. Data were analyzed using intention-to-treat. RESULTS: All treatment modalities resulted in a significant decrease in vestibular pain (p<0.001), sexual pain (p<0.05), Friedrich score (p<0.001), and an increase in the frequency of sexual intercourse (p<0.05). G3 was more effective than G1 at reducing sexual pain (G1: 5.3±3.3 vs G3: 3.2±2.7; p=0.01) and at improving sexual function (G1: 18.8±9.8 vs G3: 23.9±7.8; p=0.04). CONCLUSION: Kinesiotherapy and electrotherapy additions to amitriptyline administration as well as amitriptyline alone, were effective at improving vestibular pain in women with vulvodynia. Women receiving physical therapy had the greatest improvement in sexual function and frequency of intercourse at post-treatment and follow-up.

Factors Associated with Spontaneous Clearance of Recently Acquired Hepatitis C Virus among HIV-Positive Men in Brazil.

INTRODUCTION: The objective of the present study was to describe the clinical and epidemiological aspects of recently acquired hepatitis C virus (HCV) infection and the frequency of its spontaneous clearance in a people living with the human immunodeficiency virus (PLWH) cohort. METHODS: We reviewed the medical records from all PLWH at the human immunodeficiency virus (HIV) outpatient reference clinic affiliated with the University of São Paulo, Brazil, and identified, by immunoassays and RNA-PCR individuals who acquired HCV infection between January 2015 and December 2017. The factors associated with subsequent spontaneous clearance of the infection in this group were identified and analyzed. RESULTS: Among 3143 PLWH individuals, 362 (11.5%) were coinfected with HCV. Forty-eight (13.2%) of these subjects first became HCV-positive between January 2015 and December 2017. Spontaneous HCV clearance was documented in 23 individuals (47.9%). The majority of this latter group were male (83.3%), and the median age was 31 years (23-39). The main risk group for HCV acquisition was men who had sex with men (MSM) (89.5%). In a multivariate analysis, only an elevated CD4+ T lymphocyte count at the time of seroconversion was found to be associated with subsequent HCV clearance (p = 0.025). CONCLUSIONS: In HIV-infected individuals in Sao Paulo, Brazil, most cases of recent HCV transmission were by sexual exposure. In PLWH, particularly in MSM, the individual's CD4+ T lymphocyte count is a determinant of whether an acquired HCV infection will be prolonged or will spontaneously clear.

Impact of the COVID-19 Pandemic on Cervical Cancer Screening in São Paulo State, Brazil.

INTRODUCTION: The early identification of precursor lesions followed by appropriate treatment prevents development of cervical cancer and its consequences. OBJECTIVE: The present study evaluated the influence of the COVID-19 pandemic on cervical cancer screening by comparing the quantity of tests to detect cervical cellular changes performed in São Paulo state in 2019, prior to the detection of SARS-CoV-2 in Brazil, to the first (2020) and second (2021) years following its appearance. MATERIALS AND METHODS: Data from Fundação Oncocentro de São Paulo (FOSP), the agency that analyses approximately 220,000 Papanicolaou (Pap) tests annually, were reviewed. RESULTS: A median of 1,835 Pap tests were performed in 55 municipalities in 2019. This was reduced to 815 tests in 2020, a 56% decrease (p = 0.0026). In 2021, the median number was 1,745, a 53% increase over 2020 levels (p = 0.0233). The 26 municipalities with >1,000 tests in 2020 had a median reduction from 4,433 in 2019 to 2,580 in 2020 (p = 0. 0046). The 29 municipalities with <1,000 tests had a median reduction from 951 in 2019 to 554 in 2020 (p < 0.0001). There was a 44% reduction in the number of follow-up cytological evaluations from 2019 to 2020, followed by a 30% increase in the following year. However, the percentage of women with a normal finding or with any abnormality remained unchanged. The findings from a histological evaluation of women in São Paulo city indicated that the percent of cases positive for CIN-1 (p < 0.0410) and CIN-3 (p < 0.0012) increased in 2020 and 2021 as compared to 2019 levels. CONCLUSION: A reduction in testing for cervical cancer in the first year of the COVID-19 pandemic, accompanied by an elevated incidence of precancerous lesions in each of the first 2 years following its initiation, may portend a subsequent increased occurrence of cervical cancer in Brazil.

The Medium Is the Message: Defining a "Normal" Vaginal Microbiome in Healthy Reproductive-Age Women.

The microbial composition of the human vagina differs from that of all other mammals, likely as a consequence of the unique composition of vaginal constituents that promote the selective maintenance and proliferation of distinct bacterial species. Similarly, variations between individual healthy women in genetic, environmental, and medical variables also modify the vaginal lumen composition. The direction and magnitude of immune reactions to microorganisms present in the vagina, responses to stress and non-infectious stimuli, coupled with medical and pregnancy history and environmental exposures, can greatly differ between women. Adaptations to both internal and external pressures will determine the ability of select resident vaginal bacteria to numerically dominate and, therefore, the definition of a "normal" vaginal microbiota will substantially differ between individual healthy women.

Improved detection of hepatitis C virus-positive blood donors and determination of infection status.

BACKGROUND: In low-risk populations, variability in the sensitivity of current serological tests for Hepatitis C virus (HCV) blood donor screening may lead to the presence of false-positive results. This contributes to the unnecessary loss of blood donor samples as well as to difficulty in accurate donor counselling. The present study determined the optimal cut-off value of a chemiluminescent immunoassay for identification of HCV-reactive blood donors. STUDY DESIGN AND METHODS: In a retrospective cross-sectional analysis of 193 973 blood donations, 578 samples that were positive for HCV antibody in a chemiluminescent immunoassay and/or RNA screening tests were identified. Blood from 379 of these positive samples was available for retesting by a second confirmatory HCV immunoassay followed by a receiver operating characteristic (ROC) curve analysis. Donors were also recalled for a new analysis. RESULTS: Only 71 (18.7%) blood samples remained HCV-positive upon retesting, while 233 (61.5%) now tested negative and 75 (19.8%) yielding indeterminate results. A signal to cutoff ratio ≥4.32 was determined as the best differential threshold between a positive and negative result, increasing the positive predictive value from 27.3% to 66.7%. CONCLUSION: Using a higher threshold for an HCV-positive blood sample enhances the chemiluminescent immunoassay screening test´s accuracy and helps to improve donor counselling and notification processes.

Transcriptomic Profiling of Rectus Abdominis Muscle in Women with Gestational Diabetes-Induced Myopathy: Characterization of Pathophysiology and Potential Muscle Biomarkers of Pregnancy-Specific Urinary Incontinence.

Gestational diabetes mellitus (GDM) is recognized as a "window of opportunity" for the future prediction of such complications as type 2 diabetes mellitus and pelvic floor muscle disorders, including urinary incontinence and genitourinary dysfunction. Translational studies have reported that pelvic floor muscle disorders are due to a GDM-induced-myopathy (GDiM) of the pelvic floor muscle and rectus abdominis muscle (RAM). We now describe the transcriptome profiling of the RAM obtained by Cesarean section from GDM and non-GDM women with and without pregnancy-specific urinary incontinence (PSUI). We identified 650 genes in total, and the differentially expressed genes were defined by comparing three control groups to the GDM with PSUI group (GDiM). Enrichment analysis showed that GDM with PSUI was associated with decreased gene expression related to muscle structure and muscle protein synthesis, the reduced ability of muscle fibers to ameliorate muscle damage, and the altered the maintenance and generation of energy through glycogenesis. Potential genetic muscle biomarkers were validated by RT-PCR, and their relationship to the pathophysiology of the disease was verified. These findings help elucidate the molecular mechanisms of GDiM and will promote the development of innovative interventions to prevent and treat complications such as post-GDM urinary incontinence.