Viremia and viruria of trichodysplasia spinulosa-associated polyomavirus before the development of clinical disease in a kidney transplant recipient.

Trichodysplasia spinulosa (TS) is a rare disease associated with immunosuppression and induced by a polyomavirus denominated Tricodisplasia Polyomavirus (TSPyV). We report a case of TS 6 months after kidney transplantation in a 65 years-old woman under immunosuppression therapy with prednisone, mycophenolate and tacrolimus. The patient developed follicular papules on the face with a thickening of the skin and alopecia of the eyebrows, leading to distortion of the face and a leonine appearance characteristic of the disease. The skin biopsy confirmed the clinical diagnosis and the presence of TSPyV DNA in the skin was detected. Staining for SV40 was positive. Immunosuppression was changed: mycophenolate was withdrawn, tacrolimus reduced and everolimus added. Intravenous cidofovir and later on leflunomide were added. Although the literature has reported clinical success with topical cidofovir, we were unable to use it because this drug is not available. There was an improvement of skin lesions and on cosmetic appearance. The patient had three rejections (one clinically diagnosed and two other biopsy proven), progressed with renal failure and graft loss. Retrospective analysis of stored urine and blood samples detected TSPyV DNA in some of those samples two months before the TS clinical development. This case highlights the TSPyV detection in blood and urine samples before the development of skin lesions.

Cytokine Levels and Human Herpesviruses in Saliva from Clinical Periodontal Healthy Subjects with Peri-Implantitis: A Case-Control Study.

This study evaluated the presence of cytokines (IL-1ß, IL-2, IL-4, IL-6, MCP-1, MIP-1α, MIP-1ß, and TNF-α) and human herpesvirus (HSV1, HSV2, EBV, CMV, VZV, HHV6, HHV7, and HHV8) in saliva samples taken from subjects with and without peri-implantitis. Forty-two periodontally healthy subjects were divided according to peri-implant condition: healthy and peri-implantitis groups. The clinical parameters as probing depth, clinical attachment level, plaque index, gingival bleeding, bleeding on probing, and suppuration were evaluated. For cytokine detection, multiplex analysis was performed, and PCR assay was used to identify herpesviruses. No significant differences were found in cytokine levels between groups (p > 0.05). The presence of herpesvirus was 1.97-fold higher in patients with peri-implantitis (odds ratio, CI 0.52-7.49). The association of the presence or absence of herpesvirus with the salivary markers was statistically significant for MIP-1ß (p = 0.0087) and TNF-α (p = 0.0437) only in the peri-implantitis group. The presence of herpesviruses in patients with peri-implantitis suggests the development of a proinflammatory environment, which is characterized by increased expression of MIP-1ß and TNF-α in saliva.

Oral shedding of human herpesviruses in renal transplant recipients.

OBJECTIVE: To describe the shedding profile of human herpesviruses in the saliva of renal transplant recipients. METHODS: This is a prospective case-control study of 50 renal transplant recipients and control group of 50 individuals (non-transplanted and immunocompetent). Mouthwash samples were collected via oral rinse and then submitted to screening for the presence of eight types of herpesviruses by using multiplex PCR. Fisher's exact, chi-square, and Student t tests were used for statistical analysis, and the significance level was set at 5%. RESULTS: The mean age of the study group was 49.42 ± 12.94 years, 28/50 (56%) were female, and the time elapsed after transplantation was 68.20 ± 67.19 months. Herpes simplex virus 1 (HSV-1) (P = 0.025) and Epstein-Barr virus (EBV) (P = 0.024) were, statistically, more excreted in the saliva of renal transplant recipients compared to control group. Gender (P = 1.00) and age (P = 0.563) did not influence the salivary shedding of herpesviruses in renal transplant recipients. Individuals who excreted varicella-zoster virus in saliva had a shorter mean time of transplantation (22:00 + 2.82 months) (P < 0.001). CONCLUSION: Renal transplant recipients excreted herpesviruses more often than controls, especially HSV-1 and EBV, with salivary shedding of herpesviruses being more frequent in patients with recent kidney transplantation. CLINICAL RELEVANCE: The present findings support other longitudinal studies evaluating the relationship between oral shedding of human herpesviruses and clinical presence of active infection and renal transplant failure.

Cross reactivity of commercial anti-dengue immunoassays in patients with acute Zika virus infection.

Several countries have local transmission of multiple arboviruses, in particular, dengue and Zika viruses, which have recently spread through many American countries. Cross reactivity among Flaviviruses is high and present a challenge for accurate identification of the infecting agent. Thus, we evaluated the level of cross reactivity of anti-dengue IgM/G Enzyme-Linked Immunosorbent Assays (ELISA) from three manufacturers against 122 serum samples obtained at two time-points from 61 patients with non-dengue confirmed Zika virus infection. All anti-dengue ELISAs cross reacted with serum from patients with acute Zika infection at some level and a worrisome number of seroconversion for dengue IgG and IgM was observed. These findings may impact the interpretation of currently standard criteria for dengue diagnosis in endemic regions.

Oral shedding of human herpesviruses in patients undergoing radiotherapy/chemotherapy treatment for head and neck squamous cell carcinoma.

OBJECTIVE: Opportunistic infections may affect the oral mucosa of patients undergoing radio/chemotherapy through exacerbation of oral mucositis. The aim of this study is to evaluate the oral shedding of all eight human herpesviruses and its possible association with oral mucositis. MATERIALS AND METHODS: In this prospective cohort study, we analyzed oral rinse samples, collected weekly, from 20 patients during radiotherapy treatment. Serologic status to HSV1 and HSV2, EBV, CMV, and VZV in three different periods was performed by ELISA assay. PCR and enzymatic digestion was performed to detect HSV1, HSV2, EBV, CMV, VZV, HHV6, HHV7, and HHV8. Oral mucositis was evaluated according to the WHO criteria. RESULTS: Oral shedding of EBV, HHV6, and HHV7 was observed in all weeks of radiotherapy. Considering the episodes of shedding, the highest frequency was found in patients with EBV excretion (55.0%). No virus reactivation was observed by serological analysis. EBV oral shedding frequency was significantly higher than that of other viruses and showing a positive correlation with oral mucositis grade ≥2. CONCLUSIONS: There was a positive correlation between EBV oral shedding and oral mucositis grade ≥2, particularly after 3 weeks of radiotherapy, a period in which the severity of mucositis was statistically higher. These findings allow us to infer that the local inflammatory environment in mucositis grade ≥2 is more favorable for EBV replication. CLINICAL RELEVANCE: Mucositis is a frequent and important side effect of radio/chemotherapy treatment. Understanding the possible participation of viruses in the mechanism of this condition is important to develop strategies for treatment and prevention.

Occurrence, genotypic characterization, and patterns of shedding of human polyomavirus JCPyV and BKPyV in urine samples of healthy individuals in São Paulo, Brazil.

The objective of this study was to evaluate the prevalence, genotypic characterization, and determination of the patterns of shedding of human polyomavirus JC (JCPyV) and BK (BKPyV) in consecutive urine samples collected from healthy adults. Urine samples collected monthly over a 6 month period were screened by polymerase chain reaction (PCR) with two sets of primers complementary to the VP1 protein region specific for the JCPyV or BKPyV genome. The viral load of JCPyV and BKPyV in positive samples was determined by quantitative real time PCR. Seventy-one healthy individuals (ages between 18 and 65) were included in the study. Polyomavirus DNA urinary shedding was identified in 44 (62%) of the 71 individuals evaluated: BKPyV only in 16 (22.5%); JCPyV only in 19 (26.7%); and both in 9 (12.7%). Among the 28 individuals shedding JCPyV, the shedding was nearly continuous in 13 (46.4%) and sporadic in 15 (53.6%), whereas all BKPyV shedding was sporadic. A total of 45 (19 BKPyV and 26 JCPyV) strains were identified. Of the BKPyV strains, individuals were observed that excreted all genotypes except genotype 3 and the JCPyV strains, excretion of 5 different genotypes. Evaluating the age of individuals who excrete JCPyV and BKPyV, mostly are young adults, with a slight increase with increasing age and observing the viral load can not draw any parallel between the increase or decrease of age or excreted genotype as there was a wide variation both in the excretion of BKPyV and JCPyV. The high occurrence of isolated or simultaneous urinary shedding of JCPyV and BKPyV in healthy individuals merits further study.

Prevalence of human herpesvirus 8 infection in people living with HIV/AIDS in Pernambuco, Brazil.

This cross-sectional study aimed to estimate the seroprevalence and risk factors for Human herpesvirus 8 (HHV-8) infection among people living with HIV/AIDS in Recife, Pernambuco, Brazil. A total of 500 individuals were tested for antibodies against HHV-8 using the whole-virus ELISA. The prevalence of anti-HHV-8 was 28.6% and the frequency among 140 men who have sex with men (MSM) was 38.6%. In the univariate model, there were significant associations with male gender, detectable HIV load, travel abroad, bissexual, and homossexual orientation. The first HHV-8 seroepidemiologic study, in northeast Brazil, documents a highly prevalent HHV-8 infection among MSM living with HIV/AIDS. J. Med. Virol. 88:2016-2020, 2016. © 2016 Wiley Periodicals, Inc.

The differential clinical impact of human coronavirus species in children with cystic fibrosis.

We investigated the clinical impact of human coronaviruses (HCoV) OC43, 229E, HKU1 and NL63 in pediatric patients with cystic fibrosis (CF) during routine and exacerbation visits. A total of 408 nasopharyngeal aspirate samples were obtained from 103 patients over a 1-year period. Samples positive for HCoV were submitted for nucleotide sequencing to determine the species. Nineteen samples (4.65%) were positive for HCoV, of which 8 were positive for NL63, 6 for OC43, 4 for HKU1, and 1 for 229E. Identification of HCoV was not associated with an increased rate of respiratory exacerbations, but NL63-positive patients had higher exacerbation rates than patients who were positive for other HCoV species.

Rubella vaccination of unknowingly pregnant women: the São Paulo experience, 2001.

BACKGROUND: Rubella vaccination is contraindicated during pregnancy. During mass immunization of women of childbearing age against rubella, women unknowingly pregnant may be vaccinated. To evaluate the effects of rubella vaccination during pregnancy, the Brazilian state of São Paulo conducted a follow-up study of pregnant women vaccinated during a rubella campaign in 2001. METHODS: Women vaccinated during pregnancy were reported to a national surveillance system. In the state of São Paulo, follow-up of vaccinated women included household interviews. Serum samples from vaccinated women were tested for antirubella antibodies to classify susceptibility to rubella infection. Children born to susceptible mothers were tested for evidence of congenital rubella infection and evaluated for signs of congenital rubella syndrome. RESULTS: The São Paulo State Health Department received 6473 notifications of women vaccinated during pregnancy. Serology performed for 5580 women identified 811 (15%) that were previously susceptible. Incidence of spontaneous abortion or stillbirth among previously susceptible vaccinated women was similar to women with prior immunity. Twenty-seven (4.7%) of 580 newborns tested had evidence of congenital rubella infection; none had congenital rubella syndrome. CONCLUSIONS: Mass rubella vaccination of women of childbearing age was not associated with adverse birth outcomes or congenital rubella syndrome among children born to women vaccinated during pregnancy.

Human polyomaviruses JC and BK in the urine of Brazilian children and adolescents vertically infected by HIV.

The aim of this study was to characterize the urinary excretion of the BK (BKV) and JC (JCV) human polyomaviruses in a cohort of human immunodeficiency virus (HIV)-infected children and adolescents. One hundred and fifty-six patients were enrolled: Group I included 116 HIV-infected children and adolescents [median age = 11.4 years (y); range 1-22 y]; Group II included 40 non-HIV-infected healthy controls (median age = 11.37 y; range 7-16 y). Single urine samples from both groups were screened for the presence of JCV and BKV DNA by polymerase chain reaction at enrolment. The overall rate of JCV and BKV urinary excretion was found to be 24.4% and 40.4%, respectively (n = 156). Group I had urinary excretion of JCV and BKV in 27.6% and 54.3% of subjects, respectively. In contrast, Group II showed positive results for JCV in 17.5% of subjects and for BKV in 12.5% of subjects (p Pearson JCV = 0.20; p Pearson BKV < 0.0001). In Group I, there was no association between JCV/BKV shedding and age, gender or CD4 values. Patients with an HIV viral load < 50 copies/mL had a lower excretion of BKV (p < 0.001) and a trend of lower JCV excretion (p = 0.07). One patient in Group I (1/116, 0.9%) showed clinical and radiological features consistent with progressive multifocal leukoencephalopathy, suggesting that children with HIV/polyomavirus coinfection should be kept under surveillance.

SARS-CoV-2 reinfection caused by the P.1 lineage in Araraquara city, Sao Paulo State, Brazil.

Reinfection by the severe acute respiratory syndrome coronavirus type 2 (SARS-COV-2) has been reported in many countries, suggesting that the virus may continue to circulate among humans despite the possibility of local herd immunity due to massive previous infections. The emergence of variants of concern (VOC) that are more transmissible than the previous circulating ones has raised particular concerns on the vaccines effectiveness and reinfection rates. The P.1 lineage was first identified in December 2020 in Manaus city and is now globally spread. We report the first case of reinfection of SARS-CoV-2 caused by the P.1 variant outside of Manaus. The potential of these new variants to escape naturally and vaccine- induced immunity highlights the need for a global vigilance.

Evaluation of serological cross-reactivity between yellow fever and other flaviviruses.

OBJECTIVES: This study was performed to determine whether neutralizing antibodies against yellow fever virus (YFV) generated by YFV vaccine could interfere in the specificity of dengue virus (DENV) and Zika virus (ZIKV) IgG ELISA tests. METHODS: Seventy-nine pairs of serum samples (pre- and post-vaccination), collected during the years 1997-1998 from children with no history of yellow fever disease who had been vaccinated against YFV, were tested. The seroconversion post-vaccination was evaluated through plaque reduction neutralization test (PRNT), and four different commercial ELISA kits were used for the detection of DENV and ZIKV IgG antibodies. RESULTS: A cross-reactivity rate of 3.9% with DENV IgG antibodies was found only with the Dengue Virus IgG Dx Select kit (Focus Diagnostics). CONCLUSIONS: As several countries have local transmission of multiple arboviruses, the absence of cross-reactivity or minimum cross-reactivity of YFV neutralizing antibodies with DENV and ZIKV antigens is a relevant finding, since the interpretation of sero-epidemiological investigations would be seriously impacted in many regions where YFV vaccination is mandatory.

BK virus salivary shedding and viremia in renal transplant recipients.

OBJECTIVES: This study aimed to verify the presence of polyomavirus BK (BKPyV) in the saliva of kidney transplant recipients and to correlate it with blood viremia. MATERIAL AND METHODS: We have conducted a cross-sectional study with a sample involving 126 renal transplant recipients. 126 samples of saliva and 52 samples of blood were collected from these patients. Detection and quantification of BKPyV were performed using a real-time PCR. To compare the presence of BKPyV in blood and saliva, the binomial proportion test was used. To verify associations between salivary shedding BKPyV and post-transplant periods (in months), the Mann-Whitney test was used. Spearman's correlation was used to correlate the viral load in the saliva with blood of kidney transplant recipients. RESULTS: The mean age of the study group was 51.11±12.45 years old, and 69 participants (54.8%) were female, with a mean post-transplantation time of 4.80±6.04 months. BKPyV was quantified in several samples of saliva and blood, with medians of 1,108 cp/mL and 1,255 cp/mL, respectively. Only 16/52 (30.8%) participants presented BKPyV in blood, and 59/126 (46.8%) excreted the virus in saliva (p=0.004). BKPyV shedding was found in patients at a shorter post-transplantation period (3.86±5.25, p=0.100). A weak correlation was observed between viral quantification in saliva and blood (Spearman's correlation coefficient=0.193). CONCLUSION: The results of this study suggested that, although saliva excretes more BKPyV than blood, there is no reliable correlation between salivary shedding and blood viremia, showing two independent compartments of viral replication.

Comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus in nasopharyngeal aspirates from infants.

A total of 316 samples of nasopharyngeal aspirate from infants up to two years of age with acute respiratory-tract illnesses were processed for detection of respiratory syncytial virus (RSV) using three different techniques: viral isolation, direct immunofluorescence, and PCR. Of the samples, 36 (11.4%) were positive for RSV, considering the three techniques. PCR was the most sensitive technique, providing positive findings in 35/316 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). A sample was positive by immunofluorescence and negative by PCR, and 11 (31.4%) were positive only by RT-PCR. We conclude that RT-PCR is more sensitive than IF and viral isolation to detect RSV in nasopharyngeal aspirate specimens in newborn and infants.

Oral shedding of human herpesviruses in patients undergoing radiotherapy/chemotherapy for head and neck squamous cell carcinoma is not affected by xerostomia.

Background: Xerostomia is a very relevant and frequent complication of radiotherapy, causing the irradiated oral mucosa to be affected by bacterial, fungal and viral infections. Objective: The objective of this study was to evaluate a possible relationship between oral shedding of human herpesviruses and xerostomia in patients with squamous cell carcinoma of head and neck submitted to radio/chemotherapy. Methods: In this study, oral rinse samples were collected weekly from 20 patients during radiotherapy. The samples were submitted to PCR and enzymatic digestion for detection of human herpesviruses. Xerostomia was evaluated according to the Seminars in Radiation Oncology criteria. Results: There was a higher frequency of grade 1 xerostomia (51.4%), observed first in the 1st week of radiotherapy. In the 4th week of radiotherapy, all patients presented some degree of xerostomia. Analysis of herpesviruses showed oral shedding of EBV, HHV-6 and HHV-7 in all weeks. Considering all the periods, the highest frequency was in patients with EBV excretion (55.0%), which was significantly higher than that of other viruses. Conclusion: We observed that oral shedding of herpesviruses was not affected by xerostomia as there was a progression in their excretion, even with the evolution of xerostomia. This suggested that there is a local replication in the oral cavity that is not completely dependent of salivary excretion.

Sensitivity and specificity of three ELISA-based assays for discriminating primary from secondary acute dengue virus infection.

BACKGROUND: Discrimination between primary and secondary dengue virus infection traditionally has been performed using the hemagglutination inhibition (HI) test. However, this test has practical limitations and disadvantages. OBJECTIVE: To evaluate the ability of three ELISA-based methods (IgG avidity test, IgM/IgG ratio and IgG titer) to discriminate primary from secondary dengue infection. STUDY DESIGN: Serum samples from convalescent-phase patients with confirmed acute, primary (n=46) or secondary (n=33) dengue virus infection were tested using three ELISA-based methods. A ROC curve was employed to establish the cut-off points and to evaluate the ability of the three methods to distinguish between acute, primary and secondary dengue virus infection. RESULTS: All three assays exhibited sensitivity and negative predictive values of 100% for defining secondary infection. The specificity and positive predictive values were respectively 97.8% and 93.7% for the IgG avidity test, 95.7% and 88.2% for the IgM/IgG ratio assays, and 97.8% and 93.7% for the IgG titer assay. CONCLUSION: All three ELISA-based assays proved reliable tools for discriminating between acute, primary and secondary dengue virus infection when using serum samples from convalescent-phase patients.

CCR5 genotypes and progression to HIV disease in perinatally infected children.

The CCR5 molecule, a chemokine receptor, is the most important co-receptor for macrophage-tropic HIV-1. A 32-bp deletion in the gene encoding CCR5 (CCR5-del32) confers nearly complete resistance to HIV-1 infection in homozygotes, and slows the rate of progression to AIDS in heterozygous adults. The aim of this study was to describe the CCR5 genotypes and the characteristics of HIV disease progression in perinatally infected children. From a total of 51 children analyzed for the CCR5-del32 mutation, 18 (35%) were considered to be rapid progressors, 28 (55%) were moderate progressors and 5 (10%) were slow progressors. A portion of the CCR5 gene was amplified by PCR from genomic DNA followed by agarose gel electrophoresis. Forty-nine children (96%) carried the homozygous wild type genotype for CCR5 while 2 (4%) carried the heterozygous wt/del32 genotype. In the population studied, the CCR5 genotype was unable to account for the differences in pattern of the disease progression among the three groups (rapid, moderate and slow progressors), and the allele frequency of CCR5-del32 was too low to allow statistical comparisons with adequate resolving power. Studies on larger populations may help to further elucidate the role of this allele and other host factors in the regulation of HIV-1 pathogenesis in children.

Withdrawal of maintenance therapy for cytomegalovirus retinitis in AIDS patients exhibiting immunological response to HAART.

BACKGROUND: Before the introduction of highly active antiretroviral therapy (HAART), CMV retinitis was a common complication in patients with advanced HIV disease and the therapy was well established; it consisted of an induction phase to control the infection with ganciclovir, followed by a lifelong maintenance phase to avoid or delay relapses. METHODS: To determine the safety of CMV maintenance therapy withdrawal in patients with immune recovery after HAART, 35 patients with treated CMV retinitis, on maintenance therapy, with CD4+ cell count greater than 100 cells/mm(3) for at least three months, but almost all patients presented these values for more than six months and viral load < 30000 copies/mL, were prospectively evaluated for the recurrence of CMV disease. Maintenance therapy was withdrawal at inclusion, and patients were monitored for at least 48 weeks by clinical and ophthalmologic evaluations, and by determination of CMV viremia markers (antigenemia-pp65), CD4+/CD8+ counts and plasma HIV RNA levels. Lymphoproliferative assays were performed on 26/35 patients. RESULTS: From 35 patients included, only one had confirmed reactivation of CMV retinitis, at day 120 of follow-up. No patient returned positive antigenemia tests. No correlation between lymphoproliferative assays and CD4+ counts was observed. CONCLUSION: CMV retinitis maintenance therapy discontinuation is safe for those patients with quantitative immune recovery after HAART.

Reliability of information on varicella history in preschool children.

OBJECTIVE: To verify how reliable is the information provided by parents about the history of varicella in their children. METHODS: 204 parents of previously healthy children attending two municipal day-care centers of São Paulo city were interviewed between August 2003 and September 2005. A standardized form was filled out with information regarding age, sex, history of varicella and other diseases, drug use and antecedent of immunization, After medical history, physical examination and checking of immunization records, 5 ml of blood were collected for ELISA (in house) varicella test. Exclusion criteria were: age less than 1 year or more than 60 months, previous immunization against chickenpox, presence of co-morbidities or recent use of immunosuppressive drugs. Data were filed in a data bank using the Excel 2003 Microsoft Office Program and stored in a PC computer. The exact Fisher test was employed to calculate sensibility, specificity, positive and negative predictive values of history of varicella informed by children's parents. RESULTS: The age of the children varied from 12 to 54 months (median, 26 months; 49 (24%) children had positive history of varicella, 155 (76%) a negative or doubtful history. The predictive positive and negative values of the information were 90% and 93%, respectively (p = 0.0001). CONCLUSIONS: The degree of reliability of information about history of varicella informed by parents of children attending day care centers was high and useful to establish recommendations on varicella blocking immunization in day-care centers.

Serum from dengue virus-infected patients with and without plasma leakage differentially affects endothelial cells barrier function in vitro.

BACKGROUND: Although most of cases of dengue infections are asymptomatic or mild symptomatic some individuals present warning signs progressing to severe dengue in which plasma leakage is a hallmark. METHODOLOGY/PRINCIPAL FINDINGS: The present study used Electric Cell-substrate Impedance Sensing (ECIS®) which allows for electrical monitoring of cellular barrier function measuring changes in Transendothelial Electric Resistance (TEER) to investigate the parameters associated with dengue induced leakage. Three groups of individuals were tested: dengue-positives with plasma leakage (leakage), dengue-positives without plasma leakage (no leakage), and dengue-negatives (control). Data show that TEER values of human umbilical vein endothelial cells (HUVECs) was significantly lower after incubation with serum from subjects of the leakage group in comparison to the no leakage or control groups. The serum levels of CXCL1, EGF, eotaxin, IFN-γ, sCD40L, and platelets were significantly decreased in the leakage group, while IL-10, IL-6, and IP-10 levels were significantly increased. We also found a strong correlation between TEER values and augmented levels of IP-10, GM-CSF, IL-1α, and IL-8, as well as decreased levels of CXCL1 and platelets. CONCLUSIONS/SIGNIFICANCE: The present work shows that the magnitude of the immune response contributes to the adverse plasma leakage outcomes in patients and that serum components are important mediators of changes in endothelial homeostasis during dengue infections. In particular, the increased levels of IP-10 and the decreased levels of CXCL1 and platelets seem to play a significant role in the disruption of vascular endothelium associated with leakage outcomes after DENV infection. These findings may have important implications for both diagnostic and therapeutic approaches to predict and mitigate vascular permeabilization in those experiencing the most severe clinical disease outcomes after dengue infection.