Serum soluble mediators as prognostic biomarkers for morbidity, disease outcome, and late-relapsing hepatitis in yellow fever patients.

This study described a soluble mediator storm in acute Yellow Fever/YF infection along the kinetics timeline towards convalescent disease. The analyses of the YF Viral RNAnemia, chemokines, cytokines, and growth factors were performed in YF patients at acute/(D1-15) and convalescent/(D16-315) phases. Patients with acute YF infection displayed a trimodal viremia profile spreading along D3, D6, and D8-14. A massive storm of mediators was observed in acute YF. Higher levels of mediators were observed in YF with higher morbidity scores, patients under intensive care, and those progressing to death than in YF patients who progress to late-relapsing hepatitis/L-Hep. A unimodal peak of biomarkers around D4-6 with a progressive decrease towards D181-315 was observed in non-L-Hep patients, while a bimodal pattern with a second peak around D61-90 was associated with L-Hep. This study provided a comprehensive landscape of evidence that distinct immune responses drive pathogenesis, disease progression, and L-Hep in YF patients.

Evaluation of humoral immune response after yellow fever infection: an observational study on patients from the 2017-2018 sylvatic outbreak in Brazil.

Between 2016 and 2018, Brazil experienced major sylvatic yellow fever (YF) outbreaks that caused hundreds of casualties, with Minas Gerais (MG) being the most affected state. These outbreaks provided a unique opportunity to assess the immune response triggered by the wild-type (WT) yellow fever virus (YFV) in humans. The plaque reduction neutralization test (PRNT) is currently the standard method to assess the humoral immune response to YFV by measuring neutralizing antibodies (nAbs). The present study aimed to evaluate the humoral immune response of patients from the 2017-2018 sylvatic YF outbreak in MG with different disease outcomes by using PRNTs with a WT YFV strain, isolated from the 2017-2018 outbreak, and a vaccine YFV strain. Samples from naturally infected YF patients were tested, in comparison with healthy vaccinees. Results showed that both groups presented different levels of nAb against the WT and vaccine strains, and the levels of neutralization against the strains varied homotypically and heterotypically. Results based on the geometric mean titers (GMTs) suggest that the humoral immune response after a natural infection of YFV can reach higher levels than that induced by vaccination (GMT of patients against WT YFV compared to GMT of vaccinees, P < 0.0001). These findings suggest that the humoral immune responses triggered by the vaccine and WT strains of YFV are different, possibly due to genetic and antigenic differences between these viruses. Therefore, current means of assessing the immune response in naturally infected YF individuals and immunological surveillance methods in areas with intense viral circulation may need to be updated.IMPORTANCEYellow fever is a deadly febrile disease caused by the YFV. Despite the existence of effective vaccines, this disease still represents a public health concern worldwide. Much is known about the immune response against the vaccine strains of the YFV, but recent studies have shown that it differs from that induced by WT strains. The extent of this difference and the mechanisms behind it are still unclear. Thus, studies aimed to better understand the immune response against this virus are relevant and necessary. The present study evaluated levels of neutralizing antibodies of yellow fever patients from recent outbreaks in Brazil, in comparison with healthy vaccinees, using plaque reduction neutralization tests with WT and vaccine YFV strains. Results showed that the humoral immune response in naturally infected patients was higher than that induced by vaccination, thus providing new insights into the immune response triggered against these viruses.

Remodeling of immunological biomarkers in patients with chronic hepatitis C treated with direct-acting antiviral therapy.

The HCV treatment with DAAs has offered a unique opportunity to analyze the changes in the immune system caused by the rapid inhibition of viral replication. We sought to analyze the kinetics profiles of serum biomarkers (LuminexTM) in fifty patients with chronic hepatitis C enrolled in a longitudinal investigation carried out before (baseline), during (W2-4 and W8-12 weeks) and post-treatment (W12-24 weeks) with sofosbuvir plus daclatasvir or simeprevir. The results demonstrated a clear biomarker overproduction in HCV patients at baseline. The kinetics timeline of baseline fold changes upon DAAs treatment revealed an early decline of CXCL8, CCL4, IL-6, IL-15, IL-17, IL-9, GM-CSF and IL-7 at W8-12 and a late shift towards lower levels of CCL3, CCL2, CCL5, IL1ß, TNF-α, IL-12, IFN-γ, IL1-Ra, IL-4, IL-10, IL-13, PDGF, VEGF, G-CSF at W12-24. Our data demonstrated that HCV treatment with DAAs resulted in a clear change of the serum biomarker overproduction, hallmark of untreated HCV patients. High ALT (>69U/L), low platelet (≤150,000/mm3) and cirrhosis status at baseline were factors related to delayed immune response shift, as well as, in the kinetics of baseline fold changes in serum biomarkers. These findings added novel evidences for the immunological restoration process triggered by DAAs.

Acute-Phase Levels of CXCL8 as Risk Factor for Chronic Arthralgia Following Chikungunya Virus Infection.

The immunopathogenesis of chikungunya virus (CHIKV) infection and the role of acute-phase immune response on joint pain persistence is not fully understood. We investigated the profile of serum chemokine and cytokine in CHIKV-infected patients with acute disease, compared the levels of these biomarkers to those of patients with other acute febrile diseases (OAFD) and healthy controls (HC), and evaluated their role as predictors of chronic arthralgia development. Chemokines and cytokines were measured by flow Cytometric Bead Array. Patients with CHIKV infection were further categorized according to duration of arthralgia (≤ 3 months vs >3 months), presence of anti-CHIKV IgM at acute-phase sample, and number of days of symptoms at sample collection (1 vs 2-3 vs ≥4). Patients with acute CHIKV infection had significantly higher levels of CXCL8, CCL2, CXCL9, CCL5, CXCL10, IL-1ß, IL-6, IL-12, and IL-10 as compared to HC. CCL2, CCL5, and CXCL10 levels were also significantly higher in patients with CHIKV infection compared to patients with OAFD. Patients whose arthralgia lasted > 3 months had increased CXCL8 levels compared to patients whose arthralgia did not (p<0.05). Multivariable analyses further indicated that high levels of CXCL8 and female sex were associated with arthralgia lasting >3 months. Patients with chikungunya and OAFD had similar cytokine kinetics for IL-1ß, IL-12, TNF, IFN-γ, IL-2, and IL-4, although the levels were lower for CHIKV patients. This study suggests that chemokines may have an important role in the immunopathogenesis of chronic chikungunya-related arthralgia.

CCL3, CCL5, IL-15, IL-1Ra and VEGF compose a reliable algorithm to discriminate classes of adverse events following 17DD-YF primary vaccination according to cause-specific definitions.

In the present study, a range of serum biomarkers were quantified in suspected cases of adverse events following YF immunization (YEL-AEFI) to propose a reliable laboratorial algorithm to discriminate confirmed YEL-AEFI ("A1" class) from cases with other illnesses ("C" class). Our findings demonstrated that increased levels of CXCL8, CCL2, CXCL10, IL-1ß, IL-6 and TNF-α were observed in YEL-AEFI ("A1" and "C" classes) as compared to primary vaccines without YEL-AEFI [PV(day 3-28)] and reference range (RR) controls. Notably, increased levels of CCL3, CCL4, CCL2, CCL5, IL-1ß, IL-15, IL-1Ra and G-CSF were found in "A1" as compared to "C" class. Venn diagrams analysis allowed the pre-selection of biomarkers for further analysis of performance indices. Data demonstrated that CCL3, CCL5, IL-15 and IL-1Ra presented high global accuracy (AUC = 1.00) to discriminate "A1" from "C". Decision tree was proposed with a reliable algorithm to discriminate YEL-AEFI cases according to cause-specific definitions with outstanding overall accuracy (91%). CCL3, CCL5, IL-15 and IL-1Ra appears as root attributes to identify "A1" followed by VEGF as branch nodes to discriminate Wild Type YFV infection ("C(WT-YFV)") from cases with other illnesses ("C*"). Together, these results demonstrated the applicability of serum biomarker measurements as putative parameters towards the establishment of accurate laboratorial tools for complementary differential diagnosis of YEL-AEFI cases.

Late-Relapsing Hepatitis after Yellow Fever.

One patient presented hyporexia, asthenia, adynamia, and jaundice two months after acute yellow fever (YF) onset; plus laboratory tests indicating hepatic cytolysis and a rebound of alanine and aspartate transaminases, and total and direct bilirubin levels. Laboratory tests discarded autoimmune hepatitis, inflammatory or metabolic liver disease, and new infections caused by hepatotropic agents. Anti-YFV IgM, IgG and neutralizing antibodies were detected in different times, but no viremia. A liver biopsy was collected three months after YF onset and tested positive for YFV antigens and wild-type YFV-RNA (364 RNA-copies/gram/liver). Transaminases and bilirubin levels remained elevated for five months, and the arresting of symptoms persisted for six months after the acute YF onset. Several serum chemokines, cytokines, and growth factors were measured. A similar immune response profile was observed in the earlier phases of the disease, followed by more pronounced changes in the later stages, when transaminases levels returned to normal. The results indicated viral persistence in the liver and continual liver cell damage three months after YF onset and reinforced the need for extended follow-ups of YF patients. Further studies to investigate the role of possible viral persistence and the immune response causing relapsing hepatitis following YF are also necessary.

Molecular variants of Leishmania (Viannia) braziliensis trigger distinct patterns of cytokines and chemokines expression in golden hamster.

Cutaneous leishmaniasis (CL) mainly caused by Leishmania braziliensis is a chronic inflammatory disease widely spread in Brazil. Genetic variant strains of this parasite have been associated with atypical clinical manifestations of CL in an endemic area in Brazil. Furthermore, these strains have presented distinct biological behaviors in golden hamster, suggesting differential activation of the immune response. In the present study we proposed to evaluate the localized immune response in golden hamsters infected with known molecular variant strains of L. braziliensis, in distinct time points post-infection (PI). Detailed analyses of the mRNA expression of cytokines and chemokines in hamster-skin lesions were performed. Heat map matrix and hierarchical cluster analysis were carried out to segregate the strains due to mRNA expression. Distinct patterns of immune response were found in both time points, more evident in the recent-phase disease (30 days-PI). At this time point, the genetic variant strains expressed high levels of tnfα, il12 and tgfß whilst the non-variant strain expressed ifnγ, il6, il4, il10, il13 and ccl17. The hierarchical clustering highlights this distinct pattern in which all genetic variant strain was grouped in the cluster I and the non-variant strain grouped into the cluster II. At late-phase disease (60 days-PI) all isolates expressed high levels of il4 and il10. The non-variant strain shown a significant reduced expression of ifnγ, il6, ccl17, and ccl22 whilst distinct patterns were observed for the genetic variant strains. For the first time, a large panel of cytokines and chemokines mRNA-expression was analyzed in experimental trials using golden hamsters as animal model and genetic variant strains of L. braziliensis. Our findings suggest that genetic variant strains of L. braziliensis are able to trigger differential gene expression of cytokines and chemokines in the skin lesion from infected hamsters. The parasite intrinsic ability to activate distinct pathways in the host-parasite interaction may be associated to the large spectrum of clinical manifestation observed in CL-patients.

Duration of Humoral and Cellular Immunity 8 Years After Administration of Reduced Doses of the 17DD-Yellow Fever Vaccine.

The present study aims to determine whether 17DD-YF-specific humoral and cellular immunological memory is maintained 8-years after primary vaccination with subdoses (10,447IU;3,013IU;587IU;158IU;31IU). For this purpose, this follow-up study was carried out in a subset of volunteers (n = 98) originally enrolled in the dose-response study in 2009 and 46 non-vaccinated controls. Our results demonstrated that vaccinees, who had seroconverted following primary vaccination and had not been revaccinated, present similar neutralizing antibodies levels and YF-specific cellular memory, particularly CMCD4 and EMCD8 as compared to the reference full dose (27,476IU). Although, PRNT seropositivity rates were similar across subgroups (94, 82, 83, 94, 80, and 91%, correspondingly), only doses above 587IU elicited similar iterative proportion of seropositivity rates, calculated as a progressive decrease on seropositivity rates along time (89, 80, 80, and 91%, respectively) as compared to 158IU and 31IU (68 and 46%, respectively). Noteworthy were the strong positive correlations ("EMCD4,EMCD8" and "TNFCD8,IFNCD8") observed in most subdoses, except for 31IU. Major similarities underscored the preserved antibody titers and the outstanding levels of EMCD8, relevant correlates of protection for YF-specific immunity. These findings provide evidences to support the regular use of dose sparing strategy for YF vaccine in adults.

Corrigendum: Duration of Humoral and Cellular Immunity 8 Years After Administration of Reduced Doses of the 17DD-Yellow Fever Vaccine.

[This corrects the article DOI: 10.3389/fimmu.2019.01211.].

Sexually Transmitted Infections Detected by Multiplex Real Time PCR in Asymptomatic Women and Association with Cervical Intraepithelial Neoplasia.

OBJECTIVE: To determine the frequency of sexually transmitted infections (STIs) in asymptomatic women and the association of STIs with cervical intraepithelial neoplasia (CIN). METHODS: A cross-sectional study was performed, enrolling women examined in a general gynecology clinic and in a colposcopy referral center from October 2014 to October 2015. The colposcopy group consisted of 71 women, and the general gynecology group consisted of 55 women. Cervical samples were collected for cervical cytology and a multiplex real-time polymerase chain reaction (PCR) was developed to detect human papillomavirus (HPV) and the STIs caused by the following microorganisms: Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Neisseria gonorrhoeae. A multivariate analysis was performed by logistic regression, considering the significance level of 0.05. RESULTS: The general frequency of STIs was: 46.8% (HPV); 27.8% (C. trachomatis); 28.6% (M. genitalium); 0.8% (M. hominis); 4.8% (U. urealyticum); and 4.8% (N. gonorrhoeae). The significant risk factors for CIN were: HPV infection (odds ratio [OR] = 2.53; p = 0.024); C. trachomatis (OR = 3.04; p = 0.009); M. genitalium (OR = 2.37; p = 0.04); and HPV and C. trachomatis coinfection (OR = 3.11; p = 0.023). After the multivariate analysis, a significant association was found between HPV and CIN (OR = 2.48; 95% confidence interval [95%CI]: 1.04-5.92; p = 0.04); and between C. trachomatis and CIN (OR = 2.69; 95%CI: 1.11-6.53; p = 0.028). CONCLUSION: The frequency of STIs was high in asymptomatic patients. Infections by HPV and C. trachomatis were independently associated with the presence of CIN. The high frequency of STIs in asymptomatic women suggests the need for routine screening of these infections.

OBJETIVO: Determinar a frequência de infecções sexualmente transmissíveis (ISTs) em mulheres assintomáticas e a associação destas infecções com a neoplasia intraepitelial cervical (NIC). MéTODOS: Foi realizado um estudo transversal recrutando mulheres atendidas em uma clínica ginecológica geral e em um centro de referência para colposcopia, de outubro de 2014 a outubro de 2015. O grupo de colposcopia consistiu de 71 mulheres, e o grupo de ginecologia geral consistiu de 55 mulheres. Amostras cervicais foram coletadas para citologia cervical e uma reação em cadeia de polimerase (RCP) multiplex em tempo real para detecção do vírus do papiloma humano (HPV) e das ISTs provocadas pelos seguintes micro-organismos: Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum e Neisseria gonorrhoeae. Foi realizada uma análise multivariada por regressão logística, considerando-se o nível de significância de 0,05. RESULTADOS: A frequência geral de ISTs foi: 46,8% (HPV); 27,8% (C. trachomatis); 28,6% (M. genitalium); 0,8% (M. hominis); 4,8% (U. urealyticum); e 4,8% (N. gonorrhoeae). Os fatores de risco significantes para NIC foram: infecção pelo HPV (razão de probabilidades [RP] = 2,53; p = 0,024); C. trachomatis (RP = 3,04; p = 0,009); M. genitalium (RP = 2,37; p = 0,04); e coinfecção por HPV e C. trachomatis (RP = 3,11; p = 0,023). Após a análise multivariada, foi encontrada uma associação significante entre HPV e NIC (RP = 2.48; intervalo de confiança de 95% [IC95%]: 1,04­5,92; p = 0,04) e entre C. trachomatis e NIC (RP = 2,69; IC95%: 1,11­6,53; p = 0,028). CONCLUSõES: A frequência de ISTs foi alta em mulheres assintomáticas. Infecções por HPV e C. trachomatis foram independentemente associadas com a presença de NIC. A alta frequência de ISTs em mulheres assintomáticas sugere a necessidade de rastreamento rotineiro dessas infecções.

Sexually transmitted infections detected by multiplex real time PCR in asymptomatic women and association with cervical Intraepithelial neoplasia / Infecções sexualmente transmissíveis detectadas por PCR multiplex em tempo real em mulheres assintomáticas e associação com neoplasia intraepitelial cervical

Abstract Objective To determine the frequency of sexually transmitted infections (STIs) in asymptomatic women and the association of STIs with cervical intraepithelial neoplasia (CIN). Methods A cross-sectional studywas performed, enrollingwomen examined in a general gynecology clinic and in a colposcopy referral center fromOctober 2014 to October 2015. The colposcopy groupconsisted of 71women, and the general gynecologygroupconsisted of 55 women. Cervical samples were collected for cervical cytology and a multiplex realtime polymerase chain reaction (PCR) was developed to detect human papillomavirus (HPV) and the STIs caused by the following microorganisms: Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Neisseria gonorrhoeae. A multivariate analysis was performed by logistic regression, considering the significance level of 0.05. Results The general frequency of STIs was: 46.8% (HPV); 27.8% (C. trachomatis); 28.6% (M. genitalium); 0.8% (M. hominis); 4.8% (U. urealyticum); and 4.8% (N. gonorrhoeae). The significant risk factors for CIN were: HPV infection (odds ratio [OR] = 2.53; p = 0.024); C. trachomatis (OR = 3.04; p = 0.009); M. genitalium (OR = 2.37; p = 0.04); and HPV and C. trachomatis coinfection (OR = 3.11; p = 0.023). After the multivariate analysis, a significant associationwas found betweenHPVand CIN(OR = 2.48; 95% confidence interval [95%CI]: 1.04-5.92; p = 0.04); and between C. trachomatis and CIN (OR = 2.69; 95%CI: 1.11-6.53; p = 0.028). Conclusion The frequency of STIs was high in asymptomatic patients. Infections by HPV and C. trachomatis were independently associated with the presence of CIN. The high frequency of STIs in asymptomatic women suggests the need for routine screening of these infections.

Resumo Objetivo Determinar a frequência de infecções sexualmente transmissíveis (ISTs) em mulheres assintomáticas e a associação destas infecções com a neoplasia intraepitelial cervical (NIC). Métodos Foi realizado um estudo transversal recrutando mulheres atendidas em uma clínica ginecológica geral e em um centro de referência para colposcopia, de outubro de 2014 a outubro de 2015. O grupo de colposcopia consistiu de 71 mulheres, e o grupo de ginecologia geral consistiu de 55 mulheres. Amostras cervicais foram coletadas para citologia cervical e uma reação em cadeia de polimerase (RCP) multiplex em tempo real para detecção do vírus do papiloma humano (HPV) e das ISTs provocadas pelos seguintes micro-organismos: Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum e Neisseria gonorrhoeae. Foi realizada uma análise multivariada por regressão logística, considerando-se o nível de significância de 0,05. Resultados A frequência geral de ISTs foi: 46,8% (HPV); 27,8% (C. trachomatis); 28,6% (M. genitalium); 0,8% (M. hominis); 4,8% (U. urealyticum); e 4,8% (N. gonorrhoeae). Os fatores de risco significantes para NIC foram: infecção pelo HPV (razão de probabilidades [RP] = 2,53; p = 0,024); C. trachomatis (RP = 3,04; p = 0,009); M. genitalium (RP = 2,37; p = 0,04); e coinfecção por HPV e C. trachomatis (RP = 3,11; p = 0,023). Após a análise multivariada, foi encontrada uma associação significante entre HPV e NIC (RP = 2.48; intervalo de confiança de 95% [IC95%]: 1,04-5,92; p = 0,04) e entre C. trachomatis e NIC (RP = 2,69; IC95%: 1,11-6,53; p = 0,028). Conclusões A frequência de ISTs foi alta em mulheres assintomáticas. Infecções por HPV e C. trachomatis foram independentemente associadas com a presença de NIC. A alta frequência de ISTs em mulheres assintomáticas sugere a necessidade de rastreamento rotineiro dessas infecções.

Structural investigation of anti-Trypanosoma cruzi 2-iminothiazolidin-4-ones allows the identification of agents with efficacy in infected mice.

We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6-24. Compounds with a phenyl at position N3, 15-19, 22-24, exhibited better inhibitory properties for cruzain and against the parasite than 2-iminothiazolidin-4-one 5. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which inhibited the activity of cruzain and the proliferation of epimastigotes and was cidal for trypomastigotes but was not toxic for splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26-45 and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.