IL-23R Variant rs11805303 Is Associated With Susceptibility to the Development of Cutaneous Leishmaniasis in Leishmania guyanensis-Infected Individuals.

BACKGROUND: Emerging evidence suggests that the interleukin (IL) 17/ IL-23 axis may play a role in the pathogenesis of leishmaniasis. Our aim was to investigate whether the IL-23R variant rs11805303 is a risk factor for the development of cutaneous leishmaniasis (CL) in Leishmania guyanensis-infected individuals. METHODS: We genotyped by polymerase chain reaction-restriction fragment length polymorphism the rs11805303 C/T in 828 patients with CL and 806 healthy individuals. Plasma tumor necrosis factor-α, IL-6, interferon-γ, IL-1ß, and IL-17 were measured with the Bioplex assay. RESULTS: The distribution of the genotypes differed between patients with CL and healthy controls with a common odds ratio of 1.78 (P = 2.2 × 10-11) for the disease-associated T allele. Leishmania guyanensis-infected individuals homozygous for the T allele show a 200% increased risk of progressing to disease development, with a 95% confidence interval ranging from 81% to 400% (P = 9.9 × 10-6) in comparison to individuals homozygous for the C allele. Males homozygous for the T allele have higher plasma levels of IL-17 compared with heterozygous or homozygous CC individuals. CONCLUSIONS: The present association of the IL-23R variant rs11805303 with the development of CL suggests that the IL-17/IL-23 axis may play an important role in the pathogenesis of CL.

Duffy blood system and G6PD genetic variants in vivax malaria patients from Manaus, Amazonas, Brazil.

BACKGROUND: Over a third of the world's population is at risk of Plasmodium vivax-induced malaria. The unique aspect of the parasite's biology and interactions with the human host make it harder to control and eliminate the disease. Glucose-6-phosphate dehydrogenase (G6PD) deficiency and Duffy-negative blood groups are two red blood cell (RBC) variations that can confer protection against malaria. METHODS: Molecular genotyping of G6PD and Duffy variants was performed in 225 unrelated patients (97 with uncomplicated and 128 with severe vivax malaria) recruited at a Reference Centre for Infectious Diseases in Manaus. G6PD and Duffy variants characterizations were performed using Real Time PCR (qPCR) and PCR-RFLP, respectively. RESULTS: The Duffy blood group system showed a phenotypic distribution Fy(a + b-) of 70 (31.1%), Fy(a + b +) 96 (42.7%), Fy(a-b +) 56 (24.9%) and Fy(a-b-) 1 (0.44%.) The genotype FY*A/FY*B was predominant in both uncomplicated (45.3%) and severe malaria (39.2%). Only one Duffy phenotype Fy(a-b) was found and this involved uncomplicated vivax malaria. The G6PD c.202G > A variant was found in 11 (4.88%) females and 18 (8.0%) males, while c.376A > G was found in 20 females (8.88%) and 23 (10.22%) male patients. When combined GATA mutated and c.202G > A and c.376A > G mutated, was observed at a lower frequency in uncomplicated (3.7%) in comparison to severe malaria (37.9%). The phenotype Fy(a-b +) (p = 0.022) with FY*B/FY*B (p = 0.015) genotype correlated with higher parasitaemia. CONCLUSIONS: A high prevalence of G6PD c202G > A and c.376A > G and Duffy variants is observed in Manaus, an endemic area for vivax malaria. In addition, this study reports for the first time the Duffy null phenotype Fy(a-b-) in the population of the Amazonas state. Moreover, it is understood that the relationship between G6PD and Duffy variants can modify clinical symptoms in malaria caused by P. vivax and this deserves to be further investigated and explored among this population.

Basic biochemical and hematological parameters of structural hemoglobin variants in the postpartum women and their respective newborn from Manaus, Amazonas, Brazil.

The present study aimed at estimating the prevalence of structural hemoglobinopathies in newborn and describing the hematological and biochemical characteristics between postpartum women (PW) and their respective newborns (NB) at a public maternity hospital in Manaus, Amazonas state, Brazil. In total, 825 NB and 820 PW were included in the study. Hematological and biochemical analysis and screening of structural hemoglobinopathies were performed and compared in groups of individuals (NICU or not; hemoglobin genotypes; gestational age and prenatal). The age of PW ranged from 13 to 44 years old (mean of 23.7 ± 6.6 years), with 45.9% pregnant for the first time and 54.1% multiparous. Reported receiving prenatal care 88% and regarding the type of delivery, 47.7% had delivered by cesarean section. Among the births, 19.4% were born premature and 8.3% were admission to the neonatal intensive care unit (NICU). The male NB represented 53.4% of the total. Sickle cell trait (FAS) was found in 16 (1.94%) and heterozygous for D hemoglobin (FAD) in 6 (0.73%) newborns. A statistically significant values was found between the previous history of miscarriage and increase of Mean corpuscular volume (MCV) (p < .001), Red blood cell distribution width (RDW) (p = .003), total and indirect bilirubin concentration (p < .001) and LDL cholesterol (p = .004). Hemoglobin levels below 13.5 g/dL was found in 66% black newborns, compared with 15% of Afro-Brazilian and 5% of whites. The frequency of structural hemoglobinopathies was higher in African-Brazilian newborn babies (78%) and those who with low birth weight had a higher frequency of NICU (35.7%). Interestingly, underage mothers had a higher frequency of NB with low birth weight and premature birth. Postpartum women who had children carriers of FAS and FAD had a higher frequency of urinary tract infection (65.2%) and moderate anemia (23.8%). This study estimated for the first time the prevalence of structural hemoglobinopathies in NB in Manaus, Amazonas, Brazil. Despite the small prevalence of, we highlight the importance of early diagnosis of hemoglobin variants, contributing to the improvement of the quality of life of PW and your NB, reinforce the need to implement educational and prevention programs to raise awareness among the population and in order to counsel parents regarding the probability of having a child with abnormal hemoglobins homozygous as HbSS or HbCC.

A genetic variant in the TRAF1/C5 gene lead susceptibility to active pulmonary tuberculosis by decreased TNF-α levels.

Host genetics are important to consider in the role of resistance or susceptibility for developing active pulmonary tuberculosis (TB). Several association studies have reported the role of variants in STAT4 and TRAF1/C5 as risk factors to autoimmune diseases. Nevertheless, more data is needed to elucidate the role of these gene variants in infectious disease. Our data reports for the first time, variant rs10818488 in the TRAF1/C5 gene (found 47% of the population worldwide), is associated with susceptibility (OR = 1.51) to development TB. Multivariate analysis evidenced association between rs10818488 TRAF1/C5 and risk to multibacillary TB (OR = 4.18), confers increased bacteria load in the lung, indicates a decreased ability to control pathogen levels in the lung, and spread of the pathogen to new hosts. We showed that the "loss-of-function" variant in TRAF1/C5 led to susceptibility for TB by decreased production of TNF-α. Our results suggest the role of variant TRAF1/C5 in susceptibility to TB as well as in clinical presentation of multibacillary TB.

A Single Haplotype of IFNG Correlating With Low Circulating Levels of Interferon-γ Is Associated With Susceptibility to Cutaneous Leishmaniasis Caused by Leishmania guyanensis.

BACKGROUND: Interferon-γ (IFN-γ) plays an important role in the control of Leishmania infection. Blockade of IFN-γ signaling in mice increases lesion size and parasite load. In endemic areas of Leishmaniasis, only a fraction of the population develop the disease. This suggest that host genetics may play a role in this response. We investigated whether single nucleotide polymorphisms (SNPs) in IFNG may be associated with elevated or decrease risk in the development of cutaneous leishmaniasis (CL). METHODS: We assessed 9 SNP and cytosine-adenine (CA) repeats in IFNG by nucleotide sequencing in 647 patients with CL caused by Leishmania guyanensis and 629 controls. Circulating plasma IFN-γ levels were also assayed in 400 patients with CL and 400 controls. RESULTS: The rs2069705TT genotype is associated with elevated risk of developing CL compared with the rs2069705CC genotype (OR, 1.7; 95% CI, 1.3-2.4; P = .0008). There is a 70% chance that this genotype raises the risk of developing CL. In a dominant model, carriers of the rs2069705T allele compared with the rs2069705CC genotype showed a 50% (range, 20-100%) increased risk of developing CL (OR, 1.5; 95% CI, 1.2-2.0; P = .0004). Haplotype analysis showed 1 haplotype (H1) associated with low levels of IFN-γ presented an increased risk of 60% of developing CL (OR, 1.6; 95% CI, 1.3-1.9; P = 5 × 10-5) compared with non-H1. CONCLUSIONS: IFNG variant rs2069705 seems to be a genetic modifier of clinical outcome of Leishmania infection; individuals with the H1 haplotype, associated with low levels of IFN-γ, have a 60% risk of developing CL.

Combination of genetic polymorphisms in TLR influence cytokine profile in HCV patients treated with DAAs in the State of Amazonas.

Hepatitis C is a public health problem and affects approximately 3% of the world's population. HCV infections have a wide spectrum of clinical manifestations, and several single nucleotide polymorphisms (SNPs) in the genes of the toll-like receptors are cited to influence the clinical outcomes. A cross-sectional study was conducted in the Amazonas State, Brazil in which SNPs in TLR4 and TLR9 genes were genotyped by PCR-RFLP in 151 HCV chronic liver disease patients and 206 healthy donors. The circulating cytokines IL-6, TNF, IL-10, IL-2, IFN-γ, IL-4 and IL-17A were measured by cytometric bead array (CBA) which revealed that the combined genotypes of TLR9 -1237T/T and -1486C/T seem to influence the cytokine profile under lipopolysaccharide (LPS) stimulation of the Th17 profile, especially among patients with advanced chronic liver disease when treated with DAAs.

Increased platelet distribution width and reduced IL-2 and IL-12 are associated with thrombocytopenia in Plasmodium vivax malaria.

BACKGROUND: Thrombocytopenia in malaria involves platelet destruction and consumption; however, the cellular response underlying this phenomenon has still not been elucidated. OBJECTIVE: To find associations between platelet indices and unbalanced Th1/Th2/Th17 cytokines as a response to thrombocytopenia in Plasmodium vivax infected (Pv-MAL) patients. METHODS: Platelet counts and quantification of Th1/Th2/Th17 cytokine levels were compared in 77 patients with uncomplicated P. vivax malaria and 37 healthy donors from the same area (endemic control group - ENCG). FINDINGS: Thrombocytopenia was the main manifestation in 55 patients, but was not associated with parasitaemia. The Pv-MAL patients showed increases in the mean platelet volume (MPV), which may be consistent with larger or megaplatelets. Contrary to the findings regarding the endemic control group, MPV and platelet distribution width (PDW) did not show an inverse correlation, due the increase in the heterogeneity of platelet width. In addition, the Pv-MAL patients presented increased IL-1ß and reduced IL-12p70 and IL-2 serum concentrations. Furthermore, the reduction of these cytokines was associated with PDW values. MAIN CONCLUSIONS: Our data demonstrate that an increase in MPV and the association between reductions of IL-2 and IL-12 and PDW values may be an immune response to thrombocytopenia in uncomplicated P. vivax malaria.

A polymorphism in the IL1B gene (rs16944 T/C) is associated with cutaneous leishmaniasis caused by Leishmania guyanensis and plasma cytokine interleukin receptor antagonist.

Nod-like Receptor Protein3 (NLRP3) inflammasome in macrophages infected with Leishmania sp. enhances the secretion of IL-1ß. Excess IL-1ß production is linked to disease severity in patients with cutaneous leishmaniasis (CL) caused by L. mexicana. Blockade of the NLRP3 inflammasome in cell cultures from skin biopsies of patients with CL caused by L. braziliensis inhibited the release of IL-1ß. We hypothesized that common single nucleotide polymorphisms in the IL1B and in its receptor antagonist IL1RN genes may be predictive of CL caused by L. guyanensis. The SNPs -511T/C (rs16944) and +3954C/T (rs1143634) of the IL1B and IL1RN VNTR (rs2234663) were assessed in 881 patients with CL and 837 healthy controls by PCR-RFLP and direct PCR respectively. Plasma cytokines levels were also assayed. The plasma levels of IL-1ß were higher in patients compared to control subjects. In contrast, increased plasma levels of IL-1Ra were observed in controls. The rs16944 C/C genotype was more common among the patients (OR = 1.5 [95%CI 1.1-2.0]; P = 0.004) and the C allele suggests susceptibility to CL (OR = 1.2 [95%CI 1.1-1.4]; P = 0.003). The rs16944 C/C genotype shows a tendency to correlate with lower levels of the IL-1Ra cytokine. Low levels of IL-1Ra cytokine and rs16944 C/C genotype seem to confer susceptibility to L. guyanensis-infection in the Amazonas.

Leishmania braziliensis isolated from disseminated leishmaniasis patients downmodulate neutrophil function.

AIMS: The polymorphism observed in Leishmania braziliensis is associated with different clinical forms of leishmaniasis. Neutrophils (PMNs) participate in the pathogenesis of leishmania infection, and here, we evaluate neutrophil function after infection with isolates of L. braziliensis from cutaneous leishmaniasis (CL) or disseminated leishmaniasis (DL) patients. METHODS AND RESULTS: Neutrophils from 30 healthy subjects (HS) were infected with isolates of L. (V.) braziliensis obtained from three CL and three DL patients. They were infected at the ratio of 3:1 parasites per neutrophil, and leishmania uptake was evaluated by microscopy. The neutrophil activation markers and oxidative burst by expression of dihidrorhodamine (DHR) were evaluated by flow cytometry and cytokine production by ELISA. The frequency of infected cells and the number of amastigotes were higher in neutrophils infected with CL isolates compared to DL isolates (P < 0.05). The DHR and CD66b expression after infection with DL isolate was lower than with CL isolates. There was no difference regarding chemokine production. CONCLUSION: The L. (V.) braziliensis isolates of DL induced lower respiratory burst and neutrophils activation markers compared with CL isolates which may contribute to parasite survival and dissemination in DL patients.

Polymorphisms in TLRs influence circulating cytokines production in Plasmodium vivax malaria: TLR polymorphisms influence cytokine productions in malaria-vivax.

The efficiency of the immune system has been shaped throughout the evolutionary process allowing adaptations. In a Plasmodium vivax infection, the host attempts to develop an innate immune response to keep in check the parasite that is associated with inflammatory and regulatory processes. Production of pro-inflammatory and regulatory cytokines simultaneously appears to be a balancing mechanism for the host to prevent the onset of severe disease. Changes in the dynamics of circulating cytokines production can influence the pathogenesis, severity of the disease and episodes of recurrent Plasmodium vivax malaria (Pv-malaria). A cross-sectional study was conducted in endemic areas for Pv-malaria in the Amazonas State, Brazil. Several SNPs in TLR genes were genotyped by PCR-RFLP in 137 patients infected with P. vivax. Circulating cytokines IL-6, TNF, IL-2, IL-10, IFN-γ and IL-4 were measured by CBA. Influence of the studied SNPs on circulating cytokines was investigated by applying the Kruskal-Wallis test followed by Dunns' multiple comparison post-test. A Spearman correlation test also was performed to elaborate circulating cytokine networks and to demonstrate the level of interaction between each molecule. Individuals with genotypes A/G (TLR4 A299G), C/C (TLR6 S249P) and T/T (TLR9 -1486C/T) appear to produce less/gain IL-6, IFN-γ, IL-10, IL-2 and IL-4 compared to patients with wild-type and heterozygous genotypes. In addition, these genotypes seem to influence the interaction network between the molecules studied, causing a lower interaction, absence or even negative interaction between the cytokines. Data presented in this study suggests the influence of polymorphisms TLR4 (A299G), TLR6 (S249P) and TLR9 (-1486C/T) on the production of circulating cytokines during Pv-malaria.

Atrial fibrillation in a patient with Zika virus infection.

BACKGROUND: Zika virus is an emerging arbovirus of the family Flaviviridae and genus Flavivirus that until 2007 was restricted to a few cases of mild illness in Africa and Asia. CASE PRESENTATION: We report a case of atrial fibrillation disclosed during an acute Zika virus infection in a 49-year-old man. Different biological samples were analyzed for the molecular diagnosis of Zika by real-time PCR, however only the saliva specimen was positive. The patient's wife tested positive in the serum sample, although she was an asymptomatic carrier. Moreover, a complete overview of patient's biomarkers, including cytokines, chemokines, and growth-factors levels, was analyzed and compared to gender and age matching non-infected controls, as well as other Zika infected patients, considering the 95%CI of the mean values. Elevated levels of CXCL8, CCL11, CCL2, CXCL10, IL-1ß, IL-6, TNF-α, IFN-γ, IL-17, IL-1Ra, IL-4, IL-9, FGF-basic, PDGF, G-CSF, and GM-CSF were observed in the Atrial fibrillation patient, in contrast to uninfected controls. Furthermore, increased levels of CCL5, IL-1ß, TNF-α, IFN-γ, IL-9, G-CSF, and GM-CSF were observed only in the atrial fibrillation patient, when compared to other Zika patients. CONCLUSIONS: To our knowledge, this is the first description of this type of cardiac disorder in Zika patients which may be considered another atypical manifestation during Zika virus infection.

CYP2D6 activity and the risk of recurrence of Plasmodium vivax malaria in the Brazilian Amazon: a prospective cohort study.

BACKGROUND: CYP2D6 pathway mediates the activation of primaquine into active metabolite(s) in hepatocytes. CYP2D6 is highly polymorphic, encoding CYP2D6 isoforms with normal, reduced, null or increased activity. It is hypothesized that Plasmodium vivax malaria patients with defective CYP2D6 function would be at increased risk for primaquine failure to prevent recurrence. The aim of this study was to investigate the association of CYP2D6 polymorphisms and inferred CYP2D6 phenotypes with malaria recurrence in patients from the Western Brazilian Amazon, following chloroquine/primaquine combined therapy. METHODS: The prospective cohort consisted of P. vivax malaria patients who were followed for 6 months after completion of the chloroquine/primaquine therapy. Recurrence was defined as one or more malaria episodes, 28-180 days after the initial episode. Genotyping for nine CYP2D6 SNPs and copy number variation was performed using TaqMan assays in a Fast 7500 Real-Time System. CYP2D6 star alleles (haplotypes), diplotypes and CYP2D6 phenotypes were inferred, and the activity score system was used to define the functionality of the CYP2D6 diplotypes. CYP2D6 activity scores (AS) were dichotomized at ≤ 1 (gPM, gIM and gNM-S phenotypes) and ≥ 1.5 (gNM-F and gUM phenotypes). RESULTS: Genotyping was successfully performed in 190 patients (44 with recurrence and 146 without recurrences). Recurrence incidence was higher in individuals presenting reduced activity CYP2D6 phenotypes (adjusted relative risk = 1.89, 95% CI 1.01-3.70; p = 0.049). Attributable risk and population attributable fraction were 11.5 and 9.9%, respectively. The time elapsed from the first P. vivax malaria episode until the recurrence did not differ between patients with AS of ≤ 1 versus ≥ 1.5 (p = 0.917). CONCLUSIONS: The results suggest that CYP2D6 polymorphisms are associated with increased risk of recurrence of vivax malaria, following chloroquine-primaquine combined therapy. This association is interpreted as the result of reduced conversion of primaquine into its active metabolites in patients with reduced CYP2D6 enzymatic activity.

Chloroquine resistance is associated to multi-copy pvcrt-o gene in Plasmodium vivax malaria in the Brazilian Amazon.

BACKGROUND: The resistance of Plasmodium vivax to chloroquine has become an obstacle to control strategies based on the use of anti-malarials. The current study investigated the association between P. vivax CQ-resistance in vivo with copy number variation and mutations in the promoter region in pvcrt-o and pvmdr1 genes. METHODS: The study included patients with P. vivax that received supervised treatment with chloroquine and primaquine. Recurrences were actively recorded during this period. RESULTS: Among the 60 patients with P. vivax, 25 were CQ-resistant and 35 CQ-susceptible. A frequency of 7.1% of multi-copy pvcrt-o was observed in CQ-susceptible samples and 7.7% in CQ-resistant at D0 (P > 0.05) and 33.3% in CQ-resistant at DR (P < 0.05). For pvmdr1, 10.7% of the CQ-susceptible samples presented multiple copies compared to 11.1% in CQ-resistant at D0 and 0.0% in CQ-resistant at DR (P > 0.05). A deletion of 19 bp was found in 11/23 (47.6%) of the patients with CQ-susceptible P. vivax and 3/10 (23.1%) of the samples with in CQRPv at D0. At day DR, 55.5% of the samples with CQRPv had the 19 bp deletion. For the pvmdr-1 gene, was no variation in the analysed gene compared to the P. vivax reference Sal-1. CONCLUSIONS: This was the first study with 42-day clinical follow-up to evaluate the variation of the number of copies and polymorphisms in the promoter region of the pvcrt-o and pvmdr1 genes in relation to treatment outcomes. Significantly higher frequency of multi-copy pvcrt-o was found in CQRPv samples at DR compared to CQ-susceptible, indicating parasite selection of this genotype after CQ treatment and its association with CQ-resistance in vivo.

Analysis of the immunological biomarker profile during acute Zika virus infection reveals the overexpression of CXCL10, a chemokine linked to neuronal damage.

BACKGROUND: Infection with Zika virus (ZIKV) manifests in a broad spectrum of disease ranging from mild illness to severe neurological complications and little is known about Zika immunopathogenesis. OBJECTIVES: To define the immunologic biomarkers that correlate with acute ZIKV infection. METHODS: We characterized the levels of circulating cytokines, chemokines, and growth factors in 54 infected patients of both genders at five different time points after symptom onset using microbeads multiplex immunoassay; comparison to 100 age-matched controls was performed for statistical analysis and data mining. FINDINGS: ZIKV-infected patients present a striking systemic inflammatory response with high levels of pro-inflammatory mediators. Despite the strong inflammatory pattern, IL-1Ra and IL-4 are also induced during the acute infection. Interestingly, the inflammatory cytokines IL-1ß, IL-13, IL-17, TNF-α, and IFN-γ; chemokines CXCL8, CCL2, CCL5; and the growth factor G-CSF, displayed a bimodal distribution accompanying viremia. While this is the first manuscript to document bimodal distributions of viremia in ZIKV infection, this has been documented in other viral infections, with a primary viremia peak during mild systemic disease and a secondary peak associated with distribution of the virus to organs and tissues. MAIN CONCLUSIONS: Biomarker network analysis demonstrated distinct dynamics in concurrence with the bimodal viremia profiles at different time points during ZIKV infection. Such a robust cytokine and chemokine response has been associated with blood-brain barrier permeability and neuroinvasiveness in other flaviviral infections. High-dimensional data analysis further identified CXCL10, a chemokine involved in foetal neuron apoptosis and Guillain-Barré syndrome, as the most promising biomarker of acute ZIKV infection for potential clinical application.

Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species.

American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L (Viannia) braziliensis, and L (V) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL.

TOLLIP gene variant is associated with Plasmodium vivax malaria in the Brazilian Amazon.

BACKGROUND: Toll-interacting protein is a negative regulator in the TLR signaling cascade, particularly by impeding the TLR2 and, TLR4 pathway. Recently, TOLLIP was shown to regulate human TLR signaling pathways. Two common TOLLIP polymorphisms (rs5743899 and rs3750920) were reported to be influencing IL-6, TNF and IL-10 expression. In this study, TOLLIP variants were investigated to their relation to Plasmodium vivax malaria in the Brazilian Amazon. METHODS: This cohort study was performed in the municipalities of Careiro and, Manaus, in Western Brazilian Amazon. A total of 319 patients with P. vivax malaria and, 263 healthy controls with no previous history of malaria were included in the study. Genomic DNA was extracted from blood collected on filter paper, using the QIAamp® DNA Mini Kit, according to the manufacturer's suggested protocol. The rs5743899 and rs3750920 polymorphisms of the TOLLIP gene were typed by PCR-RFLP. RESULTS: Homozygous individuals for the rs3750920 T allele gene had twice the risk of developing malaria when compared to individuals homozygous for the C allele (OR 2.0 [95% CI 1.23-3.07]; p = 0.004). In the dominant model, carriers the C allele indicates protection to malaria, carriers of the C allele were compared to individuals with the T allele, and the difference is highly significant (OR 0.52 [95% CI 0.37-0.76]; p = 0.0006). The linkage disequilibrium between the two polymorphisms was weak (r2 = 0.037; D' = 0.27). CONCLUSIONS: These findings suggest that genes involved in the TLRs-pathway may be involved in malaria susceptibility. The association of the TOLLIP rs3750920 T allele with susceptibility to malaria further provides evidence that genetic variations in immune response genes may predispose individuals to malaria.

Chronic HDV/HBV co-infection: predictors of disease stage---a case series of HDV-3 patients.

BACKGROUND & AIMS: Chronic HDV/HBV co-infection is perhaps the most intriguing amongst all viral hepatitis. Only few studies focus deeply on this topic, particularly with patients infected with HDV-3. This study aimed to identify predictors of advanced disease, examining a cross-sectional data of 64 patients. METHODS: Histological grading was used to characterize the disease stages and viral loads were tested as predictors of necroinflammatory activity and fibrosis. RESULTS: We identified three HDV/HBV co-infection patterns: patients with predominant HDV replication (56.3%), patients with similar viral loads of both viruses (40.6%), and patients with predominant HBV replication (3.1%). Mean HDV-RNA showed a positive trend regarding inflammatory activity and grade of fibrosis. HDV viral load correlated positively with serum levels of liver enzymes and inversely with platelets count. HBV viral load showed no correlation with any of the above parameters. Advanced fibrosis was associated with age, splenomegaly, and HDV viral load of more than 2 log10. Multiple logistic regression confirmed the independent effect of HDV viral predominance. Advanced necroinflammatory activity was independently associated with HDV viral load and splenomegaly. CONCLUSIONS: HDV may possibly play an important and direct role in the establishment of necroinflammatory activity and fibrosis. Data show an indigenous HDV genotype, HDV-3, similar to those described in the Amazon region.

Detection of Herpesvirus, Enterovirus, and Arbovirus infection in patients with suspected central nervous system viral infection in the Western Brazilian Amazon.

Acute infections of the central nervous system (CNS) can be caused by various pathogens. In this study, the presence of herpesviruses (HHV), enteroviruses (EVs), and arboviruses were investigated in CSF samples from 165 patients with suspected CNS viral infection through polymerase chain reaction (PCR) and reverse transcriptase PCR. The genomes of one or more viral agents were detected in 29.7% (49/165) of the CSF samples. EVs were predominant (16/49; 32.6%) followed by Epstein-Barr virus (EBV) (22.4%), Varicella-Zoster virus (VZV) (20.4%), Cytomegalovirus (CMV) (18.4%), herpes simplex virus (HSV-1) (4.1%), (HSV-2) (4.1%), and the arboviruses (14.3%). Four of the arboviruses were of dengue virus (DENV) and three of oropouche virus (OROV). The detection of different viruses in the CNS of patients with meningitis or encephalitis highlight the importance of maintaining an active laboratory monitoring diagnostics with rapid methodology of high sensitivity in areas of viral hyperendemicity that may assist in clinical decisions and in the choice of antiviral therapy.

Variants of NOD2 in Leishmania guyanensis-infected patients with cutaneous leishmaniasis and correlations with plasma circulating pro-inflammatory cytokines.

Leishmaniases, a group of vector-borne diseases, are caused by the protozoan intracellular parasite Leishmania (L.) and are transmitted by the phlebotomine sandflies. A wide range of clinical manifestations in L- infection is observed. The clinical outcome ranges from asymptomatic, cutaneous leishmaniasis (CL) to severe mucosal leishmaniasis (ML) or visceral leishmaniasis (VL), depending on the L. species. Interestingly, only a fraction of L.-infected individuals progress to disease development, suggesting a key role of host genetics in the clinical outcome. NOD2 plays a critical role in the control of host defense and inflammation. The NOD2-RIK2 pathway is involved in developing a Th1- type response in patients with VL and C57BL/6 mice infected with L. infantum. We investigated whether variants in the NOD2 gene (R702W rs2066844, G908R rs2066845, and L1007fsinsC rs2066847) are associated with susceptibility to CL caused by L. guyanensis (Lg) in 837 patients with Lg-Cl and 797 healthy controls (HC) with no history of leishmaniasis. Both patients and HC are from the same endemic area of the Amazonas state of Brazil. The variants R702W and G908R were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and L1007fsinsC was by direct nucleotide sequencing. The minor allele frequency (MAF) of L1007fsinsC was 0.5% among the patients with Lg-CL and 0.6% in the healthy controls group. R702W genotypes frequencies were similar in both groups. Only 1% and 1.6% were heterozygous for G908R among the patients with Lg-CL and HC, respectively. None of the variants revealed any association with susceptibility to the development of Lg-CL. Correlations of genotypes with the level of plasma cytokines revealed that individuals with the mutant alleles of R702W tend to have low levels of IFN-γ. G908R heterozygotes also tend to have low IFN-γ, TNF-α, IL-17, and IL-8. Variants of NOD2 are not involved in the pathogenesis of Lg-CL.

Variants of CARD8 in Leishmania guyanensis-cutaneous leishmaniasis and influence of the variants genotypes on circulating plasma cytokines IL-1ß, TNFα and IL-8.

Nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein family (NLR) are intracellular pathogen recognition receptors mediating innate immunity, releasing proinflammatory cytokines IL-1ß and IL-18, and promoting pyroptotic cell death, upon sensing pathogenic or endogenous danger signals. In animal models, NLRP3 inflammasome has a dual role, pathogenic or protective in Leishmania-infection, depending on the Leishmania species and mice strain. Caspase recruitment containing domain 8 (CARD8) is a negative regulator of NLRP3 inflammasome and also an inhibitor of transcription factor NFĸB, a major transcription factor of proinflammatory cytokines. We investigated whether single nucleotide variants in CARD8 may partially explain why only a proportion of individuals coming from the same area of endemicity of leishmaniasis develop cutaneous leishmaniasis caused by Leishmania guyanensis. We genotyped four single nucleotide variants of the CARD8 gene by direct nucleotide sequencing in 1741 individuals from an endemic area of leishmaniasis, constituting 850 patients with CL and 891 healthy controls. The frequencies of the genotypes of the variants rs2288877 T>C, rs73944113 C>T, and rs2043211 A>T are similar among the patients with CL and HC, while the variant rs2288876 A>G) reveals an excess of the genotype AA among the patients with CL (44%) compared to 37% in the HC group. Allele A of the variant rs2288876 A>G) is associated with susceptibility to CL (OR = 1.2 [95%CI 1.03-1.4]; P = 0.01). Haplotype analysis showed that individuals harboring the haplotype CCAA have 280% odds of developing CL caused by L. guyanensis (OR = 3.8 [95% CI 2.0-7.7]; p = 0.00004). The variants rs2288877 T>C and rs2288876 A>G correlate with the plasma level of IL-8. Spearman correlation showed a significant positive correlation between the rs2288876 A>G allele A and the level of IL-8 (ρ = 0.22; p = 0.0002). CARD8 may partially contribute to the development of CL caused by L. guyanensis.