George Allan Villarouco da Silva

IFN-γ +875 microsatellite polymorphism as a potential protection marker for leprosy patients from Amazonas state, Brazil

Polymorphisms present in the first intron of IFN-γ may have an important role in the regulation of the immune response, which could have functional consequences for gene transcription. Leprosy patients are characterized by different immune responses in different clinical forms. We investigated a possible association of the +874 polymorphism and CA repeats present in the first intron of IFN-γ with susceptibility to leprosy and with the manifestation of the different clinical forms. Nucleotide sequencing was performed with samples from 108 leprosy patients and 113 controls subjects, as well as immunophenotyping of CD(4)(+), CD(8)(+) and CD(69)(+) T cells by flow cytometry. The data showed that there were no significant differences between patients and control subjects, as well as according classification of Ridley-Jopling. However, the A/A genotype was significantly increased in paucibacillary patients (p=0.028) and the microsatellite encoding 16 CA repeats were significantly associated with paucibacillary compared to multibacillary patients (p=0.019). Individuals homozygous for the +874 A allele, the mean level of CD(4)(+) and CD(69)(+) T cells was higher. Our data suggest that polymorphisms present in the first intron of IFN-γ are not associated with susceptibility to leprosy, nevertheless, the +874 polymorphism and the CA repeats number encoded in IFN-γ gene may be related to a higher cellular immune response in patients and are consistently more frequently detected in PB patients.

Decreased RNA expression of interleukin 17A in skin of leprosy

Interleukin-17A (IL-17A) is a proinflamatory cytokine that plays an important role in fighting pathogens at mucosal interfaces, by summoning neutrophils and upregulating cytoplasmatic antimicrobial peptides. So far, the presence of IL-17A in leprosy has not been demonstrated. The expression of IL-17A and related cytokines (IL-6 and IL-23p19) was addressed through RNA extraction and cDNA quantitative amplification in macerated biopsies of active lesions of 48xa0leprosy patients and 20xa0fragments of normal skin of individuals. Blood levels of IL-17A, IL-23p19 and IL-6 were determined by ELISA. We found an abrogated mRNA IL-17A response in all biopsies of leprosy patients, as compared with controls. Circulating IL-17A and IL-23p19 were undetectable in both patients and controls, but IL-6 was higher in lepromatous patients. Although at low levels, IL-17A mRNA in lepromatous patients had an inverse linear correlation with bacillary burden. Low expression of IL-17A in patients may be a constitutive genetic feature of leprosy patients or a circumstantial event induced by the local presence of the pathogen, as an escape mechanism.

Polymorphisms assessment in the promoter region of IL12RB2 in Amazon leprosy patients

Leprosy displays a wide clinical spectrum that is dependent of the type of immune response. We investigate here whether polymorphisms in the promoter region of the IL12RB2 gene are associated with susceptibility or resistance to clinical forms of leprosy. Nucleotide sequencing of the promoter region of IL12RB2 encompassing SNPs -1035 A/G, -1033 T/C, -1023 A/G, -650 del/G and -464 A/G was performed on DNA samples from 105 leprosy patients and 108 healthy controls. However, none of the SNPs were associated with susceptibility to the disease or any of its clinical forms. Similarly, haplotype analysis did not show any association. The haplotype -1035A/-1033T/-650G/-464A was prevalent, and homozygosity for this haplotype was associated to a lower distribution of CD4(+) T cells (p=0.041). Our data suggest that polymorphisms present in the promoter region of IL12RB2 may not be associated with susceptibility to leprosy or its clinical forms.

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-1β, 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.

Identification of Primary Drug Resistance to Rifampin in Mycobacterium leprae Strains from Leprosy Patients in Amazonas State, Brazil

ABSTRACT The aim of this study was to identify polymorphisms in the folp1, gyrA, and rpoB genes in leprosy patients treated in Amazonas State, Brazil. Among 197 slit-skin smear samples from untreated or relapsed patients, we found three cases of primary resistance to rifampin and one confirmed case of multidrug resistance.

Atrial fibrillation in a patient with Zika virus infection

BackgroundZika 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 presentationWe 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-1β, 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-1β, 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.ConclusionsTo 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.

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

BackgroundThe 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.MethodsThe study included patients with P. vivax that received supervised treatment with chloroquine and primaquine. Recurrences were actively recorded during this period.ResultsAmong 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 (Pu2009>u20090.05) and 33.3% in CQ-resistant at DR (Pu2009<u20090.05). 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 (Pu2009>u20090.05). A deletion of 19xa0bp 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 19xa0bp deletion. For the pvmdr-1 gene, was no variation in the analysed gene compared to the P. vivax reference Sal-1.ConclusionsThis 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.

Toll-Like Receptor-1 Single-Nucleotide Polymorphism 1805T/G Is Associated With Predisposition to Multibacillary Tuberculosis

Tuberculosis (TB), caused by mycobacterial species of the Mycobacterium tuberculosis complex, is a serious global health issue. Brazil is among the 22 countries with the highest number of TB cases, and the state of Amazonas has the highest incidence of TB cases in the country. Toll-like receptors (TLRs) are important pattern recognition receptors of the innate immunity and play a key role in orchestrating an effective immune response. We investigated whether the single-nucleotide polymorphisms (SNPs) 1805T/G TLR1, 2258G/A TLR2, 896A/G and 1196C/T of TLR4, 745T/C TLR6, and −1237A/G and −1486A/G of TLR9 are associated with the predisposition to TB and/or bacillary load. The SNPs genotyping was performed by nucleotide sequencing in 263 TB patients and 232 healthy controls residing in the state of Amazonas. Alleles and genotypes frequencies were similar between patients and healthy individuals for most of the investigated SNPs. Stratification of the TB patients according to their bacillary load showed that the genotype 1805TT TLR1 (rs5743618) was prevalent among paucibacillary patients [odds ratio (OR)u2009=u20090.38; 95% confidence interval (CI)u2009=u20090.19–0.76; pu2009=u20090.009] while the genotype 1805TG was common among multibacillary patients (ORu2009=u20093.72; CIu2009=u20091.65–8.4; pu2009=u20090.004). Comparison of demographic characteristics of patients to controls showed that TB is strongly associated with smoking (ORu2009=u20096.55; 95% CIu2009=u20093.2–13.6; pu2009<u20090.0001); alcohol use disorder (ORu2009=u20097.14; 95% CIu2009=u20093.7–13.9; pu2009<u20090.0001); and male gender (ORu2009=u20093.66; 95% CIu2009=u20092.52–5.3; pu2009<u20090.0001). Multivariate logistic regression demonstrated that alcoholism (ORu2009=u20092.93; 95% CIu2009=u20091.05–8.16; pu2009=u20090.03) and the 1805G allele (ORu2009=u20092.75; 95% CIu2009=u20091.33–5.7; pu2009=u20090.006) are predictive variables for multibacillary TB. Altogether, we suggest that the TLR1 1805G allele may be a relevant immunogenetic factor for the epidemiology of TB together with environmental, sociodemographic, and behavioral factors.

Haplotype of the Promoter Region of TNF Gene May Mark Resistance to Tuberculosis in the Amazonas State, Brazil

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is worldwide and keeps on increasing with the appearance of HIV. Host genes involved in the immune response play key role in conveying resistance or susceptibility to TB. We investigated a possible association of the different single nucleotide polymorphisms (SNPs) present in the promoter region of the TNF with either susceptibility or resistance TB. Nucleotide sequencing of the promoter region of TNF encompassing the SNPs –238 G/A, –308 G/A, –857 C/T, –863 A/C and –1031 T/C was performed on 230 TB patients and 293 control subjects. The –863 A/C and –857 C/C genotypes show protection to TB. The –308A allele is associated with protection to TB. Haplotypes analysis revealed that the haplotype –1031T/– 863C/–857C/–308A/–238G is protective to TB (p=0.024). Our data suggests that SNPs present in the promoter region of TNF are associated with resistance to the development of TB.