Tania Regina Tozetto-Mendoza

Concentration of cytokines in patients with osteoarthritis of the knee and fibromyalgia

Introduction Fibromyalgia and osteoarthritis may present a relationship with the concentration of cytokines. The aim of this study was to compare the serum concentrations of IL-12p70, tumor necrosis factor, IL-10, IL-6, IL-1β, and IL-8 in patients with knee osteoarthritis and fibromyalgia. Materials and methods The study included 53 women (71.2±7.6 years old) diagnosed with knee osteoarthritis with moderate-to-severe pain (visual analog scale >4) for at least 3 months. Sixty women (54.1±8.1 years old) diagnosed with fibromyalgia according to the American College of Rheumatology criteria and with moderate-to-severe pain (visual analog scale >4) also participated in this study. For the dosage of cytokines, blood was collected in the morning: 5 mL from the cubital vein. The material was centrifuged at 4°C, separated into 100 μL aliquots and stored at −80°C until processing. Serum concentrations of the studied cytokines were assessed using the BD Cytometric Bead Array method. Data were analyzed with Student’s t-test and the Mann–Whitney U test. Results We found higher levels of IL-6, IL-10, and IL-1β in fibromyalgia patients. After adjustment of age as a covariate, there was no statistically significant difference in the concentration of any cytokine between fibromyalgia and knee osteoarthritis patients. Conclusion Patients with knee osteoarthritis and fibromyalgia with the same duration and intensity of pain demonstrate similar concentrations of cytokines. Aging may play a role in cytokine profile, a finding not so extensively addressed in the literature and one that should be further investigated.

Identification of respiratory virus in infants with congenital heart disease by comparison of different methods

Respiratory virus infections are the main cause of infant hospitalization and are potentially severe in children with congenital heart disease (CHD). Rapid and sensitive diagnosis is very important to early introduction of antiviral treatment and implementation of precautions to control transmission, reducing the risk of nosocomial infections. In the present study we compare different techniques in the diagnosis of respiratory viruses in CHD infants. Thirty-nine samples of nasopharyngeal aspirate were obtained from CHD infants with symptoms of respiratory infection. The Multiplex PCR (Seeplex® RV 12 ACE Detection) driven to the detection of 12 respiratory viruses was compared with the direct immunofluorescence assay (DFA) and PCR, both targeting seven respiratory viruses. The positivity found by DFA, Multiplex and PCR was 33.3%, 51.3% and 48.7%, respectively. Kappa index comparing DFA and Multiplex, DFA and PCR and PCR and Multiplex PCR was 0.542, 0.483 and 0.539, respectively. The concordance between techniques was considered moderate. Both Multiplex PCR (p = 0.001) and PCR (p = 0.002) detected significantly more respiratory virus than DFA. As the performance of the tests may vary, the combination of two or more techniques may increase diagnostic sensitivity favoring the diagnosis of co-infections, early introduction of antiviral therapy and implementation of appropriate measures.

Genotypic distribution of HHV-8 in AIDS individuals without and with Kaposi sarcoma: Is genotype B associated with better prognosis of AIDS-KS?

AbstractAIDS-associated Kaposis sarcoma (AIDS-KS) caused by human herpes virus 8 (HHV-8) is the most severe and resistant form of KS tumor. Our aim was to verify whether there is an association between HHV-8 variability and development of AIDS-KS in Brazil by comparing the HHV-8 variability between individuals without and with KS. Saliva samples and blood, when available, were analyzed by polymerase chain reaction (PCR) techniques for detection of the fragments of ORF K1 of HHV-8, which were then genotyped and analyzed regarding the genetic variability. Our study described 106 positive cases for HHV-8 in the saliva from 751 AIDS patients without previous KS. In addition, we performed a phylogenetic analysis of HHV-8 in 34 of the 106 AIDS patients without KS and in 33 of the 37 patients with active KS. The distribution of HHV-8 genotypes A, B, C, and F in AIDS individuals was indistinguishable by comparing non-KS and KS groups, as well as regarding ethnicity. Considering the KS group, genotype B was associated with better prognosis of KS tumor. Interestingly, we found a particular profile of diversity within clade C and 2 recombinant patterns of HHV-8 in the saliva of AIDS individuals without KS. We emphasize the need to achieve standard genotyping protocol for ORF K1 amplification, thus allowing for substantial detection of HHV-8 variants. Our findings can shed light on the role of HHV-8 variability in the pathogenesis of AIDS-KS.

STANDARDIZATION OF PROCEDURES OF Plasmodium falciparum ANTIGEN PREPARATION FOR SEROLOGIC TESTS

The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20 degrees C up to 10 days or at -70 degrees C for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.

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

ABSTRACT 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.

Prospective study of human herpesvirus 8 oral shedding, viremia, and serological status among human immunodeficiency virus seropositive and seronegative individuals in Sao Paulo, Brazil.

ABSTRACT Human herpesvirus 8 (HHV-8) is a gamma-herpesvirus and etiological agent of all forms of Kaposi sarcoma (KS). Saliva may play an important role in HHV-8 transmission in specific populations. Little is known about HHV-8 oral shedding pattern and the possible correlation with the HHV-8 serological profile and viremia. A prospective study was conducted of HHV-8 salivary excretion among human immunodeficiency virus HIV-seronegative (n = 47) and -seropositive (n = 44) homosexual men and HIV-seropositive women (n = 32) over a 6-month period with monthly HHV-8 serologies (immunofluorescence assays to identify antibodies to latent and lytic HHV-8 viral proteins, and a whole-virus HHV-8 enzyme-linked immunosorbent assay [ELISA]), monthly HHV-8 DNA serum/plasma detection, and daily self-collected oral rinses for HHV-8-DNA detection using real-time polymerase chain reaction. HHV-8 seropositivity was 51.1%, 63.6%, and 37.5%, in the three studied groups. There was no case of HHV-8 DNA detection in serum/plasma. Intermittent detection of oral HHV-8 DNA was observed during 5.1% (110/2,160) of visits among 28% (18/64) of HHV-8-seropositive individuals, all of whom were males and HHV-8 ELISA seropositive. In immunologically controlled populations of Brazil, HHV-8 oral shedding was limited to HHV-8-seropositive men, occurred infrequently and intermittently, and was not linked to HHV-8 viremia, suggesting a limited potential for oral or blood transmission.

A50 Genotypic distribution of HHV-8 in aids individuals without and with Kaposi sarcoma

Rotaviruses of species A (RVA) are a common cause of diarrhea in children and the young of various other mammals worldwide. Interspecies transmission of RVA may lead to the emergency of novel RVA strains which may potentially affect rotavirus vaccine efficacy. The aim of this study was to investigate for possible interspecies transmission of RVAs in Uganda. Whole-genome sequencing of eighteen human (under-fives with diarrhea) and six animal (one bovine, one caprine, and four porcine) RVA strains identified in Uganda in the same geographical region, between 2012 and 2014 was undertaken using the Illumina HiSeq platform. RotaC version 2, a classification tool for RVAs was used to assign genotypes to all eleven genome segments of each isolate. Phylogenetic analysis was carried out using the maximum likelihood method in MEGA 6.06. Human RVA strains had either a Waor a DS-1-like genetic constellation. One human strain was a Wa-like mono-reassortant containing a DS-1-like VP2 gene of possible animal origin. In addition, three human RVA strains had one or two genes with possible zoonotic origin. All eleven genes of the bovine RVA strain were closely related to those of human RVAs. The caprine strain had a mixed genotype backbone, suggesting that it emerged from multiple re-assortment events involving different host species. Porcine RVA strains had mixed genotype backbones with possible multiple reassortant events with strains of human and bovine origin. Interspecies transmission of RVA strains occurred in this setting. RVA strains causing diarrhea in children are primarily transmitted from person to person. Rotavirus vaccination in children in Uganda will control rotavirus transmission. It is recommended to continue molecular surveillance of RVAs in humans and animals living in the same geographical region to understand the molecular epidemiology and evolution of RVAs in Uganda and other countries.

Role of T. cruzi exposure in the pattern of T cell cytokines among chronically infected HIV and Chagas disease patients

OBJECTIVES: The impact of Chagas disease (CD) in HIV-infected patients is relevant throughout the world. In fact, the characterization of the adaptive immune response in the context of co-infection is important for predicting the need for interventions in areas in which HIV and Chagas disease co-exist. METHODS: We described and compared the frequency of cytokine-producing T cells stimulated with soluble antigen of Trypanosoma cruzi (T. cruzi) using a cytometric assay for the following groups: individuals with chronic Chagas disease (CHR, n=10), those with Chagas disease and HIV infection (CO, n=11), those with only HIV (HIV, n=14) and healthy individuals (C, n=15). RESULTS: We found 1) a constitutively lower frequency of IL-2+ and IFN-γ+ T cells in the CHR group compared with the HIV, CO and healthy groups; 2) a suppressive activity of soluble T. cruzi antigen, which down-regulated IL-2+CD4+ and IFN-γ+CD4+ phenotypes, notably in the healthy group; 3) a down-regulation of inflammatory cytokines on CD8+ T cells in the indeterminate form of Chagas disease; and 4) a significant increase in IL-10+CD8+ cells distinguishing the indeterminate form from the cardiac/digestive form of Chagas disease, even in the presence of HIV infection. CONCLUSIONS: Taken together, our data suggest the presence of an immunoregulatory response in chronic Chagas disease, which seems to be driven by T. cruzi antigens. Our findings provide new insights into immunotherapeutic strategies for people living with HIV/AIDS and Chagas disease.