RESUMO
The multiplex molecular diagnostic assays described for severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), influenza A (IAV) and B (IBV) viruses have been mainly based on real-time reaction, which limits their access to many laboratories or diagnostic institutions. To contribute to available strategies and expand access to differential diagnosis, we describe an end-point multiplex RT-PCR targeting SARS-CoV-2, IAV and IBV with simultaneous endogenous control amplification. Initially, we looked for well-established primers sets for SARS-CoV-2, IAV, IBV and RNAse P whose amplicons could be distinguished on agarose gel. The multiplex assay was then standardized by optimizing the reaction mix and cycle conditions. The limit of detection (LoD) was determined using titrated viruses (for SARS-CoV-2 and IAV) and by dilution from a pool of IBV-positive samples. The diagnostic performance of the multiplex was evaluated by testing samples with different RNAse P and viral loads, previously identified as positive or negative for the target viruses. The amplicons of IAV (146 bp), SARS-CoV-2 (113 bp), IBV (103 bp) and RNAse P (65 bp) were adequately distinguished in our multiplex. The LoD for SARS-CoV-2, IAV and IBV was 0.02 TCID50/ml, 0.07 TCID50/ml and 10-3 from a pool of positive samples, respectively. All samples positive for SARS-CoV-2 (n=70, Ct 17.2-36.9), IAV (n=53, Ct 14-34.9) and IBV (n=12, Ct 23.9-31.9) remained positive in our multiplex assay. RNAse P from negative samples (n=40, Ct 25.2-30.2) was also amplified in the multiplex. Overall, our assay is a timely and alternative tool for detecting SARS-CoV-2 and influenza viruses in laboratories with limited access to supplies/equipment.
Assuntos
COVID-19 , Vírus da Influenza A , Vírus da Influenza B , Reação em Cadeia da Polimerase Multiplex , Ribonuclease P , SARS-CoV-2 , Humanos , Ribonuclease P/genética , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/genética , Vírus da Influenza B/isolamento & purificação , Vírus da Influenza B/genética , COVID-19/diagnóstico , COVID-19/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Diagnóstico Diferencial , Influenza Humana/diagnóstico , Influenza Humana/virologia , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Limite de Detecção , RNA Viral/genética , RNA Viral/análiseRESUMO
Papillomaviruses (PVs) have been identified in several animal species, including dogs (canine papillomaviruses, CPVs) and cattle (bovine papillomaviruses, BPVs). Although some BPVs may occasionally infect species other than cattle, to the best of our knowledge, BPVs have not been reported in dogs to date. Herein, we carried out a retrospective phylogenetic study of PVs circulating in dogs from southern Brazil between 2017 and 2022, also investigating possible mixed infections and spillover events. For this, we screened 32 canine papilloma samples by PCR using the degenerate primers FAP59/64 and/or MY09/11, which amplify different regions of the L1 gene; the genomic target often used for PV classification/typing. Out these, 23 PV DNA samples were successfully amplified and sequenced. All PVs amplified by FAP59/64 (n = 22) were classified as CPV-1. On the other hand, PVs amplified by MY09/11 (n = 4) were classified as putative BPV-1. Among these, three samples showed mixed infection by CPV-1 and putative BPV-1. One of the putative BPV-1 detected in co-infected samples had the L1 gene full-sequenced, confirming the gene identity. Furthermore, the phylogenetic classifications from the FAP59/64 and/or MY09/11 amplicons were supported by a careful in silico analysis, which demonstrated that the analysis based on them matches to the classification from the complete L1 gene. Overall, we described CPV-1 circulation in southern Brazil over the years and the potencial BPV infection in dogs (potential spillover event), as well as possible CPV/1/BPV-1 co-infections. Finally, we suggest the analysis of the complete genome of the putative BPVs detected in dogs in order to deepen the knowledge about the PV-host interactions.
Assuntos
Coinfecção , Doenças do Cão , Epidemiologia Molecular , Papillomaviridae , Infecções por Papillomavirus , Filogenia , Animais , Cães , Brasil/epidemiologia , Doenças do Cão/virologia , Doenças do Cão/epidemiologia , Infecções por Papillomavirus/veterinária , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/epidemiologia , Papillomaviridae/genética , Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Estudos Retrospectivos , Coinfecção/virologia , Coinfecção/veterinária , Coinfecção/epidemiologia , DNA Viral/genéticaRESUMO
Canine distemper virus (CDV) is a major threat to domestic dogs and wildlife worldwide. Molecular assays are the most sensitive and specific tests to diagnose the disease, however, the high CDV genetic variability may compromise laboratory diagnosis. Herein, we designed a high-coverage primer set for end-point (RT-PCR) and real-time (RT-qPCR) for CDV detection. Initially, we collected 194 complete/near-complete CDV genomes (GenBank) and analyzed them for highly conserved regions for primer design. We then assessed the in silico coverage, analytical sensitivity, specificity and diagnostic performance of RT-PCR/RT-qPCR reactions based on our primers. Furthermore, the coverage of our primers, as well as their analytical sensitivity and diagnostic performance, were compared to a commonly used primer set for CDV detection (named PP-I). Our forward (F) and reverse (R) primers fully matched 100 % (194/194) and 99 % (192/194) of the analyzed sequences, whereas the PP-I F and R primers fully matched 15 % (29/194) and 9 % (18/194) sequences, respectively. The detection limit of our RT-PCR and RT-qPCR was equivalent to that of PP-I primers (0.001 TCID50/mL). Out of 70 clinical samples tested, 38 were positive by our RT-PCR/RT-qPCR assays, whereas reactions with primers PP-I failed to detect 9/28 (32 %) positive samples selected for comparison purposes. In addition, our assays did not amplify other canine viruses associated with respiratory and neurological diseases: canine adenovirus 2, canine parainfluenza virus 2, canine herpesvirus 1 and rabies virus. Overall, we describe a high-coverage primer set for CDV detection, which represents an attractive tool for laboratory diagnosis of canine distemper.
Assuntos
Vírus da Cinomose Canina , Cinomose , Animais , Cães , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus da Cinomose Canina/genética , Sensibilidade e Especificidade , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Cinomose/diagnósticoRESUMO
BACKGROUND: The severity and distribution of dengue virus (DENV) infections have been attributed to a complex interaction among viral, host and environmental factors. Herein, we investigated the influence of chikungunya (CHIKV) and Zika (ZIKV) viruses on the epidemiological profile of dengue cases, using Recife, Pernambuco state, Brazil, as a study model. In addition, we described and compared the epidemiological profile related to each arbovirus (DENV vs. CHIKV vs. ZIKV). METHODS: All cases of dengue, chikungunya and Zika reported to the Pernambuco Health Department in 2011-2013 (DENV circulation) and 2016-2018 (DENV, CHIKV and ZIKV co-circulation) were included in our study. The cases were classified by sex, age and race/color and their distribution was analyzed by the χ2 test. Furthermore, the data were also analyzed for co-infections. Temperature, humidity and rainfall data were analyzed using one-way ANOVA and paired t-test. RESULTS: During 2011-2013, 15,315 dengue cases were diagnosed, most of them female, brown and 20-29 age group. Between 2016 and 2018, 15,870 dengue cases were described, which presented the same profile described above. In the two triennia, the female/male dengue ratio fluctuated significantly, ranging from 1.07 to 1.52. Regarding chikungunya, 7076 cases were reported, most of them female and brown. The female/male ratio also fluctuated significantly, ranging from 1.62 to 2.1. Two main age groups were observed in chikungunya: ≤ 19 years (minority of diagnoses) and ≥ 20 years (majority of diagnoses). In the same triennium, 266 Zika cases were reported to the Pernambuco Health Department, mainly in females and in the 0-9 and 20-39 age groups. In general, 119 co-infections were identified: 117 DENV-CHIKV, 1 CHIKV-ZIKV and 1 DENV-CHIKV-ZIKV. Concerning climate data, only the humidity in 2011 was significantly different from the other years. CONCLUSION: The epidemiological profile of dengue cases did not change after the introduction of CHIKV and ZIKV. Females were the most diagnosed with dengue, chikungunya or Zika, however we found important differences in the age profile of these arboviruses, which should be considered by public health policies, as well as investigated in future studies of virus-host interaction.
Assuntos
Arbovírus , Febre de Chikungunya , Vírus Chikungunya , Coinfecção , Vírus da Dengue , Dengue , Infecção por Zika virus , Zika virus , Masculino , Humanos , Feminino , Adulto Jovem , Adulto , Infecção por Zika virus/complicações , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/diagnóstico , Febre de Chikungunya/diagnóstico , Dengue/diagnóstico , Coinfecção/epidemiologiaRESUMO
INTRODUCTION: Human T-lymphotropic virus (HTLV) 1 and 2 infections can lead to neurological diseases, mainly in HIV/HTLV 1 coinfected. Furthermore, HTLV 1 infection in HIV/AIDS patients has also been associated with AIDS progression. Despite this, HTLV 1/2 infections are not of mandatory notification in Brazil. Here, we describe the prevalence of HTLV 1/2 in HIV/AIDS patients from Paraíba state, Brazil, as well as the sociodemographic characteristics of the coinfected individuals. METHODOLOGY: Information about HIV viral load and TCD4 lymphocyte count were obtained from patients' records. Data on the patients' sociodemographic characteristics were obtained by interview conducted after signing the informed consent form. The serological diagnosis for HTLV 1/2 was performed by Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB). RESULTS: A total of 401 HIV/AIDS patients participated in the study, of whom about 1.5% (6/401) were positive for antibodies against HTLV, specifically for HTLV 1, evaluated by both ELISA and WB. No risk factors were found associated with HIV/HTLV 1/2 coinfection. CONCLUSIONS: We report a 1.5% prevalence of HTLV 1 infection in HIV/AIDS patients from Paraíba state. Although we have not identified risk factors associated with HTLV 1, we describe the most observed sociodemographic characteristics in HIV/HTLV 1 coinfection.
Assuntos
Infecções por HIV/epidemiologia , Infecções por HTLV-I/epidemiologia , Brasil/epidemiologia , Coinfecção , Estudos Transversais , Feminino , Infecções por HIV/transmissão , Infecções por HTLV-I/transmissão , Humanos , Masculino , Prevalência , Fatores de RiscoRESUMO
Imbalance in the immune response is one of the main pathogenic mechanisms of diseases related with human immunodeficiency virus (HIV)/human gammaherpesvirus 8 (HHV-8) coinfection, such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD) and the Kaposi's sarcoma-associated herpesvirus inflammatory cytokine syndrome (KICS). However, significant changes in pro- and anti-inflammatory cytokine levels may be observed in HIV/HHV-8 individuals who are negative for KS, PEL, MCD, and/or KICS. In this study, serum levels of interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor nucrosis factor α (TNF-α) and interferon γ (IFN-γ) were assessed in 69 HIV and 48 HIV/HHV-8 individuals, all negatives for HHV-8-related diseases. The cytokines were measured by flow cytometry and analyzed by the Mann-Whitney test. The p < .05 and 95% confidence interval were considered in all analyzes. IL-4 (p = .0155), IL-6 (p = .0036), and IL-10 (p = .0036) levels were significantly higher in HIV/HHV-8 patients than in the HIV group. On the other hand, IL-2 (p = .2295), TNF-α (p = .1216) and IFN-γ (p = .1178) did not differ between the groups analyzed. To our knowledge, to date, this is the first report on significant differences in the levels of IL-4 and IL-6 in HIV versus HIV/HHV-8 individuals. Finally, these early findings are important as a prognostic tool and contribute to clarifying the HHV-8-host interaction.
Assuntos
Citocinas/genética , Citocinas/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 8/imunologia , Interferon gama/genética , Fator de Necrose Tumoral alfa/genética , Adulto , Estudos de Casos e Controles , Citocinas/classificação , Feminino , Infecções por HIV/sangue , Infecções por HIV/virologia , Infecções por Herpesviridae/sangue , Infecções por Herpesviridae/virologia , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: The spread of Dengue virus (DENV) infections, as well as their signs and symptoms, are the result of a complex interaction between several factors. In Brazil, especially in the Northeastern, dengue is an important public health problem. Here, we report an epidemiological analysis of dengue cases in Pernambuco state, Northeastern Brazil, during 2015-2017. METHODS: This work is a retrospective cross-sectional observational study on the epidemiological profile of all dengue cases confirmed and reported to the Health Secretary of Pernambuco between 2015 and 2017. These data cover all municipalities of Pernambuco, except Fernando de Noronha. DENV-positive individuals were classified according to the dengue type (without and with warning signs, or severe dengue), age, gender, ethnicity and intermediate geographic region of residence (Recife, Caruaru, Serra Talhada or Petrolina). The distribution of cases over the years was assessed by χ2 test. Temperature and rainfall data were evaluated by Unpaired t-test. p-value < 0.05 and CI 95% were considered in all analyses. RESULTS: Most dengue cases was without warning signs. The most observed characteristics in the less severe dengue phenotypes were: female, mulatto ethnicity and age between 20 and 39 years old; this profile was more clearly observed in 2015. In 2016 and 2017, however, the numbers of dengue without and with warning signs were more evenly distributed and the difference in cases within groups decreased significantly. Regarding severe dengue, mulattoes were the most affected, but it is possible to note a trend towards a more uniform distribution between the genders and ages. Recife was the region with the highest numbers of both total cases and incidence rates and the highest rainfall levels. Overall, over the years, there has been a decrease in dengue cases in all regions of Pernambuco. CONCLUSIONS: We identified the epidemiological profile of dengue in Pernambuco, Brazil, reporting the gender, age, ethnicity and regions most affected by different dengue types. In addition, we observed that these cases were probably more influenced by rainfall than by temperature. Finally, we believe that this epidemiological knowledge is important to direct public health policies to the reality of each population.
Assuntos
Dengue/epidemiologia , Adolescente , Adulto , Brasil/epidemiologia , Criança , Pré-Escolar , Estudos Transversais , Demografia , Dengue/etnologia , Vírus da Dengue , Feminino , Humanos , Incidência , Lactente , Masculino , Pessoa de Meia-Idade , Chuva , Estudos Retrospectivos , Dengue Grave/epidemiologia , Adulto JovemRESUMO
Human gammaherpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS), one of the most common cancers in people living with HIV/AIDS. It is believe that the course of both HIV and HHV-8 infection is associated with the imbalance of anti- and/or pro-inflammatory cytokines. Here, we evaluated the IL-6, TNF-α, IL-10, CCL2 and CXCL10 serum concentrations in HIV- and HIV/HHV-8 (without KS) individuals, and in patients with cutaneous or visceral AIDS-KS. Serum concentrations of IL-6, IL-10 and CXCL10 were significantly higher in the AIDS-KS group compared to HIV and HIV/HHV-8 individuals. Similarly, the concentrations of theses cytokines were higher in patients with visceral than in those with cutaneous AIDS-KS. The TNF-α concentration was significantly higher in the HIV group compared to HIV/HHV-8 (with and without KS) individuals, and CCL2 levels did not present significant difference among the groups. The HIV viral load was undetectable in all patients from the HIV and HIV/HHV-8 groups. On the other hand, in the AIDS-KS group, most patients had detectable HIV viral load. In this context, we believe that the cytokine levels in AIDS-KS may be result of a complex interaction between HIV, HHV-8 and immunity