Laboratory diagnosis of measles infection using molecular and serology during 2019-2020 outbreak in Brazil.

INTRODUCTION: Laboratory diagnosis of measles can be challenging, and the reintroduction of the measles virus in Brazil has brought about new issues. The aim of this study was to analyze the qPCR results of swab and urine samples and compare them with those of immunological methods for the diagnosis of measles. METHODS: This was a cross-sectional study based on a retrospective analysis of 3,451 suspected cases using laboratory test surveillance databases for qPCR (respiratory swabs and urine) and serologic tests for IgM and paired IgG. Sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and agreement through kappa and adjusted kappa coefficients (PABAK) were calculated using different diagnostic strategies. RESULTS: The swab and urine samples obtained using real-time qPCR were equivalent. Samples collected simultaneously and the combined samples showed moderate agreement between IgM ELISA and real-time qPCR; however, 48.9 % of the IgM ELISA analyses did not demonstrate detectable qPCR concentrations during simultaneous collections and 43.9 % of combined collections. The paired analysis of IgG showed an accuracy of 67.5 % for IgM and 90.7 % for real-time qPCR. CONCLUSIONS: Diagnosis based on IgM presents detection delimitation in samples collected early (1-5 days), suggesting that these individuals satisfy at least two criteria. In addition to qPCR, paired analysis of IgG using ELISA can be used to increase the sensitivity and specificity of laboratory diagnoses.

Prevalence of Chlamydia trachomatis in Women Who Are Candidates for In Vitro Fertilization in a Private Reference Service in Southern Brazil: A Cross-Sectional Study.

Introduction Chlamydia trachomatis (CT) has been related to fallopian tube damage and infertility. Its prevalence in the population that attend public services is known; however, there is scant data on this factor in private infertility clinics. The objective of this study is to verify the prevalence of CT among women attending a private in vitro fertilization (IVF) reference clinic in southern Brazil. Methods This is a cross-sectional study carried out between January 1, 2019, and August 30, 2021, at an IVF private clinic in southern Brazil. Infertile women between 18 and 50 years old, who provided a morning urinary sample for reverse transcription-polymerase chain reaction (RT-PCR) test for CT analysis, were included in the study. The variables studied included the patient's age, body mass index (BMI), duration of infertility, type of infertility, indication for IVF, and detection or not of CT in the urine. Results The prevalence of CT was 10.84% (22 out of 203; 95% CI: 7.27-15.87). Patients with secondary infertility were older and had more ovarian and tubal factors compared to cases of primary infertility. The tubal factor was the most prevalent (27.3% in women with primary infertility and 35.8% in those with secondary). Time of infertility and BMI were similar between groups. Our results are derived from a single private IVF clinic which reduces the external validity. Conclusion The prevalence of 10.84% of CT in this population raises the importance of screening for sexually transmitted infections for proper treatment and to achieve better IVF outcomes.

Comparison among plaque assay, tissue culture infectious dose (TCID50) and real-time RT-PCR for SARS-CoV-2 variants quantification.

Background and Objectives: SARS-CoV-2 variants of concern (VOC) and interest (VOI) pose a significant threat to public health because the rapid change in the SARS-CoV-2 genome can alter viral phenotypes such as virulence, transmissibility and the ability to evade the host response. Hence, SARS-CoV-2 quantification techniques are essential for timely diagnosis and follow-up. Besides, they are vital to understanding viral pathogenesis, antiviral evaluation, and vaccine development. Materials and Methods: Five isolates of SARS-CoV-2: D614G strain (B.1), three VOC (Alpha, Gamma and Delta), and one VOI (Mu) were used to compare three techniques for viral quantification, plaque assay, median tissue culture infectious dose (TCID50) and real-time RT-PCR. Results: Plaque assay showed viral titers between 0.15 ± 0.01×107 and 1.95 ± 0.09×107 PFU/mL while viral titer by TCID50 assay was between 0.71 ± 0.01×106 to 4.94 ± 0.80×106 TCID50/mL for the five SARS-CoV-2 isolates. The PFU/mL titer obtained by plaque and the calculated from TCID50 assays differed by 0.61 log10, 0.59 log10, 0.59 log10 and 0.96 log10 for Alfa, Gamma, Delta, and Mu variants (p≤0.0007), respectively. No differences were observed for the D614G strain. Real-time PCR assay exhibited titers ranging from 0.39 ± 0.001×108 to 3.38 ± 0.04×108 RNA copies/µL for all variants. The relation between PFU/mL and RNA copies/mL was 1:29800 for D614G strain, 1:11700 for Alpha, 1:8930 for Gamma, 1:12500 for Delta, and 1:2950 for Mu. Conclusion: TCID50 assay was comparable to plaque assay for D614G but not for others SARS-CoV-2 variants. Our data demonstrated a correlation among PFU/mL and E gene RNA copies/µL, units of measure commonly used to quantify the viral load in diagnostic and research fields. The results suggest that the proportion of infectious virions in vitro changes depending on the SARS-CoV-2 variant, being Mu, the variant reaching a higher viral titer with fewer viral copies.

Automated Reverse Transcription Polymerase Chain Reaction Data Analysis for Sars-CoV-2 Detection.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is a current public health concern. Rapid diagnosis is crucial, and reverse transcription polymerase chain reaction (RT-PCR) is presently the reference standard for SARS-CoV-2 detection. OBJECTIVE: Automated RT-PCR analysis (ARPA) is a software designed to analyze RT-PCR data for SARSCoV-2 detection. ARPA loads the RT-PCR data, classifies each sample by assessing its amplification curve behavior, evaluates the experiment's quality, and generates reports. METHODS: ARPA was implemented in the R language and deployed as a Shiny application. We evaluated the performance of ARPA in 140 samples. The samples were manually classified and automatically analyzed using ARPA. RESULTS: ARPA had a true-positive rate = 1, true-negative rate = 0.98, positive-predictive value = 0.95, and negative-predictive value = 1, with 36 samples correctly classified as positive, 100 samples correctly classified as negative, and two samples classified as positive even when labeled as negative by manual inspection. Two samples were labeled as invalid by ARPA and were not considered in the performance metrics calculation. CONCLUSIONS: ARPA is a sensitive and specific software that facilitates the analysis of RT-PCR data, and its implementation can reduce the time required in the diagnostic pipeline.

Predictors of severe symptomatic laboratory-confirmed SARS-CoV-2 reinfection.

OBJECTIVE: The aim of the study was to evaluate factors predicting severe symptomatic laboratory-confirmed (via Reverse transcription polymerase chain reaction, RT-PCR polymerase chain reaction) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection. STUDY DESIGN: This is a nationwide retrospective cohort study that was conducted in Mexico. METHODS: Data from 258 reinfection cases (at least 28 days between both episodes onset) were analyzed. We used risk ratios (RRs) and 95% confidence intervals (CIs) to evaluate predictors of severe (dyspnea requiring hospital admission) secondary SARS-CoV-2 infection. RESULTS: The risk of severe disease was 14.7%, and the observed overall fatality rate was 4.3%. Patients with more serious primary disease were more likely to develop severe symptoms (39.5% vs. 5.5%, P < 0.001) during reinfection. In multiple analysis, factors associated with an increased risk of severe symptomatic SARS-CoV-2 reinfection were increasing age (RRper year = 1.007, 95% CI = 1.003-1.010), comorbidities (namely, obesity [RR = 1.12, 95% CI = 1.01-1.24], asthma [RR = 1.26, 95% CI = 1.06-1.50], type 2 diabetes mellitus [RR = 1.22, 95% CI = 1.07-1.38]), and previous severe laboratory-confirmed coronavirus disease 2019 (RR = 1.20, 95% CI = 1.03-1.39). CONCLUSIONS: To the best of our knowledge, this is the first study evaluating disease outcomes in a large set of laboratory-positive cases of symptomatic SARS-CoV-2 reinfection, and factors associated with illness severity were characterized. Our results may contribute to the current knowledge of SARS-CoV-2 pathogenicity and to identify populations at increased risk of a poorer outcome after reinfection.

Exuberant bullous vasculitis associated with SARS-CoV-2 infection.

We described a case of exuberant cutaneous small-vessel vasculitis in a 27-year-old male with mild CoVID-19 in Brazil. The patient presented painful purpuric papules and vesicobullous lesions with hemorrhagic content located in the larger amount in the lower limbs and, to a lesser extent in the region of the back and upper limbs, saving palms and soles of the feet. Influenza-like syndrome with anosmia and ageusia was reported seven days before the skin lesions. A real-time reverse transcription polymerase chain reaction was positive on a nasopharyngeal swab for SARS-CoV-2. Histopathological study showed leukocytoclastic cutaneous vasculitis affecting small vessels and microthrombi occluding some vessels. The patient presented an improvement in skin lesions by the fifth day of prednisone therapy. This case highlights the importance of the SARS-CoV-2 test in investigating the etiology of cutaneous vasculitis during this pandemic.

Response to SARS-CoV-2 Pandemic in a Non-COVID-19 Designated Latin-American Neurosurgery Department.

Background: Mexico declared the first case of novel coronavirus disease (COVID-19) in February 2020. At the time we write this article, our country is facing a community spread phase, expecting a rapid increase in the number of cases and fatalities. The Fray Antonio Alcalde Civil Hospital of Guadalajara has been declared a non-COVID-19 hospital with the mission of providing care to patients already registered and also those transferred from neurosurgical departments of neighboring centers, which have been converted into COVID-19 only hospitals. Methods: An organized response regarding personnel, surgical case selection, operating room behavior, and facility reorganization were designed to prevent an internal coronavirus outbreak in the neurosurgery department at the Fray Antonio Alcalde Civil Hospital of Guadalajara. Results: Distancing actions by the staff and residents, including ward case discussions, neurosurgery rounds, and classes, will be carried out virtually. We classified neurosurgical patients into 4 groups depending on whether their condition demands care in 0-6 hours, 6-48 hours, 48 hours to 14 days, and >14 days. Subsequently, a questionnaire with epidemiologic, radiologic, clinical, and serologic criteria will be applied to determine the risk of COVID-19 infection to define to which area they are going to be transferred according to the different risk zones in our facility. Conclusions: Despite not being a COVID-19 center, we consider all patients at the neurosurgical ward and staff members as asymptomatic carriers or infected in the preclinical period. Specific measures must be taken to ensure the safety and care of neurosurgical patients and medical staff during the community spread phase.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Obtention and characterization of the recombinant simian Interleukin-15 in Escherichia coli for the preclinical assessment of an IL-15-based therapeutic vaccine.

Recombinant simian IL-15 (siIL-15) was obtained for the preclinical assessment of an anti-human IL-15 vaccine. For this purpose, the cDNA from peripheral blood mononuclear cells of a Macaca fascicularis monkey was cloned into a pIL-2 vector. The siIL-15 was expressed in Escherichia coli strain W3110 as an insoluble protein which accounted for 13% of the total cellular proteins. Inclusion bodies were solubilized in an 8 M urea solution, which was purified by ion exchange and reverse phase chromatography up to 92% purity. The protein identity was validated by electrospray ionization-mass spectrometry, confirming the presence of the amino acids which distinguish the siIL-15 from human IL-15. The purified siIL-15 stimulates the proliferation of cytotoxic T-lymphocytes line (CTLL)-2 and Kit 225 cells with EC50 values of 3.1 and 32.5 ng/mL, respectively. Antisera from modified human IL-15-immunized macaques were reactive to human and simian IL-15 in enzyme-linked immunosorbent assays. Moreover, the anti-human IL-15 antibodies from immune sera inhibited siIL-15 activity in CTLL-2 and Kit 225 cells, supporting the activity and purity of recombinant siIL-15. These results indicate that the recombinant siIL-15 is biologically active in two IL-15-dependent cell lines, and it is also suitable for the preclinical evaluation of an IL-15-based therapeutic vaccine.

Evaluation of photodynamic therapy in adhesion protein expression.

Photodynamic therapy (PDT) is a treatment modality that has clinical applications in both non-neoplastic and neoplastic diseases. PDT involves a light-sensitive compound (photosensitizer), light and molecular oxygen. This procedure may lead to several different cellular responses, including cell death. Alterations in the attachment of cancer cells to the substratum and to each other are important consequences of photodynamic treatment. PDT may lead to changes in the expression of cellular adhesion structure and cytoskeleton integrity, which are key factors in decreasing tumor metastatic potential. HEp-2 cells were photosensitized with aluminum phthalocyanine tetrasulfonate and zinc phthalocyanine, and the proteins ß1-integrin and focal adhesion kinase (FAK) were assayed using fluorescence microscopy. The verification of expression changes in the genes for FAK and ß1 integrin were performed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that HEp-2 cells do not express ß-integrin or FAK 12 h following PDT. It was concluded that the PDT reduces the adhesive ability of HEp-2 cells, inhibiting their metastatic potential. The present study aimed to analyze the changes in the expression and organization of cellular adhesion elements and the subsequent metastatic potential of HEp-2 cells following PDT treatment.

Chloroquine interferes with dengue-2 virus replication in U937 cells.

The objective of this study was to investigate the use of chloroquine (CLQ) as an antiviral agent against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of U937 cells infected with dengue virus type 2 (DENV-2). Viral replication was assessed by quantification of virus produced through detection of copy numbers of DENV-2 RNA, plaque assay and indirect immunofluorescence. qRT-PCR and plaque assays were used to quantify the DENV-2 load in infected U937 cells after CLQ treatment. It was found that a dose of 50 µg/mL of CLQ was not toxic to the cells and resulted in significantly less virus production in infected U937 cells than occurred in untreated cells. In the present work, CLQ was effective against DENV-2 replication in U937 cells, and also caused a statistically significant reduction in expression of proinflammatory cytokines. The present study indicates that CLQ may be used to reduce viral yield in U937 cells.

Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease.

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.

The potential effects of Zataria multiflora Boiss essential oil on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes of toxigenic Aspergillus parasiticus.

This study aims at evaluating the effects of Zataria multiflora (Z. multiflora) essential oil (EO) on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes. Total RNAs of Aspergillus parasiticus (A.parasiticus) ATCC56775 grown in yeast extract sucrose (YES) broth medium treated with Z. multiflora EO were subjected to reverse transcription- polymerase chain reaction (RT-PCR). Specific primers of nor-1, ver-1, omt-A and aflR genes were used. In parallel mycelial dry weight of samples were measured and all the media were assayed by high-pressure liquid chromatography (HPLC) for aflatoxinB1 (AFB1), aflatoxinB2 (AFB2), aflatoxinG1 (AFG1), aflatoxinG2 (AFG2) and aflatoxin total (AFTotal) production. The results showed that mycelial dry weight and aflatoxin production reduce in the presence of Z. multiflora EO (100 ppm) on day 5 of growth. It was found that the expression of nor-1, ver-1, omt-A and aflR genes was correlated with the ability of fungus to produce aflatoxins on day 5 in YES medium. RT-PCR showed that in the presence of Z.multiflora EO (100 ppm) nor-1, ver-1 and omtA genes expression was reduced. It seems that toxin production inhibitory effects of Z. multiflora EO on day 5 may be at the transcription level and this herb may cause reduction in aflatoxin biosynthesis pathway genes activity.

Proline-induced changes in acetylcholinesterase activity and gene expression in zebrafish brain: reversal by antipsychotic drugs.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures, cognitive dysfunctions, and schizoaffective disorders. However, the mechanisms related to these symptoms are still unclear. In the present study, we evaluated the in vivo and in vitro effects of proline on acetylcholinesterase (AChE) activity and gene expression in the zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0mM) during 1h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 µM) were tested. Long-term proline exposures significantly increased AChE activity for both treated groups when compared to the control (34% and 39%). Moreover, the proline-induced increase on AChE activity was completely reverted by acute administration of antipsychotic drugs (haloperidol and sulpiride), as well as the changes induced in ache expression. When assessed in vitro, proline did not promote significant changes in AChE activity. Altogether, these data indicate that the enzyme responsible for the control of acetylcholine levels might be altered after proline exposure in the adult zebrafish. These findings contribute for better understanding of the pathophysiology of hyperprolinemia and might reinforce the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

The potential effects of Zataria multiflora Boiss essential oil on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes of toxigenic Aspergillus parasiticus

This study aims at evaluating the effects of Zataria multiflora (Z. multiflora) essential oil (EO) on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes. Total RNAs of Aspergillus parasiticus (A.parasiticus) ATCC56775 grown in yeast extract sucrose (YES) broth medium treated with Z. multiflora EO were subjected to reverse transcription-polymerase chain reaction (RT-PCR). Specific primers of nor-1, ver-1, omt-A and aflR genes were used. In parallel mycelial dry weight of samples were measured and all the media were assayed by high-pressure liquid chromatography (HPLC) for aflatoxinB1 (AFB1), aflatoxinB2 (AFB2), aflatoxinG1 (AFG1), aflatoxinG2 (AFG2) and aflatoxin total (AFTotal) production. The results showed that mycelial dry weight and aflatoxin production reduce in the presence of Z. multiflora EO (100 ppm) on day 5 of growth. It was found that the expression of nor-1, ver-1, omt-A and aflR genes was correlated with the ability of fungus to produce aflatoxins on day 5 in YES medium. RT-PCR showed that in the presence of Z.multiflora EO (100 ppm) nor-1, ver-1 and omtA genes expression was reduced. It seems that toxin production inhibitory effects of Z. multiflora EO on day 5 may be at the transcription level and this herb may cause reduction in aflatoxin biosynthesis pathway genes activity.

The potential effects of Zataria multiflora Boiss essential oil on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes of toxigenic Aspergillus parasiticus

This study aims at evaluating the effects of Zataria multiflora (Z. multiflora) essential oil (EO) on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes. Total RNAs of Aspergillus parasiticus (A.parasiticus) ATCC56775 grown in yeast extract sucrose (YES) broth medium treated with Z. multiflora EO were subjected to reverse transcription-polymerase chain reaction (RT-PCR). Specific primers of nor-1, ver-1, omt-A and aflR genes were used. In parallel mycelial dry weight of samples were measured and all the media were assayed by high-pressure liquid chromatography (HPLC) for aflatoxinB1 (AFB1), aflatoxinB2 (AFB2), aflatoxinG1 (AFG1), aflatoxinG2 (AFG2) and aflatoxin total (AFTotal) production. The results showed that mycelial dry weight and aflatoxin production reduce in the presence of Z. multiflora EO (100 ppm) on day 5 of growth. It was found that the expression of nor-1, ver-1, omt-A and aflR genes was correlated with the ability of fungus to produce aflatoxins on day 5 in YES medium. RT-PCR showed that in the presence of Z.multiflora EO (100 ppm) nor-1, ver-1 and omtA genes expression was reduced. It seems that toxin production inhibitory effects of Z. multiflora EO on day 5 may be at the transcription level and this herb may cause reduction in aflatoxin biosynthesis pathway genes activity.(AU)

Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid ...

Diseño y aplicación de un método molecular para el diagnóstico del virus influenza A (H1N1) pandémico en Cuba / Design and implementation of a molecular method for influenza A virus (H1N1) in Cuba

INTRODUCCIÓN: entre marzo y abril de 2009 se produjeron en México brotes de enfermedad respiratoria, debido a un nuevo virus de influenza proveniente del cerdo, el cual se diseminó rápidamente mediante la transmisión humano-humano. Los métodos moleculares usados en la actualidad eran inadecuados, porque la composición del genoma del nuevo virus era muy diferente del virus influenza A (H1N1) que había circulado hasta el momento. En su composición, estaba formado por segmentos de genes de origen aviar, humano y cerdo. OBJETIVO: teniendo en cuenta las secuencias publicadas, se diseñó un juego de cebadores específicos para el gen de la hemaglutinina, con la finalidad de evaluar un nuevo ensayo de TR-RCP para detectar el nuevo virus pandémico en Cuba. MÉTODOS: se procesó un total de 3 197 muestras clínicas de casos sospechosos de infección por el virus influenza A (H1N1) pandémico (pdm) mediante un ensayo de transcripción reversa-reacción en cadena de la polimerasa. RESULTADOS: el ensayo optimizado permitió obtener una banda de 292 pb, sin reacciones inespecíficas. El nuevo método resultó ser útil en el diagnóstico y subtipado del virus de influenza H1N1 pdm. El producto amplificado fue analizado por secuenciación nucleotídica y se confirmó la identificación del virus. CONCLUSIONES: con la introducción de este nuevo ensayo para la vigilancia de influenza, se fortalece la capacidad diagnóstica del Laboratorio Nacional de Referencia.

INTRODUCTION: from March through April of 2009, Mexico notified outbreaks of respiratory illness, due to a new influenza virus of swine origin, which spread over rapidly via human-to-human transmission. The molecular methods currently in use were not suitable because the genome composition based on gene segments of swine, avian and human origin was quite different from the influenza A virus (H1N1) circulating at that time. OBJECTIVE: based on the published sequences, a set of specific primers for the HA gene was designed to evaluate a new RT-PCR assay. METHODS: the RT-PCR assay processed 3 197 clinical samples from suspected cases of pandemic influenza A (H1N1) infection. RESULTS: the novel optimized method obtained a 262 pb segment, without unspecific reactions. The new method proved to be useful in the diagnosis and subtyping of pandemic HINI influenza virus. The amplified product was verified by nucleotide sequencing, thus confirming the virus. CONCLUSIONS: the introduction of this new assay for the laboratory surveillance of influenza virus strengthens the diagnostic capacity of the National Reference Laboratory.

Viral etiology among the elderly presenting acute respiratory infection during the influenza season / Etiologia viral de infecção respiratória aguda entre idosos, durante a temporada de gripe

INTRODUCTION: Acute respiratory tract infections are the most common illness in all individuals. Rhinoviruses have been reported as the etiology of more than 50 percent of respiratory tract infections worldwide. The study prospectively evaluated 47 elderly individuals from a group of 384 randomly assigned for acute respiratory viral infections (cold or flu) and assessed the occurrence of human rhinovirus (HRV), influenza A and B, respiratory syncytial virus and metapneumovirus (hMPV) in Botucatu, State of São Paulo, Brazil. METHODS: Forty-nine nasal swabs collected from 47 elderly individuals following inclusion visits from 2002 to 2003 were tested by GenScan RT-PCR. HRV-positive samples were sequenced for phylogenetic analysis. RESULTS: No sample was positive for influenza A/B or RSV. HRV was detected in 28.6 percent (14/47) and hMPV in 2 percent (1/47). Of 14 positive samples, 9 isolates were successfully sequenced, showing the follow group distribution: 6 group A, 1 group B and 2 group C HRVs. CONCLUSIONS: The high incidence of HRV during the months of the influenza season requires further study regarding HRV infection impact on respiratory complications among this population. Infection caused by HRV is very frequent and may contribute to increasing the already high demand for healthcare during the influenza season.

INTRODUÇÃO: Infecções agudas do trato respiratório estão entre as doenças mais comuns em todas as pessoas. Os rinovírus têm sido descritos como agente etiológico de mais de 50 por cento das infecções do trato respiratório ao redor do mundo. O objetivo deste trabalho foi avaliar a ocorrência de rinovírus humano (HRV), influenza vírus A e B, vírus respiratório sincicial humano e metapneumovírus (hMPV) em uma população de idosos que apresentava sintomas de gripe ou resfriado, e que residiam na Cidade de Botucatu, Estado de São Paulo, Brasil. MÉTODOS: Foram coletados swabs nasais de 47 idosos após visitas de inclusão, entre os anos de 2002 e 2003 e que foram testadas através de GeneScan RT-PCR. RESULTADOS: HRV foi detectado em 28.6 por cento (14/47) e hMPV em 2 por cento (1/47). De 14 amostras positivas para HRV, 9 foram sequenciadas, mostrando a seguinte distribuição de grupos: grupo A: 6 amostras, grupo B: 1 amostra e grupo C: 2 amostras. CONCLUSÕES: A alta incidência de HRV durante os meses de ocorrência de gripe necessita de estudos posteriores para avaliar o impacto desse vírus entre os idosos. A alta frequência de HRV pode contribuir para o aumento da demanda por serviços de saúde durante a estação de influenza.

Estandarización de un método de detección molecular del virus influenza (H5N1) de alta patogenicidad / Standardization of a molecular detection method of highly pathogenic avian influenza virus (H5N1)

El virus de la influenza aviar H5N1 de alta patogenicidad mantiene el alerta mundial debido a su potencial zoonótico y pandémico. Surge entonces la necesidad de contar con herramientas para la detección temprana y de esta forma reducir el impacto potencial a la salud humana y animal. En este estudio se estandarizó un método de detección molecular de los genes de la matriz (M), hemaglutinina (H5) y neuraminidasa (N1) del virus de la influenza aviar H5N1 de alta patogenicidad de linaje asiático, mediante transcripción-reversa y reacción en cadena de la polimerasa en tiempo real (RRT-PCR). A partir de un ARN viral de referencia cepa A/Vietnam/1203/2004 (H5N1) se construyeron controles positivos mediante clonación de productos de PCR. Los estándares de naturaleza plasmídica se emplearon en la obtención de curvas estándar para determinar los límites de detección de la técnica. La sensibilidad observada para todos los genes analizados fue de 10² copias de ADN/μL. Las curvas mostraron una eficiencia superior al 90%, y R²>0,99. Este método puede ser útil en las campañas de monitoreo del virus en aves migratorias, así como para el tamizaje de muestras clínicas de humanos, en una emergencia de salud.

Highly pathogenic avian influenza virus (HPAI) H5N1 is a global threat due to its zoonotic and pandemic potential. Then, concern arises and the need to have early detection tools to minimize the impact on human and animal health. In this work, a molecular detection method was implemented to detect matrix (M), hemagglutinin (H5) and neuraminidase (N1) genes of HPAI avian influenza virus H5N1, based on real time RT-PCR (RRT-PCR). Positive controls were constructed from reference RNA viral A/Vietnam/1203/2004 (H5N1), cloned into plasmidic vectors and sequenced. Assay detection sensitivity was assessed with standard curves for each gene. Assay sensitivity was 10² DNA copies/μl in all cases. Curves showed amplification efficiency higher than 90% and R²>0.99. This method could be useful for bird monitoring campaigns and as a screening procedure for clinical samples.