FALSE-NEGATIVE DENGUE CASES IN RORAIMA, BRAZIL: AN APPROACH REGARDING THE HIGH NUMBER OF NEGATIVE RESULTS BY NS1 AG KITS

Serum samples from 150 NS1-negative (Platelia ELISA) patients presumptively diagnosed with dengue were analyzed by the TaqMan probed real-time reverse transcription PCR (TaqMan qRT-PCR) method. The qRT-PCR positive samples were tested for serotype by semi-nested RT-PCR and a qualitative immunochromatographic assay for IgG and IgM. Molecular detection methods showed 33 (22%) positive samples out of 150 NS1-antigen negative samples. Of these, 72% were collected up to day 2 after the onset of symptoms, when diagnostic sensitivity of NS1-antigen test assays is significantly enhanced. Most of the cases were not characterized as secondary infection. Twenty-eight samples were successfully serotyped, 75% of which for DENV-4, 14% for DENV-2, 7% for DENV-3 and 4% for DENV-1. These findings reaffirm the hyperendemic situation of the state of Roraima and suggest a lower sensitivity of the NS1 test, mainly when DENV-4 is the predominant serotype. Health care providers should therefore be aware of samples tested negative by NS1 antigen assays, especially when clinical symptoms and other laboratory data results show evidence of dengue infection.

SAINT LOUIS ENCEPHALITIS VIRUS IN MATO GROSSO, CENTRAL-WESTERN BRAZIL

The dengue virus (DENV), which is frequently involved in large epidemics, and the yellow fever virus (YFV), which is responsible for sporadic sylvatic outbreaks, are considered the most important flaviviruses circulating in Brazil. Because of that, laboratorial diagnosis of acute undifferentiated febrile illness during epidemic periods is frequently directed towards these viruses, which may eventually hinder the detection of other circulating flaviviruses, including the Saint Louis encephalitis virus (SLEV), which is widely dispersed across the Americas. The aim of this study was to conduct a molecular investigation of 11 flaviviruses using 604 serum samples obtained from patients during a large dengue fever outbreak in the state of Mato Grosso (MT) between 2011 and 2012. Simultaneously, 3,433 female Culex spp. collected with Nasci aspirators in the city of Cuiabá, MT, in 2013, and allocated to 409 pools containing 1-10 mosquitoes, were also tested by multiplex semi-nested reverse transcription PCR for the same flaviviruses. SLEV was detected in three patients co-infected with DENV-4 from the cities of Cuiabá and Várzea Grande. One of them was a triple co-infection with DENV-1. None of them mentioned recent travel or access to sylvatic/rural regions, indicating that transmission might have occurred within the metropolitan area. Regarding mosquito samples, one pool containing one Culex quinquefasciatus female was positive for SLEV, with a minimum infection rate (MIR) of 0.29 per 1000 specimens of this species. Phylogenetic analysis indicates both human and mosquito SLEV cluster, with isolates from genotype V-A obtained from animals in the Amazon region, in the state of Pará. This is the first report of SLEV molecular identification in MT.

RELATED FACTORS FOR COLONIZATION BY Candida SPECIES IN THE ORAL CAVITY OF HIV-INFECTED INDIVIDUALS

The colonization of the oral cavity is a prerequisite to the development of oropharyngeal candidiasis. Aims: The aims of this study were: to evaluate colonization and quantify Candida spp. in the oral cavity; to determine the predisposing factors for colonization; and to correlate the levels of CD4+ cells and viral load with the yeast count of colony forming units per milliliter (CFU/mL) in HIV-positive individuals treated at a University Hospital. Saliva samples were collected from 147 HIV patients and were plated on Sabouraud Dextrose Agar (SDA) and chromogenic agar, and incubated at 30 ºC for 72 h. Colonies with similar morphology in both media were counted and the result expressed in CFU/mL. Results: Of the 147 HIV patients, 89 had positive cultures for Candida spp., with a total of 111 isolates, of which C. albicans was the most frequent species (67.6%), and the mean of colonies counted was 8.8 × 10³ CFU/mL. The main predisposing factors for oral colonization by Candida spp. were the use of antibiotics and oral prostheses. The use of reverse transcriptase inhibitors appears to have a greater protective effect for colonization. A low CD4+ T lymphocyte count is associated with a higher density of yeast in the saliva of HIV patients.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160), whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

Chagas' disease: an algorithm for donor screening and positive donor counseling

Classical serological screening assays for Chagas' disease are time consuming and subjective. The objective of the present work is to evaluate the enzyme immuno-assay (ELISA) methodology and to propose an algorithm for blood banks to be applied to Chagas' disease. Seven thousand, nine hundred and ninety nine blood donor samples were screened by both reverse passive hemagglutination (RPHA) and indirect immunofluorescence assay (IFA). Samples reactive on RPHA and/or IFA were submitted to supplementary RPHA, IFA and complement fixation (CFA) tests. This strategy allowed us to create a panel of 60 samples to evaluate the ELISA methodology from 3 different manufacturers. The sensitivity of the screening by IFA and the 3 different ELISA's was 100%. The specificity was better on ELISA methodology. For Chagas disease, ELISA seems to be the best test for blood donor screening, because it showed high sensitivity and specificity, it is not subjective and can be automated. Therefore, it was possible to propose an algorithm to screen samples and confirm donor results at the blood bank.

Digoxigenin-labeled probe for rabies virus nucleoprotein gene detection

A digoxigenin-labeled probe was produced from the Pasteur virus strain for the detection of the rabies virus N gene. The probe hybridization was performed from amplified N gene obtained by reverse transcription polymerase chain reaction and the results by RT-PCR and hybridization showed 100% agreement. The hybridization, when carried out in products amplified by RT-PCR, increases the sensitivity of this technique even more and confers specificity to the diagnosis. The technique described in this work will be useful in rabies diagnosis laboratories, once the cost is compatible with traditional rabies diagnostic techniques.

G and P rotavirus genotypes in stool samples from children in Teresina, State of Piauí

A total of 123 stool specimens collected in Teresina, Piauí between 1994 and 1996, from 0 to 2-year-old children with diarrhea, were used for this study. Molecular characterization of the G and P rotavirus genotypes was performed using the reverse transcriptase polymerase chain reaction. The following results were obtained for the P genotypes: P[8] (17. 1%), P[1] (4. 9%), P[4] (3. 3%), P[6, M37] (2. 4%) and mixtures (27. 6%). The P[1]+P[8] mixture was found in 19. 5% of the samples. For the G genotypes, the results were: G1 (25. 2%), G5 (13. 8%), G2 (2. 5%), G4 (2. 5%), G9 (0. 8%) and mixtures (41. 5%). G1+G5 was the mixture most frequently found (12. 1%). Our results showed unusual combinations such as P[1]G5 and P[1]+P[8]G5. The high percentage of mixtures and unusual combinations containing mixtures of human and animal rotavirus genotypes strongly suggests the possibility of gene reassortment and interspecies transmission.

Viral etiology among the elderly presenting acute respiratory infection during the influenza season

INTRODUCTION: Acute respiratory tract infections are the most common illness in all individuals. Rhinoviruses have been reported as the etiology of more than 50% 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% (14/47) and hMPV in 2% (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.

Lessons from the epidemiological surveillance program, during the influenza A (H1N1) virus epidemic, in a reference university hospital of Southeastern Brazil

INTRODUCTION: The case definition of influenza-like illness (ILI) is a powerful epidemiological tool during influenza epidemics. METHODS: A prospective cohort study was conducted to evaluate the impact of two definitions used as epidemiological tools, in adults and children, during the influenza A H1N1 epidemic. Patients were included if they had upper respiratory samples tested for influenza by real-time reverse transcriptase polymerase chain reaction during two periods, using the ILI definition (coughing + temperature > 38ºC) in period 1, and the definition of severe acute respiratory infection (ARS) (coughing + temperature > 38ºC and dyspnoea) in period 2. RESULTS: The study included 366 adults and 147 children, covering 243 cases of ILI and 270 cases of ARS. Laboratory confirmed cases of influenza were higher in adults (50%) than in children (21.6%) ( p < 0.0001) and influenza infection was more prevalent in the ILI definition (53%) than ARS (24.4%) (p < 0.0001). Adults reported more chills and myalgia than children (p = 0.0001). Oseltamivir was administered in 58% and 46% of adults and children with influenza A H1N1, respectively. The influenza A H1N1 case fatality rate was 7% in adults and 8.3% in children. The mean time from onset of illness until antiviral administration was 4 days. CONCLUSIONS: The modification of ILI to ARS definition resulted in less accuracy in influenza diagnosis and did not improve the appropriate time and use of antiviral medication.

Molecular characterization of rabies virus isolated from non-haematophagous bats in Brazil

INTRODUCTION: Rabies is an important zoonosis that causes thousands of deaths worldwide each year. Although the terrestrial cycle, mainly transmitted by dogs, is controlled in Brazil, the aerial cycle remains a serious public health issue, besides the economic problem. In the aerial cycle, the haematophagous bat Desmodus rotundus is the main source of infection, where several different species of non-haematophagous bats can be infected and can transmit the virus. METHODS: The aim of this work was to study the epidemiological pattern of rabies using antigenic characterization with monoclonal antibodies and genetic characterization by reverse-transcriptase polymerase chain reaction followed by sequencing and phylogenetic analysis of non-haematophagous bats' and herbivorous animals' central nervous system samples from the western region of the State of São Paulo, Brazil. RESULTS: From 27 samples, 3 antigenic variants were identified: AgV-3, AgV-4, and AgV-6; and from 29 samples, 5 different clusters were identified, all belonging to the rabies virus species. CONCLUSIONS: Although only non-haematophagous bats were evaluated in the studied region, the majority of samples were from antigenic and genetic variants related to haematophagous bats Desmodus rotundus. Samples from the same antigenic variant were segregated in more than one genetic cluster. This study demonstrated the diversity of rabies virus genetic lineages presented and circulating in non-haematophagous bats in the studied region.

Evaluation of inadequate anti-retroviral treatment in patients with HIV/AIDS

INTRODUCTION: Since the emergence of antiretroviral therapy, the survival of patients infected with human immunodeficiency virus has increased. Non-adherence to this therapy is directly related to treatment failure, which allows the emergence of resistant viral strains. METHODS: A retrospective descriptive study of the antiretroviral dispensing records of 229 patients from the Center for Health Care, University Hospital, Federal University of Juiz de Fora, Brazil, was conducted between January and December 2009. RESULTS: The study aimed to evaluate patient compliance and determine if there was an association between non-adherence and the therapy. Among these patients, 63.8% were men with an average age of 44.0 ± 9.9 years. The most used treatment was a combination of 2 nucleoside reverse transcriptase inhibitors with 1 non-nucleoside reverse transcriptase inhibitor (55.5%) or with 2 protease inhibitors (28.8%). It was found that patients taking lopinavir/ritonavir with zidovudine and lamivudine had a greater frequency of inadequate treatment than those taking atazanavir with zidovudine and lamivudine (85% and 83.3%, respectively). Moreover, when the combination of zidovudine/ lamivudine was used, the patients were less compliant (χ2 = 4.468, 1 degree of freedom, p = 0.035). CONCLUSIONS: The majority of patients failed to correctly adhere to their treatment; therefore, it is necessary to implement strategies that lead to improved compliance, thus ensuring therapeutic efficacy and increased patient survival.

Genetic diversity of dengue virus serotypes 1 and 2 in the State of Paraná, Brazil, based on a fragment of the capsid/premembrane junction region

INTRODUCTION: The precise identification of the genetic variants of the dengue virus is important to understand its dispersion and virulence patterns and to identify the strains responsible for epidemic outbreaks. This study investigated the genetic variants of the capsid-premembrane junction region fragment in the dengue virus serotypes 1 and 2 (DENV1-2). METHODS: Samples from 11 municipalities in the State of Paraná, Brazil, were provided by the Central Laboratory of Paraná. They were isolated from the cell culture line C6/36 (Aedes albopictus) and were positive for indirect immunofluorescence. Ribonucleic acid (RNA) extracted from these samples was submitted to the reverse transcription polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: RT-PCR revealed that 4 of the samples were co-infected with both serotypes. The isolated DENV-1 sequences were 95-100% similar to the sequences of other serotype 1 strains deposited in GenBank. Similarly, the isolated DENV-2 sequences were 98-100% similar to other serotype 2 sequences in GenBank. According to our neighbor-joining tree, all strains obtained in this study belonged to genotype V of DENV-1. The DENV-2 strains, by contrast, belonged to the American/Asian genotypes. CONCLUSIONS: The monitoring of circulating strains is an important tool to detect the migration of virus subtypes involved in dengue epidemics.

Simultaneous circulation of all four dengue serotypes in Manaus, State of Amazonas, Brazil in 2011

INTRODUCTION: Manaus, the capital city of the state of Amazon with nearly 2 million inhabitants, is located in the middle of the Amazon rain forest and has suffered dengue outbreaks since 1998. METHODS: In this study, blood samples were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), aimed at identifying dengue virus serotypes. RESULTS: Acute phase sera from 432 patients were tested for the presence of dengue virus. Out of the 432 patients, 137 (31.3%) were found to be positive. All the four dengue virus serotypes were observed. CONCLUSIONS: The simultaneous circulation of the four dengue serotypes is described for the first time in Manaus and in Brazil.

In vitro detection of hepatitis C virus in platelets from uninfected individuals exposed to the virus

IntroductionDespite hepatocytes being the target cells of hepatitis C virus (HCV), viral ribonucleic acid RNA has been detected in other cells, including platelets, which have been described as carriers of the virus in the circulation of infected patients. Platelets do not express cluster differentiation 81 CD81, the main receptor for the virus in hepatocytes, although this receptor protein has been found in megakaryocytes. Still, it is not clear if HCV interacts with platelets directly or if this interaction is a consequence of its association with megakaryocytes. The aim of this study was to evaluate the interaction of HCV with platelets from non-infected individuals, after in vitro exposure to the virus.MethodsPlatelets obtained from 50 blood donors not infected by HCV were incubated in vitro at 37°C for 48h with serum containing 100,000IU∕mL of genotype 1 HCV. After incubation, RNA extracted from the platelets was assayed for the presence of HCV by reverse transcription – polymerase chain reaction RT-PCR.ResultsAfter incubation in the presence of virus, all samples of platelets showed HCV RNA.ConclusionsThe results demonstrate that, in vitro, the virus interacts with platelets despite the absence of the receptor CD81, suggesting that other molecules could be involved in this association.