Rhinovirus detection using different PCR-based strategies

Human rhinoviruses (HRVs) are the major cause of the common cold. HRVs were recently reclassified into the Enterovirus genus (HEV) in the Picornaviridae family. HRVs and other members of the HEV genus share many common features, including sense RNA genomes and partial nucleotide sequence identity. The aim of this study was to evaluate different HRV detection strategies. Samples from adults with acute respiratory infection (n = 291) who were treated in Sao Paulo Hospital (2001-2003) were tested using three assays. The first assay detected picornaviruses by RT-PCR and hybridization, the second detected rhinoviruses using RT-PCR/sequencing, and the third differentiated HRV from HEV using duplex semi-nested-RT-PCR. Analysis of the results obtained from the first two strategies revealed 83% concordance. Discordant samples were then evaluated by the third protocol, and 82% were negative. The picornavirus detection protocol was more sensitive but less specific than the rhinovirus detection protocols. The semi-nested protocol utilized in the present study was less sensitive and was not useful in differentiating HRV from HEV. Sequencing assays examining different genes would address the best strategy of confirming rhinovirus and enterovirus infections.

Rhinovirus detection using different PCR-based strategies.

Human rhinoviruses (HRVs) are the major cause of the common cold. HRVs were recently reclassified into the Enterovirus genus (HEV) in the Picornaviridae family. HRVs and other members of the HEV genus share many common features, including sense RNA genomes and partial nucleotide sequence identity. The aim of this study was to evaluate different HRV detection strategies. Samples from adults with acute respiratory infection (n = 291) who were treated in Sao Paulo Hospital (2001-2003) were tested using three assays. The first assay detected picornaviruses by RT-PCR and hybridization, the second detected rhinoviruses using RT-PCR/sequencing, and the third differentiated HRV from HEV using duplex semi-nested-RT-PCR. Analysis of the results obtained from the first two strategies revealed 83% concordance. Discordant samples were then evaluated by the third protocol, and 82% were negative. The picornavirus detection protocol was more sensitive but less specific than the rhinovirus detection protocols. The semi-nested protocol utilized in the present study was less sensitive and was not useful in differentiating HRV from HEV. Sequencing assays examining different genes would address the best strategy of confirming rhinovirus and enterovirus infections.

Prevalence of serological markers of hepatitis B virus in pregnant women from Paraná State, Brazil.

The prevalence of hepatitis B virus (HBV) in Brazil increases from South to North but moderate to elevated prevalence has been detected in the Southwest of Paraná State. The prevalence of serological markers of HBV was evaluated in 3188 pregnant women from different counties in Paraná State and relevant epidemiological features were described. The prevalence of HBV markers in pregnant women for the state as a whole was 18.5% (95% CI = 17.2-19.9), ranging from 7.2% in Curitiba to 38.5% in Francisco Beltrão. The endemicity of HBV marker prevalence in pregnant women was intermediate in Cascavel, Foz do Iguaçu, and Francisco Beltrão, and low in Curitiba, Londrina, Maringá, and Paranaguá. Multiple logistic regression showed that HBV marker prevalence increased with age, was higher among black women, among women of Italian and German descent, and among women who had family members in neighboring Rio Grande do Sul State. Univariate analysis showed that HBV marker prevalence was also higher among women with no education or only primary education, with a lower family income and whose families originated from the South Region of Brazil. Pregnant women not having positive HBV markers (anti-HBc, HBsAg or anti-HBs detected by ELISA) corresponded to 73.7% of the population studied, implying that HBV vaccination needs to be reinforced in Paraná State. The highest prevalence was found in three counties that received the largest number of families from Santa Catarina and Rio Grande do Sul, where most immigrants were of German or Italian ascendance. This finding probably indicates that immigrants that came to this area brought HBV infection to Southwestern Paraná State.

Prevalence of serological markers of hepatitis B virus in pregnant women from Paraná State, Brazil

The prevalence of hepatitis B virus (HBV) in Brazil increases from South to North but moderate to elevated prevalence has been detected in the Southwest of Paraná State. The prevalence of serological markers of HBV was evaluated in 3188 pregnant women from different counties in Paraná State and relevant epidemiological features were described. The prevalence of HBV markers in pregnant women for the state as a whole was 18.5 percent (95 percent CI = 17.2-19.9), ranging from 7.2 percent in Curitiba to 38.5 percent in Francisco Beltrão. The endemicity of HBV marker prevalence in pregnant women was intermediate in Cascavel, Foz do Iguaçu, and Francisco Beltrão, and low in Curitiba, Londrina, Maringá, and Paranaguá. Multiple logistic regression showed that HBV marker prevalence increased with age, was higher among black women, among women of Italian and German descent, and among women who had family members in neighboring Rio Grande do Sul State. Univariate analysis showed that HBV marker prevalence was also higher among women with no education or only primary education, with a lower family income and whose families originated from the South Region of Brazil. Pregnant women not having positive HBV markers (anti-HBc, HBsAg or anti-HBs detected by ELISA) corresponded to 73.7 percent of the population studied, implying that HBV vaccination needs to be reinforced in Paraná State. The highest prevalence was found in three counties that received the largest number of families from Santa Catarina and Rio Grande do Sul, where most immigrants were of German or Italian ascendance. This finding probably indicates that immigrants that came to this area brought HBV infection to Southwestern Paraná State.

Risk factors for poor immune response to influenza vaccination in elderly people

Influenza vaccination of elderly people is efficacious and cost effective for the prevention of influenza and its complications. Some studies have pointed out low immunogenicity in this group. Health status has been poorly investigated as a risk factor that may influence the immune response to influenza vaccine. We established an immunization response study of a highly-matched elderly population in a nursing home. One-hundred-twenty subjects of Ashkenazian origin had their vaccine-induced antibody response assessed. Good response was obtained in 30.8 percent (37/120), and 31.7 percent (38/120) did not react. A lack of good response was found to be associated with dementia (P=0.016) in a multivariate analysis. In addition to dementia, malnutrition was frequently observed among poor responders, suggesting that these factors should be considered in vaccination studies. Chemoprophylaxis in addition to vaccination for elderly presenting dementia should be considered, particularly for those people living nursing homes.

Effect of a single tetanus-diphtheria vaccine dose on the immunity of elderly people in São Paulo, Brazil.

Epidemiological data regarding tetanus and diphtheria immunity in elderly people in Brazil are scarce. During the First National Immunization Campaign for the Elderly in Brazil in April 1999, 98 individuals (median age: 84 years) received one tetanus-dyphtheria (Td) vaccine dose (Butantan Institute, lot number 9808079/G). Inclusion criteria were elderly individuals without a history of severe immunosuppressive disease, acute infectious disease or use of immunomodulators. Blood samples were collected immediately before the vaccine and 30 days later. Serum was separated and stored at -20 degrees C until analysis. Tetanus and diphtheria antibodies were measured by the double-antigen ELISA test. Tetanus and diphtheria antibody concentrations lower than 0.01 IU/mL were considered to indicate the absence of protection, between 0.01 and 0.09 IU/mL were considered to indicate basic immunity, and values of 0.1 IU/mL or higher were considered to indicate full protection. Before vaccination, 18% of the individuals were susceptible to diphtheria and 94% were susceptible to tetanus. After one Td dose, 78% became fully immune to diphtheria, 13% attained basic immunity, and 9% were still susceptible to the disease. In contrast, 79% remained susceptible to tetanus, 4% had basic immunity and 17% were fully immune. Although one Td dose increases immunity to diphtheria in many elderly people who live in Brazil, a complete vaccination series appears to be necessary for the prevention of tetanus.

Effect of a single tetanus-diphtheria vaccine dose on the immunity of elderly people in São Paulo, Brazil

Epidemiological data regarding tetanus and diphtheria immunity in elderly people in Brazil are scarce. During the First National Immunization Campaign for the Elderly in Brazil in April 1999, 98 individuals (median age: 84 years) received one tetanus-dyphtheria (Td) vaccine dose (Butantan Institute, lot number 9808079/G). Inclusion criteria were elderly individuals without a history of severe immunosuppressive disease, acute infectious disease or use of immunomodulators. Blood samples were collected immediately before the vaccine and 30 days later. Serum was separated and stored at -20°C until analysis. Tetanus and diphtheria antibodies were measured by the double-antigen ELISA test. Tetanus and diphtheria antibody concentrations lower than 0.01 IU/mL were considered to indicate the absence of protection, between 0.01 and 0.09 IU/mL were considered to indicate basic immunity, and values of 0.1 IU/mL or higher were considered to indicate full protection. Before vaccination, 18 percent of the individuals were susceptible to diphtheria and 94 percent were susceptible to tetanus. After one Td dose, 78 percent became fully immune to diphtheria, 13 percent attained basic immunity, and 9 percent were still susceptible to the disease. In contrast, 79 percent remained susceptible to tetanus, 4 percent had basic immunity and 17 percent were fully immune. Although one Td dose increases immunity to diphtheria in many elderly people who live in Brazil, a complete vaccination series appears to be necessary for the prevention of tetanus.

Risk factors for poor immune response to influenza vaccination in elderly people.

Influenza vaccination of elderly people is efficacious and cost effective for the prevention of influenza and its complications. Some studies have pointed out low immunogenicity in this group. Health status has been poorly investigated as a risk factor that may influence the immune response to influenza vaccine. We established an immunization response study of a highly-matched elderly population in a nursing home. One-hundred-twenty subjects of Ashkenazian origin had their vaccine-induced antibody response assessed. Good response was obtained in 30.8% (37/120), and 31.7% (38/120) did not react. A lack of good response was found to be associated with dementia (P=0.016) in a multivariate analysis. In addition to dementia, malnutrition was frequently observed among poor responders, suggesting that these factors should be considered in vaccination studies. Chemoprophylaxis in addition to vaccination for elderly presenting dementia should be considered, particularly for those people living nursing homes.

Sequential cytomegalovirus antigenemia monitoring in kidney transplant patients treated with antilymphocyte antibodies.

BACKGROUND: Antilymphocyte antibodies (ALA) use is related to disseminated cytomegalovirus (CMV) disease after kidney transplantation. Strict surveillance of CMV infection, preemptive antiviral treatment or concomitant ganciclovir and ALA use are proposed as an attempt to prevent related clinical complications. Our objective was to describe the pattern of CMV infection, based on sequential antigenemia detection, after ALA treatment. PATIENTS AND METHODS: Thirty renal transplant patients were prospectively screened for CMV infection after ALA treatment. CMV antigenemia (pp65 antigen detection) was monitored twice a week in the first month and weekly until 60 days after the beginning of ALA therapy. Any positive value of antigenemia was considered CMV infection. RESULTS: Twenty-eight (93.3%) patients were CMV positive (IgG) before transplantation. The mean duration of ALA treatment was 12.1+/-2.4 days. Positive antigenemia was detected in 24 (80%) patients, a mean of 52.5+/-15 days after transplant and 44.7+/-14 days after the beginning of ALA treatment. The median antigenemia count was 7 positive cells/300,000 neutrophils (range: 1-227). Antigenemia preceded clinical symptoms by 5.8 days (0-28 days). Eighteen (75%) of 24 positive patients received ganciclovir treatment: 8 patients (26.7%) for viral syndrome, 2 patients (33.3%) for invasive disease, and 8 patients (26.7%) as part of preemptive therapy, asymptomatic with high antigenemia values. Six pp65-positive patients with low counts were followed up until a negative result and remained asymptomatic without any specific treatment. CONCLUSION: CMV infection was frequent after ALA treatment in this group and generally occurred late after completion of treatment. Antigenemia was a reliable tool to guide preemptive treatment in these patients, and such strategy is an alternative option compared to the prophylactic use of ganciclovir with ALA treatment.

Single human cytomegalovirus gB genotype shed in multiple sites at the time of diagnosis in renal transplant recipients.

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in immunocompromised patients, such as renal transplant recipients. Analysis of the gene encoding the envelope glycoprotein B (gB) showed that clinical isolates adopted one of the sequence configurations, permitting the isolates to be assigned a gB genotype of 1-4. It has been suggested that HCMV gB genotypes could be correlated with tropism and pathogenesis. A number of reports in the literature refer to shedding of different gB strains, permitting follow-up of renal transplant recipients. Considering that a single strain might be responsible for the clinical expression of the disease in multiply exposed individuals, the frequency distribution of gB genotypes was examined by nested polymerase chain reaction and restriction fragment length polymorphism in 20 renal transplant recipients at the time of diagnosis. The association between gB genotypes and cellular tropism was determined using blood, saliva, and urine for each patient. HCMV gB genotype 2 was found more frequently than other genotypes (gB2, 40%; gB1, 30%; gB3, 25%; and gB4, 5%) in renal transplant recipients. The gB type did not correlate with tropism for different body sites. All the patients with HCMV infections presumably harbored a single HCMV strain at the time of diagnosis. In multiply exposed patients, the immunomodulation provided by acute HCMV infection could favor later shedding of different strains.

In vitro induced antibody production for the diagnosis of herpes simplex virus infection in immunosuppressed patients

We developed and evaluated a specific test for herpes simplex virus (HSV) infection based on the secretion of HSV-specific antibodies by lymphocytes stimulated in vitro with HSV-1 antigens. The in vitro induced antibody production (IVIAP) test was used for the diagnosis of HSV infection in 43 seropositive selected subjects: 9 healthy subjects (controls), 30 symptomatic patients (26 of them immunocompromised and 4 immunocompetent) and 4 patients with varicella zoster infection. Anti-HSV antibodies were detected by an immune assay using an anti-human IgG peroxidase conjugate. The test showed a sensitivity of 93 per cent (15/16) and specificity of 92 per cent (1/13) which were confirmed by positive culture or clinical and laboratory follow-up. One AIDS patient had a false-negative result and one false-positive result (1/9) was obtained among the healthy subjects. All patients infected with varicella zoster virus were negative to the IVIAP test. The test is rapid, inexpensive, easy to interpret and can be used for the diagnosis of HSV infections, especially in immunocompromised patients.