A. M. van Loon

Simultaneous Detection of Influenza Viruses A and B Using Real-Time Quantitative PCR

ABSTRACT Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.

Poliomyelitis outbreak in an unvaccinated community in the Netherlands, 1992-93

An outbreak of poliomyelitis occurred in the Netherlands between September, 1992, and February, 1993, after 14 years without endemic cases. The outbreak was due to poliovirus type 3 and involved 71 patients, of whom 2 died and 59 had paralysis. The patients were aged between 10 days and 61 years (median 18 years). None of the patients had been vaccinated, and all but 1 belonged to a socially and geographically clustered group of people who refuse vaccination for religious reasons. Control measures were taken within 5 days of notification of the first patient and included a wide offer of vaccination with the trivalent oral poliovirus vaccine to the population at risk. Sequence analysis of the viral genome showed closest similarity (96.7%) with a strain isolated in India in 1992, indicating that the virus probably originates from the Indian subcontinent. The difference, however, is still too large to assume direct import. Extensive outbreak investigation at schools, in the environment, at virus diagnostic laboratories, and in the general population showed no evidence of widespread circulation of the epidemic virus outside the groups at risk and area where these groups live. As in the previous outbreak in 1978, the general population, including the majority of unvaccinated people who live dispersed in the population, seemed to be well-protected against poliomyelitis.

Isolation of epidemic poliovirus from sewage during the 1992-3 type 3 outbreak in the Netherlands

To examine the extent of wild poliovirus circulation during the 1992-3 epidemic in the Netherlands caused by poliovirus type 3, 269 samples from sewage pipelines at 120 locations were examined for the presence of poliovirus. The epidemic virus strain was found in 23 samples, all from locations inside the risk area which contained communities that refuse vaccination for religious reasons. By sewage investigation, the wildtype virus was shown to be present in the early phase of the epidemic at two locations, one week before patients were reported from that area. The wild type 3 poliovirus was also detected retrospectively in a river water sample collected for other reasons three weeks before notification of the first poliomyelitis case, at a site a few kilometres upstream the home village of this patient. Oral poliovirus vaccine (OPV) virus was found at 28 locations inside or at the border of the risk area. Trivalent OPV was offered to unvaccinated or incompletely-vaccinated persons living in this region as part of the measures to control the epidemic.

Efficacy of single-dose azithromycin versus doxycycline in the treatment of cervical infections caused byChlamydia trachomatis

The efficacy of single-dose azithromycin therapy in the treatment of cervicalChlamydia trachomatis infections was compared to that of a standard seven-day course of treatment with doxycycline. Cervical samples from 60 patients reacted positively in an enzyme immunoassay for detection ofChlamydia trachomatis. In 31 patientsChlamydia trachomatis was isolated from the sample taken before treatment. Fourteen of the 31 patients were treated with doxycycline and 17 with azithromycin. All cultures of samples taken one and four weeks after the start of therapy were negative. All 31 isolates showed a similar pattern of MICs for the seven antibiotics tested, including azithromycin and doxycycline. No differences were observed between isolates of different serovars. In samples from four patients chlamydial DNA could be detected by PCR one week after the start of the therapy and in two patients also after four weeks. No difference in microbiological parameters could be observed between the two treatment groups. It is concluded that single-dose azithromycin is as effective as a seven-day course of doxycycline in the therapy of cervicalChlamydia trachomatis infections.

Conventional Influenza Vaccination Is Not Associated with Complications in Working-Age Patients with Asthma or Chronic Obstructive Pulmonary Disease

Abstract By using a nested case-control design, the authors studied the effectiveness of the influenza vaccine in reducing severe and fatal complications in 4,241 and 5,966 primary care, working-age patients aged 18–64 years who had asthma or chronic obstructive pulmonary disease during the 1998–1999 and 1999–2000 influenza epidemics in the Netherlands. Patients developing fatal or nonfatal exacerbations of lung disease, pneumonia, congestive heart failure, or myocardial infarction during either epidemic were considered cases. For each case, four age- and sex-matched controls were randomly sampled, and patient records were reviewed. Conditional logistic regression and propensity scores were used to assess vaccine effectiveness after adjustment for confounding factors. In seasons one and two, respectively, 87% (47/54) and 85% (171/202) of the cases and 74% (155/210) and 75% (575/766) of the controls had been vaccinated. After adjustments, vaccination was not associated with reductions in complications (season one: odds ratio = 0.95, 95% confidence interval (CI): 0.26, 3.48; season two: odds ratio = 1.07, 95% CI: 0.59, 1.96; pooled odds ratio = 1.07, 95% CI: 0.63, 1.80). Because influenza vaccination appeared not to be associated with a clinically relevant reduction in severe morbidity, other measures need to be explored.

Enzyme-linked immunosorbent assay for measurement of antibody against cytomegalovirus and rubella virus in a single serum dilution.

Enzyme-linked immunosorbent assays (ELISA) were developed for quantifying cytomegalovirus (CMV) and rubella antibodies using a single serum dilution (1/800) in conjunction with a standard curve. A near linear relation was found between the logarithms of absorbance values of sera at a dilution of 1/800 and the titres as determined by an end point dilution ELISA. The reproducibility of the single dilution ELISA was good; the within-test coefficients of variation averaged 7.5% for CMV antibody and 12.4% for rubella antibody. A close correlation was found between ELISA and complement-fixing (CF) antibody titres to CMV and between ELISA and haemagglutination-inhibition (HI) antibody titres to rubella virus. The titres in ELISA were 200 to 1000 times higher than in CF for CMV and 50 to 100 times higher than in HI for rubella virus.

Poliomyelitis in the Netherlands: a review of population immunity and exposure between the epidemics in 1978 and 1992

An overview of serological and virological studies on poliomyelitis in the Netherlands between two epidemics in 1978 and 1992 is given. Three unvaccinated patients acquired poliomyelitis abroad. In the Netherlands vaccination coverage with quadruple DPT-IPV vaccine is very high. The strong immunogenicity of inactivated poliovirus vaccine was confirmed in a cohort of children, reflected in age-stratified antibody profiles of the population. Adults born in the pre vaccination era appeared in general protected, but 10-25% of persons born between 1930 and 1945 lacked neutralizing antibodies. Revaccination induced a booster type of antibody response in 75-90% of such persons, indicating immunological memory and protection. Virological studies on adopted children from other countries, patients with indications for viral examination, and river waters showed that the Netherlands was regularly exposed to polio virus (PV), without signs of indigenous transmission. Persons found to carry PV or their close contacts had travelled to a PV endemic country. Most of 557 isolates were vaccine-derived, only 8% were wild type viruses. Despite their presence, up to 1992 the well-known susceptibles for PV in the Netherlands were shielded by the herd immunity of the Dutch population.

Enzyme-linked immunosorbent assay for quantitation of toxoplasma antibodies in human sera.

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of toxoplasma antibodies using a single serum dilution (1:800) in conjunction with a standard curve Antigen was prepared from Toxoplasma gondii cultivated in human cell cultures. A nearly linear relationship was found between the logarithms of the absorbance values of 120 human sera at a dilution of 1/800 and the titres as determined by an end point dilution ELISA. The reproducibility of the single dilution ELISA was excellent; the coefficients of variation for within-day and day-to-day tests were less than 15%. A close correlation was found between the results obtained with ELISA, indirect immunofluorescence (IF), and complement fixation (CF). The titres in ELISA were 20 to 40 times higher than in IF and 200 to 1000 times higher than in CF.

Immunisation status of children adopted from China

Many children adopted from China have antibody titres that do not correlate with those expected from their medical records. We have compared the concentrations of antibodies to poliomyelitis, diphtheria, and tetanus in children adopted from China, those adopted from other countries, and those vaccinated in the Netherlands and the UK. About 30% of children adopted from China did not have adequate protection from tetanus, diphtheria, or poliomyelitis, despite having complete vaccination recorded in their vaccination documents. These children, unlike those adopted from other countries who have documented evidence of adequate vaccination status, should be tested for antibody concentrations or reimmunised.

Post-transfusion non-A, non-B hepatitis in the Netherlands.

tions and hospital admissions (r=09; p<0 01). There was a tendency throughout the period, when the figures for whooping cough increased, for the number of notifications to increase proportionately more than the number of admissions. In the peak quarters of 1974 and 1978 there were 25 and 58 admissions and 159 and 666 notifications respectively. We studied Hospital Activity Analysis statistics for a range of other respiratory and infective diseases in childhood during the period but found no disease other than whooping cough for which admissions corresponded with the quarter by quarter trends in whooping-cough notifications. There were three deaths -one in 1974, one in 1977, and one in 1979-ascribed to whooping cough in the region during the period.