Evaluation of quantitative PCR for early diagnosis of Pseudomonas aeruginosa infection in cystic fibrosis: a prospective cohort study.

OBJECTIVES: Early detection of Pseudomonas aeruginosa lung positivity is a key element in cystic fibrosis (CF) management. PCR has increased the accuracy of detection of many microorganisms. Clinical relevance of P. aeruginosa quantitative PCR (qPCR) in this context is unclear. Our aim was to determine P. aeruginosa qPCR sensitivity and specificity, and to assess the possible time saved by qPCR in comparison with standard practice (culture). METHODS: A multicentre cohort study was conducted over a 3-year period in 96 patients with CF without chronic P. aeruginosa colonization. Sputum samples were collected at each visit. Conventional culture and two-step qPCR (oprL qPCR and gyrB/ecfX qPCR) were performed for 707 samples. The positivity criteria were based on the qPCR results, defined in a previous study as follow: oprL qPCR positivity alone if bacterial density was <730 CFU/mL or oprL qPCR combined with gyrB/ecfX qPCR if bacterial density was ≥730 CFU/mL. RESULTS: During follow up, 36 of the 96 patients with CF were diagnosed on culture as colonized with P. aeruginosa. This two-step qPCR displayed a sensitivity of 94.3% (95% CI 79.7%-98.6%), and a specificity of 86.3% (95% CI 83.4%-88.7%). It enabled P. aeruginosa acquisition to be diagnosed earlier in 20 patients, providing a median detection time gain of 8 months (interquartile range 3.7-17.6) for them. CONCLUSIONS: Implementing oprL and gyrB/ecfX qPCR in the management of patients with CF allowed earlier detection of first P. aeruginosa lung positivity than culture alone.

Frequency of aquagenic palmoplantar keratoderma in cystic fibrosis: a new sign of cystic fibrosis?

BACKGROUND: Aquagenic palmoplantar keratoderma (APPK), also known as aquagenic wrinkling of the palms, is characterized by oedema of palms and/or soles, whitish papules, hyperwrinkling and sometimes pruritus or pain after water immersion. Its frequency in the general population is unknown. About 40 cases have been reported to date, including some among patients with cystic fibrosis (CF) or CF heterozygotes. OBJECTIVES: To determine the frequency of APPK among patients with CF. METHODS: Twenty-seven patients from the Centre of Competence on Cystic Fibrosis of Roscoff were examined by a dermatologist after immersion of the palms in water for 2-3 min. RESULTS: The frequency of APPK was 41% (11 of 27 patients). Some patients had not previously noticed the lesions. The frequency was higher among inpatients than outpatients. We suspect that occlusion (caused by the gloves worn by inpatients) can explain this difference. The number of patients included in this study is not sufficient to draw any conclusions concerning the type of CF mutation and its impact on the frequency of APPK. CONCLUSIONS: APPK is frequent among patients with CF and, thus, should be considered a sign of CF. APPK is underdiagnosed because physicians usually do not look for it. CF screening should be considered for any patient presenting with these symptoms, followed by genetic counselling if necessary.

A 44,000 glycoprotein is involved in the attachment of echovirus-11 onto susceptible cells.

Cellular receptors play an important role in viral pathogenesis. Until now little was known on echovirus (EV) receptor. Using detergent-treated KB cell extracts as immunogen, a mouse monoclonal antibody (Mab 143) was produced that selectively blocks the attachment of EV-11 to KB and other susceptible cells. By immunoblotting, Mab 143 detected a 44,000 protein on susceptible cell lines but not on cell lines from nonprimate origin. The receptor protein complex, purified from KB cell membranes by immunoaffinity using Mab 143 as ligand, was shown to contain a single glycoprotein with apparent molecular weight of 44,000 (gp44). The role of gp44 in the attachment of EV-11 onto KB cells was demonstrated by the ability (i) of affinity-purified gp44 to reduce the infectivity of EV-11 and (ii) of rabbit polyclonal antisera raised against gp44 to protect cells from the replication of various EV, as did Mab 143.

Monoclonal antibody specific for the cellular receptor of echoviruses.

Cell lines of primate origin carry membrane receptors which are specific for echoviruses (EV). The present report describes isolation and characterization of a monoclonal antibody (Mab 143) reacting with the membrane of KB cells. The Mab was selected for its protection of different cell lines from primate origin against the CPE of EV-11. This protection was found to extend to most EV serotypes and to coxsackievirus A9, while the replication of several other picornaviruses was not affected. The fluorecein isothiocyanate labelled Mab did not react with cell lines from bovine, canine, or rabbit origin, but bound specifically to the cell lines from human or simian origin. These results suggest that a unique receptor site is used by most EV serotypes for binding onto and penetration into susceptible cells.

Detection of antibodies against initial polypeptides in patients infected with an enterovirus (EV-33).

Antibodies against initial polypeptides have been detected in sera of patients infected with an Echo virus 33. The technique is an IF test using infected cells in which the virus multiplication cycle is blocked at 2 hrs p.i.

Rapid diagnosis of echovirus 33 infection by neutralizing specific IgM antibody.

In 1982, we isolated 20 strains of echovirus type 33 (EV33) from 18 patients. We studied the humoral response to EV33 of 2,437 subjects from whom at least one serum was available during the same year. In 388 subjects with the neutralizing antibody level at 64 or more, we assayed the EV33 IgM antibodies by seroneutralization test after fractionation of sera by ion exchange chromatography. One hundred ninety-five subjects (8.0%) had a high titre (greater than or equal to 32) of EV33 IgM antibodies, which was considered as evidence of recent infection. The EV33-positive IgM fractions were assayed against five other enteroviruses. Sixty percent of the IgM fractions did not cross-react with any of the five serotypes, 8.7% cross-reacted with at least one serotype but with predominant EV33 IgM response, and 31.3% had an equivalent or greater amount of non-EV33 IgM antibodies; the type specificity of the assay was directly related to the age of the subjects. These findings suggest that determination of neutralizing specific IgM antibody is a sensitive and rapid test for the diagnosis of enterovirus infections, especially in young people.