Marcel F. Peeters

Utility of Real-Time PCR for Diagnosis of Legionnaires’ Disease in Routine Clinical Practice

ABSTRACT The main aim of our study was to determine the added value of PCR for the diagnosis of Legionnaires’ disease (LD) in routine clinical practice. The specimens were samples submitted for routine diagnosis of pneumonia from December 2002 to November 2005. Patients were evaluated if, in addition to PCR, the results of at least one of the following diagnostic tests were available: (i) culture for Legionella spp. on buffered charcoal yeast extract agar or (ii) detection of Legionella pneumophila antigen in urine specimens. Of the 151 evaluated patients, 37 (25%) fulfilled the European Working Group on Legionella Infections criteria for a confirmed case of LD (the “gold standard”). An estimated sensitivity, specificity, and overall percent agreement of 86% (32 of 37; 95% confidence interval [CI] = 72 to 95%), 95% (107 of 112; 95% CI = 90 to 98%), and 93% (139 of 149), respectively, were found for 16S rRNA-based PCR, and corresponding values of 92% (34 of 37; 95% CI = 78 to 98%), 98% (110 of 112; 95% CI = 93 to 100%), and 97% (144 of 149), respectively, were found for the mip gene-based PCR. A total of 35 patients were diagnosed by using the urinary antigen test, and 34 were diagnosed by the 16S rRNA-based PCR. With the mip gene PCR one more case of LD (n = 36; not significant) was detected. By combining urinary antigen test and the mip gene PCR, LD was diagnosed in an additional 4 (11%) patients versus the use of the urinary antigen test alone. The addition of a L. pneumophila-specific mip gene PCR to a urinary antigen test is useful in patients with suspected LD who produce sputum and might allow the early detection of a significant number of additional patients.

Detection of respiratory viruses and Legionella spp. by real-time polymerase chain reaction in patients with community acquired pneumonia

We conducted a study on throat swabs obtained from a group of hospitalized patients with community acquired pneumonia (CAP). Throat swab specimens from 242 adults admitted to hospital with CAP were tested. In total, 1 or more aetiological agents were identified by real-time PCR in 55 (23%) patients. The most frequently detected pathogens were coronavirus (17%), parainfluenza virus (6%) and influenza virus (4%). Overall, viral pathogens were identified by conventional techniques in 7 (2%) patients, and real-time PCR in 50 (21%) patients (p<0.0001). The diagnostic yield increased from 137 cases (57% of patients using conventional microbiological assays) to 158 cases (65% of patients using real-time PCR assays and conventional microbiological assays; p=0.06). A significantly higher percentage of mortality was present in patients with a mixed bacterial and viral infection. L. pneumophila PCR was positive in only 3 out of 11 cases (27%) of Legionnaires’ disease (LD). This study demonstrates that real-time PCR can increase the number of microbiological detections of respiratory pathogens, mainly as a result of detection of respiratory viruses.

Evaluation of sensitivity, specificity and cross-reactivity in Bartonella henselae serology.

Laboratory diagnosis of cat-scratch disease (CSD) mainly relies on serological testing for Bartonella henselae antibodies by indirect immunofluorescence (IFA) or enzyme-linked immunoassay (ELISA). In this study the current diagnostic value of seven B. henselae serological tests was evaluated, including both commercial and in-house IFA’s as well as in-house ELISA. Fifty patients with proven CSD, based on the presence of lymphadenopathy, positive PCR and no other diagnosis, were compared to 55 controls initially suspected for CSD but tested negative in PCR and who ultimately received a different diagnosis. Cross-reactivity was tested in serum samples positive for infection with Epstein-Barr virus (n = 141), cytomegalovirus (n = 39), Toxoplasma gondii (n = 20), Streptococcus pyogenes (n = 54), Chlamydophila pneumoniae (n = 14) and Coxiella burnetii (n = 21). Sensitivity was higher in the two IgG tests (88-98%) than in the IgM tests (50-64%). Adding genotype II strains to the IgM IFA did not improve sensitivity and resulted in higher crossreactivity. Specificity was higher in the IFA IgM tests (up to 95-96%) than in the Focus IgG tests (69%). The concurrent use of both IgM and IgG Euroimmun IFA improved sensitivity to 92% with a specificity of 84%. Cross-reactivity in lymph node diseases was 0-5% in the Houston-strain IgM IFA’s, 0-8% in IgG IFA and 5-24% in the IgM ELISA. Cross-reactivity was 0-36% in C. pneumoniae and 13-30% in C. burnetii, depending on the test. The results of this study indicate that the combined use of IgM and IgG IFA’s based on co-cultivation can be useful in serodiagnosis of CSD. 97 Sensitivity, specificity and cross-reactivity in CSD serology

An outbreak of Legionnaire's disease among visitors to a fair in Belgium in 1999

This paper describes an outbreak of Legionnaires disease at Kapellen in Belgium among visitors of the annual fair. The investigation started on 13th November 1999 after a respiratory physician notified the health authorities of the province of Antwerp of presumptive cases of legionellosis. The annual commercial fair at Kapellen, a small town in northern Belgium, was held 10 days previously and attracted 50,000 visitors. Stand employees (professionals or volunteers), technical staff of the hall and visitors at the fair were affected cases. An exploratory case-control study was conducted to trace the source of the epidemic. To complete the inventory study and to evaluate other risk factors, a cohort study of exhibitors and staff was conducted. Ninety-three people met the case definition, 41 of whom were considered as confirmed, 14 as presumptive cases and 38 as possible/clinical cases. Five people died. Further testing at the reference laboratory confirmed all strains to be Legionella pneumophila serogroup 1. The sensitivity for culture was low (29.2%), and sensitivity for seroconversion was high (90.9%). For urinary antigen test, a sensitivity with Biotest EIA of 65.6% was found, and the sensitivity of polymerase chain reaction (PCR) was 85.7%. In all cases, the individual had visited the fair. Those individuals working in the central areas of the tent, near the aerosol-producing devices, were at higher risk of disease. Legionella was detected by PCR on swabs of the surfaces of the whirlpool. Although not fully proven, an aerosol-producing device was the most probable source of the outbreak.

Low sensitivity of Bartonella henselae PCR in serum samples of patients with cat-scratch disease lymphadenitis.

Cat-scratch disease (CSD) is caused by Bartonella henselae and usually presents as self-limiting lymphadenitis. Invasive procedures are often needed to confirm the diagnosis by PCR on lymph node or other material, because culture and serological tests have low sensitivity (Bergmans et al., 1997; La Scola & Raoult, 1999; Fournier et al., 2002). Recently, Arvand & Schäd (2006) described isolation of B. henselae DNA from peripheral blood of a CSD patient 3 and 4 months after a cat scratch. They suggest that detection of B. henselae DNA in blood may prove useful, especially in cases where lymphadenectomy or biopsy is not feasible or serological results are equivocal.

Are Oropharyngeal Swabs Suitable as Samples for Legionella-Specific PCR Testing?

In a recent study by McDonough et al., a Legionella cluster was identified through retrospective PCR analysis of 240 throat swab samples from cases of pneumonia among young and otherwise healthy U.S. military recruits ([4][1]). Results were confirmed by sequence analysis. No diagnostic evidence

Infecties met Bartonella henselae; de vele gezichten van kattenkrabziekte

SummaryThe clinical spectrum of cat scratch disease caused by Bartonella henselae is much wider than initially recognised. Three patients are described. A 5 year old girl with axillar lymphadenopathy and negative serology, had a positive polymerase chain reaction (pcr) for Bartonella henselae. An 11 year old girl presenting with convulsions and coma, had Bartonella henselae encephalitis. A 7 year old girl with fever of unknown origin, turned out to have systemic cat scratch disease with liver and spleen abcesses. Bartonella henselae infections should be suspected in patients with an unclear infectious presentation. In case of negative serological results and persistent suspicion of a Bartonella henselae infection, one should obtain a pcr from relevant tissue. Antibiotic treatment is usually not necessary. In severe atypical infections, as in immuno-compromised hosts, macrolides are the drug of choice.samenvattingDoor de toegenomen mogelijkheden om infecties door Bartonella henselae te diagnosticeren, blijkt kattenkrabziekte een breed spectrum aan verschijningsvormen te hebben. We beschrijven drie patiënten. Een 5-jarig meisje met een typische lymfadenitis met negatieve serologie, bleek op grond van een positieve polymerasekettingreactie (pcr) in lymfeklierweefsel toch kattenkrabziekte te hebben. Een 11-jarig meisje met coma en convulsies bij een Bartonella henselae-encefalitis, herstelde restloos. Tot slot bleek een 7-jarig meisje met piekende koorts, lever- en miltabcessen te hebben in het kader van een systemische kattenkrabziekte. Bij bijzondere infectieuze presentaties moet aan atypische vormen van kattenkrabziekte gedacht worden. Bij negatieve serologie en blijvende klinische verdenking op kattenkrabziekte is het raadzaam een pcr op relevant materiaal te verrichten. Antibiotische behandeling van een typische lymfadenitis door een Bartonella henselae is niet nodig. Bij atypische presentatievormen en bij immuungecompromitteerde patiënten met kattenkrabziekte kan behandeling overwogen worden. In dat geval hebben macroliden de voorkeur.