Identification of Haemophilus influenzae and Haemophilus haemolyticus by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Generally accepted laboratory methods that have been used for decades do not reliably distinguish between H. influenzae and H. haemolyticus isolates. H. haemolyticus strains are often incorrectly identified as nontypeable Haemophilus influenzae (NTHi). To distinguish H. influenzae from H. haemolyticus we have created a new database on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) bio-typer 2 and compared the results with routine determination of Haemophilus (growth requirement for X and V factor), and multilocus sequence typing (MLST). In total we have tested 277 isolates, 244 H. influenzae and 33 H. haemolyticus. Using MLST as the gold standard, the agreement of MALDI-TOF MS was 99.6 %. MALDI-TOF MS allows reliable and rapid discrimination between H. influenzae and H. haemolyticus.

Dutch guideline on the laboratory detection of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has rapidly emerged worldwide, affecting both healthcare and community settings, and intensive livestock industry. The efficient control of MRSA strongly depends on its adequate laboratory detection. This guideline provides recommendations on the appropriate use of currently available diagnostic laboratory methods for the timely and accurate detection of MRSA in patients and healthcare workers. Herewith, it aims to standardise and improve the diagnostic laboratory procedures that are used for the detection of MRSA in Dutch medical microbiology laboratories.

Evaluation of Meridian TRU Legionella®, a new rapid test for detection of Legionella pneumophila serogroup 1 antigen in urine samples.

We evaluated the ability of a new antigen test (TRU Legionella® assay, Meridian Bioscience, Cincinnati, OH, USA) to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained with the Binax NOW® urinary antigen test (Binax, Portland, ME, USA). The sensitivities and specificities were 73 % [95 % confidence interval (CI), 65 to 81 %] and 100 %, respectively, for the Meridian TRU Legionella test and 77 % (95 % CI, 68 to 84 %) and 100 %, respectively, for the Binax NOW urinary antigen test. The sensitivity of the Meridian TRU Legionella test increased to 81 % if tests were re-examined after 60 min of incubation. Prolonged incubation did not affect the specificity. We conclude that both assays evaluated have similar performance characteristics and are suitable for the rapid diagnosis of Legionnaires' disease.

Evaluation of Legionella V-TesT for the detection of Legionella pneumophila antigen in urine samples.

We evaluated a new immunochromatographic assay (Legionella V-TesT, Coris BioConcept, Gembloux, Belgium) for its ability to detect Legionella pneumophila serogroup 1 antigen in urine. Test devices were read at various time points to determine the optimum incubation time regarding performance. The results were compared with those obtained with the BinaxNOW urinary antigen test. The sensitivity and specificity were 82.2% and 98.6%, respectively, for the Legionella V-TesT and 83.9% and 100%, respectively, for the BinaxNOW urinary antigen test after 15 min of incubation. When tests were examined after 60 min, the sensitivity for both tests increased to 91.5%.

Evaluation of the Oxoid Xpect Legionella test kit for detection of Legionella pneumophila serogroup 1 antigen in urine.

We evaluated a new immunochromatographic assay (Oxoid Xpect Legionella test kit) for the ability to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained with the Binax NOW urinary antigen test by following the manufacturers' instructions. The sensitivities and specificities were estimated to be 89 and 100%, respectively, for the Oxoid Xpect Legionella test kit and 86 and 100%, respectively, for the Binax NOW test.

Evaluation of the SAS Legionella Test, a new immunochromatographic assay for the detection of Legionella pneumophila serogroup 1 antigen in urine.

This study evaluated a new immunochromatographic assay (SAS Legionella Test) for its ability to detect Legionella pneumophila serogroup 1 antigen in urine. Results were compared with those obtained using the Binax Now urinary antigen test. Sensitivity and specificity were estimated as 82.9% and 99.0%, respectively, for the SAS Legionella Test, and 91.4% and 100%, respectively, for the Binax Now urinary antigen test. The sensitivity of both tests increased to 97.1% (p 0.009) and 94.2% (p 0.7), respectively, if the tests were examined after 1 h.

Identification and susceptibility testing of Staphylococcus aureus by direct inoculation from positive BACTEC blood culture bottles.

This study explored the possibility of combining direct inoculation of tube coagulase and DNase tests, and the VITEK2 system, from BACTEC blood culture bottles in order to achieve rapid identification and susceptibility testing of Staphylococcus aureus. All isolates were identified correctly as S. aureus or coagulase-negative staphylococci (CNS). Antimicrobial susceptibility testing with the VITEK2 system gave 99.6% correct category agreement, with 0.1% very major errors and 0.3% minor errors among S. aureus isolates, and 97.4% correct category agreement, with 0.9% very major errors and 1.7% minor errors among CNS isolates. The results suggested that direct identification and susceptibility testing is sufficiently accurate for immediate reporting.

Mass treatment with albendazole reduces the prevalence and severity of Oesophagostomum-induced nodular pathology in northern Ghana.

Previous surveys conducted in northern Ghana where Oesophagostomum bifurcum is endemic showed that O. bifurcum-induced nodular pathology could be detected in up to 50% of the inhabitants. The impact of albendazole-based mass treatment to control both infection and morbidity is assessed and compared with the situation in a control area where no mass treatment has taken place. A significant reduction in the prevalence of infection based on stool cultures was achieved following two rounds of mass treatment in one year: from 52.6% (361/686) pre treatment to 5.2% (22/421) 1 year later (chi(1)(2)=210.1; P<0.001). At the same time, the morbidity marker of ultrasound-detectable nodules declined from 38.2% to 6.2% (chi(1)(2)=138.1; P<0.001). There was a shift from multinodular pathology, often seen in heavy infections, to uninodular lesions. In the control villages where no treatment took place, O. bifurcum infection increased from 17.8% (43/242) to 32.2% (39/121) (chi(1)(2)=9.6; P<0.001). Nodular pathology decreased slightly from 21.5% to 19.0%, but a higher proportion of these subjects developed multinodular pathology compared with baseline (chi(1)(2)=5.5; P=0.019). It is concluded that repeated albendazole treatment significantly reduces O. bifurcum-induced morbidity.

No evidence of Legionella infection in general practice patients presenting with acute respiratory infections in The Netherlands.

The role of Legionella spp. in the aetiology of acute respiratory infections (ARIs) is largely unknown. In this case-control study, conducted in a general practitioner setting during 2000 and 2001, nose and throat samples from patients presenting with ARIs (n = 230) and controls (n = 200) were analysed for the presence of Legionella spp. by real-time PCR. Legionella DNA was not detected in any of the cases or controls. Thus, Legionella spp. do not seem to play a role in patients presenting with ARIs, nor were they present in patients who visited their general practitioner for complaints other than ARIs.

[Diagnostic image (198). Four hospital employees with itch]. / Diagnose in beeld (198). Vier ziekenhuismedewerkers met jeuk.

Four employees working on an Internal Medicine ward complained of itch after contact with an HIV-positive woman with crusted scabies.

The evaluation of nasal mupirocin to prevent Staphylococcus aureus burn wound colonization in routine clinical practice.

BACKGROUND: Staphylococcus aureus wound colonization frequently occurs in patients with burns and can cause impaired wound healing. Nasal mupirocin application may contribute to the reduction of burn wound colonization of endogenous origin, whereas colonization by the exogenous route can be reduced by blocking cross-infection from other sources. In this study we evaluated whether the implementation of routine treatment of patients and burn center personnel using nasal mupirocin ointment reduces S. aureus burn wound colonization. METHODS: We composed three study groups, consisting of a control period (Control), a mupirocin period (MUP), in which patients with burns were all receiving nasal mupirocin at admission, and a mupirocin+personnel period (MUP+P), in which we also screened the burn center personnel and decolonized S. aureus carriers by nasal mupirocin. RESULTS: The patients who carried S. aureus in their nose and did not have S. aureus burn wound colonization at admission were considered as patients susceptible for the use of nasal mupirocin. In these patients, the S. aureus burn wound colonization rate was the same in all study groups. S. aureus nasal carriage was a significant independent risk factor for burn wound colonization (OR: 3.3; 95% CI: 1.4-7.6) when analyzed within the three study groups. CONCLUSION: Although S. aureus carriage is a significant risk factor for developing burn wound colonization, the routine use of nasal mupirocin did not contribute to a reduction of burn wound colonization.

An outbreak of a multiresistant methicillin-susceptible Staphylococcus aureus (MR-MSSA) strain in a burn centre: the importance of routine molecular typing.

Here we report an outbreak among 17 patients caused by a single strain of a multiresistant methicillin-susceptible Staphylococcus aureus (MR-MSSA) in a burn centre. The MR-MSSA strains were resistant to penicillin, ciprofloxacin, erythromycin, clindamycin and co-trimoxazole. Further analysis showed an increased prevalence of MR-MSSA carriership in S. aureus colonized patients admitted to the burn centre, from 0% in 2005 (0/118), 3.3% in 2006 (4/121), 6.1% in 2007 (6/99), to 7.8% in 2008 (7/90). Molecular typing with amplified fragment length polymorphism showed that all MR-MSSA isolates derived from burn centre patients had a unique genotype, and was different compared to isolates derived from other hospital patients. All healthcare workers (HCWs) who worked in the burn centre during the outbreak were screened for nasal carriage with MR-MSSA. One HCW tested positive for a genotype of MR-MSSA that was indistinguishable from the genotype found in samples of the burned patients. No new cases of MR-MSSA colonization or infection were identified after the colonized HCW stopped working at the burn centre. The routine practice of molecular typing of collected S. aureus strains from both patients and HCWs will help to detect nosocomial spread in a burn centre, and opens the possibility of a rapid, almost pre-emptive response.

Legionella spp. and Legionnaires' disease.

Infection with Legionella spp. is an important cause of community- and hospital-acquired pneumonia, occurring both sporadically and in outbreaks. Infection with Legionella spp. ranks among the three most common causes of severe pneumonia in the community setting, and is isolated in 1-40% of cases of hospital-acquired pneumonia. There are no clinical features unique to Legionnaires' disease. Macrolides and fluoroquinolones are the most widely used drugs in treatment. The availability of a good diagnostic repertoire, suitable for accurately diagnosing LD, constitutes the basis for the early recognition and treatment of the individual patient as well as for effective measures for prevention and control. This review summarizes the available information regarding the microbiology, clinical presentation, diagnosis and treatment of LD, with an emphasis on the laboratory diagnosis of infection with Legionella spp.

Evaluation of an internally controlled real-time polymerase chain reaction assay targeting the groEL gene for the detection of Bartonella spp. DNA in patients with suspected cat-scratch disease.

Bartonella (B.) henselae is the causative agent of cat-scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. This study reports the development and evaluation of an internally controlled real-time polymerase chain reaction targeting the groEL gene for detection of Bartonella spp. DNA was extracted using the MagNA Pure system. The lower detection limit was 10-100 fg DNA and the in vitro sensitivity of the assay was not affected by duplexing with an internal control PCR. The real-time PCR assay detected DNA from all five B. henselae strains tested, and from B. birtlesii, B. vinsonii subsp. vinsonii, B. vinsonii subsp. arupensis and B. doshiae. The assay generated negative results with a selection of other bacteria, including several Mycobacterium spp., Streptococcus pyogenes and Staphylococcus aureus. Results of real-time PCR in clinical samples were compared with those of a conventional 16S rDNA-based PCR assay. During the period described in the Material and methods section, real-time PCR and conventional 16S PCR were performed on 73 clinical samples. Of these samples, 29 (40%) were found to give positive results and 44 (60%) gave negative results, both by real-time PCR and by conventional PCR, with a 100% agreement between the two tests. The PCR developed in this study is a rapid, sensitive, and simple method for the detection of Bartonella spp. in CSD and is suitable for implementation in the diagnostic laboratory.

The role of atypical respiratory pathogens in exacerbations of chronic obstructive pulmonary disease.

The aetiology of acute exacerbations of chronic obstructive pulmonary disease (COPD) is heterogeneous and still under discussion. Serological studies have suggested that Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila may play a role in acute exacerbations of COPD. The presence of these atypical pathogens in sputum samples was investigated in patients with stable COPD and with acute exacerbations of COPD using real-time PCR. The present study was part of a randomised, double-blind, single-centre study and a total of 248 sputum samples from 104 COPD patients were included. In total, 122 samples obtained during stable disease (stable-state sputa) and 126 samples obtained during acute exacerbations of COPD (exacerbation sputa) were tested. Of the 122 stable-state sputa, all samples were negative for M. pneumoniae and C. pneumoniae DNA, whereas one sample was positive for Legionella non-pneumophila DNA. Of the 126 exacerbation sputa, all samples were negative for M. pneumoniae and C. pneumoniae DNA, whereas one sample was positive for Legionella non-pneumophila DNA. The possible relationship between the presence of atypical pathogens and the aetiology of acute exacerbations in chronic obstructive pulmonary disease was investigated in patients with stable disease and in those with acute exacerbations using real-time PCR. No indication was found of a role for Legionella spp., Chlamydia pneumoniae or Mycoplasma pneumoniae in stable, moderately severe chronic obstructive pulmonary disease and in its exacerbations.

The emergence of infections with community-associated methicillin resistant Staphylococcus aureus.

Recently there have been reports indicating an increased incidence of MRSA infections, afflicting individuals with no apparent risk factors for hospital acquisition. Patients with community-associated (CA) MRSA are significantly younger and had different distributions of clinical infections compared with HA-MRSA patients. CA-MRSA infections have mostly been associated with staphylococcal strains bearing the SCCmec type IV element and PVL genes. These strains are more frequently susceptible to a variety of non-beta-lactam antibiotics. Clinicians must be aware of the wide and, in some cases, unique spectrum of disease caused by CA-MRSA. Continued emergence of MRSA in the community is a public-health problem that warrants increased vigilance in the diagnosis and management of suspected and confirmed staphylococcal infections.

Evaluation of S. aureus ID, a chromogenic agar medium for the detection of Staphylococcus aureus.

BACKGROUND: Screening for staphylococci among various patient populations has become important for appropriate therapeutic management and for control of nosocomial infections. The purpose of this study is to evaluate the in vitro sensitivity and specificity of a chromogenic agar medium, S. aureus ID (bioMérieux, France), for the identification of Staphylococcus aureus. MATERIALS AND METHODS: A well-defined collection of S. aureus and coagulase-negative staphylococci (CNS) was used. The methicillin-resistant S. aureus (MRSA) isolates were collected in The Netherlands and all had a unique typing pattern. The methicillin-susceptible S. aureus (MSSA) and CNS were isolated from cultures of blood. The isolates were inoculated on Columbia agar plates with 5% sheep blood and incubated for 24 h at 35 degrees C. From the resulting cultures, a suspension of 0.5 McFarland was made and subsequently 10 mul was streaked on a S. aureus ID plate using a sterile loop. The results were read after 24 h and 48 h of incubation at 35 degrees C. Growth of colonies showing green coloration was considered to be positive (indicating S. aureus). RESULTS: A total of 519 S. aureus strains were tested (249 MSSA, 270 MRSA). The sensitivity to detect S. aureus was 96.5% (501/519) after 24 h and 97.5% (506/519) after 48 h. A total of 478 CNS were tested. The specificity was 98.5% (471/478) after 24 h and 98.3% (470/478) after 48 h. The differences between 24 h and 48 h incubation were not statistically significant. CONCLUSION: S. aureus ID is highly sensitive and specific to differentiate between S. aureus and CNS in vitro. Since the performance does not significantly differ between 24 h or 48 h of incubation, samples need only 1 day of incubation before optimal results can be obtained.

Community-acquired pneumonia caused by Legionella longbeachae in an immunocompetent patient.

Reported here is the case of a previously healthy 67-year-old man who was admitted to the intensive care unit with pneumonia caused by Legionella longbeachae. The organism was identified in sputum and serum by 16S rRNA-based PCR assay and sequence-based typing. One acute serum sample produced a single elevated IgM antibody titer of 1:512 against non-pneumophila Legionella spp. The patient fully recovered following the initiation of appropriate antibiotic treatment. Since most current laboratory tests for Legionella spp. cannot detect infections caused by non-pneumophila Legionella spp., culture on Legionella-selective media or PCR should be considered when diagnosing severe pneumonia of unknown etiology.