Bacterial infection (BI)-INDEX: an improved and simplified rapid flow cytometric bacterial infection marker.

The aim of this study was to develop a rapid and simple flow cytometric bacterial infection marker. In this prospective comparative study, quantitative flow cytometric analysis of CD10, CD35, CD66b, CD282, and MHC Class I molecules on human neutrophils, monocytes, and B-lymphocytes from 141 hospitalized febrile patients with suspected infection and from 50 healthy controls was performed. We developed a flow cytometric marker of local and systemic bacterial infections, designated "bacterial infection (BI)-INDEX", incorporating the quantitative analysis of CD10, CD35, MHCI, CD66b, and CD282 on neutrophils, monocytes, and B-lymphocytes, which displayed 90% sensitivity and 96% specificity in distinguishing between microbiologically confirmed bacterial (n = 31) and viral infections (n = 27) within a 1-h time-frame. We propose that our novel rapid BI-INDEX test will be useful in assisting physicians to ascertain whether antibiotic treatment is required, thus limiting unnecessary antimicrobial usage.

High-throughput screening of colonization samples for methicillin-resistant Staphylococcus aureus.

BACKGROUND: We present here the first application of 2-photon excited fluorescence detection (TPX) technology for the direct screening of clinical colonization samples for methicillin-resistant Staphylococcus aureus (MRSA). METHODS: A total of 125 samples from 14 patients with previously identified MRSA carriage and 16 controls from low-prevalence settings were examined. RESULTS: The results were compared to those obtained by both standard phenotypic and molecular methods. In identifying MRSA carriers, i.e. persons with at least 1 MRSA positive colonization sample by standard methods, the sensitivity of the TPX technique was 100%, the specificity 78%, the positive predictive value 75%, and the negative predictive value 100%. The TPX assay sensitivity per colonization sample was 89%, the specificity 93%, the positive predictive value 84%, and the negative predictive value 95%. The median time for a true-positive test result was 3 h and 26 min; negative test results are available after 13 h. The assay capacity was 48 samples per test run. CONCLUSIONS: The TPX MRSA technique could provide early preliminary results for clinicians, while simultaneously functioning as a selective enrichment step for further conventional testing. Costs and workload associated with hospital infection control can be reduced using this high-throughput, point-of-care compatible methodology.

Use of complement regulators, CD35, CD46, CD55, and CD59, on leukocytes as markers for diagnosis of viral and bacterial infections.

Several complement regulatory proteins exist on self-cells to prevent damage by the serum complement system. In the present study, we aimed to perform quantitative analysis of membrane-bound complement regulators, CR1 (CD35), MCP (CD46), DAF (CD55), and MIRL (CD59), on peripheral blood neutrophils, monocytes, and lymphocytes from healthy controls (n=36) and febrile patients diagnosed with either bacterial (n=21) or viral (n=26) infections. Our results show that: (a) increased CD35 and CD55 levels on neutrophils and monocytes present potent markers of bacterial infection, (b) increased expression of CD46 on monocytes is an indicator of viral infection, and (c) increased CD59 expression on neutrophils and monocytes is a general infection marker. Additionally, CD19-positive B-lymphocytes represent practically the only lymphocyte population capable of expressing CD35. We further developed two novel clinical flow cytometric markers (indices), specifically, clinical mononucleosis (CM)-INDEX (incorporating CD35, CD55, and CD59 expression on lymphocytes) and clinical bacterial infection (CBI)-INDEX (incorporating CD35 and CD55 expression on neutrophils and lymphocytes), for the effective detection of viral mononucleosis and bacterial infection, respectively. In summary, bacterial and viral infections induce different expression patterns of membrane-bound complement regulators in human leukocytes, which may be effectively exploited in clinical differential diagnosis.

A rapid flow cytometric method for distinguishing between febrile bacterial and viral infections.

Antibiotic resistance due to the inappropriate use of antimicrobials is one of the most critical public health problems worldwide. A major factor underlying the unnecessary use of antibiotics is the lack of rapid and accurate diagnostic tests. Therefore, we aimed to develop a novel rapid flow cytometric method for distinguishing between febrile bacterial and viral infections. In this prospective comparative study, quantitative flow cytometric analysis of FcγRII/CD32, CR1/CD35, MHC Class I receptor (MHCI), and C5aR/CD88 on human phagocytes was performed in 286 hospitalized febrile patients with suspected infection. After using microbiological and serological detection methods, or clinical diagnosis, 205 patients were identified with either bacterial (n=136) or viral (n=69) infection. Receptor data from patients were compared to those of 50 healthy controls. We developed a flow cytometric marker of local and systemic bacterial infections designated "bacterial infection score (BIS)" incorporating the quantitative analysis of FcγRII/CD32, CR1/CD35, C5aR/CD88 and MHCI on neutrophils and/or monocytes, which displays 91% sensitivity and 92% specificity in distinguishing between microbiologically confirmed bacterial (n=77) and serologically confirmed viral infections (n=61) within 1h. The BIS method was effectively applied to distinguish between bacterial and viral (pandemic H1N1 influenza) pneumonia cases with 96% sensitivity and 92% specificity. We propose that the rapid BIS test can assist physicians in deciding whether antibiotic treatment is necessary, thus reducing unnecessary antimicrobial use.

Inaccuracy of the disk diffusion method compared with the agar dilution method for susceptibility testing of Campylobacter spp.

The agar dilution method has been standardized by the CLSI for the susceptibility testing of Campylobacter species, and according to these standards, the disk diffusion method should be used only in screening for macrolide and ciprofloxacin resistance. Nevertheless, the disk diffusion test is currently widely used, since it is easy to perform in clinical microbiology laboratories. In this study, the disk diffusion method was compared to the agar dilution method by analyzing the in vitro activities of seven antimicrobial agents against 174 Campylobacter strains collected in Finland between 2003 and 2008. Recommendations of the CLSI were followed using Mueller-Hinton agar plates with 5% of sheep blood. For each strain, the disk diffusion tests were performed two to four times. Of the 33 erythromycin-resistant strains (MIC, ≥16 µg/ml), 24 (73%) constantly showed a 6-mm erythromycin inhibition zone (i.e., no inhibition), while for seven strains the inhibition zone varied from 6 to 44 mm in repeated measurements. Among the 141 erythromycin-susceptible strains (MIC, <16 µg/ml), erythromycin inhibition zones varied between 6 and 61 mm. Of the 87 ciprofloxacin-resistant strains, 47 (54%) showed 6-mm inhibition zones, while 40 strains showed inhibition zones between 6 and 60 mm. Significant differences between the repetitions were observed in the disk diffusion for all antimicrobial agents and all strains except for the macrolide-resistant strains regarding the macrolides. For 17 (10%) strains, the variation in repeated measurements was substantial. These results show that the disk diffusion method may not be a reliable tool for the susceptibility testing of Campylobacter spp. Further studies are needed to assess whether the disk diffusion test could be improved or whether all susceptibilities of campylobacters should be tested using an MIC-based method.

Ribosomal mutations as the main cause of macrolide resistance in Campylobacter jejuni and Campylobacter coli.

The aim of this study was to examine macrolide resistance mutations in Campylobacter species. In 76 strains studied, point mutation A to G at position 2059 of the 23S rRNA gene was detected in 30 of the 33 erythromycin-resistant strains. An amino acid insertion in the ribosomal protein L22 was found in one resistant strain without a 23S rRNA mutation. The A2059G mutation is the main cause of macrolide resistance in Campylobacter species.

Secular trend in candidemia and the use of fluconazole in Finland, 2004-2007.

BACKGROUND: In a previous study we observed an increasing trend in candidemia in Finland in the 1990s. Our aim was now to investigate further population-based secular trends, as well as outcome, and evaluate the association of fluconazole consumption and prophylaxis policy with the observed findings. METHODS: We analyzed laboratory-based surveillance data on candidemia from the National Infectious Diseases Register during 2004-2007 in Finland. Data on fluconazole consumption, expressed as defined daily doses, DDDs, was obtained from the National Agency for Medicines, and regional prophylaxis policies were assessed by a telephone survey. RESULTS: A total of 603 candidemia cases were identified. The average annual incidence rate was 2.86 cases per 100,000 population (range by year, 2.59-3.09; range by region, 2.37-3.85). The highest incidence was detected in males aged >65 years (12.23 per 100,000 population). Candida albicans accounted for 67% of cases, and C. glabrata ranked the second (19%), both without any significant change in proportions. C. parapsilosis accounted for 5% of cases and C. krusei 3% of cases. The one-month case-fatality varied between 28-32% during the study period. Fluconazole consumption increased from 19.57 DDDs per 100,000 population in 2000 to 25.09 in 2007. Systematic fluconazole prophylaxis was implemented for premature neonates, patients with acute leukemias and liver transplant patients. CONCLUSION: The dominant proportion of C. albicans remained stable, but C. glabrata was the most frequent non-albicans species. The proportion of C. glabrata had increased from our previous study period in the presence of increasing use of fluconazole. The rate of candidemia in Finland is still low but mortality high like in other countries.

In vitro activity of azithromycin against nontyphoidal Salmonella enterica.

The in vitro activity of azithromycin against 1,237 nontyphoidal Salmonella enterica isolates collected from Finnish patients between 2003 and 2008 was investigated. Only 24 (1.9%) of the isolates tested and 15 (5.1%) of the 294 isolates with reduced fluoroquinolone susceptibility had azithromycin MICs of >or=32 microg/ml. These data show that azithromycin has good in vitro activity against nontyphoidal S. enterica, and thus, it may be a good candidate for clinical treatment studies of salmonellosis.

Metastasis in the temporal bone mimicking temporal arteritis.

This report describes a 72-year-old female patient with a previous history of cured breast cancer who presented with acute monocular visual disturbances, intense unilateral headache, painful temporal artery palpation, high erythrocyte sedimentation rate, and pain and weakness of the femoral muscles. These symptoms and signs were suggestive of temporal arteritis, but the finding of the temporal artery biopsy was negative, and the patient did not respond to corticosteroid treatment. Later, whole body bone scintigraphy revealed dissemination of malignancy throughout her skeleton including the skull. To our knowledge, this is the first report to show that a metastasis in the temporal bone can closely mimic temporal arteritis.

Antimicrobial susceptibilities of multidrug-resistant Campylobacter jejuni and C. coli strains: in vitro activities of 20 antimicrobial agents.

There is a paucity of information regarding antimicrobial agents that are suitable to treat severe infections caused by multidrug-resistant Campylobacter spp. Our aim was to identify agents that are potentially effective against multiresistant Campylobacter strains. The in vitro activities of 20 antimicrobial agents against 238 Campylobacter strains were analyzed by determining MICs by the agar plate dilution method or the Etest. These strains were selected from 1,808 Campylobacter isolates collected from Finnish patients between 2003 and 2005 and screened for macrolide susceptibility by using the disk diffusion test. The 238 strains consisted of 183 strains with erythromycin inhibition zone diameters of < or =23 mm and 55 strains with inhibition zone diameters of >23 mm. Of the 238 Campylobacter strains, 19 were resistant to erythromycin by MIC determinations (MIC > or = 16 microg/ml). Given that the resistant strains were identified among the collection of 1,808 isolates, the frequency of erythromycin resistance was 1.1%. All erythromycin-resistant strains were multidrug resistant, with 18 (94.7%) of them being resistant to ciprofloxacin (MIC > or = 4 microg/ml). The percentages of resistance to tetracycline and amoxicillin-clavulanic acid (co-amoxiclav) were 73.7% and 31.6%, respectively. All macrolide-resistant strains were susceptible to imipenem, meropenem, and tigecycline. Ten (52.6%) multiresistant strains were identified as being Campylobacter jejuni strains, and 9 (47.4%) were identified as being C. coli strains. These data demonstrate that the incidence of macrolide resistance was low but that the macrolide-resistant Campylobacter strains were uniformly multidrug resistant. In addition to the carbapenems, tigecycline was also highly effective against these multidrug-resistant Campylobacter strains in vitro. Its efficacy for the treatment of human campylobacteriosis should be evaluated in clinical trials.

Simultaneous quantitative analysis of Fc gamma RI (CD64) and CR1 (CD35) on neutrophils in distinguishing between bacterial infections, viral infections, and inflammatory diseases.

A flow cytometric quantitative analysis of receptors on neutrophils can be exploited in distinguishing between inflammatory and infectious diseases. In this prospective comparative study, simultaneous quantitative analysis of CD64 and CD35 on peripheral blood neutrophils was performed in febrile patients in order to differentiate between bacterial infections (n=89), viral infections (n=46), and inflammatory diseases (n=21). The patient data was compared to 60 healthy controls. We could divide patients into three groups depending on how they express CD35 and CD64 on neutrophils: (1) patients with a high probability of viral infection (low CD35/low CD64 and low CD35/high CD64), (2) patients with a high probability of inflammatory disease (high CD35/low CD64), and (3) patients with a high probability of bacterial infection (high CD35/high CD64). In summary, simultaneous quantitative analysis of CD64 and CD35 on neutrophils could potentially assist physicians to distinguish between inflammatory and infectious diseases.

Methicillin-resistant Staphylococcus aureus screening by online immunometric monitoring of bacterial growth under selective pressure.

Rapid, high-throughput screening tools are needed to contain the spread of hospital-acquired methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains. Most techniques used in current clinical practice still require time-consuming culture for primary isolation of the microbe. We present a new phenotypic assay for MRSA screening. The technique employs a two-photon excited fluorescence (TPX) detection technology with S. aureus-specific antibodies that allows the online monitoring of bacterial growth in a single separation-free process. Different progressions of fluorescence signals are recorded for methicillin-susceptible and -resistant strains when the growth of S. aureus is monitored in the presence of cefoxitin. The performance of the new technique was evaluated with 20 MRSA strains, 6 methicillin-susceptible S. aureus strains, and 7 coagulase-negative staphylococcal strains and two different monoclonal S. aureus-specific antibodies. When either of these antibodies was used, the sensitivity and the specificity of the TPX assay were 100%. All strains were correctly classified within 8 to 12 h, and up to 70 samples were simultaneously analyzed on a single 96-well microtiter plate. As a phenotypic method, the TPX assay is suited for screening purposes. The final definition of methicillin resistance in any S. aureus strain should be based on the presence of the mecA gene. The main benefit afforded by the initial use of the TPX methodology lies in its low cost and applicability to high-throughput analysis.

Mechanisms of resistance in nontyphoidal Salmonella enterica strains exhibiting a nonclassical quinolone resistance phenotype.

Nontyphoidal Salmonella enterica strains with a nonclassical quinolone resistance phenotype were isolated from patients returning from Thailand or Malaysia to Finland. A total of 10 isolates of seven serovars were studied in detail, all of which had reduced susceptibility (MIC > or = 0.125 microg/ml) to ciprofloxacin but were either susceptible or showed only low-level resistance (MIC < or = 32 microg/ml) to nalidixic acid. Phenotypic characterization included susceptibility testing by the agar dilution method and investigation of efflux activity. Genotypic characterization included the screening of mutations in the quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE by PCR and denaturing high-pressure liquid chromatography and the amplification of plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, qnrD, aac(6')-Ib-cr, and qepA by PCR. PMQR was confirmed by plasmid analysis, Southern hybridization, and plasmid transfer. No mutations in the QRDRs of gyrA, gyrB, parC, or parE were detected with the exception of a Thr57-Ser substitution within ParC seen in all but the S. enterica serovar Typhimurium strains. The qnrA and qnrS genes were the only PMQR determinants detected. Plasmids carrying qnr alleles were transferable in vitro, and the resistance phenotype was reproducible in Escherichia coli DH5alpha transformants. These data demonstrate the emergence of a highly mobile qnr genotype that, in the absence of mutation within topoisomerase genes, confers the nontypical quinolone resistance phenotype in S. enterica isolates. The qnr resistance mechanism enables bacteria to survive elevated quinolone concentrations, and therefore, strains carrying qnr alleles may be able to expand during fluoroquinolone treatment. This is of concern since nonclassical quinolone resistance is plasmid mediated and therefore mobilizable.

CRP/CD11b ratio: a novel parameter for detecting gram-positive sepsis.

To commence proper antibiotic treatment in sepsis, timely knowledge of whether the cause of systemic infection is gram-negative (gram(-)) or gram-positive (gram(+)) bacteria in origin would be beneficial for clinicians. In this clinical prospective study, our objective was to develop a method for distinguishing between gram(+) and gram(-) bacterial infection. In gram(-) bacterial infection (n = 21), the average amount of CD11b on neutrophils was significantly higher than in gram(+) bacterial infection (n = 22). On the contrary, serum C-reactive protein (CRP) level was significantly higher in gram(+) than in gram(-) bacterial infection. By dividing the serum CRP value by the amount of CD11b on neutrophils, we derived a novel marker of gram(+) sepsis, CRP/CD11b ratio, which displayed 76% sensitivity and 80% specificity for the detection of gram(+) sepsis (n = 17) among febrile patients with microbiologically confirmed or clinically diagnosed bacterial infection. The detection of gram(+) sepsis is possible after the combination of neutrophil CD11b data and serum CRP level. In conclusion, our findings indicate that the proposed CRP/CD11b ratio test could potentially assist physicians in determining an appropriate antibiotic treatment in patients with severe bacterial infection.

Comparison of degranulation of easily mobilizable intracellular granules by human phagocytes in healthy subjects and patients with infectious diseases.

The aim of this study was to compare degranulation of easily mobilizable secretory vesicles (SVs) or secretory vesicle-like granules (SVLGs) in neutrophils, monocytes, and eosinophils of healthy controls (n = 60) and febrile patients with microbiologically confirmed or clinically diagnosed bacterial (n = 89) and viral (n = 46) infections. For this purpose, flow cytometric immunophenotyping of isolated phagocytes was performed using monoclonal antibodies against the phagocytosis receptors CR1 (CD35) and CR3 (CD11b) that are predominantly stored in the SVs of resting neutrophils. Similar to neutrophils, monocytes contain easily mobilizable SVLGs that constitute the main intracellular reservoir of CD35 and CD11b. In both neutrophils and monocytes, activation mechanisms leading to degranulation of SV and SVLG appeared dependent on both intra- and extracellular calcium levels. The kinetics of degranulation of SVLGs in control monocytes was significantly faster than that of SVs of control neutrophils. We conclude that phagocytes in patients with bacterial infections can be arranged in order of decreasing magnitude of SV or SVLG degranulation as follows (from left to right): neutrophils > monocytes " eosinophils. However, in viral infections, the corresponding degranulation order is monocytes > neutrophils approximately eosinophils.

Reduced fluoroquinolone susceptibility in Salmonella enterica isolates from travelers, Finland.

We tested the fluoroquinolone susceptibility of 499 Salmonella enterica isolates collected from travelers returning to Finland during 2003-2007. Among isolates from travelers to Thailand and Malaysia, reduced fluoroquinolone susceptibility decreased from 65% to 22% (p = 0.002). All isolates showing nonclassical quinolone resistance were from travelers to these 2 countries.

A novel method for distinguishing between dsDNA and ssRNA virus infections.

BACKGROUND: To commence proper antiviral treatment, timely knowledge of whether the infection is caused by DNA or RNA virus would be beneficial for the clinician. OBJECTIVES: Our objective was to develop a method for distinguishing between DNA and RNA virus infections. STUDY DESIGN: In this prospective study, total and differential count of leukocytes, serum C-reactive protein level, erythrocyte sedimentation rate, and quantitative flow cytometric analysis of FcgammaRI (CD64) on neutrophils and monocytes were obtained from 289 hospitalized febrile patients. After microbiological confirmation, 89 patients (31%) were found to have either bacterial (n=46) or viral (n=43) infection. The patient data was compared to 60 healthy controls. RESULTS: For the first time ever, it was noticed that in dsDNA virus infections (n=21) the average amount of CD64 on neutrophils was over five-fold compared to ssRNA virus infections (n=22). CONCLUSIONS: DNA virus score (DNAVS) point, which incorporates quantitative analysis of CD64 on neutrophils and total and differential count of leukocytes, varied between 0 and 8, and displayed 95% sensitivity and 100% specificity in distinguishing between dsDNA and ssRNA virus infections [average (S.D.); DNAVS points: 5.4 (2.5) vs. 0.3 (0.4); p<0.001].

Long-term outcome of infective endocarditis: a study on patients surviving over one year after the initial episode treated in a Finnish teaching hospital during 25 years.

BACKGROUND: Only a few previous studies have focused on the long-term prognosis of the patients with infective endocarditis (IE). Our purpose was to delineate factors potentially associated with the long-term outcome of IE, recurrences of IE and requirement for late valve surgery. METHODS: A total of 326 episodes of IE in 303 patients were treated during 1980-2004 in the Turku University Hospital. We evaluated the long-term outcome and requirement for late valve surgery for 243 of these episodes in 226 patients who survived longer than 1 year after the initial admission. Factors associated with recurrences were analysed both for the 1-year survivors and for all 303 patients. RESULTS: The mean (SD) follow-up time for the 1-year survivors was 11.5 (7.3) years (range 25 days to 25.5 years). The overall survival was 95%, 82%, 66%, 51% and 45% at 2, 5, 10, 15 and 20 years. In age and sex adjusted multivariate analyses, significant predictors for long-term overall mortality were heart failure within 3 months of admission (HR 1.97, 95% CI 1.27 to 3.06; p = 0.003) and collagen disease (HR 2.54, 95% CI 1.25 to 5.19; p = 0.010) or alcohol abuse (HR 2.39, 95% CI 1.30 to 4.40; p = 0.005) as underlying conditions, while early surgery was significantly associated with lower overall mortality rates (HR 0.31, 95% CI 0.17 to 0.58; p < 0.001). Heart failure was also significantly associated with the long-term cardiac mortality (p = 0.032). Of all 303 patients, 20 had more than 1 disease episode. Chronic dialysis (p = 0.002), intravenous drug use (p = 0.002) and diabetes (p = 0.015) were significant risk factors for recurrent episodes of IE, but when analysed separately for the 1-year survivors, only chronic dialysis remained significant (p = 0.017). Recurrences and late valve surgery did not confer a poor prognosis. CONCLUSION: Heart failure during the index episode of IE was the complication, which significantly predicted a poor long-term outcome. Patients who underwent surgery during the initial hospitalisation for IE faired significantly better than those who did not.

Imported tungiasis in a Finnish journalist: the first case reported from the Nordic countries.

Tungiasis is a parasitic infection widely spread in tropical Africa and in South and Central America. Only a few cases involving travellers have been reported from Europe, and none from the Nordic countries. We report a case of tungiasis in a Finnish journalist returning from Uganda. In this era of increasing intercontinental travel it is important that all physicians are aware of tungiasis.