Identification of haemolytic Haemophilus species isolated from human clinical specimens and description of Haemophilus sputorum sp. nov.

Haemolytic Haemophilus strains with no requirement for X factor are regularly isolated from sputum and throat swabs and occasionally from invasive infections, but the classification of such strains is not clear. We characterized 56 strains with a phenotype concordant with Haemophilus parahaemolyticus (V, but not X factor-dependent; urease-positive; tryptophanase-negative; ornithine decarboxylase-negative) by extended phenotypic testing and 16S rRNA gene sequencing. In addition, 31 of the strains and representative type strains were investigated by multilocus sequence analysis based on 3 housekeeping gene fragments. Most strains could be assigned to H. parahaemolyticus and were characterized by expression of IgA1 protease and a negative test for ß-galactosidase. Isolation of H. parahaemolyticus from various infections and its absence among more than 300 commensal Haemophilus isolates suggests a pathogenic potential of this organism. The majority of haemolytic strains with ß-galactosidase activity did not cluster with the type strain of H. paraphrohaemolyticus, but constituted a distinct and coherent novel taxon. Ten strains of this new taxon proved to be genetically and phenotypically homogeneous. Few biochemical characters discriminate the new taxon from related Haemophilus species, but identification is easily accomplished by routine matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Genetic, biochemical, and spectrometry data show that the taxon merits recognition as a novel species of Haemophilus. The name Haemophilus sputorum is proposed, with CCUG 13788(T) (=DSM 24472(T)=NCTC 13537(T)) as the type strain.

National surveillance of fungemia in Denmark (2004 to 2009).

A 6-year nationwide study of fungemia in Denmark was performed using data from an active fungemia surveillance program and from laboratory information systems in nonparticipating regions. A total of 2,820 episodes of fungemia were recorded. The incidence increased from 2004 to 2007 (7.7 to 9.6/100,000) and decreased slightly from 2008 to 2009 (8.7 to 8.6/100,000). The highest incidences were seen at the extremes of age (i.e., 11.3 and 37.1/100,000 for those <1 and 70 to 79 years old, respectively). The rate was higher for males than for females (10.1 versus 7.6/100,000, P = 0.003), with the largest difference observed for patients >50 years of age. The species distribution varied significantly by both age and gender. Candida species accounted for 98% of the pathogens, and C. albicans was predominant, although the proportion decreased (64.4% to 53.2%, P < 0.0001). C. glabrata ranked second, and the proportion increased (16.5% to 25.9%, P = 0.003). C. glabrata was more common in adults and females than in children and males, whereas C. tropicalis was more common in males (P = 0.020). C. krusei was a rare isolate (4.1%) except at one university hospital. Acquired resistance to amphotericin and echinocandins was rare. However, resistance to fluconazole (MIC of >4 µg/ml) occurred in C. albicans (7/1,183 [0.6%]), C. dubliniensis (2/65 [3.1%]), C. parapsilosis (5/83 [6.0%]), and C. tropicalis (7/104 [6.7%]). Overall, 70.8% of fungemia isolates were fully fluconazole susceptible, but the proportion decreased (79.7% to 68.9%, P = 0.02). The study confirmed an incidence rate of fungemia in Denmark three times higher than those in other Nordic countries and identified marked differences related to age and gender. Decreased susceptibility to fluconazole was frequent and increasing.

Molecular screening for Candida orthopsilosis and Candida metapsilosis among Danish Candida parapsilosis group blood culture isolates: proposal of a new RFLP profile for differentiation.

Candida orthopsilosis and Candida metapsilosis are recently described species phenotypically indistinguishable from Candida parapsilosis . We evaluated phenotyping and molecular methods for the detection of these species among 79 unique blood culture isolates of the C. parapsilosis group obtained during the years 2004-2008. The isolates were screened by PCR amplification of the secondary alcohol dehydrogenase-encoding gene ( SADH) followed by digestion with the restriction enzyme Ban I, using C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96139 and C. metapsilosis ATCC 96144 as controls. Isolates with RFLP patterns distinct from C. parapsilosis were characterized by sequence analysis of the ITS1-ITS2, 26S rRNA (D1/D2) and SADH regions. Restriction patterns for the 3 species with each of 610 restriction enzymes were predicted in silico using 12 available sequences. By PCR-RFLP of the SADH gene alone, four isolates (5.1 %) had a pattern identical to the C. orthopsilosis reference strain. Sequence analysis of SADH and ITS (internal transcribed spacer) regions identified two of these isolates as C. metapsilosis. These results were confirmed by creating a phylogenetic tree based on concatenated sequences of SADH, ITS and 26S rRNA gene sequence regions. Optimal differentiation between C. parapsilosis, C. metapsilosis and C. orthopsilosis was predicted using digestion with NlaIII, producing discriminatory band sizes of: 131 and 505 bp; 74, 288 and 348 bp; and 131, 217 and 288 bp, respectively. This was confirmed using the reference strains and 79 clinical isolates. In conclusion, reliable discrimination was obtained by PCR-RFLP profile analysis of the SADH gene after digestion with NlaIII but not with BanI. C. metapsilosis and C. orthopsilosis are involved in a small but significant number of invasive infections in Denmark.

Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis.

Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic characterization using 100 fastidious Gram negative bacteria. Seventy-five strains represented 21 of the 26 taxa included in the Vitek 2 NH database and 25 strains represented related species not included in the database. Of the 100 strains, 31 were the type strains of the species. Vitek 2 NH identification results: 48 of 75 database strains were correctly identified, 11 strains gave `low discrimination´, seven strains were unidentified, and nine strains were misidentified. Identification of 25 non-database strains resulted in 14 strains incorrectly identified as belonging to species in the database. Partial 16S rRNA gene sequence analysis results: For 76 strains phenotypic and sequencing identifications were identical, for 23 strains the sequencing identifications were either probable or possible, and for one strain only the genus was confirmed. Thus, the Vitek 2 NH system identifies most of the commonly occurring species included in the database. Some strains of rarely occurring species and strains of non-database species closely related to database species cause problems. Partial 16S rRNA gene sequence analysis performs well, but does not always suffice, additional phenotypical characterization being useful for final identification.

Breakthrough Aspergillus fumigatus and Candida albicans double infection during caspofungin treatment: laboratory characteristics and implication for susceptibility testing.

Caspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicans and Aspergillus fumigatus detected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicans to caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1 sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatus and resistance mutations. The MICs for the C. albicans isolate were as follows: >32 microg/ml caspofungin and 0.5 microg/ml anidulafungin by Etest, 2 microg/ml caspofungin and 0.125 microg/ml anidulafungin by EUCAST methods, and 1 microg/ml caspofungin and 0.5 microg/ml anidulafungin by CLSI methods. Sequencing of the FKS1 gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P > 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatus without FSK1 resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1 resistance mutations in the Aspergillus isolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods.

Listeria monocytogenes: maternal-foetal infections in Denmark 1994-2005.

Maternal-foetal infection by Listeria monocytogenes is a rare complication in pregnancy. In the period 1994-2005, 37 culture-confirmed cases of maternal-foetal Listeria monocytogenes infections were reported in Denmark. We examined 36 patients' files in order to evaluate risk factors, clinical and laboratory findings, response to therapy, and outcome for maternal-foetal listeriosis. Patient data and bacteriological findings were divided into 2 groups for comparison: 1 group with children born alive (n=24) and another group with abortion or stillbirth (n=12). 23 of the 36 children survived the acute infection, as did all the mothers. The mothers were generally only mildly affected by the infection. In contrast, among the children born alive, 15 were diagnosed with bacteraemia/septicaemia, 3 children with pneumonia, 3 with neonatal meningitis, and 3 were unaffected. Despite the high frequency of illness only 1 of the live-born children died from the infection and none of the surviving children showed signs of permanent damage at the time they were discharged from hospital. Listeriosis during pregnancy is a serious threat to the unborn child. One-third of culture-confirmed cases of maternal-foetal infections resulted in abortion or stillbirth; however, the prognosis for live-born children is good, even in severely ill newborns.

Six cases of Aerococcus sanguinicola infection: clinical relevance and bacterial identification.

Aerococcus sanguinicola is a Gram-positive coccus first described in 2001. Infections in humans are rare but the use of 16S rRNA gene sequencing and improved phenotypic methods has facilitated the identification of A. sanguinicola. We report here 6 cases of A. sanguinicola bacteraemia, 2 of which were associated with infective endocarditis. Most patients were elderly (median age 70 y) and had underlying neurological disorders including dementia, cerebral degeneration, and myelomeningocele. The primary focus of infection was the urinary tract in 3 cases and the gallbladder in 1; no focus was detected in 2 cases. Long-term prognosis was poor reflecting the frailty of the patients. All strains were susceptible to penicillin, ampicillin, cefuroxime, vancomycin, erythromycin, and rifampicin. The optimal treatment of infection with A. sanguinicola has yet to be determined.

Case of bacteraemic cellulitis by a non-haemolytic strain of Streptococcus pyogenes.

Erysipelas and bacteraemia with what initially was diagnosed as a non-haemolytic streptococcus is reported. As neither colony morphology nor clinical picture was characteristic of non-haemolytic streptococci, the isolate was sent to a reference laboratory. 16S rRNA sequencing and phenotypic characterization identified the strain as a streptolysin S-deficient S. pyogenes..

Bedside diagnosis of imported malaria using the Binax Now malaria antigen detection test.

Malaria may be misdiagnosed in non-endemic countries when the necessary experience for rapid expert microscopy is lacking. Rapid diagnostic tests may improve the diagnosis and may play a role as a bedside diagnostic tool. In a multicentre study we recruited patients suspected of malaria over a period of 14 months. The Binax Now Malaria rapid test was used at the bedside and in the clinical microbiology laboratory. The training of clinical staff was monitored and their experience with the use of the test was recorded. 542 patients were included, 80 of whom had malaria diagnosed by microscopy. The rapid test used at the bedside had a sensitivity of 88% for the detection of P. falciparum compared to 95% when the test was performed in the microbiology laboratory. The risk of technical problems and invalid tests was highest when the test was used at the bedside. The rapid diagnostic test may be useful for the diagnosis of P. falciparum malaria when used by routine laboratory staff, but could lead to misdiagnoses when used at the bedside. Microscopy is still essential in order to identify the few missed diagnoses, to determine the degree of parasitaemia, and to ensure species diagnosis, including mixed infections.

Antibiotic susceptibility of Listeria monocytogenes in Denmark 1958-2001.

In order to see whether the susceptibility of Danish Listeria monocytogenes strains has changed over the years we examined a collection of human isolates from the period 1958-2001. We, furthermore, wanted to compare L. monocytogenes susceptibility testing using a disc diffusion assay with MIC measurements performed by the E-test. 106 strains isolated predominantly from blood cultures and cerebrospinal fluids were examined together with three reference strains. Susceptibility to the following antibiotics was tested by the E-test and by Oxoid discs using Iso-sensitest agar: penicillin G, ampicillin, meropenem, gentamicin, sulphamethoxazole, trimethoprim, ciprofloxacin, erythromycin, vancomycin, linezolid, chloramphenicol and tetracycline. The strains were in the main sensitive to all antibiotics examined using both methods, except for ciprofloxacin, where the strains were intermediate sensitive. However, for penicillin, ampicillin and sulphamethoxazole, while the disc diffusion assay found the strains to be sensitive, MIC measurements generally placed the strains one dilution above the breakpoint for sensitivity in the intermediate sensitive group. Based on the MIC measurements, the antibiotic susceptibility of L. monocytogenes has not changed in Denmark from 1958 to 2001, and the multiresistant strains found in human infections elsewhere have not been found in Denmark.

Multilocus sequence phylogenetic study of the genus Haemophilus with description of Haemophilus pittmaniae sp. nov.

The phylogeny of human isolates of Haemophilus species was estimated based on partial sequences of four separate housekeeping genes. The clustering of each set of sequences was in accordance with speciation of the strains with few exceptions: of 108 gene fragments examined, only three appeared to have been subject to recombination events across the species barrier. Housekeeping gene similarity supported previous DNA-DNA hybridization data for the genus rather than the phylogeny inferred from 16S rRNA gene sequence comparison. The similarity of sequences of Haemophilus parainfluenzae with those of Haemophilus influenzae suggested preservation of the former species in the genus Haemophilus. Three strains representing a novel taxon were unique with respect to the four investigated gene loci. 16S rRNA gene sequence analysis suggested that this taxon belonged to the Parainfluenzae cluster. DNA-DNA hybridization data supported this generic placement. Nine strains of the novel taxon were available for analysis. They were distinct from representatives of all current species of the genus Haemophilus by conventional phenotypic characterization. Genotypic and phenotypic data show that the strains merit recognition as a novel species of Haemophilus. The name Haemophilus pittmaniae sp. nov. is proposed, with HK 85T (=CCUG 48703T=NCTC 13334T) as the type strain.

Clonal relationship of recent invasive Haemophilus influenzae serotype f isolates from Denmark and the United States.

Surveillance performed after the introduction of general Haemophilus influenzae serotype b (Hib) vaccination in Denmark identified 13 cases of invasive bacteraemic H. influenzae serotype f (Hif) disease in adults over a period of 7 years. Bacteraemic respiratory tract infections accounted for 61 % of cases, but meningitis, epiglottitis and osteoarthritis were also seen. Recent Danish isolates were compared to recent American isolates, historical Hif strains and non-Hif invasive strains. Results of conventional serotyping were confirmed by PCR detection of the serotype-f-specific cap and bexA gene sequences. Multilocus enzyme electrophoresis typing revealed that recent Danish and American isolates belonged to a single Hif clone, which may be undergoing expansion. The need for accurate serotyping of H. influenzae to enable reliable monitoring for Hib replacement by other capsular types is emphasized.

Delineation of the genus Actinobacillus by comparison of partial infB sequences.

A 426 bp fragment of infB, a housekeeping gene that encodes translation initiation factor 2, was sequenced from 59 clinical isolates and type strains of Actinobacillus species and sequences were compared. Partial sequences of 16S rRNA genes were also obtained. By comparing infB sequences, Actinobacillus pleuropneumoniae, Actinobacillus equuli, Actinobacillus suis, Actinobacillus ureae, Actinobacillus arthritidis, Actinobacillus hominis and two unnamed genomospecies showed more than 85 % similarity to the type strain of the type species of the genus, Actinobacillus lignieresii. The taxonomic position of Actinobacillus capsulatus was unresolved; this species is more remotely related to A. lignieresii. The two species A. lignieresii and A. pleuropneumoniae could not be clearly separated by infB sequence analysis. The phylogeny of the genus Actinobacillus based on infB analysis was essentially congruent with relationships inferred from 16S rRNA sequence comparisons and DNA hybridization studies. Discrepancies were encountered with single strains or taxa at the periphery of the genus. Greater intraspecies variation was observed with infB sequences than with 16S rRNA gene sequences, with notable exceptions. The apparent subdivision of some species by 16S rRNA analysis was most likely caused by RNA operon heterogeneity.

Brucellosis in immigrants in Denmark.

Brucellosis is a rarely encountered infection in northern Europe. We report 4 cases of Brucella abortus bacteremia occurring in Denmark during 1999-2000. The clinical presentation was characteristically vague and brucellosis was not suspected by the attending physicians, partly because incomplete patient histories were obtained as a result of language barriers. The diagnosis was finally established by means of blood cultures, which were performed because of fever of unknown origin.

Phylogeny of the genus Haemophilus as determined by comparison of partial infB sequences.

A 453 bp fragment of infB, the gene encoding translation initiation factor 2, was sequenced and compared from 66 clinical isolates and type strains of Haemophilus species and related bacteria. Analysis of the partial infB sequences obtained suggested that the human isolates dependent on X and V factor, H. influenzae, H. haemolyticus, H. aegyptius and some cryptic genospecies of H. influenzae, were closely related to each other. H. parainfluenzae constituted a heterogeneous group within the boundaries of the genus, whereas H. aphrophilus/paraphrophilus and Actinobacillus actinomycetemcomitans were only remotely related to the type species of the genus Haemophilus H. parahaemolyticus and H. paraphrohaemolyticus took up an intermediary position and may not belong in the genus Haemophilus sensu stricto. Ambiguous results were obtained with seven isolates tentatively identified as H. segnis, which fell into two discrete clusters. The delineation of 'Haemophilus sensu stricto' as suggested by infB analysis supports previous results obtained by DNA hybridization, in contrast to the delineation inferred from 16S rRNA sequence comparison.

Evaluation of the Rapid ID 32 Strep system.

OBJECTIVE: To evaluate the performance of the Rapid ID 32 Strep system in the hands of clinical microbiologists without expert knowledge of streptococci or enterococci. METHODS: One hundred and twenty-two strains of streptococci and enterococci conventionally identified in a reference laboratory were sent under code numbers to a clinical microbiology laboratory and identified with the Rapid ID 32 Strep system. RESULTS: Regardless of whether automatic reading and identification or visual reading with identification using tables were done, 75-77% of the 122 examined strains were correctly identified, 7% were misidentified and 16-18% could not be identified with certainty to the species level. The system correctly identified the majority of the examined pyogenic streptococci and enterococci, but only two-thirds of the viridans streptococcal strains. CONCLUSIONS: In a routine laboratory, the Rapid ID 32 Strep system can be used to give a rapid preliminary identification of streptococci and enterococci, but with viridans streptococci one would have to accept a certain risk of mis-identification. The assay can, however, be used to biotype viridans streptococci in order to attempt to establish identity between separate isolates, e.g. from blood in patients suspected of having endocarditis.

Fungemia: An increasing problem in a Danish university hospital 1989 to 1994.

OBJECTIVE: To present data on episodes of fungemia in a Danish tertiary-care university hospital admitting all types of patients and to compare the data with previous findings from the same hospital. METHODS: Retrospective identification of episodes of fungemia from 1989 to 1994 and collection of data from computerized files at the Clinical Microbiology Department at Rigshospitalet and the Mycology Reference Laboratory at Statens Seruminstitut. RESULTS: The incidence of fungemia increased gradually from 19 episodes in 1989 to 57 episodes in 1994. An earlier report from the same hospital showed 20 to 25 episodes of fungemia per year between 1984 and 1988. Candida albicans was the dominating species during both periods, accounting for 73% of isolates during 1984 to 1988 and 67% during 1989 to 1994. However, in the hematology department where fluconazole has been used extensively, C. albicans constituted 47% of isolates with Candida krusei and Candida glabrata comprising 25%. CONCLUSIONS: The incidence of fungemia in our tertiary-care hospital has increased threefold from 1989 to 1994. Candida albicans was the dominating cause of fungemia but, in the hematology department, this yeast accounted for less than half of the isolates during the same time period.

Evaluation of the ATB 32 C system for identification of clinical yeast isolates.

OBJECTIVE: To compare the ATB 32 C system for routine identification of clinical yeast isolates in a clinical microbiology laboratory with identification carried out by conventional methods in a mycology reference laboratory. METHODS: A total of 113 strains initially isolated at our hospital and identified in the reference laboratory were returned in duplicate, under separate code numbers, to the microbiology laboratory where the ATB 32 C system was used for identification by: 1) visual assessment of turbidity at 72 h with use of identification table; 2) visual assessment at 72 h with use of ATB 32 C analytical profile index; and 3) automatic readings with the ATB reader at 48 h and 72 h with results of growth assessments transmitted to a computer and interpreted by the ATB 32 C software. RESULTS: Visual assessment plus identification table and visual assessment plus profile index provided correct identification in 98% and 91% of strains, respectively. Visual assessment was, however, sometimes difficult and required more experience than is usually available in a routine clinical microbiology laboratory. Automatic readings with computer identification plus supplementary tests correctly identified 87% and 86% after 48 h and 72 h, respectively. CONCLUSIONS: The ATB 32 C system with automatic readings and computer identification is a satisfactory system for identification of clinical yeast isolates in a routine clinical microbiology laboratory.