Multispacer sequence typing of Coxiella burnetii DNA from removed prosthetic heart valve material discloses first human case of infective endocarditis caused by MST_18.

INTRODUCTION: In Denmark, Q fever has previously been considered a rare and imported disease; however, recent testing of antibodies in cattle as well as humans has indicated that the infection is widespread. A 76-year-old Danish man was diagnosed with infective endocarditis and underwent open surgical aortic valve replacement with insertion of a biological valve. Due to paravalvular leakage, destruction of the aortic annulus, and an aortic root abscess, the patient underwent re-operation 3 weeks later, with replacement of the biological valve and insertion of an aortic prosthetic tube. Despite treatment with various broad-spectrum antibiotic regimes, the patient died 3.5 months after initial hospital admission. METHODS: The causative agent was probed by PCR amplification of bacterial DNA on the removed prosthetic aortic valve using broad range primers targeting the variable regions V1-V3 of the 16S RNA gene. After identification of Coxiella burnetii, multispacer sequence typing (MST) was performed by PCR amplification of 10 intergenic sequences. RESULTS: BLAST analysis of DNA from prosthetic valve material identified a 16S rRNA gene fragment almost identical to the type strain of C. burnetii (462/463 nt). Molecular typing allocated the strain to MST_18. CONCLUSIONS: Molecular methods are increasingly used to characterize isolates and to determine relationships between isolates that cause disease in different contexts and geographical areas. The present case demonstrates that identification and typing of C. burnetii is achievable without access to biosafety level 3 containment and highlights the first molecular characterization of an uncultured strain of C. burnetii causing infective endocarditis.

The effect of pneumococcal conjugate vaccines on incidence and microbiology associated with complicated acute otitis media.

OBJECTIVES: The objectives of this study were to investigate the incidence of complicated acute otitis media (cAOM) as well as the associated microbiology before and after introduction of the 7- and 13-valent pneumococcal conjugate vaccines (PCV-7 and -13), respectively. CAOM comprises "heavy" AOM (AOM demanding hospitalization), mastodismus (M) and acute mastoiditis (AM). METHODS: A retrospective cohort study of the incidence and microbiology associated with cAOM during the non-PCV era, the PCV-7 and 13 eras, respectively. Clinical and microbiological data were prospectively registered in a local database. The incidences of cAOM as well as the distribution of various bacterial strains in the three eras were compared. RESULTS: A total of 246 cases of cAOM (125 in the pre-vaccine period (2001-2006), 50 in the PCV-7 period (2007-2010) and 71 in the PCV-13 period (2011-2015)) were identified. The incidence of hAOM decreased by 62% in the PCV7-era but increased to almost pre-vaccine levels in the PCV-13 era. In the M + AM group, a decrease by almost 21% in the PCV7-era was found compared to the pre vaccine era, whereas the decrease was only 12% in the PCV13-era. The three most common findings in both hAOM and M + AM were Streptococcus pneumonia (SP), group A streptococcus (GAS) and "no growth". In the hAOM group, SP decreased from 38% in the pre-vaccine era to 31% in the PCV7-era and further to 16% in the PCV13-era. GAS decreased from 17% in the pre-vaccine era to 0% in the PCV7-era and 16% in the PCV13-era. The percentage of "no growth" increased from 12% to 38% and 44%, respectively. In the M + AM group, SP decreased to 10% in the PCV13-era compared with 44% in the pre-vaccine era and 41% in the PCV7-era. An increase in GAS from 15% in the pre-vaccine era and PCV7-era to 30% in the PCV13-era was observed. The "no growth" percentage increased from 13% in the pre-vaccine era to 26% in the PCV7-era and 33% in the PCV13-era. CONCLUSION: Introduction of PCV7 and PCV13 has been associated with an overall reduction of cAOM in Central Region Denmark. Pneumococci were still one of the two most common bacteria species related to cAOM though a decrease in pneumococci positive cases was observed. We found an increase in M + AM induced by GAS and a relatively large increase in "no growth", which might be caused by a more aggressive pre-hospital approach to treatment with antibiotics. Consequently, it is not evident whether the reduction of incidences is caused by the vaccines or a more aggressive antimicrobial attitude to manage AOM. The shift to GAS from SP is worrisome, and therefore continuous surveillance of the microbiology associated with AOM is warranted.

Intravenous antibiotics given for 2 weeks do not eradicate persistent Staphylococcus aureus clones in cystic fibrosis patients.

Staphylococcus aureus is the most commonly isolated pathogen in respiratory tract secretions from young patients with cystic fibrosis (CF), and several treatment strategies are used to control the infection. However, it is not known whether intensified treatment with antimicrobial agents causes eradication of S. aureus clones. We retrospectively determined the impact of intravenous (IV) antimicrobial agents on the suppression and eradication of S. aureus clones. One thousand and sixty-one S. aureus isolates cultured from 2526 samples from 130 CF patients during a 2-year study period were subjected to spa typing. Intervals between positive samples and the occurrence of clone replacements were calculated in relation to courses of IV antimicrobial agents. Of 65 patients chronically infected with S. aureus, 37 received 139 courses of IV antimicrobial agents with activity against S. aureus (mean duration, 15 days; range, 6-31 days). Administration of IV antibiotics increased the time to the next sample with growth of S. aureus: the mean interval between two positive samples was 68 days if IV treatment had been administered, in contrast to 49 days if no IV treatment had been administered (p 0.003). When S. aureus recurred in sputum after IV treatment, the isolate belonged to a different clone in 33 of 114 (29%) intervals, in comparison with 68 of 232 (29%) intervals where IV treatment had not been prescribed (OR 0.98, 95% CI 0.60-1.61). In conclusion, we show that 2 weeks of IV antimicrobial treatment can significantly suppress chronic staphylococcal infection in CF, but is not associated with the eradication of persistent bacterial clones.

Early treatment with inhaled antibiotics postpones next occurrence of Achromobacter in cystic fibrosis.

OBJECTIVES: In this nationwide retrospective study, we analysed species distribution, antimicrobial susceptibility and time to next occurrence of Achromobacter in Danish cystic fibrosis (CF) patients from 2000 to 2011. METHODS: Thirty-four primary isolates were identified to species level and subjected to antimicrobial susceptibility testing. Effectiveness of early antimicrobial treatment was assessed by a Kaplan-Meier estimation of time to recurrence. RESULTS: Achromobacter xylosoxidans accounted for 13 (38%) of the isolates, and an unnamed species accounted for 11 (32%) of the isolates. Meropenem, piperacillin-tazobactam and trimethoprim-sulfamethoxazole were highly active against chemotherapy-naïve Achromobacter, while ceftazidime, colistin and tobramycin were judged adequate for inhalation therapy. Fifty-five percent of 25 patients treated with inhaled ceftazidime, colistin, or tobramycin remained free of Achromobacter three years after acquisition, in contrast to 17% of 22 patients who did not receive inhaled antibiotics (P<0.01). CONCLUSIONS: Early treatment with inhaled antibiotics may prevent or postpone chronic infection with Achromobacter in CF patients.

Multilocus sequence phylogenetic analysis of Avibacterium.

This study examined 49 field isolates of the genus Avibacterium, with the 49 being allocated to 36 epidemiologically unrelated groups and one isolate from each group being examined in detail. In addition, six type and reference strains were investigated. Phylogenetic analysis of partially sequenced recN, rpoB, infB, pgi and sodA genes confirmed the existence of the species Avibacterium paragallinarum, while a species complex encompassing Avibacterium volantium, Avibacterium avium, Avibacterium gallinarum, Avibacterium endocarditis and Avibacterium sp. A could not be resolved. All isolates shared at least one identical sequence in one gene, indicating low diversity or horizontal gene transfer (HGT) between isolates. Such HGT between isolates of defined species and unclassified isolates combined with high sequence similarity can be explained as the result of an ongoing speciation process. The alternative explanation is that Av. volantium, Av. avium and Avibacterium sp. A were misclassified originally. Except for Av. paragallinarum, identification of species of Avibacterium seems problematic, even by DNA sequencing, as shown in the present investigation. The results indicate that Avibacterium probably contains only two or three species. Until the taxonomic revision is completed we recommend that isolates that do not fit with named species by genotype and phenotype be designated Avibacterium sp.

Direct identification and susceptibility testing of enteric bacilli from positive blood cultures using VITEK (GNI+/GNS-GA).

OBJECTIVE: To study the possibility of reporting results of identification and susceptibility testing of Gram-negative bacilli the same day as bacteremia is detected by using direct inoculation from positive blood cultures (Bactec 9240) into VITEK GNI+ and GNS-GA cards. METHODS: All blood cultures with Gram-negative enteric bacillus-like morphology on microscopy found to be positive on workdays between 15 June 1999 and 29 February 2000 were included. Identification and susceptibility testing were done by three methods: the direct method using a suspension made by differential centrifugation of positive blood culture broth for inoculation of the VITEK cards; the standard method using an inoculum made from an overnight culture on a solid media; and the routine method (reference method) using conventional testing. RESULTS: Of 169 isolates, the direct method resulted in 75% correct identifications, 9% misidentifications and 17% non-identifications. All misidentified isolates were Escherichia coli, of which 80% were reported as Salmonella arizonae. Five biochemical tests yielded most of the aberrant results; correcting the citrate and malonate reactions in most cases led to correct identification by the VITEK database. Despite a negative H2S reaction, 11 E. coli isolates were reported as S. arizonae. Two-thirds (69%) of identifications were reported within 6 h, and 95% of these were correct. The direct susceptibility testing method was assessable for 140 isolates. Correct results were found in 99% of isolate-antimicrobial combinations, and 85% were reported within 6 h. CONCLUSION: The direct VITEK method could correctly report identifications and susceptibility patterns within 6 h, making same-day reporting possible for almost two-thirds (63%) of bacteremic episodes with Gram-negative bacilli. These results could probably be improved by modification of the identification algorithms of the VITEK software.

Clonal origin of aminoglycoside-resistant Citrobacter freundii isolates in a Danish county.

During 1997, attention was drawn to an increased frequency of aminoglycoside-resistant Citrobacterfreundii in a Danish county, when a total of 24 resistant C. freundii isolates was detected. In this study, 15 such isolates were typed by pulsed-field gel electrophoresis, riboprinting and partial sequencing of the gene encoding translation initiation factor 2. Fourteen of the 15 isolates were identical, as evaluated by their antibiograms and by all these typing methods. This epidemic strain harboured the aminoglycoside resistance genes aac(3)-II and ant(3")-I, with the latter located in tandem with a dihydrofolate reductase gene in a class I integron. The source of the strain remains unresolved. Representative isolates were obtained from various specimens from hospitals and general practice throughout the county, with no evidence of patient-to-patient transmission.

A case of Moraxella canis-associated wound infection.

Moraxella canis was isolated from an infected foot ulcer in a patient suffering from diabetes mellitus with neuropathy. Bacteriological findings and 16S rDNA data are presented.

Identification of Enterobacteriaceae by partial sequencing of the gene encoding translation initiation factor 2.

Nucleotide sequence analysis is increasingly being used to identify bacteria. In this work, a PCR assay based on degenerate primers was used to obtain the partial sequence of infB, the gene encoding translation initiation factor 2 (IF2), in 39 clinical isolates of different Enterobacteriaceae. The partial sequence encodes the GTP-binding domain of IF2. Together with sequences from the literature, a total of 15 species, each represented by one to seven strains, was investigated. Phylogenetic analysis yielded an evolutionary tree which had a topology similar to a tree constructed using available 16S rRNA sequences. It is concluded that the inter-species variation of the infB gene fragment is sufficient for its use in the characterization of strains that have aberrant phenotypic reactions.

[Perinatal Campylobacter jejuni infection after Cesarean section]. / Perinatal Campylobacter jejuni-infektion efter sectio.

A 23 year old woman was admitted for delivery after having experienced a few episodes of loose stools. Following an unsuccessful vacuum-extraction, a Caesarean section was performed 12 hours after the membranes had ruptured. Thirty-six hours after the operation, the woman developed fever, and Campylobacter jejuni was isolated from her blood. Subsequently, Campylobacter was also recovered from the faeces of both mother and child. Though it is likely, that the Campylobacter was introduced to the uterus after rupture of the membranes, a transplacental infection can not be ruled out.

Materno-umbilical ratio of hepatitis B virus (HBV) surface antigen exceeds that of human chorionic gonadotropin in HBV-infected deliveries.

Low levels of HBV surface antigen (HBsAg) are commonly present in umbilical blood from neonates born to chronically infected mothers, but the origin and clinical significance of umbilical antigenemia is not clear. The present study was undertaken to investigate whether the umbilical HBs-antigenemia is linked to a demonstrable level of admixture with maternal blood, as evaluated by the assessment of the maternal-umbilical (M/U) ratio of human chorionic gonadotropin (hCG). The latter has a steep gradient across the placenta and is currently used as the most sensitive maternal marker in foetal sampling procedures. HBsAg and hCG were assayed in 6 paired maternal-umbilical serum samples from Kenya. In 3 cases with umbilical serum testing slightly positive for HBsAg, the M/U ratio of the antigen was around 8,000, or more than ten times the M/U ratio of hCG. In conclusion, the assessment of hCG in umbilical blood does not reveal the origin of umbilical HBsAg, unless the sample is grossly contaminated with maternal blood.

Concurrence of high levels of interferons alpha and beta in cord and maternal blood and simultaneous presence of interferon in trophoblast in an African population.

A high concentration of interferon-alpha (IFN-alpha) (> 5 U/ml) in cord blood was used as the criterion for establishing our study group. In a collection from deliveries by 269 Kenyan women, 16 such cord samples with matching maternal blood and placental biopsies were identified. These 16 were studied in detail together with 23 randomly selected among those with low cord IFN-alpha levels. The levels of IFN- in retal blood correlated with levels in their mothers for both IFN-alpha and beta but not for IFN-gamma. IFN-alpha was furthermore demonstrated in villous and decidual trophoblast from 15 (94%) placentae from donors with high IFN-alpha in the cord blood but not in the placenta of any low IFN level donors. In contrast, IFN-beta was not demonstrated in any placenta. These observations suggest simultaneous IFN induction in the three compartments, transplacental IFN transport, or trophoblast production of IFN to both circulations. Looking for IFN inducers, we did serologic tests for nonspecific indicators of inflammation and for specific virus and protozoan infections, but these showed no relation to elevated IFN levels. Immunohistology also revealed no evidence of a number of placental infections. The cause of the high levels of IFN-alpha could still be infectious but remains unexplained and may be noninfectious.

Cell-mediated transmission of human T cell lymphotropic virus type I to human malignant trophoblastic cells results in long-term persistent infection.

We investigated permissiveness of the malignantly transformed trophoblast (choriocarcinoma) cell lines JAR, BeWo and JEG-3 to the human T cell lymphotropic virus type I (HTLV-I). After co-culture with the productively infected cell line MT-2 the choriocarcinoma cell lines were analysed for infection over a period of three months. The presence of HTLV-I viral DNA was examined by PCR using primers targeting the gag, pol, env and pX specific sequences. All amplified segments were found consistently in the cell cultures throughout the period of study. Further analysis that aimed to characterize the size variation of the integrated proviral DNA by Southern blotting revealed the presence of integrated proviral sequences which consisted, for the most part, of incomplete genomes. The presence of the full-length HTLV-I genome, however, was unequivocally confirmed in clonally expanded cell cultures derived from the originally infected parental cells. In order to analyse virus expression at the transcriptional level, we used reverse transcriptase (RT)-mediated PCR that was targeted at intra-exon regions (gag, pol, env and pX), and the splicing sites of the env and pX-tax/rex mRNAs. When compared with MT-2 cells, substantially lower levels of all transcripts were found in all the cell lines analysed. We were unsuccessful in attempts to detect viral protein expression using polyvalent or Tax- and Gag-specific monoclonal antibodies by Western blot analysis or immunoprecipitation, and we could not detect any RT activity released into the supernatant of the infected cells either. Collectively, these data suggest that the trophoblastic cells may become persistently but essentially non-productively infected with HTLV-I.

Human trophoblast interferons enhance major histocompatibility complex class I antigen expression on human term trophoblast cells in culture.

The expression and regulation of major histocompatibility complex class I (MHC class I) antigens by virus-induced human trophoblast interferons (tro-IFNs) were examined in term trophoblast cultures. Flow cytometry studies using fluorescence monoclonal antibodies against MHC class I antigens revealed that isolated cytotrophoblasts can express MHC class I antigens. The expression of these antigens increased with stimulation of trophoblast cultures with tro-IFN-alpha and -beta. One hundred IU tro-IFN-alpha and -beta/ml induced no significant higher levels of MHC class I antigens as compared with the control, whereas 1000 IU tro-IFN-alpha and -beta/ml did. The tro-IFN-enhanced expression of MHC class I antigens may be important as it increases the efficiency of local and viral antigen presentation, cytotoxicity by T cell response and local inflammatory processes, thereby preventing virus spread from mother to fetus.

New compounds related to podophyllotoxin and congeners: synthesis, structure elucidation and biological testing.

4-Azido, 4-amino, 4-amido and 4-alkoxy compounds related to the lignans podophyllotoxin and 4'-demethylepipodophyllotoxin have been synthesized, and their structures elucidated. The Ritter reaction was shown to be useful in the preparation of the 4-amido compounds with the required stereochemistry. A preparative method for 4-chloro-4-deoxypicrophyllotoxin, for which all earlier synthetic attempts resulted in the two dehydrated compounds, alpha- and beta-apopicropodophyllotoxin, was developed. Supplementary preliminary studies of the biological activities of some of the compounds were performed. All compounds had pronounced inhibitory effect on the in vitro growth of human cervical cancer cells and TC-mouse cells with 4-amino-4-deoxypodophyllotoxin and 4-azido-4-deoxypodophyllotoxin showing the highest activity. Alkaline elution studies indicate that the toxicity of the 4'-demethoxy derivatives is due to protein-mediated DNA nicking. None of the compounds were found to have antiviral effect against herpes simplex type 2 (HSV-2), human immunodeficiency (HIV), and cytomegalovirus (CMV) in doses not toxic to the cells.

Bleomycin differentially inhibits picornavirus, herpesvirus and poxvirus replication in a human carcinoma cell line.

MS-757, a cervix carcinoma cell line, was exposed to bleomycin at concentrations up to 200 microM for periods of 1 to 24 hours. Bleomycin treatment caused the level of DNA synthesis in uninfected cells to drop to 13% of the level achieved in the controls. Protein synthesis fell to 50%, but RNA synthesis was not affected. Exposure of uninfected cells to 200 microM bleomycin for 24 h did not induce significant cell death measured as permeability to Trypan Blue). A tetrazolium dye-reduction assay, however, showed that cell viability measured as mitochondrial activity was reduced by 50%. In contrast, the clonogenicity of the treated cells (measured as colony-forming ability) fell to less than 1% of the level in untreated controls. Bleomycin effected a highly selective inhibition of virus replication measured as production of infectious progeny virus. The replication of the RNA virus, coxsackievirus B5, was largely unaffected by bleomycin, whereas the replication of the DNA virus, herpes simplex virus type 2, was inhibited to the same extent as cellular DNA synthesis. In contrast, bleomycin caused a 2 to 3 log10-fold suppression of the production of progeny virus after infection with the DNA virus, vaccinia virus. When an early gene product (expressed prior to virus DNA replication) of vaccinia virus was assayed, little inhibitory effect of bleomycin could be demonstrated, indicating that the early transcription and translation of vaccinia virus was only modestly influenced by bleomycin. The results argue against a DNA-based replication as the bleomycin-susceptible function independent of genome, replication enzymes, and site of synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential interferon production in human first and third trimester trophoblast cultures stimulated with viruses.

Stimulation of human placental first and third trimester trophoblast and syncytiotrophoblast cultures with viruses [Newcastle Disease Virus (NDV) and Sendai virus] led to a high interferon (IFN) production. The magnitude of the production was dependent on the gestational age of the trophoblast, type of inducer and the stage of differentiation of the trophoblast. The data obtained indicated that the first trimester trophoblast cultures produced five to sixfold more IFN than the third trimester trophoblast on per cell basis whereas syncytiotrophoblast at term produced twice as much IFN than the mononuclear term trophoblast when stimulated with the viruses. NDV and Sendai virus produced different levels and composition of IFN-alpha and -beta in both first and third trimester trophoblast and syncytiotrophoblast cultures. Purification of the virus-induced trophoblast interferons (tro-IFNs) by tandem high-performance affinity chromatography resulted in specific activities between 0.7 and 2.7 x 10(8) IU/mg of protein when assayed on human amniotic WISH cells. The tro-IFN-alpha protected both human and bovine MDBK cells from virus infection whereas the tro-IFN-beta protected only the human cell lines tested. The possible roles of the tro-IFNs are discussed in light of the observed differences in trophoblast IFN response.

In vitro infection of human placental trophoblast by wild-type vaccinia virus and recombinant virus expressing HIV envelope glycoprotein.

Short-time (< or = 7 days) cultures of trophoblast mononuclear cells isolated from term placentae were challenged with vaccinia virus. Cytopathic effects were induced in crude placental cell preparations as well as in cultures established after negative immunosorting of major histocompatibility complex class I epitope-expressing cells, i.e. cultures exclusively derived from villous cytotrophoblast according to our present state of knowledge. The trophoblast in vitro supported a full replicative cycle of both wild-type viruses and a recombinant clone serving as a vector for the human immunodeficiency virus type 1 envelope gene. Results may shed light on mechanisms involved in the rarely observed foetal damage caused by smallpox vaccination during pregnancy.