Pool-seq driven proteogenomic database for Group G Streptococcus.

Proteogenomic databases use genomic and transcriptomic information for improved identification of peptides and proteins from mass spectrometry analyses. One application of such databases is in the discovery of variants/mutations. In this study, we created a proteogenomic database that contained sequences with variants derived from Pooled sequencing experiments (137 Group G Streptococcus strains sequenced in 3 pools) and used tandem mass spectrometry (MS/MS) to analyse eight protein samples from randomly selected strains sequenced in the pools. Using the proteogenomic variant database, we identified 385 variant peptides from the eight samples, none of which could be identified from the single genome conventional database utilized, while 71.2% and 93.5% of them were identified from the databases that contained 4 complete genomes and 26 assemblies, respectively. The proteogenomic variant databases exhibited the same properties as the conventional databases in terms of the Andromeda score distributions and the posterior error probability (PEP) values of the identified peptides. SIGNIFICANCE: For bacterial populations, such as Group G Streptococcus (GGS), with substantial intra-species diversity, simultaneous sequencing of large numbers of strains and generation of proteogenomic databases from those aids in improving the discovery of peptides in mass spectrometric analyses. Therefore, generation of proteogenomic variant protein databases from Pooled sequencing experiments can be a cost-effective method to complement conventional databases and discover subtle strain wise differences.

Prevalence and Clinical Significance of Streptococcus dysgalactiae subspecies equisimilis (Groups C or G Streptococci) Colonization in Pregnant Women: A Retrospective Cohort Study.

Objectives: Little is known about the significance of Streptococcus G or C colonization in pregnant women. The objective of this study was to assess whether vaginal Streptococcus group G or C colonization detected in late pregnancy increases the infectious morbidity of the mother or newborn. Methods: A total of 15,114 rectovaginal cultures taken at 35-37 weeks of pregnancy were analyzed at Tampere University Hospital, Finland, between 2012 and 2014. From this laboratory data, all Streptococcus G or C-positive cultures were included to study maternal and neonatal infectious morbidity after delivery. This study population was compared to women with a positive Streptococcus B culture and to women with a negative culture. Results: The prevalence of Streptococcus G or C colonization was 2.9%. Significantly more postpartum endometritis was found in this study group. No association was found between colonization and neonatal bacteremia. Conclusions: Streptococcus G or C colonization is associated with postpartum endometritis. More research is needed to clarify if antibiotic prophylaxis is reasonable for this group during delivery.

Novel portable platform for molecular detection of toxigenic Clostridium difficile in faeces: a diagnostic accuracy study.

BACKGROUND: A novel portable platform for nucleic acid amplification enables rapid detection of diarrhoea causing toxigenic Clostridium difficile directly from faeces, even in resource-limited settings. We evaluated the accuracy and precision of the new commercial molecular test system. METHODS: One thousand one hundred and sixty faecal samples from patients suspected of having Clostridium difficile infection (CDI) were analysed using the Orion GenRead C. difficile test system (Orion Diagnostica Oy, Espoo, Finland) and comparative methods in three teaching hospital laboratories in Finland and France. The precision of the Orion GenRead C. difficile test system was evaluated in a reproducibility study with a set of blind-coded samples. The test system is based on a new isothermal amplification technology (Strand Invasion Based Amplification, SIBA®) and detection of the tcdB gene of C. difficile. We calculated the sensitivity, specificity, and the overall agreement according to Clinical and Laboratory Standards Institute recommendations. FINDINGS: The overall agreement of the Orion GenRead C. difficile test when compared to the comparative methods in routine use in the participating laboratories was between 96.7% and 98.8%. In the reproducibility study; the total percent agreement between three laboratories was 99.8%. INTERPRETATION: The identification of toxigenic C. difficile from faeces with the light-weight portable Orion GenRead test system was highly sensitive and specific, and the results were reproducible in the participating laboratories. This platform could enable fast and accurate molecular pathogen detection even in resource-limited or point-of-care settings.

Increased incidence of Mycoplasma pneumoniae infection in Finland, 2010-2011.

The number of cases of Mycoplasma pneumoniae infection detected by laboratory-based surveillance increased in Finland in late 2010. During 2011, the number of cases was four times higher than during the previous epidemic in 2005. The 2011 epidemic affected mostly school-age children. The increased number of cases was probably not due to changes in laboratory procedures, but public interest may have had an effect, since the number of Google queries followed closely the epidemic curve.

Streptococcus pyogenes bacteraemia, emm types and superantigen profiles.

The aim of this study was to investigate the emm types and superantigen profiles of bacteraemic group A streptococcal (GAS; Streptococcus pyogenes) isolates and to detect possible associations between the molecular characteristics of isolates and the clinical presentations of disease. In this population-based study, 87 bacteraemic GAS isolates from adult patients in Pirkanmaa Health District (HD), Finland, during the period 1995-2004 were emm typed and genotyped for superantigen (SAg) profiles. The epidemiological and clinical data of the patients were analysed with the microbiological characterisation data. Among the 87 isolates, 18 different emm types were found. emm1, emm28 and emm81 were the three most common types, covering 52% of isolates. The prevalence of specific emm types showed high variability during the 10-year study period. We could not find any association between the emm type and clinical features of bacteraemic infection, such as underlying diseases, disease manifestations or case fatality. Of nine superantigen genes examined, speA and speC were identified in 20 and 30% of the strains, respectively. No association was found between disease manifestation and the presence of single superantigen genes. The 26-valent GAS vaccine would have covered only 62% of isolates causing invasive disease in Pirkanmaa HD during the study period.

Distribution of emm types in invasive and non-invasive group A and G streptococci.

Our study describes the emm type distributions of invasive and non-invasive group A streptococci (GAS) and group G streptococci (GGS) strains in one of the biggest Health Districts in Finland. A total of 571 GAS or GGS were recovered from patients with invasive or non-invasive infections during a 1-year period in 2008-2009 in Pirkanmaa Health District in Finland. We describe here the emm type distributions of GAS and GGS collected from throat (n = 246), pus (n = 217), deep tissue (n = 56) and blood (n = 52). The most common emm types among GAS were emm77, emm1, emm28, emm89 and emm12. Among GGS, the most common emm types were stG480, stG643, stG6, stC6979 and stG485. Some emm types were found to associate with certain infection focus. In GAS, emm77 associated with pus isolates, whereas emm1 and emm12 were more frequent among throat isolates. In GGS, stG480 was more commonly found from throat isolates.

Meticillin-resistant Staphylococcus aureus (MRSA) bacteraemia in Tampere University Hospital: a case-control study, Finland October 2002 to January 2010.

Meticillin-resistant Staphylococcus aureus (MRSA) has emerged as a frequent pathogen in blood cultures in Pirkanmaa Hospital District (HD), Finland. To study risk factors for MRSA bacteraemia and the adequacy of empirical antimicrobial treatment, we retrospectively reviewed the hospital records of 102 patients, 51 with MRSA, and 51 with meticillin-sensitive Staphylococcus aureus (MSSA) bacteraemias respectively, who had been admitted to Tampere University Hospital in Pirkanmaa HD, from October 2002 to January 2010. For each patient with MRSA bacteraemia, one consecutively detected unmatched patient with MSSA bacteraemia was chosen as control. Patients with MRSA bacteraemias were significantly older (median age: 73 years vs 59 years, p=0.001), were more likely to have been transferred directly from another healthcare facility or were already in the hospital at the onset of bacteraemia (39/51 vs 26/51, p=0.007) and had a higher McCabe class than patients with MSSA bacteraemia (p=0.005). Patients with MRSA bacteraemia more seldom received adequate empirical antimicrobial therapy when compared to those with MSSA bacteraemia (13/51 vs 43/51, p<0.001). Of previously known MRSA carriers 10 of 29 received adequate empirical antimicrobial therapy for their condition. The percentage of MRSA bacteraemias among all S. aureus bacteraemias in Pirkanmaa HD is high compared to corresponding figures for the whole of Finland.

Reactive arthritis in a population exposed to an extensive waterborne gastroenteritis outbreak after sewage contamination in Pirkanmaa, Finland.

OBJECTIVES: To assess the occurrence, clinical picture, and triggering infections of reactive arthritis (ReA) associated with a large waterborne gastroenteritis outbreak. METHODS: After an extensive sewage contamination of the water supply system, an estimated 8453 of the 30 016 inhabitants of the town of Nokia fell ill. General practitioners and occupational physicians were advised to refer any patients with suspicion of new ReA to rheumatological examination including faecal culture, human leucocyte antigen (HLA)-B27 and antibody tests for Campylobacter, Salmonella, and Yersinia. RESULTS: Forty-five patients (33 females, 12 males) aged 16-77 years (median 53) were referred. ReA was diagnosed in 21, postinfectious arthralgia in 13, and other musculoskeletal conditions in 11 patients. HLA-B27 was positive in five out of 44 patients (11%). Of the 21 patients with ReA, possible triggering infections were observed in seven (33%), Campylobacter in four, Yersinia in three, and Salmonella in one, who also had Campylobacter infection. ReA was mild in all but one patient who presented with persistent Salmonella enterica serotype enteritidis infection. CONCLUSIONS: Taking into account the large population contaminated with potentially arthritogenic agents, the occurrence of ReA was rare and mild in character.

Plasma level of soluble urokinase-type plasminogen activator receptor as a predictor of disease severity and case fatality in patients with bacteraemia: a prospective cohort study.

OBJECTIVES: Urokinase-type plasminogen activator receptor (uPAR) is expressed on a variety of different immune cells and vascular endothelial cells during inflammation. Previous studies indicate that a high plasma concentration of the soluble form of the receptor (suPAR) predicts poor outcome in infectious diseases. DESIGN: A prospective cohort study. SUBJECTS AND METHODS: Plasma suPAR levels were measured in 132 patients with bacteraemia caused by Staphylococcus aureus, Streptococcus pneumoniae, ß-haemolytic streptococcae or Escherichia coli using a commercial enzyme-linked immunosorbent assay (ELISA). Values were measured on days 1-4 after a positive blood culture, on days 13-18 and on recovery. RESULTS: The maximum suPAR values on days 1-4 were markedly higher in nonsurvivors compared to survivors (15.8 vs. 7.3 ng mL(-1) , P < 0.001) and the area under the receiver operating characteristic curve (AUC(ROC) ) in the prediction of case fatality was 0.84 (95% confidence interval (CI) 0.76-0.93, P < 0.001). At a cut-off level of 11.0 ng mL(-1) , the sensitivity and specificity of suPAR for fatal disease was 83% and 76%, respectively. A high level of suPAR (≥ 11 ng mL(-1) ) was associated with hypotension (mean arterial pressure < 70 mmHg) (odds ratio (OR) 6.5; 95% CI 2.9-14.6) and high sequential organ failure assessment score (≥ 4) (OR 9.3; 95% CI 4.0-21.9). A high suPAR level remained an independent risk factor for case fatality in a logistic regression model adjusted for potential confounders. CONCLUSION: Plasma suPAR level is a sensitive and specific independent prognostic biomarker in patients with bacteraemia.

An extensive gastroenteritis outbreak after drinking-water contamination by sewage effluent, Finland.

An inappropriate cross-connection between sewage- and drinking-water pipelines contaminated tap water in a Finnish town, resulting in an extensive waterborne gastroenteritis outbreak in this developed country. According to a database and a line-list, altogether 1222 subjects sought medical care as a result of this exposure. Seven pathogens were found in patient samples of those who sought treatment. To establish the true disease burden from this exposure, we undertook a population-based questionnaire investigation with a control population, infrequently used to study waterborne outbreaks. The study covered three areas, contaminated and uncontaminated parts of the town and a control town. An estimated 8453 residents fell ill during the outbreak, the excess number of illnesses being 6501. Attack rates were 53% [95% confidence interval (CI) 49.5-56.4] in the contaminated area, 15.6% (95% CI 13.1-18.5) in the uncontaminated area and 6.5% (95% CI 4.8-8.8) in the control population. Using a control population allowed us to differentiate baseline morbidity from the observed morbidity caused by the water contamination, thus enabling a more accurate estimate of the disease burden of this outbreak.

Deep, respiratory tract and ear infections caused by Pseudallescheria (Scedosporium) and Microascus (Scopulariopsis) in Finland. A 10-year retrospective multi-center study.

Deep, respiratory tract and ear infections due to Microascaceae (Pseudallescheria, Scedosporium, Microascus or Scopulariopsis) were studied nationwide in Finland during 1993-2002. The data were based on 52,000 fungal cultures that represented about 50% of all such specimens in Finland and included all Finnish cases of profound immunosuppression. There were 39 cases that were re-evaluated as clinically significant, i.e., three pneumonias, two deep pedal infections and five wound infections, 11 sinusitis and 18 ear infections. The pedal infections and most pneumonias occurred in immunocompromised patients. Most cases, except the ear infections, were due to Pseudallescheria boydii. Two patients had lethal P. boydii pneumonia and a deep P. boydii infection of the foot contributed to a third lethal case. Two of the patients with lethal outcomes had received an allogeneic haematopoietic stem cell transplantation (AHSCT). Two patients with haematological malignancies were cured of deep site infections by a prolonged course of itraconazole. Wound, sinus and ear infections were cured or improved by local surgery or topical therapy. There were 0.8-1.7 cases of any type of infection per million inhabitants per year (MY) and 3.4 cases/1000 AHSCT. Mortality associated with Microascaceae in any type of patient was 0.06-0.12 MY.

Factors predisposing to acute and recurrent bacterial non-necrotizing cellulitis in hospitalized patients: a prospective case-control study.

Acute non-necrotizing cellulitis is a skin infection with a tendency to recur. Both general and local risk factors for erysipelas or cellulitis have been recognized in previous studies using hospitalized controls. The aim of this study was to identify risk factors for cellulitis using controls recruited from the general population. We also compared patients with a history of previous cellulitis with those suffering a single episode, with regard to the risk factors: length of stay in hospital, duration of fever, and inflammatory response as measured by C-reactive protein (CRP) level and leukocyte count. Ninety hospitalized cellulitis patients and 90 population controls matched for age and sex were interviewed and clinically examined during the period April 2004 to March 2005. In multivariate analysis, chronic oedema of the extremity, disruption of the cutaneous barrier and obesity were independently associated with acute cellulitis. Forty-four (49%) patients had a positive history (PH) of at least one cellulitis episode before entering the study. Obesity and previous ipsilateral surgical procedure were statistically significantly more common in PH patients, whereas a recent (<1 month) traumatic wound was more common in patients with a negative history (NH) of cellulitis. PH patients had longer duration of fever and hospital stay, and their CRP and leukocyte values more often peaked at a high level than those of NH patients. Oedema, broken skin and obesity are risk factors for acute cellulitis. The inflammatory response as indicated by CRP level and leukocyte count is statistically significantly more severe in PH than NH patients.

Clinical presentations and epidemiology of beta-haemolytic streptococcal bacteraemia: a population-based study.

In this population-based study, all 314 episodes of beta-haemolytic streptococcal bacteraemia in adult patients in the Pirkanmaa area, Finland, during the 10-year period 1995-2004 were retrospectively reviewed. Altogether, 92 cases of bacteraemia caused by Lancefield group A beta-haemolytic streptococci (GAS), 76 caused by group B beta-haemolytic streptococci (GBS), 18 caused by group C beta-haemolytic streptococci (GCS) and 128 caused by group G beta-haemolytic streptococci (GGS) were identified. The most important finding was that the incidence of GGS increased during the study period. Disruption of the cutaneous barrier was a very common predisposing factor in GAS and GGS bacteraemias. Skin infections were the presenting clinical manifestations in two-thirds of GAS and GGS bacteraemias.

Gene-environment interaction between MBL2 genotype and smoking, and the risk of gram-positive bacteraemia.

Mannose-binding lectin (MBL) insufficiency caused by point mutations in the MBL2 gene has been associated with increased susceptibility to bacteraemic infections. We here investigated the effect of MBL2 polymorphisms on the susceptibility and clinical course of bacteraemia. The study cohort comprised 145 patients with bacteraemia and 400 controls. In the case of patients with bacteraemia, laboratory findings and clinical data were registered on admission and during six consecutive days. MBL2 structural polymorphisms at codons 52 (CGT-->TGT; designated D or O), 54 (GGC-->GAC; B or O) and 57 (GGA-->GAA; C or O) in exon 1 of the MBL2 gene and promoter region polymorphisms at position -221 (G-->C, designated Y or X alleles) were determined. No difference in MBL2 genotype frequencies between the bacteraemic patients and controls was detected, and MBL2 genotype had no independent effect on mortality, nor disease severity. However, smoking proved a significant risk factor for Gram-positive (Staphylococcus aureus, Streptococcus pneumoniae or beta-haemolytic streptococci) bacteraemia in patients carrying the variant O allele (53% current smokers in Gram-positive bacteraemia patients compared with only 21% in controls, odds ratios 4.2, 95% confidence intervals 2.0-9.0; P < 0.001), while it did not have an effect in those homozygous for the A allele. The same effect was not detected in Escherichia coli bacteraemia. In conclusion, MBL2 genotypes representing MBL insufficiency were not associated with the overall risk of bacteraemia or disease severity, but smoking in carriers of the structural variant O allele may have a deleterious effect increasing the risk of Gram-positive bacteraemia.

Effects of mode of delivery and necrotising enterocolitis on the intestinal microflora in preterm infants.

To investigate the effects of mode of delivery and of necrotising enterocolitis on the faecal microflora, 140 infants born before 33 weeks of gestation were followed up for symptoms of necrotising enterocolitis. Stool samples for gas-liquid chromatography and culture were collected twice weekly, and, when necrotising enterocolitis was suspected, for 2 months. For each infant with necrotising enterocolitis (n=21), two control infants matched for birth weight and gestational age were selected from the remaining study population. In gas-liquid chromatography analysis, the faecal bacterial microflora of infants born via caesarean section differed significantly from the gut microflora of those born via the vaginal route. The intestinal microflora showed a significant alteration in the necrotising enterocolitis group at time of diagnosis. At the onset of necrotising enterocolitis, faecal colonisation with Enterococcus species and Candida albicans was significantly more frequent in symptomatic infants than in controls. In infants with positive blood cultures and positive intestinal biopsy cultures, concomitant stool samples revealed the same microbial pathogens. In conclusion, the intestinal microbial colonisation in preterm infants born by caesarean section differs from that in preterm infants born via the vaginal route. A significant change in faecal microbial colonisation seems to occur at the onset of necrotising enterocolitis. Pathogens detected in the stools at that time might have a causative role in the development of the disease.

Cefuroxime, rifampicin and pulse lavage in decontamination of allograft bone.

The risk of bacterial infection through allogenic bone transplantation is one of the major problems facing tissue banks. Different screening methods and decontamination procedures are being used to achieve a safe surgical result. The purpose of this study was to investigate the contamination rate in fresh frozen bone allografts after treating them with different decontamination methods. The allografts were contaminated by rubbing on the operating theatre floor for 60 min, after which they were rinsed either with sterile physiological saline, cefuroxime or rifampicin solution or they were washed with low-pressure pulse lavage of sterile physiological saline. Our findings show that low-pressure pulse lavage with sterile saline solution is very effective in removing bacteria from bone allograft, when compared with the antibiotic solutions tested.

Pulse lavage washing in decontamination of allografts improves safety.

We analyzed the bacterial contamination rate of 140 femoral head allografts after rinsing the allografts in different decontamination solutions. Bacterial screening methods and cleansing effect of antibiotics (cefuroxime and rifampicin) and pulse lavage were compared. Swabbing and taking small pieces of bone for culture were the screening methods used. Both methods proved to be quite unreliable. Approximately one-fourth of the results were false negative. Culturing small pieces of bone gave the most accurate and reliable results and, therefore, can be recommended as a bacterial screening method. The use of antibiotics in allograft decontamination is controversial. In prophylactic use antibiotics include risks of allergic reactions and resistant development and our results in the present study show that antibiotics do not improve the decontamination any better than low-pressure pulse lavage with sterile saline solution. Therefore, pulse lavage with sterile saline solution can be recommended for allograft decontamination. Our results demonstrate that it decreases bacterial bioburden as effectively as the antibiotics without persisting the disadvantages.

High-pressure saline washing of allografts reduces bacterial contamination.

60 fresh-frozen bone allografts were contaminated on the operating room floor. No bacterial growth was detected in 5 of them after contamination. The remaining 55 grafts had positive bacterial cultures and were processed with three methods: soaking in saline, soaking in antibiotic solution or washing by high-pressure saline. After high-pressure lavage, the cultures were negative in three fourths of the contaminated allografts. The corresponding figures after soaking grafts in saline and antibiotic solution were one tenth and two tenths, respectively. High-pressure saline cleansing of allografts can be recommended because it improves safety by reducing the superficial bacterial bioburden.

Vulvar symptoms in paediatric and adolescent patients.

Vulvovaginal symptoms in children and young adolescents are not yet very well understood, nor is the actual incidence known. This study evaluates the character and possible infectious aetiology of vulvar symptoms of females aged up to 16 y. The signs, symptoms and bacteriological findings of 68 consecutive cases were studied. The study was conducted in the University Hospital of Tampere at a special gynaecological consultation clinic for children and adolescents. Sixty-eight patients were included in the study: 48 girls (71%) were prepubertal, at Tanner stage M1P1, 26 patients were 2-4-y-old and 15 were 5-7-y-old. The duration of symptoms was known for 46 patients: 41% had had symptoms for >1 mo and 20% for >6 mo. Forty-eight patients had abnormal clinical findings on examination and 16 (33%) of them had an infectious aetiology. Streptococcus pyogenes infection was identified in 11 (16%) patients, all of whom had symptoms. Candida was identified in 6 (9%) patients. No infectious aetiology was found among 26 patients who had symptoms and abnormal clinical findings. Vulvovaginal symptoms during childhood are more common among younger children (<7 y). In 67% of patients no infectious aetiology could be found. Samples for microbiological culture should be taken from symptomatic patients and symptomatic areas. Cultures of Candida and bacteria are necessary but usually sufficient. If a microbiological aetiology is established, treatment can be assigned accordingly. Patients with vulvar symptoms and findings but with an unclear aetiology need support and advice on proper hygiene and can intermittently use mild corticosteroids locally.