Abstract Introduction Occurrence of bacteraemia and fungemia is a serious condition with high mortality and the incidence is increasing worldwide. The aim of this study was to survey the occurrence of bacteria and fungi in blood cultures from adult patients domiciled in the county of Gävleborg during one year and also to calculate the incidence and mortality in the same geographical area. Method This is a descriptive epidemiologic study, based on all episodes of blood cultures analyzed at the Microbiology laboratory, Gävle hospital during 2005. Patients from 20 years of age, domiciled in the county of Gävleborg at the date of drawing the blood culture, where included in the study. Criteria of exclusion were negative blood cultures and cultures which were classified as contaminants. Results Altogether there were 4 564 blood cultures analyzed, resulting in 524 (11 %) positive cultures for further study. There were 442 patients (48 % women) involved in 499 episodes with confirmed bacteraemia or fungemia. Gram positive bacteria represented 52 %, gram negative 45 % and fungi 3 %. The most frequently isolated bacterium was Escherichia coli followed by Staphylococcus aureus. In women, Escherichia coli was the most common bacterium, and there was a significant difference between the genders (p= 0.004). In men, Staphylococcus aureus was the dominant species (p= 0.027). Streptococcus pneumoniae was more common in women (p= 0.005). The incidence of bacteraemia and fungemia in the county of Gävleborg was 235/100 000 inhabitants above the age of 20 (women, 223/100 000 men, 247/100 000). The incidence increased with age and the mean age was 70.2 years. The mortality within 30 days after the last positive blood culture was 22 % (97 patients). Escherichia coli was the most common bacteria diagnosed among those who died. The mortality in fungemia was 66 %. There was no significant difference in incidence or mortality between the two provinces Gästrikland and Hälsingland. Patients with bacteraemia and fungemia were initially cared for at all medical care units at the three hospitals in the county. Conclusion The incidence of bacteraemia/fungemia in the county of Gävleborg was 235/100 000 inhabitants. The most common bacteria in patients with confirmed bacteraemia were Escherichia coli and Staphylococcus aureus. Increasing age was a contributing risk factor. Patients with fungemia had considerably higher mortality compared to patients with bacteraemia. There where no significant differences in mortality between the two provinces. / Introduktion Fynd av bakterier, bakteriemi, och svampar, fungemi, i blodbanan är ett allvarligt tillstånd med hög mortalitet och incidensen ökar i världen. Syftet med denna studie var att kartlägga vilka bakterier och svampar som förekom i alla blododlingar tagna under ett år från vuxna patienter i Gävleborgs län, samt att analysera incidens och mortalitet för bakteriemi och fungemi i länet. Metod Det är en deskriptiv epidemiologisk studie som utgår från analyserade blododlingar under år 2005 vid Enheten för Klinisk Mikrobiologi Laboratoriemedicin vid Gävle sjukhus. Till studien inkluderades personer från 20 års ålder som var mantalsskrivna i Gävleborgs län det datum som blododlingen utfördes. Exklusionskriterierna var negativa odlingssvar och svar som bedömdes som kontamination. Resultat Totalt analyserades 4 564 blododlingar, av vilka 524 (11 %) var positiva och bearbetades i denna studie. Det blev 442 patienter (48 % kvinnor) med 499 episoder av säkerställd bakteriemi eller fungemi. De grampositiva bakterierna stod för 52 %, gramnegativa bakterier 45 % och svampar 3 %. De enskilt vanligaste bakterierna var Escherichia coli och Staphylococcus aureus. För kvinnorna var Escherichia coli vanligast och det fanns en signifikant skillnad mellan könen (p= 0,004 ), för männen var Staphylococcus aureus vanligast (p= 0,027). Streptococcus pneumoniae visade högre förekomst bland kvinnorna än männen (p= 0,005). Incidensen för bakteriemi och fungemi i Gävleborgs län var 235/100 000 invånare äldre än 20 år (kvinnor, 223/100 000 och män, 247/100 000). Incidensen ökade med åldern och medelåldern var 70,2 år. Mortaliteten inom 30 dagar efter utförd blododling var 22 % (97 patienter). Escherichia coli var vanligast hos de avlidna. För patienter med fungemi var mortaliteten 66 %. Det påvisades ingen signifikant skillnad beträffande incidens eller mortalitet mellan länets båda landskap Gästrikland och Hälsingland. Patienter med bakteriemi och fungemi vårdades initialt på samtliga vårdenheter på länets tre sjukhus. Konklusion Incidensen för bakteriemi/fungemi i Gävleborgs län var 235/100 000 invånare. De vanligaste fynden vid säkerställd bakteriemi var Escherichia coli och Staphylococcus aureus. Ökande ålder var en riskfaktor. Patienter med fungemi hade avsevärt högre mortalitet än de med bakteriemi. Ingen skillnad påvisades mellan de två landskapen beträffande mortalitet.

Introduktion: Sepsis är utan behandling ett livshotande tillstånd, en korrekt behandling behöver en snabb och tillförlitlig artidentifiering. Olika prepareringsmetoder av positiva blododlingar inför direkt identifiering med MALDI-TOF finns. MBT Sepsityper® IVD Kit är en CE-märkt metod. För att följa IVDR-direktivet behöver Sepsityper jämföras med den nuvarande in-house-metoden. Dessa två metoder jämfördes utifrån förmåga att ge ett tillförlitligt, snabbt resultat där även den praktiska metoden samt ekonomiska aspekten undersöktes Material och metod: Alla positiva blododlingar som undersöktes preparerades med in- house-metoden och Sepsityper innan analys med MALDI-TOF MS. Med in-house-metoden användes saponin för att lysera de humana cellerna medans Sepsityper använde lyseringsbuffert. Resultatets tillförlitlighet presenteras som scorevärde där >2 är tillförlitligt till speciesnivå, 1,7–1,99 till genusnivå och <1,7 är ej tillförlitligt. Resultat: 135 positiva blododlingar undersöktes där in-house-metoden gav 115 med ett scorevärde över 1,7 medans Sepsityper® gav 100 prover med ett scorevärde över 1,7. Bland dessa prover var det 62 gramnegativa bakterier och 53 grampositiva. Slutsats: En skillnad mellan antalet identifieringar med scorevärde över 1,7 sågs men var ej signifikant enligt CHI2 test där antalet var högre för house metoden. Den data som samlats ger ej inte tillräckligt med stöd för att inte avvika från IVDR och fortsätta med in-house- metoden. / Background: Sepsis without treatment is a life-threatening condition, a rapid as well as reliable identification of the bacteria is needed. Different preparation methods of positive blood cultures for rapid identification are available. MBT Sepsityper® IVD Kit is one of them and is a CE-marked method. To comply with the IVDR directive, Sepsityper® needs to be tested and compared with the current in-house method. These two methods were compared based on the ability to provide fast and reliable results. Material and method: All the positive blood samples that were examined were prepared by the two methods then analysed with MALDI-TOF MS. The in-house-method used saponin to lyse the blood cells, Sepsityper used a lysisbuffert. How reliable the results are presented as score value, >2 means that the result is reliable to species identification, between 1.7-1.99 to genus identification and <1.7 is not reliable. Results: 135 positive blood cultures were examined and the in-house-method gave 115 with a score value above 1.7 while Sepsityper® gave 100 bottles a score value over 1.7. Among the bottles there were 62 gram-negative bacteria and 53 gram-positive bacteria. Conclusion: A difference between the number of identifications with a score value above 1.7 were seen where the number was higher for the in-house method, but the difference was not significant according to the Chi2 test. The data collected do not provide sufficient support for not following IVDR and not switch to Sepsityper®.

Background: Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration dependently. Results: Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24–48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8–12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12–24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release. Conclusions: Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse.

I performed two randomised controlled trials (RCTs) to determine the best treatments for enteric fever in Kathmandu, Nepal, an area with a high proportion of nalidixic acid resistant S. Typhi and S. Paratyphi A isolates. I recruited 844 patients with suspected enteric fever to compare chloramphenicol versus gatifloxacin. 352 patients were culture confirmed. 14/175 patients treated with chloramphenicol and 12/177 patients treated with gatifloxacin experienced treatment failure (HR=0.86 (95% CI 0.40 to 1.86), p=0.70). The median times to fever clearance were 3.95 and 3.90 days, respectively (HR=1.06 [CI 0.86 to 1.32], p=0.59). The second RCT compared ofloxacin versus gatifloxacin and recruited 627 patients. Of the 170 patients infected with nalidixic acid resistant strains, the number of patients with treatment failure was 6/83 in the ofloxacin group and 5/87 in the gatifloxacin group (Hazard Ratio, HR=0.81, 95% CI 0.25 to 2.65; p=0.73); the median times to fever clearance were 4.7 and 3.3 days respectively (HR=1.59 [CI 1.16 to 2.18], p=0.004). I compared conventional blood culture against an electricity free culture approach. 66 of 304 patients with suspected enteric fever were positive for S. Typhi or S. Paratyphi A, 55 (85%) isolates were identified by the conventional blood culture and 60 (92%) isolates were identified by the experimental method. The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% and 96.0%, respectively. This electricity free blood culture system may have utility in resource-limited settings or potentially in disaster relief and refugee camps. I performed a literature review of RCTs of enteric fever which showed that trial design varied greatly. I was interested in the perspective of patients and what they regarded as cure. 1,481 patients were interviewed at the start of treatment, 860 (58%) reported that the resolution of fever would mean cure to them. At the completion of treatment, 877/1,448 (60.6%) reported that they felt cured when fever was completely gone. We suggest that fever clearance time is the best surrogate for clinical cure in patients with enteric fever and should be used as the primary outcome in future RCTs for the treatment of enteric fever.

A Brucella canis é a espécie de Brucella que acomete os cães, acarretando problemas reprodutivos e prejuízos econômicos aos proprietários de canis comericias, além do risco que representa para os manipuladores e os proprietários dos cães, os quais podem adquirir a infecção pelo contato com os animais infectados e/ou materiais contaminados por B. canis. Vários métodos de diagnósticos foram desenvolvidos com o intuito de diagnosticar a brucelose nos cães, no entanto cada teste apresenta um desempenho diferente em relação à sensibilidade, especificidade, rapidez e praticidade na identificação dos cães acometidos. Estudos indicam que a PCR em tempo real tende a apresentar maior sensibilidade e especificidade, além da rapidez na execução. Diante disso o objetivo deste trabalho foi desenvolver, padronizar e validar a reação em cadeia da polimerase (PCR) em tempo real para diagnóstico da brucelose canina causada por B. canis utilizando amostras de sangue total de cães. Um total de 56 cães de canis comercias e animais domiciliados, foram submetidos à hemocultura, sorodiagnóstico, PCR convencional (PCRc) e PCR em tempo real (PCRtr) e posteriormente os resultados obtidos em cada teste foram comparados por meio do coeficiente Kappa. Na padronização das reações de PCRtr foram testados três conjuntos de primers e sondas (PCRtr-A, B e C), utilizando-se diferentes temperaturas de annealing e concentrações de primers. A PCRtr-B teve melhor desempenho sob temperatura de annealing de 59° C, utilizando 900 nM de primers tendo detectado um maior número de cães positivos em relação à hemocultura e PCRc. Porém o teste não se apresentou confiável, uma vez que ocorreram reações inespecíficas em amostras provenientes de cães não infectados, sugerindo uma baixa especificidade do teste. Portanto, as técnicas de hemocultura e PCRc apresentaram melhor desempenho do que a PCRtr avaliada para o diagnóstico de B. canis. / Brucella canis is the species that affects dogs, causing reproductive problems and economic losses mainly for the owners of commercial kennels, besides the risk that it represents for the manipulators and the owners of dogs that can acquire the infection by the contact with infected animals or contaminated materials. Several diagnostic methods have been developed for the diagnosis of the infection in dogs. However, the performance of the tests vary regarding sensitivity, specificity, speed and practicality for the identification of infected dogs. Studies indicated that the real-time PCR (rtPCR) might show higher sensitivity and specificity, as well as speed of execution. Therefore, the objective of this study was to develop, standardize and validate a rtPCR assay for the diagnosis of canine brucellosis caused by B. canis using whole blood samples from dogs. A total of 56 dogs from commercial kennels and domiciled animals were tested through blood culturing, serological test, conventional PCR (cPCR) and rtPCR, and subsequently the results obtained in each test were compared using the Kappa coefficient. During the standardization of the rtPCR, three sets of primers and probes (rtPCR-A, B and C) were tested using different annealing temperatures and primer concentrations. rtPCR-B showed better performance under the annealing temperature of 59° C and using 900 nM of primers having detected a higher number of positive dogs when compared to blood culturing and PCRc. However, the test was considered not reliable to be used in canine brucellosis diagnosis, since non-specific reactions occurred in samples from uninfected dogs, suggesting a low specificity of the test. Therefore, the blood culturing and cPCR techniques showed a better performance than the developed rtPCR assay to be used in the diagnosis of B. canis infection in dogs.

The IDSA Community-Acquired Pneumonia (CAP) Guideline recommends ceftriaxone in combination with doxycycline as an alternative to combination therapy with ceftriaxone and azithromycin for non-intensive care unit (ICU) patients hospitalized with CAP. This is an attractive alternative regimen due to recent concerns of increased cardiovascular risk associated with azithromycin. The objective of this study was to compare the clinical outcomes of azithromycin and doxycycline each in combination with ceftriaxone for non-ICU Veterans hospitalized with CAP.

A Brucella canis é a espécie de Brucella que acomete os cães, acarretando problemas reprodutivos e prejuízos econômicos aos proprietários de canis comericias, além do risco que representa para os manipuladores e os proprietários dos cães, os quais podem adquirir a infecção pelo contato com os animais infectados e/ou materiais contaminados por B. canis. Vários métodos de diagnósticos foram desenvolvidos com o intuito de diagnosticar a brucelose nos cães, no entanto cada teste apresenta um desempenho diferente em relação à sensibilidade, especificidade, rapidez e praticidade na identificação dos cães acometidos. Estudos indicam que a PCR em tempo real tende a apresentar maior sensibilidade e especificidade, além da rapidez na execução. Diante disso o objetivo deste trabalho foi desenvolver, padronizar e validar a reação em cadeia da polimerase (PCR) em tempo real para diagnóstico da brucelose canina causada por B. canis utilizando amostras de sangue total de cães. Um total de 56 cães de canis comercias e animais domiciliados, foram submetidos à hemocultura, sorodiagnóstico, PCR convencional (PCRc) e PCR em tempo real (PCRtr) e posteriormente os resultados obtidos em cada teste foram comparados por meio do coeficiente Kappa. Na padronização das reações de PCRtr foram testados três conjuntos de primers e sondas (PCRtr-A, B e C), utilizando-se diferentes temperaturas de annealing e concentrações de primers. A PCRtr-B teve melhor desempenho sob temperatura de annealing de 59° C, utilizando 900 nM de primers tendo detectado um maior número de cães positivos em relação à hemocultura e PCRc. Porém o teste não se apresentou confiável, uma vez que ocorreram reações inespecíficas em amostras provenientes de cães não infectados, sugerindo uma baixa especificidade do teste. Portanto, as técnicas de hemocultura e PCRc apresentaram melhor desempenho do que a PCRtr avaliada para o diagnóstico de B. canis. / Brucella canis is the species that affects dogs, causing reproductive problems and economic losses mainly for the owners of commercial kennels, besides the risk that it represents for the manipulators and the owners of dogs that can acquire the infection by the contact with infected animals or contaminated materials. Several diagnostic methods have been developed for the diagnosis of the infection in dogs. However, the performance of the tests vary regarding sensitivity, specificity, speed and practicality for the identification of infected dogs. Studies indicated that the real-time PCR (rtPCR) might show higher sensitivity and specificity, as well as speed of execution. Therefore, the objective of this study was to develop, standardize and validate a rtPCR assay for the diagnosis of canine brucellosis caused by B. canis using whole blood samples from dogs. A total of 56 dogs from commercial kennels and domiciled animals were tested through blood culturing, serological test, conventional PCR (cPCR) and rtPCR, and subsequently the results obtained in each test were compared using the Kappa coefficient. During the standardization of the rtPCR, three sets of primers and probes (rtPCR-A, B and C) were tested using different annealing temperatures and primer concentrations. rtPCR-B showed better performance under the annealing temperature of 59° C and using 900 nM of primers having detected a higher number of positive dogs when compared to blood culturing and PCRc. However, the test was considered not reliable to be used in canine brucellosis diagnosis, since non-specific reactions occurred in samples from uninfected dogs, suggesting a low specificity of the test. Therefore, the blood culturing and cPCR techniques showed a better performance than the developed rtPCR assay to be used in the diagnosis of B. canis infection in dogs.

Background: Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration dependently. Results: Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24–48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8–12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12–24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release. Conclusions: Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse.