- Volume 57, Issue 9, 2008
Volume 57, Issue 9, 2008
- Review
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Acanthamoeba and the blood–brain barrier: the breakthrough
More LessAcanthamoeba granulomatous encephalitis is a rare disease that almost always proves fatal. Death occurs mainly due to neurological complications; however, the pathogenesis and pathophysiology associated with this disease remain incompletely understood. Haematogenous spread is a key step in the development of Acanthamoeba encephalitis, but it is not clear how circulating amoebae breakthrough the blood–brain barrier to gain entry into the central nervous system to produce the disease. This review of the literature describes the parasite factors and immune-mediated mechanisms involved in the blood–brain barrier dysfunction leading to neuropathogenesis.
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- Pathogenicity And Virulence
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Differential glycosaminoglycan binding of Chlamydia trachomatis OmcB protein from serovars E and LGV
More LessWe recently showed that OmcB protein from Chlamydia trachomatis serovar LGV1 functions as an adhesin. In this study, we produced Escherichia coli expressing OmcB from serovar E and compared this OmcB to OmcB from serovar LGV1. Infectivity inhibition assays carried out with serovars LGV1 and E of C. trachomatis in the presence of recombinant OmcB showed considerable (∼60 %) inhibition of infectivity. In the presence of heparan sulphate, there was significant inhibition (68 %) of adherence of E. coli expressing OmcB from serovar LGV1 only. In a further experiment, recombinant OmcB from serovar LGV1 showed minimal binding to glycosaminoglycan (GAG)-deficient cells, whilst to the same cells, recombinant OmcB from serovar E showed binding equal to that to the wild-type cells. Our experiments strongly suggest that OmcB from serovar E, in contrast to that from serovar LGV1, is not binding to host cells through a GAG-dependent mechanism.
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Differences in Helicobacter pylori CagA tyrosine phosphorylation motif patterns between western and East Asian strains, and influences on interleukin-8 secretion
More LessHelicobacter pylori strains from East Asia have an ‘East Asian’ type of CagA that is more active and predominantly comprises a single type. Strains from other countries have a ‘western’ type of CagA, which is less active and comprises many different types generated by intragenomic recombination. Co-culture of AGS gastric epithelial cells with isolates of western strains that displayed microevolution in CagA showed that isolates with additional copies of the C motif induced significantly more interleukin (IL)-8 secretion. Co-culture of AGS cells with western and East Asian strains, each expressing CagA with a single copy of the C or D motif, showed that East Asian strains induced significantly more IL-8 secretion. Analysis of the different CagA types from data deposited in GenBank and from the literature showed that western CagA is significantly more likely to undergo duplication of tyrosine phosphorylation motif C than East Asian CagA is of the corresponding D motif. Taken together, the data suggest that the already highly active East Asian CagA with one D motif has no requirement to increase its virulence, whereas the less active western CagA displays flexibility in its capacity to increase its number of tyrosine phosphorylation motifs to become more virulent.
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- Host Response
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Identification of virulence determinants in uropathogenic Proteus mirabilis using signature-tagged mutagenesis
The Gram-negative bacterium Proteus mirabilis causes urinary tract infections (UTIs) in individuals with long-term indwelling catheters or those with functional or structural abnormalities of the urinary tract. Known virulence factors include urease, haemolysin, fimbriae, flagella, DsbA, a phosphate transporter and genes involved in cell-wall synthesis and metabolism, many of which have been identified using the technique of signature-tagged mutagenesis (STM). To identify additional virulence determinants and to increase the theoretical coverage of the genome, this study generated and assessed 1880 P. mirabilis strain HI4320 mutants using this method. Mutants with disruptions in genes vital for colonization of the CBA mouse model of ascending UTI were identified after performing primary and secondary in vivo screens in approximately 315 CBA mice, primary and secondary in vitro screens in both Luria broth and minimal A medium to eliminate mutants with minor growth deficiencies, and co-challenge competition experiments in approximately 500 CBA mice. After completion of in vivo screening, a total of 217 transposon mutants were attenuated in the CBA mouse model of ascending UTI. Following in vitro screening, this number was reduced to 196 transposon mutants with a probable role in virulence. Co-challenge competition experiments confirmed significant attenuation for 37 of the 93 transposon mutants tested, being outcompeted by wild-type HI4320. Following sequence analysis of the 37 mutants, transposon insertions were identified in genes including the peptidyl-prolyl isomerases surA and ppiA, glycosyltransferase cpsF, biopolymer transport protein exbD, transcriptional regulator nhaR, one putative fimbrial protein, flagellar M-ring protein fliF and hook protein flgE, and multiple metabolic genes.
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Characterization of T-cell immunogenicity of two PE/PPE proteins of Mycobacterium tuberculosis
More LessThe PE and PPE proteins of Mycobacterium tuberculosis form a source of antigenic variation among different strains of this bacterium. Two of the PE_PGRS protein-encoding genes, rv3812 and rv3018c, are expressed in pathogenic mycobacteria and are implicated, respectively, in the persistence of the organism in macrophages and in virulence. Peptides derived from these proteins have been predicted to bind major histocompatibility complex (MHC) class I with high affinity on the basis of immunoinformatics analysis, suggesting a possible role for these proteins in antimycobacterial immunity. In the present work, using DNA constructs containing the rv3812 and rv3018c genes of M. tuberculosis, the immunogenicity of these proteins was demonstrated in BALB/c mice. Immunization with either DNA construct induced a significant number of CD8+-type T cells and a strong Th1-type response, with high gamma interferon (IFN-γ) and low interleukin-4 responses. Three nonameric peptides of Rv3812 and two of Rv3018c elicited a strong T-cell response in an MHC-restricted manner. An epitope-specific response was demonstrated by the lysis of peptide-pulsed antigen-presenting cells, release of perforin and IFN-γ production. Experimentally, these peptides bound with high affinity to MHC H-2Kd and showed low dissociation rates of peptide–MHC complexes. This study suggests that the identified T-cell epitopes may contribute to immunity against tuberculosis if included in a vaccine.
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Serum antibodies to West Nile virus in naturally exposed and vaccinated horses
More LessA polyvalent ELISA and plaque reduction neutralization tests (PRNTs) were used to measure serum antibodies to West Nile virus (WNV) in horses naturally exposed to or vaccinated against this flavivirus in Connecticut and New York State, USA. Relying on a PRNT as a ‘gold standard’, the main objective was to validate a modified ELISA containing a recombinant WNV envelope protein antigen. It was also important to assess specificity by testing sera from horses that had other, undiagnosed illnesses. Sera for the latter study were obtained from 43 privately owned horses during 1995–1996. Analyses by an ELISA and a PRNT confirmed the presence of WNV antibodies in 21 (91 %) of 23 sera from naturally exposed horses and in 85 % of the 20 vaccinated subjects; overall results for both study groups were highly concordant (91 % agreement). Humoral responses of naturally exposed and immunized horses were similar. Both serological tests were useful in confirming past infections with WNV, but there was no evidence that horses with undiagnosed illnesses were exposed to WNV prior to a 1999 outbreak in Connecticut, USA.
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- Diagnostics, Typing And Identification
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Novel hypertonic saline–sodium hydroxide (HS–SH) method for decontamination and concentration of sputum samples for Mycobacterium tuberculosis microscopy and culture
This study evaluated a new decontamination and concentration (DC) method for sputum microscopy and culture. Sputum samples from patients with suspected pulmonary tuberculosis (TB) (n=106) were tested using the proposed hypertonic saline–sodium hydroxide (HS–SH) DC method, the recommended N-acetyl-l-cysteine–sodium citrate–sodium hydroxide (NALC-NaOH) DC method and unconcentrated direct smear (Ziehl–Neelsen) techniques for the presence of mycobacteria using Löwenstein-Jensen culture and light microscopy. Of 94 valid specimens, 21 (22.3 %) were positive in culture and were further characterized as Mycobacterium tuberculosis. The sensitivity for acid-fast bacilli (AFB) smears was increased from 28.6 % using the direct method to 71.4 % (HS–SH) and 66.7 % (NALC-NaOH) using DC methods. Both concentration techniques were highly comparable for culture (kappa=0.794) and smear (kappa=0.631) for AFB. Thus the proposed HS–SH DC method improved the sensitivity of AFB microscopy compared with a routine unconcentrated direct smear; its performance was comparable to that of the NALC-NaOH DC method for AFB smears and culture, but it was methodologically simpler and less expensive, making it a promising candidate for evaluation by national TB control programmes in developing countries.
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Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays
More LessRapid identification of the two major species of Cryptosporidium associated with human infections, Cryptosporidium hominis and Cryptosporidium parvum, is important for investigating outbreaks of cryptosporidiosis. This study reports the development and validation of a real-time PCR TaqMan procedure for detection of Cryptosporidium species and identification of C. hominis and C. parvum in stool specimens. This procedure comprised a generic TaqMan assay targeting the 18S rRNA for sensitive detection of Cryptosporidium species, as well as two other TaqMan assays for identification of C. hominis and C. parvum. The generic Cryptosporidium species assay can be duplexed with the C. parvum-specific assay. The generic Cryptosporidium species assay was able to detect ten Cryptosporidium species and did not cross-react with a panel of ten other protozoan parasites. The generic Cryptosporidium species assay could detect 1–10 oocysts in a 300 μl stool specimen, whilst each of the species-specific TaqMan assays had detection sensitivities that were approximately tenfold higher. The 18S rRNA assay was found to detect Cryptosporidium species in 49/55 DNA extracts from stool specimens containing either C. hominis or C. parvum. The C. hominis TaqMan assay correctly identified C. hominis in 24/31 validation panel specimens containing this species. The C. parvum-specific assay correctly identified C. parvum in 21/24 validation panel specimens containing this species. This real-time PCR procedure was used to detect and identify C. hominis and C. parvum in stool specimens from outbreak investigations in the USA and Botswana, resulting in identification of C. hominis and/or C. parvum in 66/67 stool specimens shown to be positive for these species using other techniques. From the outbreak specimens tested, the TaqMan procedure was found to have a specificity of 94 %. This TaqMan PCR procedure should be a valuable tool for the laboratory diagnosis of cryptosporidiosis caused by C. hominis and C. parvum during outbreak investigations.
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Comparative prevalence of superantigenic toxin genes in meticillin-resistant and meticillin-susceptible Staphylococcus aureus isolates
A total of 118 meticillin-resistant Staphylococcus aureus (MRSA) and 140 meticillin-susceptible S. aureus (MSSA) isolates from different patients in the same time period were comprehensively searched using a multiplex PCR for the classical and recently described superantigenic toxin gene family comprising the staphylococcal enterotoxin genes sea to ser and the toxic shock syndrome toxin 1 gene, tst-1. Both MRSA and MSSA isolates carried a number of superantigenic toxin genes, but the MRSA isolates harboured more superantigenic toxin genes than the MSSA isolates. The most frequent genotype of the MRSA isolates was sec, sell and tst-1 together with the gene combination seg, sei, selm, seln and selo, which was found strictly in combination in 69.5 % of the isolates tested. In contrast, possession of the sec, sell and tst-1 genes in MSSA isolates was significantly less than in MRSA (2.1 vs 77.1 %, respectively), although they also often contained the combination genes (25.0 %). This notable higher prevalence in MRSA isolates indicated that possession of the sec, sell and tst-1 genes in particular appeared to be a habitual feature of MRSA. Moreover, these were mainly due to the fixed combinations of the mobile genetic elements type I νSa4 encoding sec, sell and tst-1, and type I νSaβ encoding seg, sei, selm, seln and selo. Analysis of the relationship between toxin genotypes and the toxin gene-encoding profiles of mobile genetic elements has a possible role in determining superantigenic toxin genotypes in S. aureus.
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- Antimicrobial Agents And Chemotherapy
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Human recombinant lactoferrin acts synergistically with antimicrobials commonly used in neonatal practice against coagulase-negative staphylococci and Candida albicans causing neonatal sepsis
More LessNeonatal sepsis causes significant mortality and morbidity. Coagulase-negative staphylococci (CoNS) and Candida frequently cause neonatal sepsis at >72 h of age. Lactoferrin, which is present in human milk, is a component of innate immunity and has broad-spectrum antimicrobial activity. The synergistic effects of lactoferrin with antibiotics against neonatal isolates have not been systematically evaluated. Here, eight clinical strains (seven neonatal) of CoNS and three strains (two neonatal) of Candida albicans were studied. MIC50 and MIC90 values of human recombinant lactoferrin (talactoferrin; TLF), vancomycin (VAN) and nafcillin (NAF) against CoNS, and of TLF, amphotericin B (AMB) and fluconazole (FLC) against C. albicans, were evaluated according to established guidelines. Antimicrobial combinations of TLF with NAF or VAN against CoNS, and TLF with AMB or FLC against C. albicans, were evaluated by a chequerboard method with serial twofold dilutions. Synergy was evaluated by the median effects principle, and combination indices and dose reduction indices were reported at 50, 75 and 90 % inhibitory effect at several drug-dose ratios. It was found that TLF acted synergistically with NAF and VAN against CoNS, and with AMB and FLC against C. albicans, at multiple dose effects and drug-dose ratios with few exceptions. In synergistic combinations, drug reduction indices indicated a significant reduction in doses of antibiotics, which may be clinically relevant. Thus TLF acts synergistically with anti-staphylococcal and anti-Candida agents commonly used in neonatal practice and is a promising agent that needs to be evaluated in clinical studies.
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Penicillin resistance in the intestinal spirochaete Brachyspira pilosicoli associated with OXA-136 and OXA-137, two new variants of the class D β-lactamase OXA-63
More LessPenicillin resistance mediated by β-lactamase activity has been reported previously in the anaerobic intestinal spirochaete Brachyspira pilosicoli, and a novel class D β-lactamase (OXA-63) hydrolysing oxacillin was described recently in a resistant human strain from France. In the current study, 18 B. pilosicoli strains from Australia and Papua New Guinea were tested for ampicillin and oxacillin susceptibility, and investigated for the presence of the class D β-lactamase gene bla OXA-63 using PCR. PCR products were amplified from seven human and four porcine strains that were penicillin resistant, but not from seven penicillin-sensitive strains. Sequence analysis of the whole gene amplified from seven of the resistant strains from humans and pigs revealed only minor nucleotide differences among them, but there were significant differences compared with bla OXA-63. The predicted amino acid sequence of the enzyme from all seven strains had the same key structural motifs as the previously reported OXA-63, but two variants with 94–95 % identity with OXA-63 were identified. OXA-136 had an additional amino acid and 12 other consistent amino acid substitutions compared with OXA-63. OXA-137 had the same differences compared with OXA-63 as OXA-136, but had an additional amino acid substitution at position 16. No structures consistent with integrons or transposons were found in the nucleotide sequences in the vicinity of bla OXA-136 in partially sequenced B. pilosicoli strain 95/1000, and the GC content (25.2 mol%) of the gene was similar to that of the whole genome. The gene encoding OXA-136 from B. pilosicoli strain Cof-10 conferred penicillin resistance on Escherichia coli. This study shows that penicillin resistance in human and porcine B. pilosicoli strains from Australia is associated with the production of two variants of OXA-63, and that susceptible strains lack the genes encoding OXA-63 or the variants.
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Enhancement of the antituberculosis activity of weak acids by inhibitors of energy metabolism but not by anaerobiosis suggests that weak acids act differently from the front-line tuberculosis drug pyrazinamide
More LessMycobacterium tuberculosis is uniquely susceptible to weak acids compared with other mycobacteria or bacteria. The antituberculosis activity of the front-line drug pyrazinamide (PZA), a weak acid (pyrazinoic acid) precursor, can be enhanced by inhibitors of energy metabolism and anaerobiosis. Here, we investigated the effect of inhibitors of energy metabolism and anaerobiosis on weak acid activity against M. tuberculosis in general. The susceptibility of M. tuberculosis to benzoic acid (BA) esters and amides was determined alone and in the presence of inhibitors of energy metabolism such as N,N′-dicyclohexylcarbodiimide (DCCD) and azide and also under anaerobic conditions in the form of MIC and drug exposure followed by colony count. Some BA esters such as propyl hydroxybenzoic acid and 4-dodecyloxylbenzoic acid had significant activity whereas amides of BA had no activity. As for PZA, inhibitors of energy metabolism DCCD and azide enhanced the antituberculosis activity of weak acids under normal atmospheric oxygen tension. However, unlike PZA, weak acids did not show antituberculosis activity and the inhibitors of energy metabolism did not enhance the weak acid activity under anaerobic conditions. The enhancement of weak acid activity by inhibitors of energy metabolism for M. tuberculosis was not seen in other bacterial species such as Helicobacter pylori. These results suggest that while the antituberculosis activity of weak acids can be enhanced by inhibitors of energy metabolism as for PZA, weak acids act differently from PZA in that they were inactive against M. tuberculosis under anaerobic conditions. The significance of these findings is discussed in the context of the unique physiology of M. tuberculosis and the development of new tuberculosis drugs.
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Effect of triclosan on the formation of crystalline biofilms by mixed communities of urinary tract pathogens on urinary catheters
More LessThe crystalline bacterial biofilms that encrust Foley catheters compromise the care of many elderly and disabled patients. The aim of this study was to examine whether the biocide triclosan can prevent encrustation by the mixed flora of uropathogens that commonly infect patients undergoing long-term catheterization. Models of the catheterized bladder were inoculated with communities of organisms isolated from patients who were experiencing catheter blockage. The catheter retention balloons were inflated with water or triclosan (3 g triclosan l−1 in 0.1 M sodium carbonate) and urine was supplied to the models for up to 7 days. The effect of triclosan was recorded on the viable cell populations, the pH of the residual urine and the times that catheters took to block. The extent of encrustation of the catheters was visualized by scanning electron microscopy. In models inoculated with communities containing Proteus mirabilis, triclosan prevented the rise in urinary pH that drives crystalline biofilm formation and catheter blockage. The biocide had no effect on populations of Enterococcus faecalis and Pseudomonas aeruginosa, but Proteus mirabilis, Escherichia coli and Klebsiella pneumoniae were eliminated from the residual urine and the catheters drained freely for the 7-day experimental period. In models inoculated with a mixed community containing Providencia rettgeri, catheters inflated with triclosan continued to block rapidly. Although K. pneumoniae and Proteus vulgaris were eliminated from the residual urine, there was no effect on the viability of Providencia rettgeri. The results indicate that the triclosan strategy should be limited to the treatment of patients who are infected with Proteus mirabilis.
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- Epidemiology
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Enteroaggregative Escherichia coli associated with a foodborne outbreak of gastroenteritis
This study investigated two foodborne outbreaks of gastroenteritis that occurred 10 days apart among individuals who had meals at the restaurant of a farm holiday resort. Mild gastrointestinal symptoms were reported and none of the patients needed hospitalization. Mean incubation times were 45 and 33 h, and the overall attack rates were 43.5 and 58.3 %, respectively. Stool sample examination was negative for common enteric pathogens in both outbreaks. Specimens from 13 people involved in the second outbreak and 3 restaurant staff were examined for diarrhoeagenic Escherichia coli. An enteroaggregative E. coli (EAEC) strain of serotype O92 : H33 was isolated from six participants and one member of staff. In particular, the EAEC strain was isolated from five of the six cases of diarrhoea examined. The strain showed an aggregative pattern of adherence to HEp-2 cells, did not produce a biofilm and possessed the virulence-related genes aat, aggR, aap and set1A, but not the astA gene. A retrospective cohort study indicated a pecorino cheese made with unpasteurized sheep milk as the possible source (P<0.001). Samples of the cheese had E. coli counts higher than 106 c.f.u. g−1, but the outbreak EAEC strain was not isolated. This report confirms that EAEC infections are probably underdiagnosed because of the limited availability of laboratories capable of identifying this group of pathogenic E. coli.
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- Clinical Microbiology And Virology
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Influence of automated screening and confirmation of extended-spectrum β-lactamase-producing members of the Enterobacteriaceae on prescribing of antibiotics
More LessThis study investigated the clinician response to the extended-spectrum β-lactamase (ESBL) confirmation report generated by an automated detection system, MicroScan Walkaway. The study compared two cohorts (pre- and post-automated detection) of patients with an ESBL-producing Escherichia coli or Klebsiella species non-urinary infection over the period October 2001–December 2006. Acceptance of the report, as defined by the initiation of carbapenem therapy, was observed in 69.2 % of the post-automated detection cohort (n=78) versus 20 % in the pre-automated detection period (n=15) (P ≤0.001). The utilization of a carbapenem increased progressively over the course of the study. Moreover, the time to initiation of carbapenem therapy was reduced from 15.7±4.9 to 0.1±2.0 days (P ≤0.001) after implementation of this automated detection system. Overall, clinicians responded positively to the ESBL automated detection report, as gauged by the increased utilization of a carbapenem and the earlier initiation of appropriate therapy; however, reductions in length of stay and mortality were not observed in this infected population.
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- Veterinary Microbiology
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Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes
Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats and humans. The mechanisms involved in adherence of M. canis to epidermis have never been investigated. Here, a model was developed to study the adherence of M. canis to feline corneocytes through the use of a reconstructed interfollicular feline epidermis (RFE). In this model, adherence of arthroconidia to RFE was found to be time-dependent, starting at 2 h post-inoculation and still increasing at 6 h. Chymostatin, a serine protease inhibitor, inhibited M. canis adherence to RFE by 53 %. Moreover, two mAbs against the keratinolytic protease subtilisin 3 (Sub3) inhibited M. canis adherence to RFE by 23 %, suggesting that subtilisins, and Sub3 in particular, are involved in the adherence process.
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- Case Reports
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Spondylodiscitis and paraspinal abscess caused by β-haemolytic group G streptococci spreading from infected leg ulcers
More LessWe report a case of spondylodiscitis due to Streptococcus dysgalactiae subsp. equisimilis spreading from infected leg ulcers. The route of infection could be unequivocally demonstrated by culturing identical isolates from leg wounds, blood culture and intra-surgery specimens from the spine. The present case illustrates the pathogenic potential of group G streptococci also for non-diabetic adults.
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Brucellosis complicating chronic non-infectious disorders: diagnostic and therapeutic dilemmas
There is little information in the literature on the clinical progress of brucellosis in patients affected by other non-infectious diseases; however, the infection can often trigger an exacerbation of existing underlying conditions in certain target organs. In this report we present four cases of brucellosis complicating previous diseases, and the difficulties in relation to their diagnosis and treatment. The study involved four patients with the following disorders: polycythaemia vera, pulmonary fibrosis, cirrhosis of the liver and arthritis of the knee. Brucellosis was diagnosed by classical serological and bacteriological methods. The strains involved could be isolated only in three of the four patients: two strains were Brucella abortus biovar 1 and one was Brucella suis biovar 1. Two patients relapsed 10 and 7 months after admission, another presented chronic brucellosis and received various therapy schemes, and one died. Since the best selection of antibiotics and the optimal duration of therapy remain unknown for patients having brucellosis complicated by previous pathologies, these remain at the discretion of the attending physician. Management of our patients was controversial in terms of the selection of antibiotics, duration of treatment and decision regarding surgery.
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Graft versus host disease-related Hafnia alvei colonization and probable infection
We describe the case of a graft versus host disease (GvHD) patient, in whom Hafnia alvei was cultured as a single organism, and at high bacterial counts from stool samples, from the onset of the disease until its resolution. This case is a further example of the contentious role of this species in causing human intestinal disease. Furthermore, it focuses on enteric damage by GvHD as a risk factor for acquiring H. alvei colonization, and probably infection.
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First case of post-endoscopic retrograde cholangiopancreatography bacteraemia caused by Acinetobacter ursingii in a patient with choledocholithiasis and cholangitis
More LessWe describe what we believe to be the first case of biliary sepsis caused by Acinetobacter ursingii. The patient was a healthy woman with no comorbidities who presented with choledocholithiasis and cholangitis. The performance of an endoscopic cholangiopancreatography was the trigger for A. ursingii bacteraemia. This report highlights the inadequacies of conventional phenotypic tests usually available in clinical microbiology laboratories for the identification of Acinetobacter species.
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Volumes and issues
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