- Volume 51, Issue 8, 2002

This paper reports a case of Haemophilus segnis polymicrobial bacteraemia and a case of H. segnis monomicrobial bacteraemia identified by 16S ribosomal RNA gene sequencing. In the first case, a gram-negative aerobic coccobacillus was isolated with Streptococcus intermedius and S. sanguis from the blood culture of a 32-year-old intravenous drug addict with left thoracic empyema. In the second case, a gram-negative aerobic coccobacillus was isolated from the blood culture of an 82-year-old woman with Clostridium difficile colitis and septicaemic shock. Both gram-negative coccobacilli grew on chocolate agar as colonies of 1 mm in diameter after incubation for 24 h at 37°C in air with CO2 5%, but only to pinpoint sizes on blood agar under the same incubation conditions. Both strains were factor V-dependent, but not factor X-dependent. For the first isolate, the Vitek system (NHI) showed that it was 56% likely to be Actinobacillus actinomycetemcomitans and 40% Neisseria subflava; whereas the API system (NH) showed that it was 58% likely to be H. aphrophilus/paraphrophilus and 42% H. parainfluenzae. For the second isolate, the Vitek system (NHI) showed that it was 95% likely to be H. influenzae VIII; whereas the API system (NH) showed that it was 58% likely to be H. aphrophilus/paraphrophilus and 42% H. parainfluenzae. 16S rRNA gene sequencing showed that there were four base differences between isolate 1 and H. segnis and two base differences between isolate 2 and H. segnis, indicating that both isolates most closely resembled a strain of H. segnis. Only two cases of H. segnis bacteraemia were found in the English scientific literature, one in a case of infective endocarditis and the other in a case of pancreatic abscess. Including the present two cases, the overall mortality of H. segnis bacteraemia was 50%.

A genome walking technique was applied to borrelial DNA to clone the gene encoding decorin-binding protein B (DbpB) in Borrelia garinii and B. afzelii. Sequence analysis showed 62–67% identity of the predicted amino acid sequences of DbpB between the B. afzelii and B. garinii strains and B. burgdorferi sensu stricto. Within subspecies, the sequences were 99–100% identical. The respective recombinant DbpBs (rDbpBs) were produced and tested as antigens in an enzyme-linked immunosorbent assay (ELISA) for Lyme borreliosis (LB). In IgG ELISA, with rDbpBs as antigens, 11 (73%) of 15 adult patients with Lyme arthritis and 9 (64%) of 14 with neuroborreliosis were positive. Of children with Lyme arthritis, 40 (77%) of 52 were positive. All adult and paediatric patients with disseminated LB had high titres of anti-flagellin IgG antibodies. Seropositivity against rDbpB from B. garinii predominated, 39 (65%) of 60 of the positive samples reacting with rDbpB from B. garinii. In patients with erythema migrans, IgM antibodies to rDbpB were detected in 1 (4%) of 23 and IgG antibodies in 6 (26%) of 23. These results indicate that DbpB may be a useful antigen in the IgG serology for disseminated LB. The high inter-species sequence heterogeneity observed indicates that a combination of the variant DbpBs should be included in the antigen set to cover all the relevant borrelial subspecies in the serodiagnosis of LB.

A comparative study of human sera was conducted to determine which purified preparations of 11 recombinant antigens of Borrelia burgdorferi sensu stricto were diagnostically most important in enzyme-linked immunosorbent assays (ELISAs). To assess sensitivity, 20 serum samples obtained 1–6 weeks after onset of illness from 20 persons who had physician-diagnosed erythema migrans (EM) were tested for IgM and IgG antibodies. In tests for IgM antibody, seropositivity of ≥25% was recorded when ELISAs had separate preparations of protein (p) 37, p41-G, outer-surface protein (Osp) C, OspE, OspF or VlsE antigens. Sera reacted most frequently (80% positive) with VlsE antigen in analyses for IgG antibodies. When results of both class-specific assays were considered for VlsE, OspC or OspF, 90% of the EM cases were serologically confirmed. Results of specificity testing with a further 59 sera from persons who had syphilis, louse-borne relapsing fever, oral infections, rheumatoid arthritis or human granulocytic ehrlichiosis and 28 normal sera indicated no false positive reactions when VlsE antigen was used in tests for IgM antibody. One of the 11 louse-borne relapsing fever sera cross-reacted with VlsE antigen in tests for IgG antibodies. Minor cross-reactivity also occurred when p37, OspC, OspE or OspF antigens were used. Overall, VlsE was the most suitable antigen for laboratory diagnosis of Lyme borreliosis during the early weeks of B. burgdorferi infection because of its high sensitivity and specificity.

The diagnosis of B. canis infection in dogs is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. Bacteriological studies are the only methods that have been considered specific but, as intermittent periods of abacteraemia may occur, a negative blood culture cannot be used as a criterion for excluding canine brucellosis. Close contact between people and infected dogs increases the risk of transmission; however, its impact on public health is probably underestimated due to lack of reporting and inadequate diagnostic services. This paper describes an indirect enzyme-linked immunoassay (IELISA) procedure for the diagnosis of brucellosis caused by B. canis in a population of normal and infected dogs previously screened by the buffered plate antigen test (BPAT) and rapid slide agglutination test (RSAT). The serological survey was performed with 446 field sera. The 270 sera from the asymptomatic group found negative by BPA, RSAT and blood culture showed IELISA specificities of 96.7% and 100%, respectively, when cut-off values of OD 0.237 and 0.281 were selected. For 52 sera from culture-positive dogs, IELISA sensitivity was 100% with cut-off values of OD414 0.237 and 0.281. OD414 0.281 was selected because this value provided the highest accuracy with minimal false-negative and false-positive results. This cut-off value was used to study 124 blood culture-negative but RSAT positive sera. IELISA produced 107 positive results; the 17 sera that were negative by IELISA presented a wide range of reactivities by RSAT (2 were RSAT positive at 1 in 2 dilution and 15 were weakly positive with pure serum). These samples were probably from animals at an early stage of the infection or were false-positive results. The IELISA described here detects IgG and IgA antibodies that are useful for evaluating the clinical status of dogs. Although RSAT is a practical screening test, a supplementary technique such as IELISA should be used on all positive RSAT samples to ensure diagnostic specificity. Furthermore, people in contact with infected dogs could be investigated for possible transmission. The procedure described in this study was relatively simple and could have widespread applications.

To identify antigens of Brucella spp. that are potentially involved in stimulating a protective T-cell-mediated immune response, previous studies identified 10 clones from a Brucella abortus 2308 genomic library with primed lymphocytes as probes. One selected positive clone (182) contained an insert of 1.2 kb which was identified, sequenced and characterised. The deduced amino acid sequence of the open reading frame (ORF) revealed 82% and 81% identity to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes from Agrobacterium tumefaciens and Xanthobacter flavus, respectively. Southern blot analysis demonstrated that the gap gene is present in only one copy in the Brucella genome. B. abortus GAPDH was then expressed in Escherichia coli as a fusion protein with the maltose-binding protein (MBP). To demonstrate the functional activity of Brucella GAPDH, E. coli gap mutants were transformed with a Brucella pMAL-gap construct. Genetic complementation was achieved and as a result E. coli mutants were able to grow on glucose or other carbon source medium. The humoral and cellular immune responses to the recombinant (r) GAPDH were characterised. In Western blots, sera from naturally infected cattle and sheep showed antibody reactivity against rGAPDH. In response to in-vitro stimulation by rGAPDH, splenocytes from mice vaccinated with rGAPDH or B. abortus S19 were able to produce γ-interferon and tumour necrosis factor-α but not interleukin (IL)-4. Furthermore, gap associated with murine IL-12 gene in a DNA vaccine formulation partially protected mice against experimental infection.

The influence of major liver resection in obstructive jaundice on bacterial translocation was evaluated in rats that were colonised predominantly with a genetically labelled strain of Escherichia coli. The strain, JNW14, originally isolated from rat faeces, was labelled with bacitracin, neomycin and streptomycin resistance markers. Fifty-two specific-pathogen-free male Wistar rats were divided into three experimental groups and were treated as follows: group 1 (n = 8), sham ligation of common bile duct; group 2 (n = 7), common bile duct ligation (CBDL); and group 3 (n = 37), 70% hepatectomy 7 days after CBDL. The rats were treated with the above antibiotics and then given E. coli strain JNW14 in their drinking water. Translocation of E. coli JNW14 from the gastrointestinal tract to the mesenteric lymph nodes (MLNs), lungs, liver, spleen and portal vein was evaluated in each group. In group 3 (CBDL plus hepatectomy), the incidence of translocation of E. coli JNW14 to the liver and spleen after hepatectomy was significantly higher than in groups 1 and 2. This result indicates that major liver resection in obstructive jaundice promotes bacterial translocation to systemic organs. Furthermore, the numbers of viable E. coli JNW14 in the MLNs in the lung culture-positive rats were significantly higher than those in the lung culture-negative rats, suggesting that lymphatic-thoracic duct systemic circulation is a major route of bacterial translocation.

The purpose of this study was to evaluate the aetiology and susceptibility of different Candida species originating from blood cultures received from different clinical wards of the University Hospital in Szeged, Hungary, from 1996 to 2000. A total of 145 episodes of fungaemia occurred in 68 patients. In 73.5% of the patients the infections were due to Candida albicans, 7.3% to C. parapsilosis, 5.9% to C. krusei, 4.4% to C. tropicalis and 3% each to C. glabrata, other Candida spp. and Cryptococcus neoformans. There were no appreciable differences in the distribution of yeast species during the 5-year period: C. albicans remained the predominant species causing bloodstream infections in this hospital, similar to the results of other studies (Norway, SENTRY Program in USA, Canada and South America). Most of the Candida isolates (39.3%) were from blood cultures of patients hospitalised in surgical wards, 28.3% were from adult intensive care units (ICUs), 13.8% from paediatric ICUs, 11% from haematology and 7.6% from cardiology departments. MICs for amphotericin B, fluconazole and itraconazole were determined for 83% of the isolates. All isolates were susceptible to amphotericin B. The percentage of yeast isolates with decreased susceptibility or resistance to fluconazole was smaller (15.7%) than that for itraconazole (24%).

The seroprevalence and genotypes of hepatitis C virus (HCV) were studied in 283 patients attending six haemodialysis units in Jordan. In all, 98 (34.6%) patients were anti-HCV-positive by EIA, 92 (93.9%) of whom were also reactive in an immunoblot assay. The prevalence of anti-HCV was correlated with a history of blood transfusion before the introduction of blood donor screening for HCV and with duration of haemodialysis. HCV RNA was detected in 30 (30.6%) of 98 anti-HCV-positive sera. HCV viraemia was not associated with a particular antibody for the six HCV antigens studied by the immunoblot assay, although reactivity to the core antigens was greater in the HCV RNA-positive sera than in negative sera. Two HCV genotypes (1 and 4) were identified for the first time in Jordan by restriction fragment length polymorphism analysis of HCV 5′-NCR. The predominant genotype was HCV 1a (12 of 30). Genotypes 1b and 4 were detected in 10 and 8 patients, respectively. The antibody response to HCV antigens was genotype-dependent, with a wider range of antibody specificities detected in the immunoblot assay in the 12 patients with genotype 1a infection than in the 8 patients with genotype 4. However, there was no significant difference in the prevalence of antibodies to HCV antigens among patients infected with either genotype 1a or 1b. In conclusion, the prevalence of anti-HCV, blood transfusion, duration of dialysis and HCV genotypes suggest possible nosocomial HCV transmission among patients which needs confirmation by phylogenetic analysis of subgenomic HCV regions.