- Volume 48, Issue 12, 1999

An outbreak of infantile diarrhoea was investigated in 32 children, all <2 years old, in the tropical north of Australia. Rotavirus (63%) and enteropathogenic Escherichia coli (EPEC) (59%) were the most common pathogens identified. Of the 19 EPEC isolates, 14 (74%) were of serotype O126:H12, hitherto unreported as an EPEC serotype. Other pathogens isolated included Salmonella spp. (16%), Campylobacter spp. (3%), Giardia (3%) and Shigella spp. (3%). EPEC-related gastro-enteritis is an uncommon but recognised cause of diarrhoeal outbreaks in Australia and clinicians need to be aware of the possibility of this serotype being implicated. This report highlights the disadvantages of relying on serotyping alone for the recognition of EPEC.

Neisseria meningitidis is an important pathogen because it causes life-threatening infections. The rapid course of meningococcal disease and the capacity of some serogroups to cause large-scale epidemics necessitates the use of sensitive, reliable and rapid typing methods to characterise strains. Molecular typing techniques for N. meningitidis are used for epidemiological purposes to investigate outbreaks and the spread of organisms and to examine the population genetic structure of the organism to understand better its variation and evolution. Many investigators have employed molecular typing methods and shown that meningococcal disease is associated with a variety of different epidemiological patterns. The choice of a typing method is dependent upon the epidemiological questions to be answered and on the population genetics of the organism under investigation. With highly clonal populations comprising independent non-recombining lineages such as serogroup A meningococci, ribotyping, multilocus enzyme electrophoresis (MLEE), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR with arbitrary primers (RAPD) or with other gene-based primers each provides a constant measure of the relationship between strains. A more restricted portfolio of molecular methods – PFGE, MLEE and MLST – is appropriate for the investigation of less clonal serogroup B and C meningococci from localised outbreaks. If a thorough evaluation of the overall population is sought to determine the relationship between new isolates and members of hyper-endemic clonal complexes then quantitative methods such as MLEE and MLST are necessary. Several PCR-based methods are used for the detection and typing of meningococcal strains, many requiring rigorous standardisation before they can be considered suitable for rapid and reliable differentiation between clones. This review examines strain characterisation by molecular techniques and non-culture-based subtyping of meningococci in clinical specimens. It assesses the importance of these techniques and examines the epidemiological questions that they answer and also their limitations.

The secretion of interleukin-12 (IL-12) following intracellular infection with virulent Brucella abortus strain 2308 was investigated in CD-1 mice and in CD-1 cultured peritoneal macrophages. Bioactive IL-12p70 and free non-immunoactive p40 subunits (IL-12p40) were determined by enzyme-linked immunosorbent assays. In CD-1 mice, B. abortus 2308 was a potent inducer of IL-12p40 (maximum levels were 5.9 and 3.4 ng/ml in sera and spleen homogenates, respectively). Secretion of IL-12p70 was also demonstrated in vivo, although at much lower levels (216.6 and 198.9 pg/ml in sera and spleen homogenates, respectively). Production of IL-12 over the first 7 days after infection was accompanied by active multiplication of B. abortus in the spleens of infected mice. CD-1 cultured peritoneal macrophages secreted only IL-12p40 (878.4 pg/107 macrophages) in response to B. abortus infection and no production of IL-12p70 was observed. In contrast, CD-1 peritoneal macrophages secreted detectable amounts of IL-12p70 (16.2 pg/107 macrophages) in response to purified lipopolysaccharide (S-LPS) from B. abortus 2308. The macrophages also secreted significant amounts of interferon-γ (IFN-γ) (520.1 pg/107 macrophages) in response to intracellular B. abortus. These results indicate that B. abortus 2308 is not a potent inducer of IL-12p70 production, whereas purified S-LPS from B. abortus 2308 induces the secretion of this bioactive form of IL-12 in cultured peritoneal macrophages. CD-1 peritoneal macrophages were able to secrete IFN-γ, as well as high amounts of IL-12p40, in response to intracellular infection by B. abortus.

Streptococcus pneumoniae grows well and generally exhibits typical morphology on Columbia blood agar, whereas Haemophilus influenzae requires a more complex medium to meet its growth requirements - usually chocolated blood agar – on which S. pneumoniae is less easily recognisable. Therefore, a single medium that produces typical morphology of S. pneumoniae and facilitates the growth of H. influenzae would have considerable potential advantages. It has been claimed that blood agar supplemented with nicotinamide adenine dinucleotide (NAD) is such a medium. However, despite its routine use in several large diagnostic laboratories its performance has never been properly evaluated. In the present study, 1724 sputum samples were examined in four laboratories. The isolation rates of H. influenzae and S. pneumoniae on NAD-supplemented blood agar (SBA) were compared with those on a two-plate combination of plain blood (BA) and chocolated blood agar (CBA). The two-plate combination performed significantly better for both organisms; isolation rates for H. influenzae were increased from 8.16% on SBA to 11.07% on BA plus CBA and for S. pneumoniae from 4.18% to 4.68%. Isolation rates were also compared after incubation for 24 and 48 h. With the two-plate combination, isolation rates for H. influenzae and S. pneumoniae were increased by 0.98% and 0.16%, respectively, and for SBA by 0.57% and 0.32% after 48 h. However, despite this increase, SBA still performed less well than the two-plate combination.

The mechanisms of resistance to ciprofloxacin and grepafloxacin were studied in 54 clinical isolates of Streptococcus pneumoniae. Restriction fragment length polymorphism analysis following Hinfl digestion was used with DNA sequencing to identify mutations in the quinolone resistance-determining regions (QRDRs) of the parC and gyrA genes. Ciprofloxacin MICs up to 16 mg/L were not associated with mutations to these genes in approximately half of the isolates. In other isolates, moderate levels of ciprofloxacin resistance (MIC 8-16 mg/L) were associated with an alteration of ParC, most commonly entailing replacement of serine-79 by phenylalanine. High-level ciprofloxacin resistance (MIC 32–128 mg/L) entailed the additional mutation of GyrA with substitution of serine-83 by phenylalanine. Grepafloxacin MICs >4 mg/L were associated with this GyrA mutation alone; no relationship was detected between grepafloxacin MICs and mutation of the QRDR of parC.

An animal model of disseminated aspergillosis was used to test the in-vivo activity of itraconazole against four isolates of Aspergillus fumigatus. Two reference isolates of A. fumigatus known to be resistant to itraconazole in vitro and in vivo were used as control isolates, and two new isolates were tested under the same conditions. For each isolate MICs for itraconazole and amphotericin B were determined by an NCCLS-based method. Mice infected intravenously were treated either with itraconazole 100 mg/kg/day or amphotericin B 4.5 mg/kg/day for 10 days. Amphotericin B showed good in vivo activity against all four isolates. For one strain, which had a low in-vitro MIC for itraconazole, in-vivo therapy with itraconazole prolonged the survival of mice and reduced fungal burdens in organs compared with untreated controls. In mice infected with a strain with a high MIC of >16 mg/L, itraconazole neither prolonged survival nor reduced fungal load in organs compared with controls. It is concluded that there is a relationship between MIC and treatment outcome in mice for A. fumigatus infection.