- Volume 57, Issue 2, 2008

Yersinia outer protein P (YopP) induces cell death in macrophages and dendritic cells (DC). In DC this YopP-dependent cell death coincides with the inhibition of nuclear factor-kappa B (NF-κB) activation. However, as shown by measurement of propidium iodide uptake via disrupted cellular membranes, the preincubation of DC with several NF-κB inhibitors prior to infection with Yersinia did not restore the death-inducing capacity of a YopP-deficient Yersinia mutant. These results suggest that in contrast to macrophages, in DC the YopP-dependent inhibition of NF-κB activation is not causative for the induction of cell death. Instead, in DC, the inhibition of mitogen-activated protein kinases (MAPKs), in particular, p38 and c-Jun N-terminal kinase, prior to infection with a YopP-deficient Yersinia mutant substituted the death-inducing capacity of the Yersinia wild-type strain, indicating that the YopP-dependent inhibition of MAPKs mediates Yersinia-induced DC death. The differences between DC and macrophages in the mechanisms of cell death induction by YopP presented herein might be crucial for the function of these antigen-presenting cells.

The obligate intracellular pathogen Anaplasma phagocytophilum is transmitted by ticks and causes human granulocytic anaplasmosis, tick-borne fever of ruminants, and equine and canine granulocytic anaplasmosis. In a previous study, the perilipin (PLIN) gene was identified as one of the genes differentially expressed in human promyelocytic HL-60 cells in response to infection with A. phagocytophilum. PLIN is a major adipocyte lipid droplet-associated phosphoprotein that plays a central role in lipolysis and cholesterol synthesis. Host cholesterol and other lipids are required by A. phagocytophilum for infection and multiplication in human cells. In this study, it was hypothesized that PLIN may be involved in infection of human HL-60 cells by A. phagocytophilum. To test this hypothesis, a combination of real-time RT-PCR, immunofluorescence and RNA interference was used to study the expression of PLIN. The results of these studies demonstrated that A. phagocytophilum modulates lipid metabolism by increasing PLIN mRNA levels and facilitates infection of HL-60 cells. The results of these studies expand our knowledge of the role of lipid metabolism in A. phagocytophilum infection and multiplication in HL-60 cells and suggest a mechanism by which A. phagocytophilum modulates lipid metabolism.

Streptococcus pneumoniae expresses at least 91 different polysaccharide (PS) capsules and the currently available serotyping methods are tedious to perform. We have been developing a rapid pneumococcal serotyping assay (named the ‘multibead assay’) based on the capacity of pneumococcal lysates to inhibit the ability of 24 different anti-capsule antibodies to bind to latex beads coated with 24 different PSs (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 23F, 2, 8, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F). Because the polyclonal antibodies used for 10 serotypes (2, 8, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F) had limited serotype specificity, we replaced them with monoclonal antibodies for the 10 serotypes. To extend the serotype coverage beyond the 24 serotypes, we have adapted multiplexed PCR for five additional serotypes (15A, 15C, 16F, 35B and 38) to be useful with the pneumococcal lysates prepared for the multibead assay. We then validated the combined assay with 157 clinical isolates from the Centers for Disease Control and Prevention and found that the new combined assay produced results that are concordant with the quellung reaction. The combined assay is robust and could be used to rapidly identify the serotypes of the majority of pneumococci (∼90 %). In addition, the assay validation study suggests the presence of serological subtypes within serotype 11A.

Rifampicin-resistant Mycobacterium leprae is regularly reported and drug resistance is a major threat for the elimination of leprosy. There is an urgent need for a simple method that can detect rifampicin resistance in clinical isolates. This study developed a multiple-primer PCR amplification refractory mutation system, a simple, reliable and economical method for clinical specimens that allowed the rapid detection of mutations in the nucleotides of the codon for Ser425 of the M. leprae rpoB gene, mutation of which to Leu, Met or Phe is associated with rifampicin resistance. The approach involved a multiple-primer PCR in which both mutant-specific and normal sets of primers were included in the reaction. The mutant-specific primer was complementary to the corresponding sequence of the wild-type gene except for one additional deliberate mismatch at the fourth nucleotide from the 3′-OH terminus. A single mismatch has little influence on the yield of PCR products, but if there are two mismatches as a result of mutation at the position being tested, the mutant-specific primer will not function in PCR under appropriate conditions, leading to no yield of PCR product from the mutant allele. The assay was evaluated successfully using a panel of plasmids and M. leprae reference strains carrying the wild-type or known rpoB mutations. The assay was subsequently applied to M. leprae DNA extracts from skin biopsies taken from patients. In all biopsy samples, the wild-type allele was detected for Ser425. The PCR results correlated with rifampicin susceptibility, as also measured by the traditional in vivo mouse footpad technique.

Data on the prevalence of pneumooccal nasopharyngeal carriage and its risk factors among adolescents are scarce. The aim of this study was to provide such information. A cross-sectional, population-based prospective study was conducted. Participants were 1013 adolescents (age range 10–19 years) randomly recruited in 22 public schools. Those schools were randomly chosen among 307 public schools from 11 Sanitary Districts of Salvador, Brazil. Nasopharyngeal samples were assessed by standard procedures to recover and identify Streptococcus pneumoniae. Data on potential risk factors were gathered by confidential interview based on a standardized questionnaire. Pneumococci were recovered from 8.2 % [83/1013, 95 % confidence interval (CI) 6.6–10.0]. By stepwise logistic regression, pneumococcal colonization was independently associated with younger age [odds ratio (OR) 0.85, 95 % CI 0.77–0.94, P=0.001], being male (OR 1.78, 95 % CI 1.11–2.85, P=0.02), exposure to passive smoke in the household (OR 1.76, 95 % CI 1.10–2.79, P=0.02), having an upper respiratory infection during recruitment (OR 2.67, 95 % CI 1.67–4.28, P<0.001) and having a history involving an episode of acute asthma during the last year (OR 2.89, 95 % CI 1.18–7.08, P=0.03). The estimated probability of pneumococcal colonization decreased with age (χ 2 for trend=8.52, P=0.003). These findings provide tools for increasing the use of prevention strategies for pneumococcal diseases, such as pneumococcal vaccination among asthmatic patients and public health measures to stop smoking.

A retrospective review was conducted of patients with external ventricular drains (EVDs) in situ in order to ascertain the utility of daily cerebrospinal fluid (CSF) analysis in such patients. All laboratory requests for CSF analysis, which were sent to the Microbiology Department, Auckland City Hospital, New Zealand, were reviewed to identify patients with EVDs in situ. The patients' clinical records were reviewed and information was obtained regarding their age, ethnicity, indication for EVD, duration of EVD, CSF analysis results, daily temperatures, Glasgow Coma Scale (GCS) and the presence of other infections. For CSF samples that grew organisms, the patients' notes were reviewed to ascertain whether the organism was a contaminant or was representative of EVD-associated ventriculitis. A total of 454 CSF specimens from 60 patients were reviewed. Of the 56 CSF specimens that were culture-positive, 40 (71 %) were found to reflect clinical infection. Routine CSF analysis identified nine episodes of EVD-associated ventriculitis. Coagulase-negative staphylococci and Staphylococcus epidermidis were the most common isolates and were associated with ventriculitis approximately half of the time. Gram-negative isolates were less frequently isolated, but, when present, were always associated with ventriculitis. This study found that patient temperature and GCS did not allow early prediction of EVD-associated ventriculitis.

Campylobacter jejuni is a major cause of diarrhoea in humans. However, the pathogenesis of C. jejuni diarrhoea is poorly understood due to the lack of a good animal model of infection. Many animals have been tried with limited success, but a mouse lung model of infection has been found to be satisfactory previously; however, the lung pathology of this model has not been studied. For the purpose of characterizing the histopathological and ultrastructural lesions in the lung of the mouse pulmonary model of C. jejuni infection, C. jejuni strain 81-176 or sterile PBS was intranasally inoculated into BALB/c mice. The infection resulted in a mild illness only, and in an initial predominance of polymorphonuclear cells, followed by the accumulation of macrophages and later the prominence of epithelioid cells. Focal peribronchial pneumonia appeared on day 3, granuloma-like reaction on day 4 and bronchopneumonia on day 5 post-infection. These features developed until day 5 post-infection, but were less consistent afterwards when histopathology was monitored up to 9 days post-infection. Intracellular structures resembling bacteria were observed on days 3 and 5 post-infection, but not on day 7 post-infection. On days 3 and 5 post-infection, degenerative changes were also observed by transmission electron microscopy. The histological changes were not associated with acid-fast bacteria or any fungal elements. The infection was systemic as C. jejuni was isolated from blood and all organ homogenates (lung, spleen, liver, and small and large intestines) at 24 h post-infection. Thereafter, the organism was recovered from the intestine only, thus indicating its predilection for this location. This characterization of pathology should contribute to a better understanding of the animal model and pathogenesis of C. jejuni infection.

Immunofluorescence microscopy-based identification of presumptive Propionibacterium acnes isolates, using the P. acnes-specific mAb QUBPa3, revealed five organisms with an atypical cellular morphology. Unlike the coryneform morphology seen with P. acnes types I and II, these isolates exhibited long slender filaments (which formed large tangled aggregates) not previously described in P. acnes. No reaction with mAbs that label P. acnes types IA (QUBPa1) and II (QUBPa2) was observed. Nucleotide sequencing of the 16S rRNA gene (1484 bp) revealed the isolates to have between 99.8 and 99.9 % identity to the 16S rRNA gene of the P. acnes type IA, IB and II strains NCTC 737, KPA171202 and NCTC 10390, respectively. Analysis of the recA housekeeping gene (1047 bp) did reveal, however, a greater number of conserved nucleotide polymorphisms between the sequences from these isolates and those from NCTC 737 (98.9 % identity), KPA171202 (98.9 % identity) and NCTC 10390 (99.1 % identity). Phylogenetic investigations demonstrated that the isolates belong to a novel recA cluster or lineage distinct from P. acnes types I and II. We now propose this new grouping as P. acnes type III. The prevalence and clinical importance of this novel recA lineage amongst isolates of P. acnes remains to be determined.

Coexistence of two illnesses in the same patient may result in atypical manifestations of either or both diseases. A case of hepatitis B virus-related cirrhosis in a patient who presented with a pharyngeal mucosal mass lesion as a manifestation of superadded Leishmania infection is presented here. The clue to the diagnosis was the origin of the patient from an area highly endemic for leishmaniasis and the presence of unexplained polyclonal hypergammaglobulinaemia. The patient responded very well to therapy with amphotericin B with complete disappearance of the mucosal lesion.