- Volume 49, Issue 10, 2000

Microbial antigen in clinical specimens can be detected rapidly by commercial test-card latex agglutination, but poor sensitivity is a potential difficulty. Antigen detection by immuno-agglutination of coated latex micro-particles can be enhanced in comparison with the conventional test-card method in both rate and sensitivity by the application of a non-cavitating ultrasonic standing wave. Antibody-coated micro-particles suspended in the acoustic field are subjected to physical forces that promote the formation of agglutinates by increasing particle–particle contact. This report reviews the application of ultrasound to immuno-agglutination testing with several commercial antibody-coated diagnostic micro-particles. This technique is more sensitive than commercial card-based agglutination tests by a factor of up to 500 for fungal cell-wall antigen, 64 for bacterial polysaccharide and 16 for viral antigen (in buffer). The detection sensitivity of meningococcal capsular polysaccharide in patient serum or CSF has been increased to a stage where serotyping by ultrasound-enhanced agglutination is comparable to that achievable with the PCR, but is available more rapidly. Serum antigen concentration as measured by ultrasonic agglutination has prognostic value. Increasing the sensitivity of antigen detection by increasing the acoustic forces that act on suspended particles is considered. Employing turbidimetry to measure agglutination as part of an integrated ultrasonic system would enable the turnover of large numbers of specimens. Ultrasound-enhanced latex agglutination offers a rapid, economical alternative to molecular diagnostic methods and may be useful in situations where microbiological and molecular methods are impracticable.

Following characterisation by phenotypic tests and amplified ribosomal DNA restriction analysis (ARDRA), 50 tetracycline-resistant (MIC≥165mumg/L) Acinetobacter strains from clinical (n=35) and aquatic (n=15) samples were analysed by PCR for tetracycline resistance (Tet) determinants of classes A–E. All the clinical strains were A. baumannii; most (33 of 35) had Tet A (n=16) or B (n=17) determinants, and only two did not yield amplicons with primers for any of the five tetracycline resistance determinants. The aquatic strains belonged to genomic species other than A. baumannii, and most (12 of 15) did not contain determinants Tet A–E. Strains negative for Tet A–E were also negative for Tet G and M; further analysis of two aquatic strains with specific primers for Tet O and Tet Y and degenerate primers for Tet M-S-O-P(B)-Q also showed negative results. Transfer of tetracycline resistance was tested for 20 strains with three aquatic Acinetobacter strains and Escherichia coli K-12 as recipients. Transfer of resistance was demonstrated between aquatic strains from distinct ecological niches, but not from clinical to aquatic strains, nor from any Acinetobacter strain to E. coli K-12. Most transconjugants acquired multiple relatively small plasmids (<36 kb). Transfer did not occur when DNA from the donor strains was added to the recipient cultures and was not affected by deoxyribonuclease I, suggesting a conjugative mechanism. It is concluded that Tet A and B are widespread among tetracycline-resistant A. baumannii strains of clinical origin, but unknown genetic determinants are responsible for most tetracycline resistance among aquatic Acinetobacter spp. These differences, together with the inability of clinical strains to transfer tetracycline resistance in vitro to aquatic strains, contra-indicate any important flow of tetracycline resistance genes between clinical and aquatic acinetobacter populations.

Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans are believed to play an important role in adult periodontitis, but the significance of their relative numbers and progress of the disease is still unclear. Traditional quantitative methods are generally time-consuming and inaccurate. The aim of this study was to develop a sensitive, quantitative PCR technique that would be useful for enumerating P. gingivalis, Pr. intermedia and A. actinomycetemcomitans in subgingival plaque samples from subjects with adult periodontitis. Primers to the following genes were employed: the fimbrial gene (fimA) of P. gingivalis, the 16S rRNA gene of Pr. intermedia and the leukotoxin-A (lktA) gene of A. actinomycetemcomitans. Competitive templates were constructed either by sequence deletion between primer binding sites or by annealing of the primer binding sites to an appropriate DNA core so as to yield products of a different size from that obtained with the target template. Co-amplification of target and competitive templates yielded products of expected size and non-specific recognition by the primers was not found. The sensitivity of the designed primers was 100 cells of P. gingivalis, 100 cells of Pr. intermedia and 10 cells of A. actinomycetemcomitans. The three species were found in subgingival plaque samples collected from both healthy and diseased sites by the quantitative-competitive (QC)-PCR method and the technique was more sensitive than cultural methods. For determining the proportions of each of the three periodontopathogens, the total number of bacteria in the samples was enumerated by quantitative-PCR with 16S rRNA universal primers (27f and 342r). The findings indicate that QC-PCR is a useful method for enumerating bacteria in clinical oral specimens and the technique could play a role in the investigation of disease progression.

This report describes the first isolation of Mycobacterium shimoidei in Finland from a sputum specimen obtained from an elderly female patient. M. shimoidei, a potential lung pathogen, is difficult to identify by routine methods and only a few cases have been reported. The present study demonstrated that M. shimoidei has a characteristic pattern for fatty acids and alcohols in gas liquid chromatography. This chromatogram and the pattern of mycolic acids on thin-layer chromatography allow it to be distinguished routinely. The unique sequence of the 16S rRNA gene and the 16S–23S rDNA spacer region allows identification by molecular methods.

The risk of obtaining false-negative results in serological assays in serum and CSF specimens with only one strain of Borrelia burgdorferi sensu lato as antigen was investigated in 79 patients with neuroborreliosis with specimens obtained at initial presentation. Serum antibodies were assessed by immunoblotting; the criteria of Hauser et al. were used to evaluate the test. The intrathecal synthesis of borrelial-specific IgM and IgG antibodies was examined by enzyme immunoassay (EIA). Strains of B. burgdorferi sensu stricto (BbZ160), B. garinii (Bbii50) and B. afzelii (PKO) served as sources of antigen in both assays. All patients produced either a positive IgM or IgG test in serum with at least one strain of B. burgdorferi sensu lato. Reactivity of IgM or IgG antibodies, or both, with antigens of all three strains was demonstrated in 67 (85%) of 79 sera. The correlation of results of immunoblotting with different strains was significantly better for IgG (85%) than for IgM antibodies (54%). The variability of positive IgM reactions in 18 specimens was mainly due to the fact that the antibodies were directed to the relevant variable outer-surface protein C (p23). Intrathecal synthesis of IgG antibodies was demonstrated in 58 patients (81%) of 72 and of IgM antibodies in 25 of 58 patients. No patient had isolated intrathecal synthesis of IgM antibodies. The majority of CSF samples (56 of 58) were assessed as IgG antibody-positive, independent of the borrelial strain used as antigen in EIA, whereas only 10 of 25 IgM antibody-positive CSF specimens reacted with all three strains. All patients in the study had intrathecal antibody synthesis demonstrable at 6-week follow-up. From this study it is concluded that there is a small, but real, risk of false-negative serological findings at the time of initial clinical presentation in patients with typical symptoms of neuroborreliosis. In these patients a negative serological result with one strain should prompt the repetition of the test with other strains of B. burgdorferi sensu lato.

The Burkholderia cepacia complex consists of at least five well-documented bacterial genomovars, each of which has been isolated from the sputum of different patients with cystic fibrosis (CF). Although the world-wide prevalence of this opportunist pathogen in CF patients is low (1–3%), ‘epidemic’ clusters occur in geographically isolated regions. Prevalence in some of these clusters is as high as 30–40%. The majority of CF B. cepacia isolates belong to genomovar III, but the relationship between genomovar and virulence has not yet been defined. Because the initial stage of infection involves bacterial binding to host tissues, the present study investigated differences in the binding of representative isolates of all five genomovars to fixed nasal sections of UNC cftr (−/−) and (+/+) mice and to human lung explants, biopsy and autopsy tissue of CF and non-CF patients. Binding was highest for isolates of genomovar III, subgroup RAPD type 2, but only if the isolates expressed the cable pili phenotype. Antibodies to the 22-kDa adhesin of cable pili virtually abolished binding. Binding occurred only to cftr (−/−) nasal sections or to CF lung sections and was negligible in cftr (+/+) or human non-CF, histologically normal lung sections. Unlike normal epithelia, the hyperplastic epithelia of CF bronchioles were enriched in cytokeratin 13, a 55-kDa protein that has previously been shown to act as a receptor in vitro for cable-piliated B. cepacia. These findings may help to explain the high transmissibility of Cbl-positive, genomovar III strains of B. cepacia among CF patients.