- Volume 46, Issue 3, 1997

Nucleic acid amplification technology is examined from the critical viewpoint of a clinical microbiologist working in a routine diagnostic bacteriology laboratory. Widely recognised limitations of amplification technology include those of false-positive and false-negative results, the difficulty of obtaining quantitative results, the problem of using this technology for susceptibility testing, and the difficulty of detecting routinely the wide range of possible pathogens contained in a clinical sample. On the positive side, amplification technology brings welcome new possibilities for rapid detection of specific pathogens in a sample, including viruses, slowly growing bacteria, fastidious or uncultivable bacteria, fungi and protozoa. Other possible applications include screening normally sterile clinical samples for non-specific bacterial contamination and the use of amplification-based DNA fingerprinting methods for identification and typing of microorganisms. Nevertheless, it is predicted that–in contrast to research and reference facilities–routine bacteriology laboratories will continue to rely on culture as the preferred ‘amplification method’ for most diagnostic applications.

The present study examined the need for neutrophils and tumour necrosis factor-α (TNFα) for early defence against gut infection with the enteroinvasive, facultative intracellular bacterial pathogen, Listeria monocytogenes. Mice were treated with a neutrophil-depleting monoclonal antibody (MAb) or a MAb directed against TNFα, and the consequences of these treatments on the course of orally initiated infection with the pathogen were monitored. By day 3, orally initiated L. monocytogenes infection in mice treated with either MAb was severely exacerbated to the extent that up to 5000-fold more listeriae were recovered from the walls of the stomach, small intestine, caecum or large intestine of treated mice than from controls. Systemic infection resulting from the ingestion of L. monocytogenes was also severely enhanced in mice treated with these MAbs. Therefore, the results showed that neutrophils and TNFα have a critical role in the early defence against enteroinvasive L. monocytogenes infection initiated by a natural (in this case the oral) route, as well as in the control of subsequent systemic infection.

Biotyping, antibiograms, bacteriophage typing, plasmid profile analysis and SDS-PAGE protein profiles were used to determine the relatedness of 44 Staphylococcus epidermidis and four S. haemolyticus isolates from 14 patients. A selection of these were further characterised by ribotyping. Biotyping classified the isolates into three major groups but was considered a poor strain marker. Although antibiograms classified the S. epidermidis isolates into 20 groups, some changes in the susceptibility patterns of related isolates from a single patient were demonstrated. Bacteriophage typing was the least discriminatory of the methods used. SDS-PAGE gave highly related patterns for the majority of S. epidermidis isolates. Plasmid profile analysis and ribotyping, with a minimum of two restriction endonucleases, were the most discriminatory methods for typing S. epidermidis. Nonetheless, some isolates from the same patient – probably representing a single strain – varied in plasmid profile indicating plasmid instability. One of six related isolates from a single patient lacked two bands from the ribotyping pattern of the other isolates. Although no single method proved entirely satisfactory on all occasions, the combination of typing methods was sufficient to provide evidence of the relatedness of S. epidermidis isolates from individual patients.

Lymph node biopsy specimens, obtained from 297 paediatric and adult patients with tuberculous lymphadenitis at Madurai, were transported in selective Kirchner’s liquid medium (KL-T) to the Tuberculosis Research Centre, Madras and processed for culture. Mycobacterium tuberculosis was isolated from 201 (68%) specimens. Of the 192 specimens within 4 days of resection, 134 (69.8%) yielded M. tuberculosis on culture and of the 105 specimens after 5 days, 67 (63.8%) were culture positive; the difference was not statistically significant. By incubating KL-T alone further, after removing the gland for processing, it was found that mere contact with the excised node during transportation was enough to retrieve 77 (38.3%) of the total of 201 positive isolates obtained, the delay did not affect the culture positivity rate. Thus, lymph node specimens for culture of tubercle bacilli can be stored in the refrigerator for up to 15 days and transported in KL-T at ambient temperature for 18–20 h without any loss in culture positivity.

The susceptibility of male Swiss white mice (MF1) to Staphylococcus aureus-induced arthritis was investigated with wild-type strain allelic replacement mutants. Comparison was made with a known mouse arthritogenic strain. The development and severity of arthritis were dependent both on the numbers of live bacteria injected intravenously and also on the mutant used; the ID50 ranged from (5 X 106) – (1 X 108) cfu. The results indicate that expression of the genes associated with virulence, including those for protein A and α-haemolysin, play a major role in the pathogenesis of staphylococcal septic arthritis. When either virulence component was carried by the S. aureus variant, a greater degree of inflammation, pannus formation and cartilage destruction was detected histologically. Loss of one or more virulence factors lowered the septic arthritis severity score based on clinical and histological parameters.

Twenty-four cerebrospinal fluid (CSF) samples from 19 AIDS patients with neurological signs were analysed by the polymerase chain reaction (PCR) for the presence of JC virus (JCV). Eleven of the 19 patients tested presented with progressive multifocal leucoencephalopathy (PML). Two specific JCV target sequences were used for the PCR analysis: a sequence specific for the T antigen genes from both BK virus (BKV) and JCV (PCR1) and a sequence specific for the large T antigen gene from JCV (PCR2). The JCV genome was detected in 10 of 11 patients with PML by the PCR1 method and in all 11 patients by the PCR2 method. With samples from the eight patients without PML, one positive result was obtained with the PCR1 method and this sample and another gave positive results with PCR2. Multiple CSF samples were collected from three patients with PML at different times, including after intrathecal cytarabine treatment, and were tested by the PCR2 method for the presence of the JCV genome. The PCR result became negative for two of the three patients during the cytarabine treatment. However, the absence of a PCR signal was not associated with clinical improvement in these patients. The PCR method is useful for the detection of JCV in CSF samples and in the diagnosis of PML. However, the application of PCR for monitoring the effect of treatment remains to be established.