During October 1999 to November 2000, 98 blood culture specimens from the same number of febrile episodes originating from 49 patients with haematological malignancies were examined for the presence of eubacteria and fungi based on 16S rRNA gene and the 5.8, 18 and 28S rRNA combined with in vitro PCR amplification and sequencing, in addition to conventional blood culture laboratory techniques. Nineteen of the samples were associated with positive blood cultures. Eubacterial (16S rRNA) PCR detected bacterial DNA in 26 febrile episodes, i.e. in an additional seven febrile episodes than blood-culture alone. The species identified by partial 16S Rrna gene sequencing were as follows Staphylococcus spp (n=6), Staphylococcus epidermidis (n=5), Acinetobacter spp (n=5), Escherichia coli (n=2), Enterobacter agglomerans (n=2), Campylobacter spp (n=1), Citrobacter spp (n=1), Corynebacterium spp (n=1), Enterobacter faecium (n=1), Ralstonia spp (n=1), Acidovorax spp. (n=1) and Stenotrophomonas maltophilia (n=1). Gram-positive bacteria were found in 12/27 (44.6%) and Gram-negative bacteria were found in 15/27 (55.6%). After optimization of a PCR-based fungal detection method, none of the febrile episodes were shown to be attributable to fungi. The results of this study suggest that fungi are not common causal agents of febrile episodes in patients with a haematological malignancy at this centre and that molecular techniques can augment cultural methods in the diagnosis of causal agents of bacteraemia in patients, so that appropriate antibiotic regimens may be commenced in patients with culture-negative episodes of infection.

Bacterial culture was allowed to dry to completeness on Columbia agar base with defibrinated horse blood. Following 6 months storage at room temperature, microbial DNA was extracted and successfully amplified by PCR. This storage technique has the advantage over other methods of not requiring (i) a DNA extraction protocol prior to storage and (ii) refrigeration and/or freezing. This technique maybe useful in the transportation of bacterial genomic DNA in nonviable cells as well as reliable method for the storage of DNA in underdeveloped countries.

The aim of this investigation was to identify the bacterial microflora in monetary coinage from 17 countries by the employment of PCR sequenced-based molecular identification of rDNA from bacterial cultures. Silver, bronze and other alloy coins (approx. 300g) from 17 currencies were enriched individually by culturing aerobically in tryptone soya broth for 72h at 30oC. Following this, 20μl broth was inoculated onto Columbia blood agar supplemented with 5% [v/v] defibrinated horse blood for 72h at 30oC and resulting colonies were purified by further subculture, as detailed above for a further 72h. All colonies were identified by initial PCR amplification of a partial region of the 16S rRNA gene locus, which was then sequenced and the sequence aligned using the BLASTn algorithm. Twenty five isolates were obtained from the coinage, and of these, 25/25 (100%) were Gram positive and the most prevalent genus observed was Bacillus [B. megaterium, B. lentus, B. litoralis, B. subtilis, B. circulans and other Bacillus spp.] accounting for 10/25 (40%) isolates and was isolated from 10/17 (58.8%) countries. This was followed by Staphylococcus spp. [Staph. aureus, Staph. epidermidis, Staph. hominis, Staph. schleiferi], which accounted for 7/25 (28%) of isolates and was isolated from 7/17 (41.2%) countries. Given the organisms identified in this study, it is not believed that monetary coinage presents any additional risk to public health, however we support the principles of basic hygiene in terms of proper hand washing, avoidance of handling money when working with food, wounds and skin lesions. In conclusion, this study demonstrated that money from 17 countries was contaminated by environmental Gram positive flora, in particular Bacillus spp. and that universal 16S rDNA-PCR approach coupled with automated direct sequencing provides a rapid means of identifying the contaminant organisms present.

To employ partial 16S rDNA PCR and automated sequencing technique to identify non-culturable causal agents of bacterial meningitis, 73 peripheral blood samples and 413 culture-negative and eight culture-positive CSF clinical specimens from patients with suspected acute meningitis were examined for the presence of bacterial genomic DNA employing broad range 16S rDNA PCR followed by sequencing of the amplicons. In blood samples, 63/73 specimens were PCR positive (86.3%) and after direct sequencing of the PCR amplicons, only 12.7% (8/63) gave clear sequencing results and 55/63 (87.3%) were mixed with more than one organism detected. The mixed PCR amplicons were separated by using PAGE and mixed amplicons from 29/55 (52.7%) specimens were successfully identified through sequencing. Of the CSF samples, 8/8 culture-positive samples were also PCR positive and 45/413 (10.9%) of culture-negative gave a strong PCR signal and 88/413 (21.3%) specimens yielded a weak PCR signal. The remaining 280 culture-negative specimens were also PCR negative. Nested PCR was set up for the 88 weak positive samples and yielded 72/88 (81.8%) strong positives, with the remainder failing to amplify 133/413 (32.2%) culture-negative samples were PCR positive. In this study, the most common bacteria identified from blood specimens were Neisseria meningitidis, 13/63 (20.6%); Streptococcus spp, 5/63 (7.9%); Acinetobacter spp and Pseudomonas spp 4/63 (6.3%). From culture-negative CSF, the pattern was different in that Staphylococcus spp (13/58, 22.4%), Neisseria meningitidis (9/58, 15.52%) and Pseudomonas spp (8/58, 14.79%), were the most frequent. Overall, 16S rRNA broad-range PCR combined with direct DNA sequencing is a valuable molecular tool to aid with the detection as well as identification of non-culturable aetiological agents of acute bacterial meningitis and can augment cultural methods in the diagnosis of central nervous system infections in patients who have been treated with antibiotics. However, this study demonstrates that contamination is an important complication of the molecular assay, which should be attempted to be eliminated through careful laboratory controls. Hence there should be careful interpretation of any molecular finding, in tandem with other laboratory findings, such as culture, immunological and biochemical markers, and the clinical scenario of the patient.

Optimum detection of the Burkholderia cepacia complex (BCC) from sputum of patients with cystic fibrosis (CF) is essential in preventing patient-to-patient transmission of this organism. The aim of this study was to develop an improved PCR assay with reference to sensitivity for the direct detection of BCC organisms from CF sputum employing the recA locus. The sensitivity results of three recA PCR assays were compared using various combinations of previously published primers. These included (i) a single-round approach using the primer set BCR1/BCR2, yielding a 1036-bp product, (ii) a single-round approach using the primer set BCR1/Mr, yielding a 465-bp product, and (iii) a semi-nested PCR (SN-PCR) approach using the primer set BCR1/BCR2 followed by BCR1/Mr. The sensitivity of these assays were determined by spiking B. cepacia-free sputum with known numbers of four strains of BCC, namely, genomovar II [B. multivorans] (C1576), genomovar IIIa (C5424, C6433) and genomovar IIIb (C1394). Following optimization, the chosen assay was performed on 14 patients. Employment of the singleround assay with BCR1/BCR2 was the least sensitive with a detection threshold of 107 cfu/g sputum for GIIIa and GIIIb, and 108 cfu/g sputum for GII. Sensitivity was improved by targeting the smaller amplification region of the recA locus (465 bp) employing the BCR1/Mr primer pair, in combination with a single-round approach, whereby the detection threshold was improved by 1 log for each genomovar. Employment of the semi-nested assay demonstrated optimum sensitivity, whereby the detection threshold increased to 101 and 102 cfu/g sputum for genomovar IIIa/IIIb and genomovar II, respectively. Subsequent genomovar characterisation can be performed by sequencing of the PCR amplicon without the need for culture which may be beneficial in patients in the initial stages of colonisation or who are transiently colonised and who may be culture-negative for BCC.