In vitro volatile organic compound profiling using GC×GC-TOFMS to differentiate bacteria associated with lung infections: a proof-of-concept study.

Chronic pulmonary infections are the principal cause of morbidity and mortality in individuals with cystic fibrosis (CF). Due to the polymicrobial nature of these infections, the identification of the particular bacterial species responsible is an essential step in diagnosis and treatment. Current diagnostic procedures are time-consuming, and can also be expensive, invasive and unpleasant in the absence of spontaneously expectorated sputum. The development of a rapid, non-invasive methodology capable of diagnosing and monitoring early bacterial infection is desired. Future visions of real-time, in situ diagnosis via exhaled breath testing rely on the differentiation of bacteria based on their volatile metabolites. The objective of this proof-of-concept study was to investigate whether a range of CF-associated bacterial species (i.e. Pseudomonas aeruginosa, Burkholderia cenocepacia, Haemophilus influenzae, Stenotrophomonas maltophilia, Streptococcus pneumoniae and Streptococcus milleri) could be differentiated based on their in vitro volatile metabolomic profiles. Headspace samples were collected using solid phase microextraction (SPME), analyzed using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and evaluated using principal component analysis (PCA) in order to assess the multivariate structure of the data. Although it was not possible to effectively differentiate all six bacteria using this method, the results revealed that the presence of a particular pattern of VOCs (rather than a single VOC biomarker) is necessary for bacterial species identification. The particular pattern of VOCs was found to be dependent upon the bacterial growth phase (e.g. logarithmic versus stationary) and sample storage conditions (e.g. short-term versus long-term storage at -18 °C). Future studies of CF-associated bacteria and exhaled breath condensate will benefit from the approaches presented in this study and further facilitate the production of diagnostic tools for the early detection of bacterial lung infections.

Development of a multiplexed bead-based immunoassay for the simultaneous detection of antibodies to 17 pneumococcal proteins.

Presently, several pneumococcal proteins are being evaluated as potential vaccine candidates. Here, we gather novel insights in the immunogenicity of PLY, PsaA, PspA, PspC, NanA, Hyl, PpmA, SlrA, Eno, IgA1-protease, PdBD, BVH-3, SP1003, SP1633, SP1651, SP0189 and SP0376. We developed a multiplex bead-based immunoassay (xMAP(®) Technology, Luminex Corporation) to simultaneously quantify antibodies against these 17 pneumococcal proteins in serum. The median fluorescence intensity (MFI) values obtained for human pooled serum with the multiplex assay were between 82% and 111% (median 94%) of those obtained with the singleplex assays. For IgG, the coefficient of variation (CV) in serum ranged from 2% to 9%, for IgA, the CV ranged from 3% to 14% and for IgM, the CV ranged from 11% to 15%. Using this immunoassay, we showed that anti-pneumococcal antibody levels exhibited extensive inter-individual variability in young children suffering from invasive pneumococcal disease. All proteins, including the proteins with, as yet, unknown function, were immunogenic. In conclusion, the multiplex Streptococcus pneumoniae immunoassay based on proteins is reproducible. This assay can be used to monitor anti-S. pneumoniae antibody responses in a material- and time-saving manner.

Emergence of multidrug-resistant Salmonella enterica serotype Typhi with decreased ciprofloxacin susceptibility in Bangladesh.

During 1989-2002, we studied the antimicrobial resistance of 3928 blood culture isolates of Salmonella enterica serotype Typhi (S. Typhi) in Dhaka, Bangladesh. Overall 32% (1270) of the strains were multidrug-resistant (MDR, resistant to chloramphenicol, ampicillin and trimethoprim-sulphamethoxazole); first detected in 1990 (rate of 8%), increased in 1994 (44%), declined in 1996 (22%, P<0.01 compared to 1994) and re-emerged in 2001 (36%) and 2002 (42%, P<0.01 compared to 1996). An increased MIC of ciprofloxacin (0.25 microg/ml) indicating decreased susceptibility to ciprofloxacin was detected in 24 (18.2%) out of 132 randomly selected strains during 1990-2002; more frequently in MDR than susceptible strains (46.3% vs. 5.5%, P<0.001), and the proportion of them rose to 47% in 2002 from 8% in 2000 (P<0.01). Ciprofloxacin (5 microg) disk diffusion zone diameters of < or =24 mm as break-point had 98% sensitivity and 100% specificity when compared with a ciprofloxacin MIC of 0.25 microg/ml as break-point for decreased susceptibility; being a useful and easy screen test. All strains were susceptible to ceftriaxone. The emergence of MDR S. Typhi with decreased ciprofloxacin susceptibility will further complicate the therapy of typhoid fever because of the lack of optimum treatment guidelines.

Decline in epidemic of multidrug resistant Salmonella typhi is not associated with increased incidence of antibiotic-susceptible strain in Bangladesh.

Since 1987, multidrug resistant (MDR) strains of Salmonella Typhi, resistant simultaneously to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole, have caused epidemics of severe typhoid fever in Asia and Africa. A retrospective analysis of blood culture results (1989-96) in a Diarrhoea Treatment Centre in Dhaka, Bangladesh detected MDR strains in 0.3% (8 of 2793) of samples in 1990. The isolation rate peaked to 3.2% (240 of 7501) in 1994 (P < 0.01) and decreased to 1.8% (165 of 9348) in 1995 and further to 1.0% (82 of 8587) in 1996 (P < 0.01 compared to 1994) indicating the emergence and decline of MDR typhoid epidemic. Ten of 15 MDR strains tested had a 176 kb conjugative R plasmid that mediates resistance to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole to Escherichia coli K12. Unlike MDR strains, the isolation rate (approximately 3.3%) of susceptible S. Typhi remained remarkably unchanged during the study. The significant decrease in isolation of MDR strains suggests that cheaper and effective first-line antibiotics may re-emerge as drugs of choice for the treatment of typhoid fever in Bangladesh.

Rapid detection of Haemophilus influenzae type b in Bangladeshi children with pneumonia and meningitis by PCR and analysis of antimicrobial resistance.

A polymerase chain reaction (PCR) assay with primers from 'bexA' gene was compared with culture for the detection of Haemophilus influenzae type b (Hib) in clinical samples from children with pneumonia and meningitis. Of 200 sera (180 from pneumonia, 20 from non-pneumonia patients) tested by PCR (serum-PCR), Hib was detected in 15 of 16 blood culture-positive and in 6 blood culture-negative pneumonia cases. When compared with the results of blood culture, serum-PCR had sensitivity, specificity, and accuracy index of 93.7%, 96.7%, and 96.5% respectively. Of 120 cerebrospinal fluid (CSF) samples from meningitis patients tested by culture and PCR (CSF-PCR), the latter method could detect Hib in all 15 culture-positive and in 8 of 105 culture-negative cases, showing sensitivity, specificity, and accuracy index of 100%, 92.4%, and 94.4% respectively. The PCR result was available within a day. Antimicrobial susceptibility of Hib was determined by the disc-diffusion method. High rate of resistance to ampicillin (54.8%), chloramphenicol (48.4%), and co-trimoxazole (80.6%) was observed among 31 invasive Hib isolates with resistance to all 3 drugs (multiresistance) in 48.4% of the isolates. All the Hib isolates were susceptible to ceftriaxone. The study has shown that PCR is a rapid, sensitive and specific diagnostic test for Hib from clinical samples, and a combination of culture and PCR is necessary for the detection of Hib infections to the maximum extent for case management to reduce morbidity, mortality, and complications of the invasive Hib infections. A high prevalence of multiresistant Hib strains is a matter of concern.