A. H. J. Kolk

Clinical utility of the polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis.

This study examines the diagnostic utility of the polymerase chain reaction (PCR) in 156 patients (five human immunodeficiency virus (HIV) seropositive) suspected of extrapulmonary tuberculosis. The results of PCR in 226 samples from 11 different sites were compared with the results of microscopy and culture. Positive culture results were predicted in 86% of samples by PCR but in only 31% by microscopy. Specificity of PCR was 92%. In cases with culture-proven tuberculosis, PCR identified all 11 microscopy positive cases and 19 of 24 (79%) of the microscopy-negative cases. In four patients, PCR excluded the diagnosis of tuberculosis in microscopy-positive samples, which were later shown to contain mycobacteria other than Mycobacterium tuberculosis or laboratory contaminants. In 20 patients (microscopy, PCR and culture negative) a trial of antituberculous drugs was given, but patients showed no improvement and treatment was stopped. In 17 patients, all culture negative (in nine PCR was positive, three of whom also had positive microscopy) the diagnosis was probable tuberculosis based on clinical findings and response to treatment. This polymerase chain reaction has a much higher sensitivity than microscopy and can facilitate therapeutic decisions for those with suspected extrapulmonary tuberculosis.

A fast method for the identification of Mycobacterium tuberculosis in sputum and cultures based on thermally assisted hydrolysis and methylation followed by gas chromatography–mass spectrometry

A fast gas chromatography-mass spectrometry (GC-MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM). The THM-GC-MS procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potential markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM-GC-MS method. No false positives were found by THM-GC-MS and all patients who were diagnosed with TB were also found positive using our newly developed THM-GC-MS method. These results show that the new fast and sensitive THM-GC-MS method holds great potential for the diagnosis of TB.

Proton Transfer Reaction Mass Spectrometry detects rapid changes in volatile metabolite emission by Mycobacterium smegmatis after the addition of specific antimicrobial agents

The metabolic activity of plants, animals or microbes can be monitored by gas headspace analysis. This can be achieved using Proton Transfer Reaction Mass Spectrometry (PTR-MS), a highly sensitive detection method for trace gas analysis. PTR-MS is rapid and can detect metabolic responses on-line as they occur. Here, we study the headspace of actively growing cultures of paired ciprofloxacin sensitive and resistant bacterial strains (Mycobacterium smegmatis in Middlebrook M7H9 liquid media) after the addition of the antibiotics ciprofloxacin and gentamicin in real time. Following the emission patterns of the mycobacteria over time allowed volatile markers specific for the bacterial response to each antibiotic to be detected. A proportion of the measured responses were very rapid, occurring within three hours after the addition of the compounds and varied between isolates with different resistance phenotypes. Specifically, we observed a two fold increase of m73 (unidentified C4 compound) within 10h after the addition of ciprofloxacin and a threefold increase of m45 (acetaldehyde) within 4h after the addition of gentamicin as compared to values before the addition. Monitoring the emission of specific volatiles into the culture headspace thus has the potential for rapid drug susceptibility testing. Moreover, these and other differences in the measured responses to the two tested compounds provide evidence that monitoring multiple compounds may also give an indication of the mechanism of action of the compound added.

The volatile metabolic fingerprint of ventilator-associated pneumonia

Dear Editor, The diagnostic approach for ventilator-associated pneumonia (VAP) needs to be improved [1]. Volatile organic compounds (VOCs), produced either by invading respiratory pathogens or the patient’s pulmonary defense system, could serve as early diagnostic markers for VAP [2]. Electronic nose (eNose) technology integratively captures complex VOC mixtures to create a ‘VOC fingerprint’ using an array of semi-selective sensors [3]. We hypothesized that an eNose would be able to discriminate patients with VAP from those without VAP based on analysis of headspace air from tracheal aspirates (TAs). In a prospective cohort study we collected TAs every third day from 45 intensive care unit (ICU) patients who were ventilated for more than 7 days. Fourteen patients developed VAP, 14 patients had airway colonization but did not develop VAP and 17 patients developed neither VAP nor airway colonization (study methodology and patient characteristics are given in the Electronic Supplementary Material). The eNose was able to accurately discriminate patients with VAP from those without VAP in both a cross-sectional and longitudinal analysis (Fig. 1, upper panels), and the use of a ‘VOC fingerprint’ was found to improve the diagnostic accuracy of the Clinical Pulmonary Infection Score (Fig. 1, lower panels) in this small cohort of patients. Notably, discrimination by the eNose was not affected by airway colonization, and the findings were independent of the number of colony forming units in the TAs. Our study has several limitations. First, we were not able to identify which VOCs differentiate between patients with VAP and those without VAP. Furthermore, this study was performed in a highly selected cohort of patients, and the sample size was rather small, thereby limiting generalization of our findings. Indeed, the results of our study need to be confirmed in robust and larger studies. Of interest, the results of our study suggest that the observed changes in VOC-fingerprints are not solely the result of the presence or absence of bacteria in TAs. VOC-fingerprints can change with the bacterial ecology from colonization to infection, which

A Broad Set of Different Llama Antibodies Specific for a 16 kDa Heat Shock Protein of Mycobacterium tuberculosis

Background Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. Methodology/Principal Findings Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4×10−10 M. Conclusions/Significance A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.

Alterations in exhaled breath metabolite-mixtures in two rat models of lipopolysaccharide-induced lung injury

Exhaled breath contains information on systemic and pulmonary metabolism, which may provide a monitoring tool for the development of lung injury. We aimed to determine the effect of intravenous (iv) and intratracheal (IT) lipopolysaccharide (LPS) challenge on the exhaled mixture of volatile metabolites and to assess the similarities between these two models. Male adult Sprague-Dawley rats were anesthetized, tracheotomized, and ventilated for 6 h. Lung injury was induced by iv or IT administration of LPS. Exhaled breath was monitored continuously using an electronic nose (eNose), and hourly using gas chromatography and mass spectrometry (GC-MS). GC-MS analysis identified 34 and 14 potential biological markers for lung injury in the iv and IT LPS models, respectively. These volatile biomarkers could be used to discriminate between LPS-challenged rats and control animals within 1 h after LPS administration. Electronic nose analysis resulted in a good separation 3 h after the LPS challenge. Hexanal, pentadecane and 6,10-dimethyl-5,9-undecadien-2-one concentrations decreased after both iv and IT LPS administration. Nonanoic acid was found in a higher concentration in exhaled breath after LPS inoculation into the trachea but in a lower concentration after iv infusion. LPS-induced lung injury rapidly changes exhaled breath metabolite mixtures in two animal models of lung injury. Changes partly overlap between an iv and an IT LPS challenge. This warrants testing the diagnostic accuracy of exhaled breath analysis for acute respiratory distress syndrome in clinical trials, possibly focusing on biological markers described in this study.

SOME PROBLEMS CONCERNING THE ASSAY OF ERYTHROPOIETIN USING THE HAEMAGGLUTINATION INHIBITION KIT

With the introduction of a coiiimercially available Hemagglutination-Inhibition (HAI) assay kit for Erythroid Stimulating Factors (ESF) (J.C.L. Clinical Research Co., Knoxville, Tennessee), we have attempted to compare the results obtained by this method with those obtained using established bioassays-the polycythaemic mouse assay in Amsterdam, and the in vitro mouse foetal liver cell assay in Cardiff (Dunn et ul, 1975). Due to the technical problems which arose in connection with the kit this was not possible. The methods used, including the inactivation and absorption of the test sera, were as described in the manual provided by the manufacturers. Cooke or Eppendorf pipettes were used for all dilutions. The difficulties, which were not specific to any one of six kits tested, can be summarized.

Potential biomarkers for identification of mycobacterial cultures by proton transfer reaction mass spectrometry analysis

RATIONALE Several mycobacterial species can produce serious infections in humans, and the treatment required depends on the infecting species. Fast identification, ideally with minimal manipulation of the infecting species, is therefore critical; here, we propose a method potentially allowing cultures to be identified by headspace analysis and use it to screen for differences between mycobacterial species based on the volatiles released during growth. METHODS Short-chain volatile organic compound emissions from two non-tuberculosis slow growing mycobacterial species, Mycobacterium avium and Mycobacterium kansasii, and a non-pathogenic fast growing species, Mycobacterium smegmatis, in Middlebrook M7H9 culturing media were followed online with a proton transfer reaction quadrupole mass spectrometer. RESULTS Measurable differences between the headspace of the two slow growing mycobacteria M. kansasii and M. avium were found, as well as differences with respect to the faster growing mycobacteria M. smegmatis. Three compounds, attributed to sulfur-containing volatiles--dimethyl sulfide, propanethiol and dimethyl disulfide--were found to be specific to M. avium. CONCLUSIONS Clear differences were detected in the low molecular weight volatile emissions compounds of the mycobacterial species under study, without the need for sample manipulation. Further studies with other mycobacterial species will reveal if the differences observed are specific to the species studied here. Furthermore, the use of an ion trap as a mass analyzer with the same ionization technique, allowing molecular detection over a wider molecular range, could allow the detection of additional biomarkers thus capturing a wider molecular range.

Validation of biomarkers for distinguishing Mycobacterium tuberculosis from non-tuberculous mycobacteria using gas chromatography-mass spectrometry and chemometrics.

Tuberculosis (TB) remains a major international health problem. Rapid differentiation of Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM) is critical for decisions regarding patient management and choice of therapeutic regimen. Recently we developed a 20-compound model to distinguish between MTB and NTM. It is based on thermally assisted hydrolysis and methylation gas chromatography-mass spectrometry and partial least square discriminant analysis. Here we report the validation of this model with two independent sample sets, one consisting of 39 MTB and 17 NTM isolates from the Netherlands, the other comprising 103 isolates (91 MTB and 12 NTM) from Stellenbosch, Cape Town, South Africa. All the MTB strains in the 56 Dutch samples were correctly identified and the model had a sensitivity of 100% and a specificity of 94%. For the South African samples the model had a sensitivity of 88% and specificity of 100%. Based on our model, we have developed a new decision-tree that allows the differentiation of MTB from NTM with 100% accuracy. Encouraged by these findings we will proceed with the development of a simple, rapid, affordable, high-throughput test to identify MTB directly in sputum.

Direct detection of Mycobacterium tuberculosis in sputum using combined solid phase extraction-gas chromatography-mass spectrometry.

Recently, thermally-assisted hydrolysis and methylation followed by gas chromatography-mass spectrometry (THM-GC-MS) in combination with chemometrics has been used to develop a 20-compound model for fast differentiation of Mycobacterium tuberculosis (MTB) from Non-tuberculous mycobacteria (NTM) in bacterial cultures. This model provided better than 95% accuracy. In our current work a hexane/methanol/water extraction followed by a solid phase extraction (SPE) clean-up procedure was developed for use before THM-GC-MS, to make the test suitable for the identification of mycobacteria in sputum. The 20 biomarker model had to be adapted since many compounds were also found in the sputum of non-tuberculosis patients. An algorithm was established based on tuberculostearic acid, hexacosanoic acid and mycoserosates. The detection limit of the method was approximately 1×10(4) bacteria/mL sputum. Sputum specimens from 32 patients from South Africa who were suspected of having tuberculosis were blindly tested using the new method. Eight of the nine culture-positive sputum specimens were detected by the new SPE-THM-GC-MS method, resulting in a sensitivity of 89%. The specimen that was missed by the new method was also microscopy negative. The specificity of the test was 100%; all 23 microscopy- and culture-negative specimens were correctly identified as negative by SPE-THM-GC-MS.