Case report: persistently seronegative neuroborreliosis in an immunocompromised patient.

BACKGROUND: Infection with Borrelia burgdorferi sensu lato complex (B. b. sl) spirochetes can cause Lyme borreliosis, manifesting as localized infection (e.g. erythema migrans) or disseminated disease (e.g. Lyme neuroborreliosis). Generally, patients with disseminated Lyme borreliosis will produce an antibody response several weeks post-infection. So far, no case of neuroborreliosis has been described with persistently negative serology one month after infection. CASE PRESENTATION: We present a patient with a history of Mantle cell lymphoma and treatment with R-CHOP (rituximab, doxorubicine, vincristine, cyclofosfamide, prednisone), with a meningo-encephalitis, who was treated for a suspected lymphoma relapse. However, no malignant cells or other signs of malignancy were found, and microbial tests did not reveal any clues, including Borrelia serology. He did not recall being bitten by ticks, and a Borrelia PCR on CSF was negative. After spontaneous improvement of symptoms, he was discharged without definite diagnosis. Several weeks later, he was readmitted with a relapse of symptoms of meningo-encephalitis. This time however, a Borrelia PCR on CSF was positive, confirmed by two independent laboratories, and the patient received ceftriaxone upon which he partially recovered. Interestingly, during the diagnostic process of this exceptionally difficult case, a variety of different serological assays for Borrelia antibodies remained negative. Only P41 (flagellin) IgG was detected by blot and the Liaison IgG became equivocal 2 months after initial testing. CONCLUSIONS: To the best of our knowledge this is the first case of neuroborreliosis that is seronegative on repeated sera and multiple test modalities. This unique case demonstrates the difficulty to diagnose neuroborreliosis in severely immunocompromised patients. In this case, a delay in diagnosis was caused by broad differential diagnosis, an absent known history of tick bites, negative serology and the low sensitivity of PCR on CSF. Therefore, awareness of the diagnostic limitations to detect Borrelia infection in this specific patient category is warranted.

The dynamics of the pulmonary microbiome during mechanical ventilation in the intensive care unit and the association with occurrence of pneumonia.

RATIONALE: Ventilator-associated pneumonia (VAP) is the most common nosocomial infections in patients admitted to the ICU. The adapted island model predicts several changes in the respiratory microbiome during intubation and mechanical ventilation. OBJECTIVES: We hypothesised that mechanical ventilation and antibiotic administration decrease the diversity of the respiratory microbiome and that these changes are more profound in patients who develop VAP. METHODS: Intubated and mechanically ventilated ICU-patients were included. Tracheal aspirates were obtained three times a week. 16S rRNA gene sequencing with the Roche 454 platform was used to measure the composition of the respiratory microbiome. Associations were tested with linear mixed model analysis and principal coordinate analysis. MEASUREMENTS AND MAIN RESULTS: 111 tracheal aspirates were obtained from 35 patients; 11 had VAP, 18 did not have VAP. Six additional patients developed pneumonia within the first 48 hours after intubation. Duration of mechanical ventilation was associated with a decrease in α diversity (Shannon index; fixed-effect regression coefficient (ß): -0.03 (95% CI -0.05 to -0.005)), but the administration of antibiotic therapy was not (fixed-effect ß: 0.06; 95% CI -0.17 to 0.30). There was a significant difference in change of ß diversity between patients who developed VAP and control patients for Bray-Curtis distances (p=0.03) and for Manhattan distances (p=0.04). Burkholderia, Bacillales and, to a lesser extent, Pseudomonadales positively correlated with the change in ß diversity. CONCLUSION: Mechanical ventilation, but not antibiotic administration, was associated with changes in the respiratory microbiome. Dysbiosis of microbial communities in the respiratory tract was most profound in patients who developed VAP.

Changes in microbiota during experimental human Rhinovirus infection.

BACKGROUND: Human Rhinovirus (HRV) is responsible for the majority of common colds and is frequently accompanied by secondary bacterial infections through poorly understood mechanisms. We investigated the effects of experimental human HRV serotype 16 infection on the upper respiratory tract microbiota. METHODS: Six healthy volunteers were infected with HRV16. We performed 16S ribosomal RNA-targeted pyrosequencing on throat swabs taken prior, during and after infection. We compared overall community diversity, phylogenetic structure of the ecosystem and relative abundances of the different bacteria between time points. RESULTS: During acute infection strong trends towards increases in the relative abundances of Haemophilus parainfluenzae and Neisseria subflava were observed, as well as a weaker trend towards increases of Staphylococcus aureus. No major differences were observed between day-1 and day 60, whereas differences between subjects were very high. CONCLUSIONS: HRV16 infection is associated with the increase of three genera known to be associated with secondary infections following HRV infections. The observed changes of upper respiratory tract microbiota could help explain why HRV infection predisposes to bacterial otitis media, sinusitis and pneumonia.

Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection.

Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method that can be applied in a rapid vaccination schedule. We vaccinated C3H/HeN mice with B. afzelii strain PKo OspC (outer-surface protein C) using a codon-optimized DNA vaccine tattoo and compared this with recombinant protein vaccination in a 0-2-4 week vaccination schedule. We also assessed protection by DNA tattoo in a 0-3-6 day schedule. DNA tattoo and recombinant OspC vaccination induced comparable total IgG responses, with a lower IgG1/IgG2a ratio after DNA tattoo. Two weeks after syringe-challenge with 5 × 10(5) B. afzelii spirochetes most vaccinated mice had negative B. afzelii tissue DNA loads and all were culture negative. Furthermore, DNA tattoo vaccination in a 0-3-6 day regimen also resulted in negative Borrelia loads and cultures after challenge. To conclude, DNA vaccination by tattoo was fully protective against B. afzelii challenge in mice in a rapid vaccination protocol, and induces a favorable humoral immunity compared to recombinant protein vaccination. Rapid DNA tattoo is a promising vaccination strategy against spirochetes.

Using online periodontal case-based discussions to synchronize theoretical and clinical undergraduate dental education.

BACKGROUND: Clinical experience is important in undergraduate dental education, but (suitable) patients to learn from are often lacking. Online case-based discussions were introduced to overcome patient dependency and to synchronize theoretical with clinical education. MATERIALS AND METHODS: Undergraduate dental students in groups of 5-7 discussed online clinical case reports presenting either minor (2nd year) or complex periodontal pathology (3rd year). Each case consisted of a brief patient history, extra- and intra-oral clinical pictures, periodontal chart, peri-apical and/or orthopantomographic radiographs. Students had to discuss diagnosis and treatment planning. Questionnaires assessed students' and supervisors' general appreciation (score on 20), time investment and opinions about organisation, relation case/course content, future planning, learning effect and online environment (5-point Likert scale). A crossover design with three tests (pre-test, test in between and post-test) was used to investigate whether the frequency of case introduction (one case per week vs. one case element per week) had an effect on learning. Data was analysed with descriptive statistics (questionnaires) and repeated measures ANOVA (crossover design). RESULTS: Students (n=119) and supervisors (n=9) highly appreciated the exercise. Students reported spending on average 74 min per week to read a case, prepare and post messages. Supervisors' total time investment was 342 min per semester to create a case, provide online feedback and to prepare a live-discussion. No significant differences in test-scores were found between the two modalities of case introduction. CONCLUSION: Online case-based discussions, in conjunction with a theoretical course, are valuable additions to the dental curriculum, especially to reinforce the transition from theory to clinical practice.

Use of the modified Robbins device to study the in vitro biofilm removal efficacy of NitrAdine, a novel disinfecting formula for the maintenance of oral medical devices.

AIMS: To evaluate the use of the modified Robbins device (MRD) to test disinfection strategies against biofilms that form on oral medical devices and to test the biofilm removal efficacy of NitrAdine, a disinfectant for the maintenance of oral medical devices. METHODS AND RESULTS: Biofilms were grown on discs using the MRD and biofilms formed in this system were used to evaluate the efficacy of NitrAdine and to determine the optimal disinfection conditions. Our data indicate that the use of the MRD allows for the rapid and reproducible formation of high-density biofilms. Determination of the efficacy of NitrAdine revealed high activity against biofilms tested (e.g. >3 log reduction for Candida albicans and Staphylococcus aureus) and allowed the determination of the optimal conditions for its use. CONCLUSION: The high reproducibility and flexibility of the MRD make it an excellent candidate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices. Using this system, we were able to demonstrate that NitrAdine exhibits high activity against biofilms formed by the micro-organisms tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that our procedure is appropriate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices.

Successful prevalidation of the slug mucosal irritation test to assess the eye irritation potency of chemicals.

A previous 'in house' validation study showed that the SMI assay can be used as an alternative to the in vivo Draize eye irritation test. The aim of this multi-centre study with four participating laboratories was to assess the transferability and inter-laboratory variability of the assay using 20 reference chemicals covering the whole irritancy range. The eye irritation potency of the chemicals was assessed by measuring the amount of mucus produced during a 60-min contact period with a 1% dilution, and a second 60-min treatment with a 3.5% dilution. After each contact period the protein release from the mucosal surface was measured. Linear discriminant equations were used to convert the results into the corresponding EU eye irritation categories (NI, R36 and R41). All the non-irritants were predicted correctly by the four laboratories resulting in a 100% specificity. For the R36 compounds a correct classification rate of 89% (VITO) and 100% (SPL, JNJ and UGent) was obtained. The R41 compounds were classified correctly in 78% of the cases for VITO, 89% for SPL and JNJ and 100% for UGent. We can conclude that the SMI assay is a relevant, easily transferable and reproducible alternative to predict the eye irritation potency of chemicals.

Development of an internally controlled real-time PCR assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system.

A real-time PCR assay with a DNA purification and inhibition control (internal control; IC) was developed to detect Chlamydophila psittaci DNA in human clinical samples. Novel C. psittaci-specific primers targeting the ompA gene were developed. The IC DNA contained the same primer-binding sites and had the same length and nucleotide content as the C. psittaci DNA amplicon, but had a shuffled probe-binding region. The lower limit of detection was 80 target copies/PCR, corresponding to 6,250 copies/mL in a clinical sample. Specificity was tested using reference strains of 30 bacterial species. No amplification was observed from any of these samples. Respiratory samples from eight patients were positive with this PCR. Six of these patients were confirmed as positive for C. psittaci with serological testing. Two patients had increasing antibody titres, but did not fulfil criteria proposed previously for serologically proven Chlamydia spp. infection. The real-time PCR described in this paper is a sensitive, specific and rapid method to detect C. psittaci DNA in human clinical respiratory samples.

Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals.

This multicentre study aimed at evaluating the reliability (reproducibility) and relevance (predictivity) of a new commercially available human corneal epithelial (HCE) model (SkinEthic Laboratories, Nice, France) to assess acute ocular irritation. A prevalidation approach (protocol optimisation, transfer and performance) was followed and at each of the four participating laboratories, 20 coded reference chemicals, covering the whole range of irritancy, were tested. The compounds were applied topically to the HCE cultures and the level of cytotoxicity (tissue viability and histological analysis) was determined. Once a standardised protocol was established, a high level of reproducibility between the laboratories was observed. In order to assess the capability of the HCE model to discriminate between irritants (I) and non-irritants (NI), a classification prediction model (PM) was defined based on a viability cut-off value of 60%. The obtained in vitro classifications were compared with different in vivo classifications (e.g. Globally Harmonised System) which were calculated from individual rabbit data described in the ECETOC data bank. Although an overall concordance of 80% was obtained (sensitivity = 100% and specificity = 56%), the predictivity of the HCE model substantially increased when other sources of in vivo and in vitro data were taken into account.

Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals.

A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals.

Ticking the right boxes: classification of patients suspected of Lyme borreliosis at an academic referral center in the Netherlands.

To provide better care for patients suspected of having Lyme borreliosis (LB) we founded the Amsterdam Multidisciplinary Lyme borreliosis Center (AMLC). The AMLC reflects a collaborative effort of the departments of internal medicine/infectious diseases, rheumatology, neurology, dermatology, medical microbiology and psychiatry. In a retrospective case series, characteristics of 200 adult patients referred to the AMLC were recorded, and patients were classified as having LB, post-treatment LB syndrome (PTLBS), persistent Borrelia burgdorferi sensu lato (s.l.) infection despite antibiotic treatment or no LB. In addition, LB, PTLBS and persistent B. burgdorferi s.l. infection cases were classified as 'definite,' 'probable' or 'questionable.' Of the 200 patients, 120 (60%) did not have LB and 31 (16%) had a form of localized or disseminated LB, of which 12 were classified as definite, six as probable and 13 as questionable. In addition, 34 patients (17%) were diagnosed with PTLBS, of which 22 (11%) were probable and 12 (6%) questionable. A total of 15 patients (8%) were diagnosed with persistent B. burgdorferi s.l. infection, of which none was classified as definite, three as probable and 12 as questionable. In conclusion, in line with previous studies, the number of definite and probable (persisting) LB cases was low. The overall high number of questionable cases illustrates the fact that it can sometimes be challenging to either rule out or demonstrate an association with a B. burgdorferi s.l. infection, even in an academic setting. Finally, we were able to establish alternative diagnoses in a large proportion of patients.

Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification.

Two primer sets were chosen for the detection of Haemophilus influenzae in cerebrospinal fluid by polymerase chain reaction (PCR) DNA amplification. One primer set was selected from sequences encoding a capsulation-associated protein and reacted with target DNA from all 15 capsulate H. influenzae strains (all serotypes) examined. The other primer set was selected from the DNA sequence of a gene encoding for outer-membrane protein P6 and reacted with the 15 capsulate and 10 non-capsulate strains of H. influenzae tested. This primer set also reacted with the closely related species H. haemolyticus and H. aegyptius, and with two of nine H. parainfluenzae strains. In reconstruction experiments, PCR DNA amplification was able to detect as few as five H. influenzae cells when 40 cycles of amplification were used. Two hundred cerebrospinal fluid (CSF) samples collected consecutively from patients suffering from meningitis were investigated by PCR; 40 were culture-positive for H. influenzae and 39 of these were also clearly positive in the PCR test with both primer sets. Contamination occurred to some extent with 40 cycles of amplification but was completely eliminated when the number of cycles was reduced to 35. We conclude that the two primer sets are appropriate for the detection of H. influenzae by PCR, each having its own specificity. When these two primer sets are used, PCR is a technique of equivalent sensitivity to culture for the detection of H. influenzae in CSF.

Analysis of interleukin-1alpha (IL-1alpha) and interleukin-8 (IL-8) expression and release in in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sensitization.

In the present study, reconstructed human epidermis (RHE) was used as an in vitro model to discriminate 1-chloro-2,4-dinitrobenzene (DNCB), nickel sulfate (NiSO(4)), oxazolone (OXA), 2,4-dinitrofluorobenzene (DNFB) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) as skin sensitizers from benzalkonium chloride (BC), benzoic acid (BA) and sodium lauryl sulfate (SLS) as skin irritants. Our criteria were (a) the differential IL-1alpha and IL-8 synthesis and release (b) cytotoxicity assessment by MTT assay. When the RHE are topically treated with the sensitizers, very low levels of extra- and intracellular IL-1alpha are observed although they induce significant cytotoxicity. In contrast, they exhibit a sharp maximum of IL-8 release. In the presence of the tested irritants, we observe the inverse cytokine release profile, although they induce dose-dependent cytotoxicity profiles similar to those observed with the sensitizers. Finally, IL-1alpha mRNA upregulation is observed after topical application of both sensitizers and irritants, but only the latter significantly increase extracellular IL-1alpha. In conclusion, our results suggest that the associated determination of IL-8, with IL-1alpha, and MTT conversion are at least necessary to discriminate and classify, in a single assay, irritant and sensitizing agents and represent a potential in vitro alternative to two classical in vivo assays.

The epidermal architecture of an in vitro reconstructed human skin equivalent (Advanced Tissue Sciences Skin2 Models ZK 1300/2000).

We investigated the epidermal ultrastructure of an organotypic skin culture model over a culture period of one week. At delivery, the stratified keratinizing epidermis consists of a basal layer with hemidesmosomes, a spinous layer with desmosomes, a granular layer with keratohyalin and membrane coating granules, a horny layer enclosing many lipid droplets. At the epidermal-dermal junction laminae lucida and densa are evident. After 7 days of culture the multilayered cornifying aspect persists; the number of viable cell layers remains constant. Spinous cells display abundant glycogen deposits. In the granular layer keratohyalin granules are more abundant. Both the size and number of hemidesmosomes increase. Anchoring fibrils are observed. From a structural point of view the model remains stable and well differentiated in culture and, therefore, may be well suited for pharmacological purposes.

Expression of type 1 5alpha-reductase and metabolism of testosterone in reconstructed human epidermis (SkinEthic(R)): a new model for screening skin-targeted androgen modulators.

The steroid 5alpha-reductase isoenzymes (5alphaR) transform testosterone into 17beta-hydroxy-5alpha-androstan-3-one (5-dihydrotestosterone, DHT), which exerts a much stronger biological activity than does testosterone. Briefly, the two 5alphaR isoenzymes are differentially expressed in the two major target organs of steroid action, the prostate (isoenzyme 2, 5alphaR2) and the skin (isoenzyme 1, 5alphaR1). We analysed the potential of a human epidermal tissue reconstituted by cell culture (RHE, provided by SkinEthic Laboratories, Nice, France) as a model for assessing 5alphaR activity. The epidermal model was found to express the type-1 (skin) isoform of 5alphaR and thus could be used as an enzyme source for the screening of 5alphaR modulators for dermatological/cosmetic purposes. A reproducible and convenient assay method was developed, allowing both the evaluation of testosterone transformation into DHT (5alphaR activity) and an outlook on the general metabolism process of testosterone. This could be important for the detection of any compound that could act mainly on another target enzyme than 5alphaR. The assay gave evidence of the inhibitory activity of finasteride against type-1 5alphaR, which is now established both in vitro and in clinical studies. In addition to enzyme inhibitors, this in situ cellular assay can detect transcriptional modulators of 5alphaR gene expression, or any compound that could modulate enzyme processing or post-translational activation. RT-PCR analysis of RNA samples from RHE failed to show any notable effect of finasteride, testosterone, or DHT treatment on the expression of 5alphaR1 at the transcriptional level.

Cosmetics Europe multi-laboratory pre-validation of the SkinEthic™ reconstituted human corneal epithelium test method for the prediction of eye irritation.

Cosmetics Europe, The Personal Care Association, known as Colipa before 2012, conducted a program of technology transfer and assessment of Within/Between Laboratory (WLV/BLV) reproducibility of the SkinEthic™ Reconstituted Human Corneal Epithelium (HCE) as one of two human reconstructed tissue eye irritation test methods. The SkinEthic™ HCE test method involves two exposure time treatment procedures - one for short time exposure (10 min - SE) and the other for long time exposure (60 min - LE) of tissues to test substance. This paper describes pre-validation studies of the SkinEthic™ HCE test method (SE and LE protocols) as well as the Eye Peptide Reactivity Assay (EPRA). In the SE WLV study, 30 substances were evaluated. A consistent outcome with respect to viability measurement across all runs was observed with all substances showing an SD of less than 18%. In the LE WLV study, 44 out of 45 substances were consistently classified. These data demonstrated a high level of reproducibility within laboratory for both the SE and LE treatment procedures. For the LE BLV, 19 out of 20 substances were consistently classified between the three laboratories, again demonstrating a high level of reproducibility between laboratories. The results for EPRA WLV and BLV studies demonstrated that all substances analysed were categorised similarly and that the method is reproducible. The SkinEthic™ HCE test method entered into the experimental phase of a formal ECVAM validation program in 2010.

Cosmetics Europe multi-laboratory pre-validation of the EpiOcular™ reconstituted human tissue test method for the prediction of eye irritation.

Cosmetics Europe, The Personal Care Association (known as Colipa before 2012), conducted a program of technology transfer and within/between laboratory reproducibility of MatTek Corporation's EpiOcular™ Eye Irritation Test (EIT) as one of the two human reconstructed tissue test methods. This EIT EpiOcular™ used a single exposure period for each chemical and a prediction model based on a cut-off in relative survival [ ≤60%=irritant (I) (GHS categories 2 and 1); >60%=no classification (NC)]. Test substance single exposure time was 30 min with a 2-h post-exposure incubation for liquids and 90 min with an 18-h post-exposure incubation for solids. Tissue viability was determined by tetrazolium dye (MTT) reduction. Combinations of 20 coded chemicals were tested in 7 laboratories. Standardized laboratory documentation was used by all laboratories. Twenty liquids (11 NC/9 I) plus 5 solids (3 NC/2 I) were selected so that both exposure regimens could be assessed. Concurrent positive (methyl acetate) and negative (water) controls were tested in each trial. In all, 298 independent trials were performed and demonstrated 99.7% agreement in prediction (NC/I) across the laboratories. Coefficients of variation for the% survival for tissues from each treatment group across laboratories were generally low. This protocol has entered in 2010 the experimental phase of a formal ECVAM validation program.

Serum and serotonin induce retraction of calf aortic smooth muscle (CASM) cells in vitro: inhibition by ketanserin, a 5-HT2 receptor antagonist.

Calf aortic smooth muscle (CASM) cells cultured in vitro at high cell density (4 x 10(4) cells/cm2) on bacteriological petri dishes in the presence of serum pile up in clusters and create open spaces in the monolayer. This phenomenon is clearly visible 6 days after plating and is markedly enhanced by the addition of fetal calf serum. Serotonin is essential for the serum-induced retraction since (1) dialyzed serum has no effect, (2) of all the vasoactive agents we tested, only serotonin induced a similar degree of retraction, and (3) the serum-induced retraction was completely blocked by preincubating the cells with serotonin 5-HT2 receptor blockers such as ketanserin and ritanserin but not by preincubation with adrenergic-alpha 1 blockers or histamine antagonists. Serotonin caused CASM cell retraction in a dose-dependent way, with a maximum effect at 10(-6) M. The serotonin-induced retraction was reversible in time and was effectively blocked by ketanserin (IC50 = 1.2 x 10(-9) M). It is therefore concluded that serotonin induces retraction of CASM cells, mediated by the serotonin 5-HT2 receptor.

Genetic typing in a cluster of Legionella pneumophila infections.

Legionella pneumophila strains isolated from six patients, three air-conditioning- and cooling tower-derived strains, and three hot water supply-derived strains were analyzed by three genetic typing methods. The results of the whole-cell DNA restriction endonuclease analysis and the restriction patterns based on genes coding for rRNA correlated with each other and demonstrated that the patient isolates were indistinguishable from the air-conditioning- and cooling tower-derived isolates but differed markedly from the hot water supply-derived isolates. The patient and air-conditioning- and cooling tower-derived strains contained plasmids of the same molecular weight; the hot water supply-derived strains were plasmidless. These results indicated that the cooling tower or the air-conditioning system was the environmental source for the examined cluster of Legionnaires disease strains.