Evaluation of different standard and modified two-tier testing strategies for the laboratory diagnosis of lyme borreliosis in a European setting.

BACKGROUND: Diagnosis of Lyme borreliosis (LB) relies on clinical symptoms and detection of Borrelia-specific antibodies. Guidelines recommend a two-tier testing (TTT) strategy for disseminated LB: serological screening with a sensitive enzyme immunoassay (EIA) and confirmation with a specific immunoblot. Searching for the most sensitive and specific approach, this retrospective study evaluated standard (STTT) and modified (MTTT) strategies using a well-defined study population. METHODS: Cases included patients with active Lyme neuroborreliosis (LNB; n = 29) or Lyme arthritis (LA; n = 17). Controls comprised patients treated for LNB (n = 36) or LA (n = 8), healthy individuals who were either untreated (n = 75) or treated for LB (n = 15) in the past, and patients with potentially cross-reactive diseases (n = 16). Sera were subjected to three EIAs and two immunoblots. Reactive screening results were confirmed by immunoblot (STTT) or EIA (MTTT). Solitary IgM results in the screening assay and effects of antibiotic treatment on isotype-specific seropositivity rates were also assessed. RESULTS: Sensitivities of STTT strategies ranged from 90%-97% for LNB and were 100% for LA. MTTT strategies were 100% sensitive. Specificities ranged from 89%-95% for STTT and from 88%-93% for MTTT strategies. Differences between STTT and MTTT strategies were not statistically significant. Solitary IgM reactivity was common among controls. Antibiotic treatment significantly reduced IgM/IgG positivity for LNB patients; for LA patients, a decline was only observed for IgM. CONCLUSION: In conclusion, MTTT strategies showed a slightly higher sensitivity and similar specificity compared to STTT strategies. Since EIAs are more time- and cost-efficient, MTTT strategies seem more favorable for clinical use. IgG testing enhances specificity with minimal sensitivity loss.

Combined throat/nasal swab sampling for SARS-CoV-2 is equivalent to nasopharyngeal sampling.

PURPOSE: PCR on a nasopharyngeal sample is the reference method for the detection of SARS-nCoV-2. However, combined throat/nasal sampling as a testing method has several advantages. We compared the combined throat/nasal sampling with nasopharyngeal sampling for detection of SARS-CoV-2 in healthcare workers suspected of COVID-19. METHODS: In 107 healthcare workers with symptoms of COVID-19, combined throat/nasal sampling and nasopharyngeal sampling was performed. Detection of SARS-CoV-2 was performed by RT-PCR targeting. RESULTS: A total of 80 healthcare workers (74.8%) tested negative with both sampling methods, and 25 healthcare workers (23.4%) tested positive with both sampling methods. There were two discrepant results with positive PCR in combined throat/nasal swabs and negative PCR in nasopharyngeal swabs (1.9%). The κ index for concordance between the 2 sampling methods was high (0.95). The median cycle threshold (Ct) value of PCR on nasopharyngeal samples was significantly lower than the Ct value of PCR on combined throat/nasal samples (19 (IQR 17-20) versus 21 (IQR 18-29) cycles, p value 0.01). CONCLUSION: Combined throat/nasal swabs yield a similar sensitivity to detect SARS-CoV-2 as nasopharyngeal swabs and are a good alternative sampling method, despite a lower Ct value for the nasopharyngeal samples.

Prospective comparison of two enzyme-linked immunosorbent spot assays for the diagnosis of Lyme neuroborreliosis.

Commercial cellular tests are used to diagnose Lyme borreliosis (LB), but studies on their clinical validation are lacking. This study evaluated the utility of an in-house and a commercial enzyme-linked immunosorbent spot (ELISpot) assay for the diagnosis of Lyme neuroborreliosis (LNB). Prospectively, peripheral blood mononuclear cells (PBMCs) were isolated from patients and controls and analysed using an in-house Borrelia ELISpot assay and the commercial LymeSpot assay. B. burgdorferi B31 whole cell lysate and a mixture of outer surface proteins were used to stimulate the PBMCs and the numbers of interferon-gamma-secreting T cells were measured. Results were evaluated using receiver operating characteristic (ROC) curve analysis. Eighteen active and 12 treated LNB patients, 10 healthy individuals treated for an early (mostly cutaneous) manifestation of LB in the past and 47 untreated healthy individuals were included. Both assays showed a poor diagnostic performance with sensitivities, specificities, positive and negative predictive values ranging from 44.4-66.7%, 42.0-72.5%, 21.8-33.3% and 80.5-87.0%, respectively. The LymeSpot assay performed equally poorly when the calculation method of the manufacturer was used. Both the in-house and the LymeSpot assay are unable to diagnose active LNB or to monitor antibiotic treatment success.

An Enzyme-Linked Immunosorbent Spot Assay Measuring Borrelia burgdorferi B31-Specific Interferon Gamma-Secreting T Cells Cannot Discriminate Active Lyme Neuroborreliosis from Past Lyme Borreliosis: a Prospective Study in the Netherlands.

Two-tier serology testing is most frequently used for the diagnosis of Lyme borreliosis (LB); however, a positive result is no proof of active disease. To establish a diagnosis of active LB, better diagnostics are needed. Tests investigating the cellular immune system are available, but studies evaluating the utility of these tests on well-defined patient populations are lacking. Therefore, we investigated the utility of an enzyme-linked immunosorbent spot (ELISpot) assay to diagnose active Lyme neuroborreliosis. Peripheral blood mononuclear cells (PBMCs) of various study groups were stimulated by using Borrelia burgdorferi strain B31 and various recombinant antigens, and subsequently, the number of Borrelia-specific interferon gamma (IFN-γ)-secreting T cells was measured. We included 33 active and 37 treated Lyme neuroborreliosis patients, 28 healthy individuals treated for an early manifestation of LB in the past, and 145 untreated healthy individuals. The median numbers of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs did not differ between active Lyme neuroborreliosis patients (6.0; interquartile range [IQR], 0.5 to 14.0), treated Lyme neuroborreliosis patients (4.5; IQR, 2.0 to 18.6), and treated healthy individuals (7.4; IQR, 2.3 to 14.9) (P = 1.000); however, the median number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs among untreated healthy individuals was lower (2.0; IQR, 0.5 to 3.9) (P ≤ 0.016). We conclude that the Borrelia ELISpot assay, measuring the number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs, correlates with exposure to the Borrelia bacterium but cannot be used for the diagnosis of active Lyme neuroborreliosis.

Disagreement between the results from three commercial tests for the detection of Borrelia-specific serum antibodies in the Netherlands associated with antibiotic treatment for Lyme borreliosis: a retrospective study.

The diagnosis of Lyme borreliosis is challenging because of the often non-specific symptoms and persisting antibodies after infection. We investigated the diagnostic characteristics of two enzyme-linked immunosorbent assays (ELISAs) and an immunoblot for the detection of Borrelia-specific serum antibodies using different test strategies in individuals with and without antibiotic treatment for Lyme borreliosis. This retrospective study included healthy individuals, patients with active Lyme neuroborreliosis and patients treated for Lyme neuroborreliosis. Two ELISAs were compared: the C6 ELISA and the SERION ELISA. Equivocal and positive results were confirmed by immunoblot. We included 174 healthy individuals, of whom 27 (15.5%) were treated for Lyme borreliosis in the past, 36 patients were treated for Lyme neuroborreliosis and 27 patients had active Lyme neuroborreliosis. All the active Lyme neuroborreliosis patients were reactive in both ELISAs (100% sensitivity); less reactivity was seen in the other three groups (range 17.7% to 69.4%). The concordance between the ELISA results was high in active Lyme neuroborreliosis patients (26/27; 96.3%) and healthy individuals (131/147; 89.1%), but lower in treated healthy individuals (18/27; 66.7%) and treated Lyme neuroborreliosis patients (18/36; 50.0%) (p ≤ 0.005). This study showed that antibiotic treatment against Lyme borreliosis was strongly associated with discordant ELISA and test strategy results (odds ratio: 10.52; p < 0.001 and 9.98; p = 0.014, respectively) suggesting antibiotic treatment influences the pace at which the various antibodies directed to the different antigens used in both ELISAs wane. Among treated neuroborreliosis patients, the SERION ELISA stayed positive for a longer period after infection compared to the C6 ELISA. This should be taken into consideration when requesting and/or interpreting Lyme serology.

Seropositivity to tick-borne pathogens in nature management workers in the Netherlands.

The incidence of tick-borne infections other than Lyme borreliosis and tick-borne encephalitis is rising in Europe, including the Netherlands. Nature management workers, being highly exposed to ticks, serve as valuable sentinels for seroprevalence studies on tick-borne pathogens (TBPs). This study assessed nature management workers' seropositivity to TBPs including Anaplasma phagocytophilum, Babesia divergens, B. microti, Borrelia burgdorferi s.l., Rickettsia conorii and R. typhi in the Netherlands. In addition, the study examined coexposure to multiple TBPs and identified risk factors for B. burgdorferi s.l.- and A. phagocytophilum-seropositivity. The study included 525 nature management workers who donated serum and completed a questionnaire. Sera were analysed for exposure to A. phagocytophilum, B. divergens, B. microti, R. conorii and R. typhi using immunofluorescence assays. For B. burgdorferi s.l. antibody detection, the recommended two-tier testing strategy was used. Risk factor analysis was performed using logistic regression modelling. Seropositivity was 30.9 % for B. burgdorferi s.l.; 16.4 % for A. phagocytophilum; 6.5 % for R. conorii; 2.3 % for R. typhi; 4.2 % for B. divergens; and 0.4 % for B. microti. Almost half (49.3 %) of the participants demonstrated seropositivity for one or more pathogens. Risk factors for B. burgdorferi s.l.-seropositivity included being male, increasing age and tick bite frequency. For A. phagocytophilum-seropositivity, increasing age and working in North Holland province were significant risk factors. This study illustrates the exposure to TBPs in the Netherlands, emphasizing the need for ongoing vigilance and international collaborations to better understand and address the growing threat of TBPs in regions with demonstrated environmental TBP circulation.

Integrated surveillance of human respiratory viruses in addition to SARS-CoV-2 in a public testing facility in the Netherlands.

BACKGROUND: SARS-CoV-2 prevention measures impact the circulation of other respiratory viruses. Surveillance in the network of general practitioners is hampered by widespread testing for SARS-CoV-2 in public testing facilities. OBJECTIVES: To evaluate integrated community surveillance of SARS-CoV-2 and other respiratory viruses and describe epidemiological trends. STUDY DESIGN: Respiratory surveillance was set up within an existing SARS-CoV-2 public testing facility. Community-dwelling (a)symptomatic persons provided consent for completion of a questionnaire and additional testing on residual material from swabs taken for SARS-CoV-2 RT-PCR (Allplex Seegene). Daily, a random subset was tested for sixteen respiratory viruses by multiplex realtime PCRs (Seegene). RESULTS: Between October 6th (week 40) 2021 and April 22nd (week 16) 2022, 3,969 subjects were tested. The weekly median age ranged from 23 to 39 years. The prevalence of respiratory symptoms ranged from 98.5% (week 40) to 27.4% (week 1). The prevalence of detection of any respiratory virus (including SARS-CoV-2), ranged from 19.6% in week 49 to 75.3% in week 14. SARS-CoV-2 prevalence ranged from 2.2% (week 40) to 63.3% (week 14). Overall, SARS-CoV-2 was detected most frequently (27.3%), followed by rhinoviruses (14.6%, range 3.5-47.8%) and seasonal coronaviruses (3.7%, range 0-10.4%, mostly 229E and OC43). Influenzavirus was detected in 3.0% of participants from week 6 onwards. CONCLUSIONS: Integrated respiratory viral surveillance within public testing facilities is feasible and informative. Prevalences may be affected by changes in SARS-CoV-2 prevention and testing policies. Population characteristics help to interpret trends over time. Integrated surveillance may inform policymakers and hospitals for adequate response measures during respiratory seasons.

Decontamination of the digestive tract and oropharynx in ICU patients.

BACKGROUND: Selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) are infection-prevention measures used in the treatment of some patients in intensive care, but reported effects on patient outcome are conflicting. METHODS: We evaluated the effectiveness of SDD and SOD in a crossover study using cluster randomization in 13 intensive care units (ICUs), all in The Netherlands. Patients with an expected duration of intubation of more than 48 hours or an expected ICU stay of more than 72 hours were eligible. In each ICU, three regimens (SDD, SOD, and standard care) were applied in random order over the course of 6 months. Mortality at day 28 was the primary end point. SDD consisted of 4 days of intravenous cefotaxime and topical application of tobramycin, colistin, and amphotericin B in the oropharynx and stomach. SOD consisted of oropharyngeal application only of the same antibiotics. Monthly point-prevalence studies were performed to analyze antibiotic resistance. RESULTS: A total of 5939 patients were enrolled in the study, with 1990 assigned to standard care, 1904 to SOD, and 2045 to SDD; crude mortality in the groups at day 28 was 27.5%, 26.6%, and 26.9%, respectively. In a random-effects logistic-regression model with age, sex, Acute Physiology and Chronic Health Evaluation (APACHE II) score, intubation status, and medical specialty used as covariates, odds ratios for death at day 28 in the SOD and SDD groups, as compared with the standard-care group, were 0.86 (95% confidence interval [CI], 0.74 to 0.99) and 0.83 (95% CI, 0.72 to 0.97), respectively. CONCLUSIONS: In an ICU population in which the mortality rate associated with standard care was 27.5% at day 28, the rate was reduced by an estimated 3.5 percentage points with SDD and by 2.9 percentage points with SOD. (Controlled Clinical Trials number, ISRCTN35176830.)

An outbreak of severe respiratory tract infection caused by human metapneumovirus in a residential care facility for elderly in Utrecht, the Netherlands, January to March 2010.

Recognition of infections with human metapneumovirus (HMPV) among institutionalised elderly is rising. When HMPV was found to be the causative agent of an outbreak of pneumonia in a residential care facility for elderly in the Netherlands, an elaborate outbreak investigation was set up, including active surveillance for new cases. From clinical cases, defined by fever (> 38°C) and symptoms of respiratory tract infections, respiratory samples for analyses of viral pathogens by real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) and blood samples for determination of HMPV-specific IgM and IgG antibody titres were taken. Five staff members and 18 residents fulfilled the clinical case definition. Of those, five residents tested positive for HMPV by rRT-PCR. The combination of rRTPCR and serology identified nine confirmed cases, six probable cases, six possible cases and ruled out two persons as cases. Among residents, the outbreak of HMPV had an attack rate, ranging from 5% for laboratory- confirmed cases, to 13% for clinical cases. This outbreak investigation shows that HMPV is a potential serious pathogen for institutionalised elderly.

Short-course aminoglycosides as adjunctive empirical therapy in patients with Gram-negative bloodstream infection, a cohort study.

OBJECTIVE: Short-course aminoglycosides as adjunctive empirical therapy to ß-lactams in patients with a clinical suspicion of sepsis are used to broaden antibiotic susceptibility coverage and to enhance bacterial killing. We quantified the impact of this approach on 30-day mortality in a subset of sepsis patients with a Gram-negative bloodstream infection. METHODS: From a prospective cohort study conducted in seven hospitals in the Netherlands between June 2013 and November 2015, we selected all patients with Gram-negative bloodstream infection (GN-BSI). Short-course aminoglycoside therapy was defined as tobramycin, gentamicin or amikacin initiated within a 48-hour time window around blood-culture obtainment, and prescribed for a maximum of 2 days. The outcome of interest was 30-day all-cause mortality. Confounders were selected a priori for adjustment using a propensity score analysis with inverse probability weighting. RESULTS: A total of 626 individuals with GN-BSI who received ß-lactams were included; 156 (24.9%) also received aminoglycosides for a median of 1 day. Patients receiving aminoglycosides more often had septic shock (31/156, 19.9% versus 34/470, 7.2%) and had an eight-fold lower risk of inappropriate treatment (3/156, 1.9% versus 69/470, 14.7%). Thirty-day mortality was 17.3% (27/156) and 13.6% (64/470) for patients receiving and not receiving aminoglycosides, respectively; yielding crude and adjusted odds ratios for 30-day mortality for patients treated with aminoglycosides of 1.33 (95% CI 0.80-2.15) and 1.57 (0.84-2.93), respectively. CONCLUSIONS: Short-course adjunctive aminoglycoside treatment as part of empirical therapy with ß-lactam antibiotics in patients with GN-BSI did not result in improved outcomes, despite better antibiotic coverage of pathogens.

An outbreak of non-typeable MRSA within a residential care facility.

In a household setting within a residential care facility for visually and intellectually disabled people, a resident (index case) was diagnosed with dermal abscesses caused by a methicillin-resistant Staphylococcus aureus (MRSA) which was non-typeable by standard pulsed-field gel electrophoresis. All residents and staff in contact with the index case (a total of 200 people) were screened for MRSA.

Interferon-gamma release assays during follow-up of tuberculin skin test-positive contacts.

SETTING: Following a large-scale contact investigation, individuals with a positive tuberculin skin test (TST) result were offered preventive tuberculosis treatment. OBJECTIVE: To investigate the effect of isoniazid (INH) treatment and the effect of time on interferon gamma release assay (IGRA) results during follow-up. DESIGN: TST-positive subjects (n = 122) detected during the large-scale contact investigation were included in the study. Blood was obtained every 6 months over 2 years to perform both tests. RESULTS: Preventive INH treatment was completed by 36 of the 122 (29.5%) subjects, 71 (58.2%) were followed up with 6-monthly X-ray screening and 15 (12.3%) did not complete INH treatment. The overall percentage of individuals with a positive result remained stable during the 2 years, at approximately 45-50%, but individual responses varied over time. The majority of initially low IGRA results remained below the cut-off value, initially high IGRA results remained positive, while initially intermediate IGRA results were followed by more dynamic patterns. CONCLUSION: This study showed a highly variable pattern of IGRA responses over time and suggests limited value for their use during follow-up of latently infected individuals. However, the significance of different kinetic patterns observed among subjects with intermediate initial IGRA results warrants further study.

Development of diagnostic prediction tools for bacteraemia caused by third-generation cephalosporin-resistant enterobacteria in suspected bacterial infections: a nested case-control study.

OBJECTIVES: Current guidelines for the empirical antibiotic treatment predict the presence of third-generation cephalosporin-resistant enterobacterial bacteraemia (3GCR-E-Bac) in case of infection only poorly, thereby increasing unnecessary carbapenem use. We aimed to develop diagnostic scoring systems which can better predict the presence of 3GCR-E-Bac. METHODS: A retrospective nested case-control study was performed that included patients ≥18 years of age from eight Dutch hospitals in whom blood cultures were obtained and intravenous antibiotics were initiated. Each patient with 3GCR-E-Bac was matched to four control infection episodes within the same hospital, based on blood-culture date and onset location (community or hospital). Starting from 32 commonly described clinical risk factors at infection onset, selection strategies were used to derive scoring systems for the probability of community- and hospital-onset 3GCR-E-Bac. RESULTS: 3GCR-E-Bac occurred in 90 of 22 506 (0.4%) community-onset infections and in 82 of 8110 (1.0%) hospital-onset infections, and these cases were matched to 360 community-onset and 328 hospital-onset control episodes. The derived community-onset and hospital-onset scoring systems consisted of six and nine predictors, respectively. With selected score cut-offs, the models identified 3GCR-E-Bac with sensitivity equal to existing guidelines (community-onset: 54.3%; hospital-onset: 81.5%). However, they reduced the proportion of patients classified as at risk for 3GCR-E-Bac (i.e. eligible for empirical carbapenem therapy) with 40% (95%CI 21-56%) and 49% (95%CI 39-58%) in, respectively, community-onset and hospital-onset infections. CONCLUSIONS: These prediction scores for 3GCR-E-Bac, specifically geared towards the initiation of empirical antibiotic treatment, may improve the balance between inappropriate antibiotics and carbapenem overuse.

Enterobacter colonisation in newborn infants: predictors, follow-up and implications for infection control.

Outbreaks with Enterobacter spp. have been described frequently in neonatal intensive care units (NICUs). This study investigated the factors that determine whether a neonate becomes colonised with Enterobacter spp., how long colonisation continues and whether the termination of isolation measures leads to spread of the organism. Neonates transferred from the NICUs of tertiary care hospitals were screened for the presence of Enterobacter spp. and any potential predictors for colonisation recorded. Those infected were monitored during their hospital stay and colonised neonates were screened every month for six months. Isolation infection control precautions were lifted and all neonates were screened for the presence of Enterobacter spp. six and 12 months later. Fifteen colonised neonates and 33 non-colonised controls were identified for study. Multivariate analysis showed that antibiotic therapy for more than three days and an Apgar score of <8 after 1 min were independently associated with Enterobacter spp. colonisation. Molecular typing using single-enzyme amplified-fragment length polymorphism (seAFLP) analysis revealed 22 different seAFLP genotypes. Three infants remained colonised with the same Enterobacter genotype after discharge; however, most neonates lost their strain or became colonised with another genotype. Lifting infection control measures for neonates colonised with Enterobacter spp. in a neonatal ward did not lead to increased incidence of colonisation and none of the infants became infected. Isolating neonates with susceptible Enterobacter spp. was not found to be necessary.

Quantifying within-household transmission of extended-spectrum ß-lactamase-producing bacteria.

OBJECTIVES: Patients can acquire extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae during hospitalization, and colonized patients may transmit these bacteria after discharge, most likely to household contacts. In this study, ESBL transmission was quantified in households. METHODS: Faecal samples were longitudinally collected from hospitalized patients colonized with ESBL-producing bacteria and from their household members during hospitalization of the index patient and at 3, 6, 12 and 18 months. A mathematical household model was developed, which allowed for person-to-person transmission, acquisition from other sources (background transmission), and losing carriage. Next, a deterministic population model with a household structure was created, informed by parameter values found in the household model. RESULTS: In all, 74 index patients and 84 household members were included. In more than half of the household members ESBL-producing bacteria were demonstrated at some time during follow up. Person-to-person transmission occurred at a rate of 0.0053/colonized person/day (0.0025-0.011), background transmission at 0.00015/day (95% CI 0.00002-0.00039), and decolonization at 0.0026/day (0.0016-0.0040) for index patients and 0.0090/day (0.0046-0.018) for household members. The estimated probability of transmission from an index patient to a household contact was 67% and 37% vice versa. CONCLUSION: There is frequent transmission of ESBL-producing bacteria in households, which may contribute to the observed endemicity of ESBL carriage in the Netherlands. However, the population model suggests that there is not a single dominant acquisition route in the community.

Molecular analysis of hematopoietic colonies derived from chronic myeloid leukemia patients: interphase fluorescence in situ hybridization compared with RT-PCR.

In this study we compared interphase fluorescence in situ hybridization (I-FISH) with reverse transcription polymerase chain reaction (RT-PCR) for the molecular analysis of hematopoietic colonies derived from patients with chronic myeloid leukemia (CML). Molecular analysis of individual colonies is often performed to monitor purging efficacy in CML. We harvested individual colony-forming unit granulocyte-macrophage (CFU-GM) colonies. One half was analyzed with I-FISH, for the presence of bcr-abl fusion gene. The other half was analyzed with RT-PCR for the presence of the bcr-abl mRNA. We wanted to address the following questions: (1) is the bcr-abl gene always expressed in CFU-GM colonies and (2) which technique has to be preferred to analyze individual CFU-GM colonies? In total, 133 colonies, derived from six CML patients, could be analyzed both with I-FISH and RT-PCR. We found a positive correlation in 89% of the cases: 118 colonies showed the same results with both techniques. However, 15 of the 106 I-FISH-positive colonies were negative in the RT-PCR. Serial analysis of the cDNA derived from 22 colonies showed in each round of amplification 21-29% RT-PCR-negative but I-FISH-positive colonies. However, all I-FISH-positive colonies showed at least one positive RT-PCR, either in the first, second or third round of amplification. These results indicate that the bcr-abl gene is probably always transcriptionally active in CFU-GM colonies. Reliable analysis with RT-PCR is possible but likely to generate false negative results. We conclude that: (1) I-FISH offers a reliable alternative to RT-PCR for analyzing individual hematopoietic colonies and (2) results obtained with RT-PCR should only be interpreted with caution.

Hypersensitivity of bcr-abl-positive progenitors to hyperthermia in patients with chronic myeloid leukemia.

In this study, we evaluated the effect of hyperthermia on hematopoietic progenitors from six chronic myeloid leukemia (CML) bone marrow (BM) samples at diagnosis and four peripheral blood stem cell (PBSC) samples from CML patients after stem cell mobilisation. CD34-positive cells, isolated from these samples, were incubated for 2 h at 37, 42 or 43 degrees C and were plated in the colony-forming unit granulocyte-macrophage (CFU-GM) and the long-term culture initiating cell (LTCIC) assay. To evaluate purging, individual colonies from these assays were analyzed for the presence of the bcr-abl gene with interphase fluorescence in situ hybridization (FISH) and/or RT-PCR. BM samples showed a significant higher sensitivity both at the CFU-GM and LTCIC level, after treatment at 42 degrees C, as compared to the control BM samples obtained from healthy volunteers. The four BM samples of CML patients with a low leukocyte number at diagnosis harbored a mixture of bcr-abl-negative and positive colonies and an increase in the percentage of bcr-abl-negative colonies was observed in all cases. CML patients with a high leukocyte count at diagnosis, however, showed only bcr-abl-positive progenitors even after hyperthermia. PBSCs showed a significant higher sensitivity at the LTCIC level but not at the CFU-GM level, after treatment at 42 degrees C, as compared to the control PBSC samples obtained from nonhematologic cancer patients. Molecular analysis of individual colonies demonstrated an increase of bcr-abl-negative progenitors after thermic treatment in two out of three samples. When comparing both stem cell sources, PBSCs showed a decreased thermic sensitivity as compared to the BM samples at the CFU-GM level, whereas at the LTCIC level an increased thermic sensitivity was observed, both for the controls and the CML samples. In conclusion, both for BM and PBSCs samples, CML progenitors are more sensitive to hyperthermia than control cells, especially at the LTCIC level. In agreement with these results, an increase of bcr-abl-negative progenitors in six out of seven samples could be demonstrated either at the CFU-GM level, LTCIC level or both. Hyperthermia should be explored further as a possible purging modality in CML.

[A patient with Legionella pneumophila serogroup-3 pneumonia, detected by PCR]. / Een patient met een pneumonie door Legionella pneumophila serogroep 3, vastgesteld door PCR.

A 59-year-old man was hospitalised because of dyspnoea, productive cough, fever, chills and malaise. Severe community-acquired pneumonia was diagnosed. Legionella urinary antigen testing, which can only detect serogroup 1, and the first culture ofa bronchoalveolar lavage (BAL) fluid sample were negative for Legionella. However, L. pneumophila DNA was detected by PCR in the BAL washing sample. Eventually, L. pneumophila serogroup 3 was isolated from this specimen by repeated culture. Although, in The Netherlands, legionellosis is caused by L. pneumophila serogroup 1 in more than 90% of all cases, this case demonstrates that a negative result of urinary antigen testing does not necessarily exclude this diagnosis. It is therefore advocated to expand the diagnostics to a Legionella PCR on respiratory material of patients with clinical signs of Legionella pneumonia in whom the urinary antigen test is negative.

The role of BCL-2 and bax protein in monocyte-mediated apoptosis in human leukemic cell lines.

Monocytes or monocyte-derived supernatants are able to kill leukemic cells via apoptosis, thereby preferentially effecting more mature leukemic cells. In the present study, the relationship between apoptosis and the apoptosis related proteins, bcl-2 and bax, was investigated in a number of human leukemic cell lines. Monocyte-derived supernatant induces extensive apoptosis in U937 myeloid leukemia cells and minor apoptosis in HL60 cells. No apoptosis was seen in four other cell lines (THP1, HL60-D3, KG1, and K562). The expression of bcl-2 and bax protein was determined in both groups of leukemic cell lines by flow cytometry (bcl-2 and bax) and Western blotting (bcl-2) at baseline level and after incubation with monocyte supernatant after different time periods. No clear relation was found between baseline bcl-2 or bax protein expression and the occurrence of apoptosis after incubation with monocyte supernatant. After different incubation time periods, no change was found in bcl-2 protein expression in U937 and K562 cells, whereas in KG1, HL60, and especially in THP1 cells, a significant decrease could be noticed. On the other hand, there was an increase in bcl-2 expression in HL60-D3 cells. Bax protein expression, measured at the same time points, remained essentially unchanged in HL60-D3 cells, decreased significantly in U937, HL60, and THP1 cells and slightly in K562 cells, and increased significantly in KG1 cells. Also, the ratio bax/bcl-2 decreased in HL60D3, but especially in U937 and HL60 cells, increased slightly in THP1 and KG1 cells, and remained essentially unchanged in K562 cells. Rh-tumor necrosis factor-alpha (TNF-alpha), the main mediator of monocyte mediated cytotoxicity, induced apoptosis in U937, HL60, and THP1 cells, thereby showing changes in bcl-2 expression similar to those found for monocyte derived supernatants. We concluded that in human leukemic cell lines, there is no relation between either bcl-2 or bax protein expression or the ratio of both, and apoptosis mediated by monocyte derived supernatant or TNF-alpha.