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Background: While influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are recognised as a cause of severe illness and mortality, clinical interest for respiratory syncytial virus (RSV), rhinovirus and human metapneumovirus (hMPV) infections is still limited., Methods: We conducted a retrospective database study comparing baseline characteristics and 30-day mortality in a large cohort of adult patients admitted for an overnight stay or longer with an influenza virus (A/B), rhinovirus, hMPV, RSV or SARS-CoV-2 infection. For non-SARS-CoV-2 viruses, data were included for the period July 2017-February 2020. For SARS-CoV-2, data between March 2020 and March 2022 were included., Results: Covariate-adjusted 30-day mortality following RSV, hMPV or rhinovirus infections was substantial (crude mortality 8-10%) and comparable with mortality following hospitalisation with an influenza A virus infection. Mortality following a SARS-CoV-2 infection was consistently higher than for any other respiratory virus, at any point in time (crude mortality 14-25%). Odds of mortality for SARS-CoV-2 compared with influenza A declined from 4.9 to 1.7 over the course of the pandemic. Patients with SARS-CoV-2 infection had less comorbidity than patients with other respiratory virus infections and were more often male. In this cohort, age was related to mortality following hospitalisation, while an association with comorbidity was not apparent., Conclusions: With the exception of SARS-CoV-2 infections, we find the clinical outcome of common respiratory virus infections requiring hospitalisation more similar than often assumed. The observed mortality from SARS-CoV-2 was significantly higher, but the difference with other respiratory viruses became less distinct over time., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. No external funding was received to conduct this study., (© 2024 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Pneumocystis jirovecii (Pj) is a fungal pathogen that can cause severe and potential fatal pneumonia (Pneumocystis pneumonia, PCP) in immunocompromised patients. Microbiological diagnosis is necessary to confirm PCP, for which mainly real-time PCR assays are used by detecting Pj from bronchoalveolar lavage (BAL) specimens. In this study, we evaluate the performance of the CE-IVD PneumoGenius® assay and CE-IVD RealStar® Pneumocystis jirovecii PCR assay in comparison to the lab developed test (LDT) that is used in routine diagnostics. Comparison was done by including 100 BAL specimens: 25 retrospective specimens, selected based on results obtained with LDT (15 positive/10 negative), and 75 prospectively collected specimens. LDT (targeting MSG) was performed according to local procedures and the PneumoGenius® (targeting mtLSU and DHPS fas) and RealStar® assays (targeting mtLSU) according to the manufacturer's instructions. Combining results of retrospective and prospective analysis, sensitivity was 69.7, 100 and 100% for the LDT, PneumoGenius® and RealStar®, respectively. Specificity was 100% for LDT and Pneumogenius®, whereas RealStar® showed a specificity of 97%. Correlation of fungal loads found with the PneumoGenius® and RealStar® assays was high (R2: 0.98). The PneumoGenius® and RealStar® assays performed comparable, and both showed high sensitivity in comparison to the LDT. For optimal diagnosis of PCP, the LDT has to be replaced by another, more sensitive assay., Lay Summary: In this study, we evaluated the performance of two commercially available CE-IVD cleared real-time PCR assays to detect Pneumocystis jirovecii in comparison to the lab-developed test as used in routine diagnostics. Performance of the CE-IVD real-time PCR assay was superior to the lab-developed test., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Background: A large outbreak of three epidemic vancomycin-resistant Enterococcus faecium (VRE) clones affected the study hospital for almost two years., Aim: To describe the strategy to successfully control this outbreak and eradicate VRE from the study hospital., Methods: Infection control interventions started after detection of VRE in three patients. Hospital-wide surveillance was started after ongoing transmission despite isolation precautions, cleaning and contact tracing. Hygiene education and discipline were enhanced. Despite these interventions, additional measures were required to control the outbreak, such as ward disinfection with hydrogen peroxide vapour and the introduction of a VRE quarantine ward. Ultimately, ciprofloxacin prophylaxis for haematological patients on chemotherapy was abandoned., Findings: Over a 22-month period, 242 VRE carriers were identified. Of these, 128 (53%) patients were detected by hospital-wide surveillance alone. Three epidemic clones were detected: ST494-vanA (N = 160), ST78-vanA (N = 23) and ST117-vanB (N = 32). In total, 5614 possible contacts were identified. VRE transmission occurred on 13 out of 23 wards. VRE was cultured from clinical specimens in 22 patients (seven with bacteraemia). Since January 2014, no further transmission of these VRE clones has been observed., Conclusion: Infection control measures according to international guidelines were insufficient to expose the outbreak to its full extent and control it. Its full extent only became apparent after sustained hospital-wide screening. Successful control of this hospital-wide VRE outbreak was feasible, but required great effort. Final containment and eradication of the epidemic clones was achieved by environmental decontamination with hydrogen peroxide vapour, strict isolation precautions, a VRE quarantine ward and antimicrobial stewardship., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) are common sexual transmitted infections (STI). However, most STI screening programmes do not include routinely detection of these pathogens. Consequently, epidemiological data about MG and TV in the general population is lacking. The current study aims to give insight into the prevalence of both infections, thereby guiding decisions whether testing for these pathogens should be included routinely., Methods: Between February 2013 and August 2015, all samples sent to the laboratory of Diakonessenhuis Utrecht for STI testing (i.e. testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG)) were additionally examined for the presence of MG and TV by means of a laboratory-developed RT-PCR. Samples were collected by our hospital or by regional general practitioners., Results: A total of 5628 PCR's were evaluated. In 7.5%, one or more STI were detected. CT was found in 5% and MG was positive in 1.9%. NG was detected in 0.5% and TV was detected in 0.6% of the samples. CT was found more often in primary care than in hospital setting (9.7% vs. 3.0%, p < .05). The same was shown for NG (1.1% vs. 0.2%, p < .05). More men than women were positive for CT (11.2% vs. 3.8%, p < .05) and NG (1.4% vs. 0.3%, p < .05)., Conclusion: MG is more prevalent than NG and TV in a regional Dutch population. Furthermore, TV is equally common as NG. Based on our prevalence data, including MG and TV in STI testing protocols should be considered in the future.

Background: Of all hospitalised community-acquired pneumonias (CAPs) only a few are known to be caused by Chlamydia psittaci. Most likely the reported incidence, ranging from of 0% to 2.1%, is an underestimation of the real incidence, since detection of psittacosis is frequently not incorporated in the routine microbiological diagnostics in CAP or serological methods are used., Methods: C. psittaci real-time polymerase chain reaction (PCR) was routinely performed on the sputum of 147 patients hospitalised with CAP, who participated in a clinical trial conducted in two Dutch hospitals. In 119/147 patients the paired complement fixation test (CFT) was also performed for the presence of Chlamydia antibodies. Positive CFTs were investigated by micro- Immunofluorescence for psittacosis specificity. Case criteria for psittacosis were a positive PCR or a fourfold rise of antibody titre in CFT confirmed by micro- Immunofluorescence. Furthermore, we searched for parameters that could discriminate psittacosis from CAPs with other aetiology., Results: 7/147 (4.8%) patients were diagnosed with psittacosis: six with PCR and one patient with a negative PCR, but with CFT confirmed by micro- Immunofluorescence. Psittacosis patients had had a higher temperature (median 39.6 vs. 38.2 °C;) but lower white blood cell count (median 7.4 vs. 13.7 x 109/l) on admission compared with other CAP patients., Conclusion: In this study, C. psittaci as CAP-causing pathogen was much higher than previously reported. To detect psittacosis, PCR was performed on all CAP patients for whom a sputum sample was available. For clinical use, PCR is a fast method and sputum availability allows genotyping; additional serology can optimise epidemiological investigations.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that has been responsible for major nosocomial epidemics worldwide. For infection control programs, rapid and adequate detection of MRSA is of great importance. We developed a rapid and high-throughput molecular screening approach that consists of an overnight selective broth enrichment, followed by mecA, mecC, and S. aureus-specific (SA442 gene) real-time PCR assays, with subsequent confirmation using a staphylococcal cassette chromosome mec element (SCCmec)-orfX-based real-time PCR assay (GeneOhm MRSA assay) and culture. Here, the results of the screening approach over a 2-year period are presented. During this period, a total of 13,387 samples were analyzed for the presence of MRSA, 2.6% of which were reported as MRSA positive. No MRSA isolates carrying the mecC gene were detected during this study. Based on the results of the real-time PCR assays only, 95.2% of the samples could be reported as negative within 24 h. Furthermore, the performance of these real-time PCR assays was evaluated using a set of 104 assorted MRSA isolates, which demonstrated high sensitivity for both the combination of mecA and mecC with SA442 and the BD GeneOhm MRSA assay (98.1% and 97.1%, respectively). This molecular screening approach proved to be an accurate method for obtaining reliable negative results within 24 h after arrival at the laboratory and contributes to improvement of infection control programs, especially in areas with a low MRSA prevalence., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The genotypic diversity of Coxiella burnetii in clinical samples obtained from the Dutch Q fever outbreak episodes of 2007-2010 was determined by using a 6-locus variable-number tandem repeat analysis panel. The results are consistent with the introduction of one founder genotype that is gradually diversifying over time while spreading throughout The Netherlands.

Background: In 2007, 2008, and 2009 outbreaks of Q-fever occurred in The Netherlands with increasing magnitude. The 2009 outbreak with 2354 reported cases is the largest human Q-fever outbreak ever recorded. To assess the extent of infection and the safety of donated blood, we tested local blood donations for presence of Coxiella burnetii antibodies and DNA., Study Design and Methods: Starting May 2009, more than 40,000 serum samples were collected from all consenting blood donors in the areas with high Q-fever incidence. The 1004 samples from the areas with the highest number of reported cases were tested for C. burnetii DNA by polymerase chain reaction; seroprevalence and incidence were determined using enzyme-linked immunosorbent assay and immunofluorescence assays (IFAs) in the subset of 543 donors of whom a follow-up sample was available., Results: A total of 6 of 1004 donor samples tested reactive for C. burnetii DNA. Confirmatory testing (IFA) on the index and follow-up samples demonstrated seroconversion in two donors, high-level preexisting antibodies in one donor, and no seroconversion in three donors. Immunoglobulin (Ig)G testing of the 543 serum pairs showed that 66 were reactive in the latest sample, of which 10 represented seroconversions., Conclusion: In the area with highest incidence during a large Q-fever outbreak, 3 of 1004 blood donations contained C. burnetii DNA (0.3%; 95% confidence interval, 0.1%-1.0%). A total of 66 of 543 (12.2%) donors tested positive for anti-Coxiella IgG. Ten seroconversions were detected, resulting in an incidence of 5.7% per year, which is more than 10-fold higher than the local number of reported clinical cases (0.47% per year)., (© 2011 American Association of Blood Banks.)

Background: Human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. The B27 allele is present in 90% of patients with this disease, whereas it is present in only 9% of Caucasians. Molecular detection of HLA-B27 is traditionally based on allele specific amplification of exon 2 (Olerup method) or exon 3 (Dominguez method) by PCR, followed by gel analysis., Methods: We developed a real-time TaqMan PCR based on the Dominguez method with a β-Globin PCR as internal control., Results: A total of 544 clinical samples were used to compare the real-time TaqMan PCR with the traditional Dominguez PCR, the traditional Olerup PCR and a commercial Olerup based HLA-B27 detection kit (Olerup SSPTM HLA-B27, GenoVision). While 542 samples gave concordant results, two samples showed discrepancies and were further analyzed. One sample that showed a discrepancy was negative with the traditional Olerup method and positive with the three other procedures. Sequencing analysis showed the presence of HLA-B*2712 in this sample. The other sample, positive with both Olerup based PCRs and negative with both Dominguez based methods, turned out to be positive for HLA-B*2707 by sequence analysis., Conclusions: With a correct result for 543 out of 544 samples (99.8%), we consider our real-time HLA-B27 PCR is a reliable method to detect HLA-B27 in the Dutch population, with reduced hands-on time and contamination risk compared to traditional PCR methods.

Contamination of an in-house diagnostic real-time PCR for Q fever was traced back to a commercially obtained PCR Master Mix. It was established that this Master Mix contained DNA from Coxiella burnetii, probably as a result of the use of compounds of animal origin such as bovine serum albumin.

In the Netherlands, there is an ongoing and unparalleled outbreak of Q fever. Rapid and reliable methods to identify patients infected with Coxiella burnetii, the causative agent of Q fever, are urgently needed. We evaluated the performance of different DNA extraction methods and real-time PCR assays that are in use in seven diagnostic or reference laboratories in the Netherlands. A low degree of variation in the sensitivities of most of the developed real-time PCR assays was observed. However, PCR assays amplifying short DNA fragments yielded better results than those producing large DNA fragments. With regard to DNA extraction, the automated MagNA Pure Compact system and the manual QIAamp DNA mini kit consistently yielded better results than either the MagNA Pure LC system and NucliSens EasyMag (both automated) or the High Pure viral nucleic acid kit (manual). The present study shows that multiple combinations of DNA extraction kits and real-time PCR assays offer equivalent solutions to detect C. burnetii DNA in serum samples from patients suspected to have Q fever.

The world's largest Q fever outbreak is ongoing in The Netherlands with around 3,000 confirmed cases since the first half of 2007. Increased awareness has resulted in early referral of patients for diagnostics. An important drawback to serological diagnosis of acute Q fever is the lag phase in antibody response. Therefore, we evaluated the performance of a real-time PCR for detection of Coxiella burnetii DNA using serum samples from patients with acute Q fever. PCR, targeting IS1111, was retrospectively performed on acute-phase and follow-up convalescent-phase serum samples from 65 patients with acute Q fever as diagnosed by immunofluorescence assay. The results obtained by PCR were related to disease stage as defined by subsequent appearance of phase II IgM, phase II IgG, phase I IgM, and phase I IgG (IgM-II, IgG-II, IgM-I, and IgG-I, respectively) antibodies and time since onset of disease. In addition, we analyzed seronegative acute-phase serum samples from patients with inconclusive Q fever serology, because no convalescent-phase serum samples were available. PCR was scored positive in 49/50 (98%) seronegative sera, 9/10 (90%) sera with isolated IgM-II antibodies, 3/13 (23%) sera with IgM-II/IgG-II antibodies, 2/41 (5%) sera with IgM-II/IgG-II/IgM-I antibodies, 0/15 (0%) sera with IgM-II/IgG-II/IgM-I/IgG-I antibodies, and 0/1 (0%) serum sample with IgM-II/IgG-II/IgG-I antibodies. The latest time point after onset of disease in which C. burnetii DNA could be detected was at day 17. In patients with inconclusive Q fever serology, PCR was positive in 5/50 (10%) cases. We conclude that real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever. C. burnetii DNA becomes undetectable in serum as the serological response develops.

Polymorphisms leading to deficiency of mannose-binding lectin (MBL) are associated with predisposition to infection. However, MBL deficiency can be protective against intracellular pathogens that use MBL to enter host cells. The role of MBL genotype and activity in infection with the intracellular pathogen Legionella pneumophila was studied in a large outbreak of legionellosis at a Dutch flower show. A total of 141 patients, 65 exposed asymptomatic exhibition staff members and 670 unexposed blood bank donors were included for the study of MBL2 genotypes and MBL-mediated complement activation. Genotypic MBL deficiency was equally prevalent in patients and controls. Deficient MBL-mediated complement activation was more prevalent in patients. Even in patients with genotypes that confer MBL sufficiency, 20.6% lacked MBL-mediated complement activation. In most patients with MBL-sufficient genotypes who lacked MBL-mediated activation at the acute phase of disease, lectin pathway functionality was restored at convalescence. In conclusion, genotypic MBL deficiency was not a risk factor for legionellosis. However, patients with legionellosis displayed deficient MBL-mediated complement activation even with MBL-sufficient genotypes. Together, these genotypical and functional data suggest that the observed deficiency of lectin pathway activation is an effect of legionellosis rather than a risk factor for acquiring it.

A 37-year-old man was admitted with cough and fever. Three days after admission he was tested using a newly developed real-time PCR technique that detects the DNA of Chlamydophila psittaci. The result was positive; serological investigation was not positive until 14 days later. Psittacosis is a potentially life-threatening infectious disease. Laboratory diagnosis relies mainly on the assessment of paired sera, but this approach has obvious disadvantages in the acute setting. Routine use of the real-time PCR technique led to the rapid diagnosis of psittacosis in 6 other patients. All 7 patients recovered after antibiotic treatment. This PCR technique is a valuable adjuvant to serological testing for the rapid diagnosis of psittacosis.

Detection of Shiga toxin-producing Escherichia coli (STEC) in The Netherlands is traditionally limited to serogroup O157. To assess the relative importance of STEC, including non-O157 serogroups, stool samples submitted nationwide for investigation of enteric pathogens or diarrhoea were screened with real-time PCR for the presence of the Shiga toxin genes. Patients were selected if their stool contained blood upon macroscopic examination, if they had a history of bloody diarrhoea, were diagnosed with haemolytic uraemic syndrome, or were aged <6 years (irrespective of the bloody aspect of the stool). PCR-positive stools were forwarded to a central laboratory for STEC isolation and typing. In total, 4069 stools were examined, with 68 (1.7%) positive PCR results. The highest prevalence was for stools containing macroscopic blood (3.5%), followed by stools from patients with a history of bloody diarrhoea (2.4%). Among young children, the prevalence (1.0%) was not significantly higher than among random, non-bloody, stool samples from diarrhoeal patients (1.4%). STEC strains were isolated from 25 (38%) PCR-positive stools. Eleven O-serogroups were detected, including five STEC O157 strains. As serogroup O157 represented only 20% of the STEC isolates, laboratories should be encouraged to use techniques enabling them to detect non-O157 serogroups, in parallel with culture, for isolation and subsequent characterisation of STEC strains for public health surveillance and detection of outbreaks.

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.

Chlamydophila (formerly Chlamydia) psittaci genotypes A, B, C, and a new genotype most similar to the 6BC type strain were found in 10 humans with psittacosis by outer membrane protein A gene sequencing. Genotypes B (n = 3) and C (n = 1) are endemic in nonpsittacine European birds. These birds may represent an important part of the zoonotic reservoir.

An outbreak of psittacosis in a veterinary teaching hospital was recognized in December 2004. Outbreak management was instituted to evaluate the extent of the outbreak and to determine the avian source. Real-time PCR, serologic testing and sequencing of the ompA gene of Chlamydophila psittaci were performed. Sputum samples from patients, throat-swab samples from exposed students and staff, and faecal specimens from parrots and pigeons were tested. In this outbreak, 34 % (10/29) of the tested individuals were infected. The clinical features of the infection ranged from none to sepsis with multi-organ failure requiring intensive-care-unit admission. C. psittaci genotype A was identified as the outbreak strain. Parrots, recently exposed to a group of cockatiels coming from outside the teaching facility, which were used in a practical class, appeared to be the source of the outbreak. One of the tested pigeons harboured an unrelated C. psittaci genotype B strain. The microbiological diagnosis by real-time PCR on clinical specimens allowed for rapid outbreak management; subsequent genotyping of the isolates identified the avian source. Recommendations are made to reduce the incidence and extent of future outbreaks.

Human L-ficolin (FCN) is a serum lectin characterized by a collagen-like and a fibrinogen-like domain that can activate the lectin pathway of complement. Structural and functional similarities to mannose-binding lectin (MBL) suggest a role for L-ficolin in innate immunity. Structural polymorphisms in the MBL2 gene lead to functional deficiency of MBL. Polymorphisms in the FCN2 gene have not been studied previously. We developed 10 denaturing gradient gel electrophoresis (DGGE) assays to screen a total of 188 Dutch Caucasians for polymorphisms in FCN2. Total gene screening in this large cohort revealed 10 single nucleotide polymorphisms (SNPs). Interestingly, two conserved coding SNPs were found in exon 8, leading to amino acid substitutions within the fibrinogen-like domain. Fibrinogen-like domains are highly conserved among several proteins in many species. As this domain is responsible for binding of L-ficolin, these newly found coding polymorphisms could alter the affinity of the protein for its substrates and possibly alter the ability of L-ficolin to recognize invading microorganisms.

Use of stereolithographic models of the resection areas in mandibular tumor surgery allows for preoperative adapting of resection plates, and shortening operating time. Adapting reconstruction plates along the deformed resection contour, however, recreates the existing, unfavorable contour. The computer-aided procedure that generates the stereolithographic model, nevertheless, allows for digital manipulation of the data, to reestablish the required contour. Here are two cases that benefited from this simple method.

Tumors of different metastatic behavior possibly differ in genomic constitution. We identified molecular cytogenetic differences between a group of metastasized and nonmetastasized primary tongue tumors by comparative genomic hybridization. Most frequent chromosome copy number changes for metastasized and nonmetastasized tumors were +8q (100% and 71%, respectively) and +3q (56% and 43%, respectively). Metastasized tumors showed significantly more chromosome copy number changes than nonmetastasized tumors. High copy number gains were exclusively found in metastasized tumors for 3q23-qter, 5p, 12p and 13q21-q22. Genomic imbalances occurring in metastasized tumors but not in nonmetastasized tumours were +7q21 (44%), +14q (33%), and -15q (33%). The genetic constitution of primary tongue tumors that metastasize differs from tongue tumors that do not metastasize. Our data, although obtained from a relative small group of tumors, spotlights copy number gain of chromosome region 7q21 as a potential marker for metastatic behavior.

Tumours need vessels to grow into clinically significant dimensions, and to metastasize. Varying results have been reported for the relation between angiogenic activity in oral carcinomas, and the occurrence of metastasis. Quantification of microvessels in tissue sections is mostly used to assess angiogenesis, but appears subject to biases as reflected in contradicting reports. Especially the choice of immunohistochemical staining technique appears pivotal. Although microvessel density measurements (MVD) appear promising for other tumour types, in oral carcinomas no unambiguous relation could be established. Furthermore, MVD assessment appears more elaborate than expected. New, more time efficient techniques may replace MVD as we know it.

The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.

A real-time PCR for the ABI Prism 7000 system targeting the 23S-5S spacer of Legionella spp. was developed. Simultaneous detection and differentiation of Legionella spp. and Legionella pneumophila within 90 min and without post-PCR melting-curve analysis was achieved using two TaqMan probes. In sputum samples from 23 controls and 17 patients with legionellosis, defined by positive culture, urinary antigen testing, or seroconversion, 94% sensitivity and 100% specificity were observed.

Background: Quantification of microvessels in solid malignancies is regarded as a potential test to predict their clinicobiologic behavior. However, discordant results have been reported for head and neck cancer that may be explained by varying methods., Methods: In this retrospective study, we therefore quantified the microvasculature in 20 nonmetastasized and 20 metastasized squamous cell carcinomas of the tongue, using recently developed methods. For immunohistochemical visualization of the vessels, we used anti-CD34 with a signal amplification step based on the catalyzed deposition of biotinylated tyramine. This protocol results in enhanced staining quality compared with standard protocols. For each tumor, a representative tissue section was systematically sampled with 40 to 60 standardized test fields. True color image analysis system was used to measure microvessel density (MVD) and to obtain additional information with regard to size categories of vessels., Results: Remarkably, in the group of nonmetastasized tumors, the MVD was greater than in the metastasized tumors (p =.007). However, the microvessels with a diameter in the range of 10 to 15 microm predominated in the group of metastasized tongue carcinomas (p =.03). A logistic regression model based on the percentage of vessels smaller than 5 microm, classified 85% of patients with a metastasized tumor correctly and 75% of patients with a nonmetastasized tumor, independently of the clinical stage of the tumor., Conclusions: These results suggest that only vessels with a diameter larger than 10 microm, consistent with functional vessels, play a role in the process of metastasis. Further research more specifically into structural and functional characterization of blood and lymphatic vessels might help provide more insight into the relationship between microvasculature and the pathogenesis of metastasis in tongue carcinomas., (Copyright 2002 Wiley Periodicals, Inc. Head Neck)

Background: Predicting the presence of metastasis, based on tumor or tumor-related characteristics is of utmost importance. The authors studied the significance of tumor DNA features and tumor-related angiogenesis to predict the occurrence of metastasis in squamous cell carcinomas (SCCs) of the tongue., Methods: Paraplast blocks from resection specimens of 20 metastasized and 20 nonmetastasized SCCs of the tongue with a minimum follow-up of 24 months were used. Tissue sections were stained with anti-CD34 monoclonal antibodies for vessel visualization, and according to Feulgen to stain DNA. Using image analysis, data from both stainings were computed for each of the 40 carcinomas. A logistic regression model to predict the presence of metastasis, based on vascular and nuclear morphology features, was developed., Results: The intratumor variation of chromatin condensation and the percentage vessels smaller than 5 microm in diameter were selected for the model. The model correctly predicted metastasis in 90% of patients and excluded metastasis correctly in 75% of nonmetastasized tumors. Taking into account the prevalence of metastasis in SCC of the tongue of between 30% and 60%, this means a predictive value for a negative outcome of between 95% and 83%., Conclusions: The proposed model shows an improvement of predictive values compared with previous models with single parameters. Therefore, a multiparameter model appears to predict the multiparameter process of metastasis better., (Copyright 2001 American Cancer Society.)

The aims of this study of head and neck tissue samples were to develop an immunohistochemical protocol based on the catalysed reporter deposition (CARD) technique to enhance staining results for use in automated true colour image analysis, to assess the reproducibility of systematic tissue sampling in the angiogenic hot spot selection, and quantification of microvessel density (MVD) and other vessel characteristics. The latter data were compared between six metastasised tongue squamous cell carcinomas, vs. four non-metastasised. In comparison to the standard immunohistochemical protocol with anti-CD34 antibodies, CARD amplification resulted in both more intensely stained and larger numbers of vessels. Averaging the 10 most vascularised fields of the 40 to 60 systematically sampled fields in a tissue section resulted in an overall acceptable interobserver reproducibility for most assessed vessel parameters (r> or =0.76 and p< or =0.01). The percentage vessels with diameter <5 microm was significantly higher in the non-metastasised tongue carcinomas (p=0.02). However, for a number of tumours the effect of tissue sampling was significant. We conclude that CARD amplification is needed for reliable segmentation of vessels by image analysis systems, and that tumour heterogeneity is a limiting factor for all procedures in which tumour vascularity is assessed in a single tissue section. Figures on http://www.esacp.org/acp/2001/22-4/hannen.htm.

Polymorphous low grade adenocarcinomas (PLGAs) are thought to be indolent tumours that are localised preferentially to the palate and affect the minor salivary glands almost exclusively. Metastases to locoregional lymph nodes occur in only 6-10% of cases. Recently, two cases of PLGA with microscopically confirmed distant metastases have been reported. This study reports a third case of PLGA with histologically and immunohistochemically confirmed distant metastases. It is the first case with multiple pleural, as well as pulmonary parenchymal, metastases and metastases in cervical and paraoesophageal lymph nodes. In most cases, PLGAs are salivary gland tumours with limited potential to metastasis and a good prognosis after local treatment. However, the recently reported cases reveal that the tumour can give rise to widely spread metastases. To obtain more information about the incidence of distant metastases, periodic chest x ray examination during follow up is desirable.

The genetic distances between several strains of Scenedesmus obliquus (Turp.) Kütz., S. acutus Hortobagyi, and S. naegelii Chod. calculated from ITS-2 sequences were found to be smaller than the genetic distances within other strains of Scenedesmus-that is, in S. acuminatus (Lagerh.) Chod. and S. pectinatus Meyen. These results confirm that the studied strains were not properly identified and should be renamed S. obliquus, as already suggested in other studies.

Correlations between the biomass of phytoplankton and the biomass of bacteria and between the biomass of bacteria and the biomass of protozoans suggest that there is coupling between these compartments of the "microbial loop." To investigate this coupling on the species level, bacteria and protozoans from untreated lake water inocula were allowed to grow on detritus of the green alga Ankistrodesmus falcatus or the cyanobacterium Oscillatoria limnetica in continuous-flow systems for 1 month. Denaturing gradient gel electrophoresis (DGGE) of the 16S and 18S rRNA genes was used to monitor the development of the bacterial community structure and the eukaryotic community structure, respectively. Nonmetric multidimensional scaling of the DGGE profiles revealed the changes in the microbial community structure. This analysis showed that significantly different bacterial communities developed on the green algal detritus and on the cyanobacterial detritus. Although similar results were obtained for the eukaryotic communities, the differences were not significant. Hence, our findings indicate that the origin of detritus can affect the structure of at least the bacterial community. A phylogenetic analysis of 20 18S ribosomal DNA clones that were isolated from the continuous cultures revealed that many sequences were related to the sequences of bacterivorous protozoans (members of the Ciliophora, Rhizopoda, Amoeba, and Kinetoplastida). One clone grouped in a recently established clade whose previously described members are all parasites. The affiliations of about 20% of the clones could not be determined.

During an experiment in two laboratory-scale enclosures filled with lake water (130 liters each) we noticed the almost-complete lysis of the cyanobacterial population. Based on electron microscopic observations of viral particles inside cyanobacterial filaments and counts of virus-like particles, we concluded that a viral lysis of the filamentous cyanobacteria had taken place. Denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments qualitatively monitored the removal of the cyanobacterial species from the community and the appearance of newly emerging bacterial species. The majority of these bacteria were related to the Cytophagales and actinomycetes, bacterial divisions known to contain species capable of degrading complex organic molecules. A few days after the cyanobacteria started to lyse, a rotifer species became dominant in the DGGE profile of the eukaryotic community. Since rotifers play an important role in the carbon transfer between the microbial loop and higher trophic levels, these observations confirm the role of viruses in channeling carbon through food webs. Multidimensional scaling analysis of the DGGE profiles showed large changes in the structures of both the bacterial and eukaryotic communities at the time of lysis. These changes were remarkably similar in the two enclosures, indicating that such community structure changes are not random but occur according to a fixed pattern. Our findings strongly support the idea that viruses can structure microbial communities.

In a retrospective case-control study on 46 metastasized and 34 non-metastasized primary tongue carcinomas, the nuclear morphology and chromatin pattern were assessed in 3 microns thick, formalin-fixed, paraffin-embedded, and Feulgen-stained tissue sections of surgical resection specimens, by means of high-resolution computer-assisted image analysis. The aim of this study was to disclose differences in karyometric features, such as nuclear size-, shape-, and chromatin-pattern features, between these groups, with a view to developing a discriminant function that can predict the occurrence of metastasis for the individual patient. In addition, the lymph node metastases of 31 patients and the normal tongue epithelium of 21 patients were also assessed, to study the possible differences between these two groups and primary tumours. In the metastasized tumours, the chromatin was significantly more condensed (P = 0.01) and exhibited significantly less variation in chromatin condensation (P < 0.001) than in the group of non-metastasized carcinomas. Comparison of lymph node metastases with their primary tumours disclosed only minor differences in chromatin pattern. These findings suggest that only minor genetic differences exist between primary tongue carcinomas and their metastases. Tumour cells of tongue carcinomas showed highly significant differences from cells of normal tongue mucosa for most karyometric features. Logistic regression analysis resulted in a classifier, based on the circularity of the nucleus (CIRC) and the standard deviation of the chromatin condensation (SD COND), to predict the occurrence of lymph node metastases. After cross-validation, the percentages of correct classifications in the group of metastasized and non-metastasized tumours were 72 and 62 per cent, respectively. These results are comparable to the classification results obtained from a classifier based on the clinical T-stage, but our karyometric classification results show a much more equal distribution between the sensitivity and specificity. Karyometric features appeared to be more appropriate to predict metastases than biomarkers such as p53, bcl-2, and Ki-67.

The influence of copper on embryonic development and hatching of Sepia offinalis was investigated. Copper exerts a profound effect on both hatching stage and time-to-hatching. At high copper concentrations (50-200 ppb Cu2+), the embryos hatch earlier than the controls but have a lower survival potential. No external morphological malformations were found. Whereas copper does not accumulate in the embryo or in the vitellus, it is absorbed by the envelope and/or the chorion.