DNA identification of species of the Anopheles maculipennis complex and first record of An. daciae in Belgium.

The present study aimed at identifying the members of the Anopheles maculipennis complex (Diptera: Culicidae) occurring in Belgium. Therefore, the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) loci were sequenced in 175 and 111 specimens, respectively, collected between 2007 and 2019. In parallel, the suitability of two species-diagnostic PCR-RFLP assays was tested. The identified specimens included: An. maculipennis s.s. (N = 105), An. daciae (N = 62), An. atroparvus (N = 6) and An. messeae (N = 2). Each species was characterized by unique ITS2 haplotypes, whereas COI only supported the monophyly of An. atroparvus, a historical malaria vector in Belgium. Species identification results were further supported by unique PCR-RFLP banding patterns. We report for the first time An. daciae in Belgium, where it was found to co-occur with An. maculipennis s.s. The latter was the most prevalent in the collection studied (60%) and appears to have the widest distribution in Belgium. As in other studies, An. daciae and An. messeae appeared the most closely related species, up to the point that their species status remains debatable, while their ecological differences, including vector competences, need further study.

A new tool for the molecular identification of Culicoides species of the Obsoletus group: the glass slide microarray approach.

Culicoides species of the Obsoletus group (Diptera: Ceratopogonidae) are potential vectors of bluetongue virus serotype 8 (BTV 8), which was introduced into central Western Europe in 2006. Correct morphological species identification of Obsoletus group females is especially difficult and molecular identification is the method of choice. In this study we present a new molecular tool based on probe hybridization using a DNA microarray format to identify Culicoides species of the Obsoletus group. The internal transcribed spacer 1 (ITS1) gene sequences of 55 Culicoides belonging to 13 different species were determined and used, together with 19 Culicoides ITS1 sequences sourced from GenBank, to design species-specific probes for the microarray test. This test was evaluated using the amplified ITS1 sequences of another 85 Culicoides specimens, belonging to 11 species. The microarray test successfully identified all samples (100%) of the Obsoletus group, identifying each specimen to species level within the group. This test has several advantages over existing polymerase chain reaction (PCR)-based molecular tools, including possible capability for parallel analysis of many species, high sensitivity and specificity, and low background signal noise. Hand-spotting of the microarray slide and the use of detection chemistry make this alternative technique affordable and feasible for any diagnostic laboratory with PCR facilities.

Compassionate use programme of irinotecan in colorectal cancer patients in The Netherlands.

BACKGROUND: Irinotecan is an effective treatment for metastatic colorectal cancer. However, its use may be associated with troublesome adverse effects such as delayed diarrhoea, acute cholinergic syndrome and neutropenic infection. The manufacturer decided to release irinotecan for compassionate use in The Netherlands prior to its regulatory approval (June 1998) and first introduction for second-line treatment of metastatic colorectal cancer. In view of the drug's adverse effect profile this was done in a carefully controlled manner. METHODS: Irinotecan was made available to patients with colorectal cancer with elaborate precautions. Treating physicians requesting irinotecan for compassionate use received a protocol, providing recommendations for the proper use and the prevention/management of potentially troublesome adverse events. Limited demographic, toxicity and efficacy data were collected. RESULTS: Between June 1997 and September 1998, 112 patients were registered for this programme, 103 of whom actually received irinotecan. The percentage of patients experiencing grade 3-4 adverse effects was relatively low: delayed diarrhoea in 17%, nausea and vomiting 17%, acute cholinergic syndrome 6%, febrile neutropenia 4% and neutropenic infection 2%. Five partial tumour responses and a high proportion of patients with 'no change' were noted. CONCLUSIONS: The carefully controlled release of irinotecan for compassionate use with a very detailed protocol for guidance and advice on safety precautions seems to have contributed to the relatively safe use of the drug outside the setting of a formal clinical trial.

Coordination and stability of one-legged hopping patterns in patients with anterior cruciate ligament reconstruction: preliminary results.

OBJECTIVES: To investigate whether the intralimb coordination of the lower extremity during one-legged hopping in patients with anterior cruciate ligament reconstruction is different and less stable compared to control subjects. DESIGN: Measures of coordination dynamics are applied to study the coordination and stability of movement patterns of the knee and ankle in patients and control subjects. BACKGROUND: Due to several factors, such as loss of proprioceptive function and muscle weakness, the intralimb coordination of the lower extremity might be altered. METHODS: Seven patients and 13 healthy control subjects were instructed to hop in place on one leg for 10 seconds at a comfortable frequency for each leg separately. RESULTS AND CONCLUSIONS: The one-legged hopping movement pattern of patients with anterior cruciate ligament reconstruction one-year post-operative is different and less stable compared to healthy subjects, and thus is still impaired. RELEVANCE: This paper shows that patients, one year after reconstruction of the anterior cruciate ligament, have a different coordination pattern of the lower limb compared to a matched control group. Intersegmental coordination and stability, features that are often observed by eye during a rehabilitation process, are objectified in this study.

Prognostic impact of urokinase-type plasminogen activator receptor (uPAR) in cytosols and pellet extracts derived from primary breast tumours.

Using a previously developed enzyme-linked immunosorbent assay (ELISA), the levels of the receptor for urokinase-type plasminogen activator (uPAR) were determined in cytosols and corresponding membrane pellets derived from 878 primary breast tumours. The levels of uPAR in the pellet extracts were more than 3-fold higher than those measured in the cytosols (P< 0.001). Moreover, the uPAR levels in the two types of extracts were weakly, though significantly, correlated with each other (rS= 0.20, P< 0.001). In Cox univariate analysis, high cytosolic levels of uPAR were significantly associated with reduced overall survival (OS) and relapse-free survival (RFS). The levels of uPAR in pellet extracts appeared not to be related with patient survival. In multivariate analysis, elevated levels of uPAR measured in cytosols and pellet extracts were found to be independent predictors of poor OS, not RFS. The prediction of poor prognosis on the basis of high uPAR levels emphasizes its important role in plasmin-mediated degradation of extracellular matrix proteins during cancer invasion and metastasis.

The analgesic efficacy of tramadol is impaired by concurrent administration of ondansetron.

Tramadol has weak opioid properties, and an analgesic effect that is mediated mainly by inhibition of the reuptake of norepinephrine and serotonin (5-hydroxytryptamine [5-HT]) and facilitation of 5-HT release (1,2) at the spinal cord. Because 5-HT3 receptors play a key role in pain transmission at the spinal level (3), the 5-HT3 antagonist ondansetron may decrease the efficacy of tramadol, as suggested in an abstract by Maroof et al. In that study, a small dose of 1 mg/kg tramadol was administered along with ondansetron 0.1 mg/kg or placebo, 15 min before the induction of anesthesia. Early postoperative pain scored differed significantly between the test groups. We therefore tested the hypothesis that the tramadol requirement by patient-controlled analgesia (PCA) may be increased when ondansetron is administered for antiemetic prophylaxis.

Closed intramedullary tibial nailing using the Marchetti-Vicenzi nail.

We present the results of using the Marchetti-Vicenzi nail in the management of 41 closed and 16 type I-III open tibial fractures in 56 patients. The treatment goals were to achieve stability and fracture alignment allowing early functional treatment of the knee and ankle by immediate partial or full weight-bearing. Early callus formation and rapid mobilisation were obtained in 84% of the patients. The mean time to clinical union was 9.8 weeks (radiological union 28.4 weeks) for closed fractures and also 9.8 weeks for open fractures (radiological union 28.7 weeks). No late rotational deformities were observed. We argue that intramedullary nailing with a Marchetti-Vicenzi nail is a quick and excellent method of treating selected closed and open tibial fractures.

Prognostic value of tissue-type plasminogen activator (tPA) and its complex with the type-1 inhibitor (PAI-1) in breast cancer.

The prognostic value of tissue-type plasminogen activator (tPA) measured in samples derived from 865 patients with primary breast cancer using a recently developed enzyme-linked immunosorbent assay (ELISA) was evaluated. Since the assay could easily be adapted to the assessment of the complex of tPA with its type-1 inhibitor (PAI-1), it was investigated whether the tPA:PAI-1 complex also provides prognostic information. To this end, cytosolic extracts and corresponding detergent extracts of 100,000 g pellets obtained after ultracentrifugation when preparing the cytosolic fractions for routine steroid hormone receptor determination were assayed. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient r(s) = 0.75, P < 0.001 for tPA and r = 0.50, P < 0.001 for tPA:PAI-1 complex). In both Cox univariate and multivariate analysis elevated levels of (total) tPA determined in the pellet extracts, but not in cytosols, were associated with prolonged relapse-free (RFS) and overall survival (OS). In contrast, high levels of the tPA:PAI-1 complex measured in cytosols, but not in the pellet extracts, were associated with a poor RFS and OS. The prognostic information provided by the cytosolic tPA:PAI-1 complex was comparable to that provided by cytosolic (total) PAI-1. Furthermore, the estimated levels of free, uncomplexed tPA and PAI-1, in cytosols and in pellet extracts, were related to patient prognosis in a similar way as the (total) levels of tPA and PAI-1 respectively. Determination of specific forms of components of the plasminogen activation system, i.e. tPA:PAI-1 complex and free, uncomplexed tPA and/or PAI-1, may be considered a useful adjunct to the analyses of the separate components (tPA and/or PAI-1) and provide valuable additional prognostic information with respect to survival of breast cancer patients.

Prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) in cytosols and pellet extracts derived from 892 breast cancer patients.

To evaluate the clinical relevance of urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) measured by a recently developed enzyme-linked immunosorbent assay (ELISA), we analysed both components in samples derived from 892 patients with primary breast cancer (median follow-up 99 months). The assays were performed in cytosolic extracts as well as in corresponding detergent extracts of pellets obtained after ultracentrifugation, which was carried out when preparing the cytosolic fractions for routine steroid hormone receptor determination. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient r = 0.60, P < 0.0001 for uPA and r = 0.65, P < 0.0001 for PAI-1). Furthermore, strong correlations were found between the levels of both uPA (r = 0.85, P < 0.0001) and PAI-1 (r = 0.90, P< 0.0001) in the cytosols and their levels previously measured with ELISAs based on commercial reagents. In both Cox univariate and multivariate analysis, high cytosolic levels of uPA or PAI-1 were significantly associated with increased rates of relapse and death. The levels of uPA and PAI-1 in the pellet extracts also provided prognostic information, although to a lesser extent compared with the cytosolic extracts. The prediction of prognosis on the basis of uPA and PAI-1 assessed by an alternative ELISA once again emphasizes the established prognostic role and usefulness of these parameters in selection of breast cancer patients at high or low risk of recurrence.

External quality assessment of trans-European multicentre antigen determinations (enzyme-linked immunosorbent assay) of urokinase-type plasminogen activator (uPA) and its type 1 inhibitor (PAI-1) in human breast cancer tissue extracts.

High levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in breast cancer tissue extracts have been associated with rapid disease progression. In these studies, different enzyme-linked immunosorbent assay (ELISA) kits have been applied for the quantification, and consequently the ranges of uPA and PAI-1 levels reported differ considerably. Therefore, the Receptor and Biomarker Study Group (RBSG) of the European Organization for Research and Treatment of Cancer (EORTC) and a consortium of the BIOMED-1 project 'Clinical Relevance of Proteases in Tumor Invasion and Metastasis' initiated three collaborative between-laboratory assessment trials aimed at controlling uPA and PAI-1 antigen analyses. For this purpose, two control preparations were produced from different sources: pooled human breast cancer specimens (QC-240893) and human breast cancer xenografts raised in nude mice (QC-101094). The lyophilized preparations were stable for prolonged times (at least 3 and 27 months respectively) at 4 degrees C. Furthermore, a good parallelism following dilution was found for uPA and PAI-1. The data from QC trial no. 1 clearly indicated that acceptable between-laboratory coefficients of variation (CVs) for uPA (<8.2%) and PAI-1 (<16.6%) in QC-240893 could be achieved when the same type of ELISA kit (American Diagnostica) was used. From the second trial, in which ten EORTC laboratories each received five identical lyophilized QC-101094 samples, it appeared that the within-laboratory variations for uPA and PAI-1 determinations obtained by 'experienced' laboratories were lower (<12.9%) than those from non-experienced laboratories (<36.4%). In a third QC trial, five BIOMED-1 laboratories, all of which employed ELISA procedures for uPA and PAI-1, participated in six subsequent quality assessment rounds receiving five samples of QC-101094. Although for each laboratory the within-run CVs for uPA as well as for PAI-1 were low (<7.8%), the between-run CVs were found to be considerably higher (up to 56.2% for uPA and to 27.6% for PAI-1). Consequently, because of the different ELISA formats used, the absolute analyte values measured in the different laboratories varied substantially. The use of 'common external standards' in the different ELISAs resulted in a significant reduction of the between-laboratory CVs from 61.3% to 15.7% (uPA) and from 42.1% to 19.1% (PAI-1). The present data demonstrate that in multicentre studies the same ELISA kit should be used, and that external quality assurance (QA) is mandatory. Furthermore, it appears from the present study that standardization of the protein assay as a tissular parameter is imperative.

Tramadol reduces the sweating, vasoconstriction, and shivering thresholds.

UNLABELLED: The analgesic tramadol inhibits the neuronal reuptake of norepinephrine and 5-hydroxytryptamine, facilitates 5-hydroxytryptamine release, and activates mu-opioid receptors. Each of these actions is likely to influence thermoregulatory control. We therefore tested the hypothesis that tramadol inhibits thermoregulatory control. Eight volunteers were evaluated on four study days, on which they received no drugs, tramadol 125 mg, tramadol 250 mg, and tramadol 250 mg with naloxone, respectively. Skin and core temperatures were gradually increased until sweating was observed and then decreased until vasoconstriction and shivering were detected. The core temperature triggering each response defined its threshold. Tramadol decreased the sweating threshold by -1.03 +/- 0.67 degrees C microgram-1.mL (r2 = 0.90 +/- 0.12). Tramadol also decreased the vasoconstriction threshold by -3.0 +/- 4.0 degrees C microgram-1.mL (r2 = 0.94 +/- 0.98) and the shivering threshold by -4.2 +/- 4.0 degrees C microgram-1.mL(r2 = 0.98 +/- 0.98). The sweating to vasoconstriction interthreshold range nearly doubled from 0.3 +/- 0.4 degree C to 0.7 +/- 0.6 degree C during the administration of large-dose tramadol (P = 0.04). The addition of naloxone only partially reversed the thermoregulatory effects of tramadol. The thermoregulatory effects of tramadol thus most resemble those of midazolam, another drug that slightly decreases the thresholds triggering all three major autonomic thermoregulatory defenses. In this respect, both drugs reduce the "setpoint" rather than produce a generalized impairment of thermoregulatory control. Nonetheless, tramadol nearly doubled the interthreshold range at a concentration near 200 ng/mL. This indicates that tramadol slightly decreases the precision of thermoregulatory control in addition to reducing the setpoint. IMPLICATIONS: The authors evaluated the effects of the analgesic tramadol on the three major thermoregulatory responses: sweating, vasoconstriction, and shivering. Tramadol had only slight thermoregulatory effects. Its use is thus unlikely to provoke hypothermia or to facilitate fever.

Desflurane reduces the febrile response to administration of interleukin-2.

BACKGROUND: Intraoperative fever is relatively rare considering how often pyrogenic causes are likely to be present and how common fever is postoperatively. This low incidence suggests that general anesthesia per se inhibits the normal response to pyrogenic stimulation. The authors therefore tested the hypothesis that desflurane-induced anesthesia produces a dose-dependent inhibition of the febrile response. METHODS: Eight volunteers were studied, each on 3 study days. Each was given an intravenous injection of 50,000 IU/ kg of interleukin-2 (elapsed time, 0 h), followed 2 h later by 100,000 IU/kg. One hour after the second dose, the volunteers were assigned randomly to three doses of desflurane to induce anesthesia: (1) 0.0 minimum alveolar concentration (MAC; control), (2) 0.6 MAC, and (3) 1.0 MAC. Anesthesia continued for 5 h. Core temperatures were recorded from the tympanic membrane. Thermoregulatory vasoconstriction was evaluated using forearm-minus-fingertip skin temperature gradients; shivering was evaluated with electromyography. Integrated and peak temperatures during anesthesia were compared with repeated-measures analysis of variance and Scheffé's F tests. RESULTS: Values are presented as mean +/- SD. Desflurane reduced the integrated (area under the curve) febrile response to pyrogen, from 7.7 +/- 2.0 degrees C x h on the control day to 2.1 +/- 2.3 degrees C x h during 0.6 MAC and to -1.4 +/- 3.1 degrees C x h during 1.0 MAC desflurane-induced anesthesia. Peak core temperature (elapsed time, 5-8 h) decreased in a dose-dependent fashion: 38.6 +/- 0.5 degrees C on the control day, 37.7 +/- 0.7 degrees C during 0.6 MAC and 37.2 +/- 1.0 degrees C during 1.0 MAC desflurane anesthesia. Rising core temperature was always associated with fingertip vasoconstriction and often with shivering. CONCLUSIONS: Desflurane-induced anesthesia produced a dose-dependent decrease in integrated and peak core temperatures after administration of pyrogen, with 1.0 MAC essentially obliterating fever. Anesthetic-induced inhibition of the pyrogenic response is therefore one reason that fever is an inconsistent clinical response to inflammation during surgery.

Post-operative effects of tramadol administered at wound closure.

The aim of this prospective, randomized and double-blind study was to assess the effects of a high dose of the analgesic tramadol administered at the conclusion of surgery on extubation time, sedation, and post-anaesthetic shivering. Forty adult patients, ASA physical status I or II, underwent laparoscopic surgery of about 1 h duration and received a standardized anaesthesia that was maintained with isoflurane in O2/N2O. Tramadol 3 mg kg-1 (n = 20) was administered intravenously at the beginning of wound closure, and was compared with saline (n = 20). Post-anaesthetic shivering did not occur in any patient who received tramadol, whereas it occurred in 60% of the control group (P < 0.001). There were no adverse effects on time to extubation and sedation, and discharge-ready time was shorter in the tramadol group (P < 0.05 compared with control). Pain scores in the post-anaesthesia care unit (PACU) were statistically not different between the two groups, but significantly more supplemental medication was administered in the control group to treat shivering and/or pain. In conclusion, administration of a high dose of tramadol at the end of surgery prevents post-anaesthetic shivering without prolongation of extubation time, and shortens the PACU/discharge-ready time.

The status of genetic material and genetic information in The Netherlands.

The moral status of genetic material and information, and the ethics of controlling and manipulating them, is a topic of hot debate in many European countries, including The Netherlands. That heat is due partly to the complexity of the topic, and partly to researchers' fear that their investigations will be hampered by restrictions on the use of personal data or body material. But there is little doubt that manifold diverging interpretations about the status of the human body, body materials, and personal information in Dutch law, written and unwritten, contribute to the intensity of the debates. This article intends to structure the debate by creating more clarity at the conceptual level. By carefully examining relevant articles of the Constitution and Civil Codes, as well as policy documents and authoritative publications, notably in reference to prominent legal concepts such as property, ownership and privacy, and answer should be provided to the following crucial question: is the status of genetic material and information in any sense special in comparison with other body parts and other kinds of information about a person? This paper first discusses the status of human body materials and personal information in general, and then continues with a more specific discussion about the status of genetic material and information. It concludes that the Dutch legislature had carefully avoided (or not felt the need to employ) the concept of ownership in regulating biomedical research; rather, privacy is found to be the prime regulatory concept.

Ownership of genetic material and information.

As a result of the International Human Genome Project genetic information is rapidly multiplying. To avoid some of the problems regarding the availability and use of genetic information, it is sometimes suggested to apply the concept of ownership. This article focuses on the clarification of the status of genetic material and genetic information, obtained as a result of screening and counseling of individual patients. First, some philosophical theories of ownership are examined for a justification of the use of the concept of ownership with regard to the human body. Next, arguments with regard to ownership of the human body are examined. The results of this analysis are applied to genetic material and genetic information.

Tramadol in the treatment of postanesthetic shivering.

BACKGROUND: As an inhibitor of the reuptake of serotonin and norepinephrine in the spinal cord, the mechanism of action of tramadol resembles that of nefopam, which has been used in the treatment of postanesthetic shivering. METHODS: In a randomized, placebo-controlled, double-blind study, we assessed the effects of tramadol (0.5 mg.kg-1, 1 mg.kg-1 and 2 mg.kg-1 i.v.) or normal saline on shivering after a standardized general anesthesia in 40 adult patients, ASA physical status I or II (group 1), and in 64 adult patients regardless of the foregoing general anesthesia and ASA physical status (group 2). RESULTS: Tramadol 1 mg.kg-1 or more abolished shivering completely 5 min after treatment in all patients of groups 1 and 2. In group 1, the three dosages of tramadol were not statistically different in lowering the severity and prevalence of postanesthetic shivering. Tramadol 0.5 mg.kg-1 was significantly slower than tramadol 1 or 2 mg.kg-1 in tempering the severity as well as lowering the prevalence of postanesthetic shivering in group 2. CONCLUSION: Tramadol's distinct features in the treatment of shivering reside in its high safety profile and weak sedative properties, particularly in patients with poor cardiorespiratory reserve, in outpatients and on recurrence of shivering.

Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): report of an EORTC/BIOMED-1 workshop.

The urokinase-type plasminogen activator (uPA) is considered to play a key role in the process of invasion and metastasis. In several independent studies, in a variety of cancer types (e.g. of the breast, colon, stomach, lung, ovary), high antigen levels of uPA in tumour extracts have been associated with rapid disease progression. In these studies, different sets of antibodies and standards (often as commercially available uPA ELISA kits) have been used. The standards provided with the different uPA ELISA kits are different from each other in both composition and source. In addition, the different uPA ELISA kits use antibodies which differ in specificity and affinity for the various forms of uPA including pro-uPA, HMW-uPA, LMW-uPA, the aminoterminal fragment (ATF) and complexes with inhibitors (PAI-1 and PAI-2) and the receptor (uPAR). Further, the composition of tumour tissue extraction buffers differ significantly among the published studies. Thus, it is not surprising that the ranges of cytosolic uPA levels reported differ considerably even when measured within the same tumour type. These discrepancies led the EORTC Receptor and Biomarker Study Group, in conjunction with the BIOMED-1 consortium on 'Clinical Relevance of Proteases in Tumour Invasion and Metastasis', to organise a workshop to study the characteristics associated with six different uPA immunoassays (ELISA) used in clinical studies reported in the literature. Although the absolute uPA antigen values measured with the respective uPA ELISA kits differed, high correlations were obtained for any two of the four uPA ELISA kits finally applied to sets of breast cancer cytosol preparations. The preparations used at present as standards in the various uPA ELISA kits are not representative of actual human breast cancer cytosols. Thus absolute standardisation is only possible by using a common reference sample (breast cancer cytosol) and similarly composed ELISA uPA kits. Then it will be possible to generate comparable data on clinical tissue as well as to check for batch-to-batch variations within particular ELISA kits.

Endocarditis of native aortic and mitral valves due to Corynebacterium accolens: report of a case and application of phenotypic and genotypic techniques for identification.

Endocarditis of native aortic and mitral valves due to an organism identified as Corynebacterium accolens developed in a 73-year-old patient without predisposing factors. The organism was identified as C. accolens by biochemical identification, amplified rRNA gene restriction analysis, and DNA-DNA hybridization. This is the first case of C. accolens endocarditis reported, adding to the increasing number of Corynebacterium-related cases of endocarditis.

Technical note: the use of arterialized arm veins in infrainguinal bypass.

In order to augment the vein donation area of arm veins for infrainguinal bypass, we performed an arteriovenous AV fistula prior to the revascularization procedure. This technique was used in 5 patients. The diameter enlargement of the vein obtained by this technique enabled us to construct completely autologous grafts with gratifying results.