Correction of underquantification of human immunodeficiency virus type 1 load with the second version of the Roche Cobas AmpliPrep/Cobas TaqMan assay.

Initial evaluations of the Cobas AmpliPrep/Cobas TaqMan human immunodeficiency virus type 1 (HIV-1) test (CAP/CTM) demonstrated good performance but, afterwards, reports about underquantification were published. We investigated whether the problem was solved with a second version of this assay, the Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0 (CAP/CTM v2.0). The remaining plasma of 375 consecutive HIV-1 positive samples with a viral load of >or=4,000 copies/ml was collected in three laboratories. The samples were diluted and retested with our routine method Cobas AmpliPrep/Cobas Amplicor HIV-1 monitor test v1.5 in ultrasensitive mode (CAP/CA PHS), as well as with the CAP/CTM and CAP/CTM v2.0 tests. An absolute difference between the results of two methods of >or=0.71 log(10) copies/ml was defined as moderately discrepant, and an absolute difference of >or=0.93 log(10) copies/ml was defined as severely discrepant. In addition, criteria for considering the new methods equivalent to the routine method were formulated. (i) For CAP/CTM compared to CAP/CA PHS, 36 (9.5%) and 20 (5.3%) samples were, respectively, considered moderately and severely underquantified by CAP/CTM. The mean difference between CAP/CTM and CAP/CA PHS was -0.32 log(10) copies/ml. Eight of nineteen of the severely underquantified samples were from patients infected with HIV-1 subtype B strain. (ii) For CAP/CTM v2.0 compared to CAP/CA PHS, no sample was moderately or severely underquantified by CAP/CTM v2.0. A mean difference of 0.08 log(10) copies/ml was found with CAP/CTM v2.0 compared to CAP/CA PHS. The underquantification problem of the CAP/CTM kit was clearly demonstrated. The criteria for the equivalence of CAP/CTM v2.0 to the routine test CAP/CA PHS were fulfilled.

Prevalence of subtilase cytotoxin in verocytotoxin-producing Escherichia coli isolated from humans and raw meats in Belgium.

Recently, subtilase cytotoxin (SubAB) was detected in verocytotoxin-producing Escherichia coli (VTEC) that do not carry the Locus of Enterocyte Effacement (LEE) pathogenicity island. The distribution of the subA gene in VTEC isolated from patients with the hemolytic uremic syndrome, patients with diarrheal disease and raw meats from ruminants and wildlife in Belgium was investigated with PCR. The subA gene was detected more frequently (χ (2) = 10.2; d.f. = 1; P = 0.001) in VTEC from raw meats (10 of 87 strains) than in those from humans (8 of 274 strains), and never in serogroups O157, O26, O103, O111 and O145. This virulence marker could play a role in the development of HUS after infection with LEE-negative VTEC but was only found in one O178:H19 isolate out of 36 HUS-associated VTEC strains.

Outbreak of multidrug-resistant Acinetobacter baumannii in a Belgian university hospital after transfer of patients from Greece.

Multidrug-resistant (MDR) Acinetobacter baumannii are emerging as important nosocomial pathogens. These organisms have a capacity for long-term survival in the hospital environment. The purpose of this study was to describe the course and control of an outbreak with MDR A. baumannii in a Belgian university hospital after transfer of two trauma patients from Greece. Wounds in both patients were colonised with MDR A. baumannii. Over an 11 month period from September 2004 to July 2005, carbapenem-non-susceptible A. baumannii (producing carbapenem-hydrolysing oxacillinase OXA-58) were isolated from 28 patients, despite early implementation of contact precautions. MDR A. baumannii was detected in routine clinical diagnostic samples from 26 patients and in screening specimens from an additional two patients. Twenty patients (71.4%) were colonised or infected during their stay in intensive care. Twenty-four (85.7%) respiratory samples were positive for MDR A. baumannii. Careful review of all procedures related to the respiratory tract did not identify a common route of transmission. Outbreak control required multiple interventions, including contact isolation of colonised and infected patients, monitoring the practice of personnel, screening of asymptomatic patients, use of isolation rooms and enhanced environmental disinfection. Introduction of single-use ventilator circuits was considered but the outbreak was controlled before implementation.

Biologically active bisbenzylisoquinoline alkaloids from the root bark of Epinetrum villosum.

Methanol and water extracts of the root of Epinetrum villosum (Exell) Troupin (Menispermaceae) were found to exhibit antimicrobial and antiplasmodial activities. Investigation of the active methanol fraction led to the isolation of four bisbenzylisoquinoline alkaloids, i.e., cycleanine, cycleanine N-oxide, isochondodendrine and cocsoline. Structures were established by spectroscopic methods. Cocsoline displayed antibacterial and antifungal activities (MIC values of 1000-15.62 and 31.25 microg/ml, respectively). Isochondodendrine was found to have the most potent antiplasmodial activity (IC50 = 0.10 microg/ml), whereas the IC50 on HCT-116 human colon carcinoma cells was 17.5 microg/ml (selectivity index 175). Cycleanine acted against HIV-2 (EC50=1.83 microg/ml) but was at least 10-fold less active against HIV-1. Cycleanine N-oxide showed no activity towards all tested microorganisms.

The clinical impact of early gram-positive bacteremia and the use of vancomycin after allogeneic bone marrow transplantation.

BACKGROUND: Gram-positive bacteremia (GPB) is an increasing infection after allogeneic bone marrow transplantation (BMT). Our purpose was to identify risk factors for GPB, to evaluate its impact on early mortality and morbidity, and to compare prophylactic with empirical intravenous vancomycin. METHODS AND RESULTS: We studied 89 consecutive BMTs in adult patients. Early GPB occurred in 29% of posttransplantation episodes. T-cell depletion (odds ratio [OR]: 0.18) and vancomycin-prophylaxis (OR: 0.28) reduced the risk of GPB. Mortality at 6 weeks was not significantly different in patients with GPB (15% vs. 9.5%, P = 0.669). GPB was associated with the development of major complications, the use of amphotericin B, and prolonged neutropenia. Vancomycin prophylaxis led to an increased risk of early renal dysfunction (OR: 18.7). CONCLUSION: GPB contributes to overall morbidity during the early post-BMT episode but has no impact on mortality. Vancomycin prophylaxis is effective to reduce GPB but has a negative effect on renal function.

Contribution of the 13C-urea breath test to the detection of Helicobacter pylori gastritis in children.

Serology, 13C-urea breath test, histology, Campylobacter-like organism testing, and culture were performed in 95 consecutive children to evaluate the contribution of these tests to the detection of Helicobacter pylori infection. In analyses considering any combination of three positive tests as "gold standard" for diagnosing H pylori infection, 26 children were Helicobacter positive (27%), which is only one patient more than the number of children with only a positive culture. The accuracy of culture was excellent when "any combination of three positive tests" was used as the gold standard (sensitivity 96%, specificity 100%, positive predictive value 100% [false positivity 0%], negative predictive value 99% [false-negative results 1%]). The results of invasive and noninvasive tests were comparable. When culture was considered as "gold standard," the sensitivity of serology and 13C-urea breath test was 96%; the specificity was 96% and 93%, respectively; the positive predictive value was 89% and 83% (false-positive results in 11% and 17%); and the negative predictive value for both was 99% (false-negative results in 1%). It is concluded that culture can be used as gold standard, but that non-invasive tests such as serology and/or 13C-urea breath test can be used to diagnose H pylori infection in children, since each has at least 95% sensitivity and 92% specificity.

PCR-mediated DNA typing of Campylobacter jejuni isolated from patients with recurrent infections.

The polymerase chain reaction (PCR) and two primers aiming at the enterobacterial repetitive intergenic consensus (ERIC) and arbitrary DNA sequences, respectively, were used to fingerprint the genomic DNA of 24 Campylobacter jejuni strains isolated from five patients with recurrent C. jejuni infections. Results were compared with biotyping and serotyping. The latter two methods, when combined, distinguished 9 different types, whereas PCR-mediated DNA analysis discriminated 14 different patterns. For six strains, the results of PCR-mediated typing led to different interpretations. This method is proposed as an additional tool to further discriminate between C. jejuni strains that appear related by conventional typing methods. In view of its rapidity and simplicity, this method is a potential candidate to replace the relatively slow and laborious conventional methods. However, further study is needed to assess the sensitivity and specificity of PCR-mediated DNA analysis and to investigate the usefulness of this method as an epidemiological tool in outbreaks of Campylobacter infections.

Chronic Ureaplasma urealyticum amnionitis associated with abruptio placentae.

Ureaplasma urealyticum is a common inhabitant of the lower genital tract of women. It is unclear whether or not the microorganism plays a role in provoking spontaneous abortion. Reported is a U urealyticum infection of placenta and amniotic fluid in an immunologically competent host resulting in abruptio placentae and spontaneous abortion during the second trimester of pregnancy. U urealyticum was isolated from peripheral maternal blood twice. Immunologic alterations, namely a transiently reversed OKT4/OKT8 rate and a decrease in immunoglobulin G levels, were detected in the patient. U urealyticum must be considered as a pathogen able to interfere with normal fetal development.

Impact of primary prevention on the incidence of toxoplasmosis during pregnancy.

Until now, it was assumed that primary prevention of congenital toxoplasmosis was possible by means of specific hygienic measures. A prospective survey of pregnant women was made at a hospital in Brussels over the period 1979-1986 to assess the impact of such a prevention program. In the first study period (1979-1982), when no prophylactic measures were taught, 2986 consecutive women demonstrated a seroconversion rate of 1.43% among the nonimmunized subjects; 1.07% of the seropositive patients had high antibody levels in their first serum sample. In the second study period (1983-1986), all 3563 patients were instructed to adopt prophylactic measures. The seroconversion rate in seronegative patients and the percentage of patients with high initial antibody level were 0.95 and 1.26%, respectively. Although the percentage of seroconversion was reduced by 34% in the second study period, this difference did not attain significance. These results indicate that the impact of a primary prevention program aimed at reducing congenital toxoplasmosis is limited.

Isolation of HIV-1 RNA from plasma: evaluation of eight different extraction methods.

The efficacy of eight different methods for the extraction of HIV-1 RNA from plasma was compared. The RNA preparation method that gave the best results by RT-PCR was the one described by Chomczynski and Sacchi (1987, Anal. Biochem. 162, 156-159). This method consists of a guanidine thiocyanate treatment followed by three phenol-chloroform-isoamylalcohol extractions and an ethanol precipitation. The disadvantage of this method is that it is time consuming and less suitable for the extraction of large series of samples. Moreover, due to the large number of procedural steps, there is a greater risk of sample mix-up or contamination. Of the single-step RNA purification methods, good results were obtained with the TRIzol method (Gibco Life Technologies, Paisley, UK) and with the extraction method offered by the NASBA kit (Organon Teknika, Turnhout, Belgium). The above single-step methods are recommended since both are sensitive enough to detect low copy numbers of HIV-RNA in the plasma of asymptomatic patients, and require only 2 h for completion. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extraction varied between 17.3 and 47.3%). Inter-laboratory reproducibility was evaluated only for the TRIzol-method and found to be low (mean variation coefficient 63.4).

Blinded, multicenter quality control study for the quantification of human immunodeficiency virus type 1 RNA in plasma by the Belgian AIDS reference laboratories.

OBJECTIVE: In order to evaluate the interlaboratory variation of HIV-1 RNA measurements in plasma, the Belgian AIDS reference laboratories organized a blinded multicenter quality control study. METHODS: Atest panel of coded spiked HIV-1 plasma samples reflecting the dynamic range of the assay was composed and distributed. The HIV-1 RNA concentration of these samples was determined by the eight Belgian AIDS reference laboratories by means of the Amplicor HIV-1 Monitor version 1.5 assay. RESULTS: Analysis of the results demonstrated that there was little interlaboratory variation for the high concentration range (4.0-5.7 log10 copies/mL), never exceeding 0.2 log10 copies/mL. However the standard deviation for the low concentration range (2.6-3.9 log10 copies/mL) reached up to 0.22 log10 copies/mL. CONCLUSIONS: Since interlaboratory variability never reached 0.5 log10 copies/mL and each of the laboratories was able to detect four-fold differences in plasma HIV-1 RNA levels, the Amplicor assay can be used in multicenter studies without a centralized analysis of samples. Furthermore, this well-characterized proficiency panel of spiked plasma samples could be used as a standard in the study of interassay comparisons.

Recovery of microorganisms in semen and relationship to semen evaluation.

Semen samples from 120 consecutive unselected men attending our fertility clinic were studied to determine the bacterial isolation rate of semen and its influence on semen quality. Each sample was cultured aerobically, anaerobically, and for Ureaplasma urealyticum and Mycoplasma hominis. The following were analyzed for each semen specimen: motility, morphologic features, and number of sperm cells and viscosity of the ejaculate. Four of the 120 samples had negative results; 101 cultures yielded one or more aerobic organisms (the majority with less than 20,000 microorganisms/ml); 26 yielded anaerobic organisms; and 40 yielded U. urealyticum. No single aerobic or anaerobic organism could be related to abnormal semen samples. Only the presence of U. urealyticum correlated significantly with abnormal semen samples (P less than 0.005). The most affected parameters were the number of spermatozoa (P less than 0.005) and motility (P less than 0.05). We conclude that routine aerobic and anaerobic bacterial cultures of semen are not useful in the clinical evaluation of male infertility. The usefulness of routine screenings for U. urealyticum must be investigated further.

Neonatal infections with Pseudomonas aeruginosa associated with a water-bath used to thaw fresh frozen plasma.

In our 15-bed neonatal intensive care unit (NICU), four new-borns were found to be colonized or infected with Pseudomonas aeruginosa within a period of one week. To identify the outbreak source, three independent studies were performed: epidemiological investigation, environmental surveillance and genotypic typing of isolates. Although epidemiological investigation by a case-control study revealed no conclusive results, the transfusion of fresh frozen plasma (FFP) and human albumin (HA) appeared to be the factor with highest risk. Environmental surveillance and random amplification of polymorphic DNA (RAPD) of isolates identified a water-bath used to warm FFP and HA as the likely reservoir for the outbreak. Further spread of the organism did not occur after elimination of this water-bath from the NICU. RAPD identified in addition an isolate from an infant hospitalized in the NICU five months before the outbreak with a pattern matching the one of the outbreak cluster.

The national prevalence survey of nosocomial infections in Belgium, 1984.

A national one-day prevalence survey of nosocomial infections was carried out in March 1984 in 106 Belgian acute-care hospitals involving 8723 patients of whom 6130 had undergone surgery. Three infections were studied: surgical wound infection, bacteraemia and urinary-tract infection. One or more of these three infections was recorded in 9.3% of all patients and in 11.8% of surgical patients. Prevalences increased with increasing duration of hospital stay and with higher ages, but the association of HAI with age was no longer significant after correction for duration of hospital stay. Prevalences varied considerably in different specialties. After adjustment for age and duration of stay, there was no association between perioperative antibiotic prophylaxis and the prevalence of the infections studied, but bias due to selection of higher risk patients in the antibiotic group was probable. Larger hospitals had a higher overall prevalence, but populations differed according to the size of the hospital. Bacteraemia was strongly associated with the presence of an intravenous catheter, and urinary-tract infection with a urinary catheter.

In vitro susceptibility of Enterococcus faecium isolated from food to growth-promoting and therapeutic antibiotics.

A total of 76 E. faecium strains, isolated at retail level from raw poultry meat, cheese, raw pork, and preparations of cheese and raw pork, were tested for their susceptibility and resistance to growth-promoting antibacterials used in animals and antibiotics used therapeutically in humans. All strains were uniformly susceptible to the growth promoters bambermycin and avilamycin. Resistance against bacitracin, virginiamycin and narasin was high among strains from poultry meat. With tylosin, a macrolide antibiotic used therapeutically and for growth promotion, resistance was mainly detected in strains originating from poultry meat, though also in some strains from pork and from pork and cheese preparations. The therapeutic antibiotic dalfopristin/quinupristin did not show full cross-resistance with the growth-promoting antibiotic virginiamycin. With dalfopristin/quinupristin two different levels of resistance were found. Only one E. faecium strain isolated from poultry was resistant to the glycopeptides avoparcin and vancomycin. Only one poultry meat strain was highly resistant to ampicillin. However, nearly all poultry meat strains showed decreased sensitivity. Only 3 out of 24 poultry strains were susceptible to minocycline, while all strains from other origins were susceptible to this tetracycline antibiotic. High-level streptomycin resistance was seen in strains of all origins, though infrequently. High-level gentamicin resistance was not found.

Rapid diagnosis of bacterial meningitis.

The diagnostic value of several investigations which may demonstrate bacteria or bacterial products in cerebrospinal fluid (CSF) samples from patients with meningitis are discussed. Estimation of CSF lactate and lactate dehydrogenase levels was found to be of value in the differential diagnosis of viral, bacterial and fungal meningitis and the detection of endotoxin by the Limulus amoebocyte lysate test was shown to be strongly suggestive of Gram-negative meningitis. The demonstration of bacterial capsular polysaccharides in CSF by counterimmuno-electrophoresis, latex agglutination and ELISA was of value in establishing a precise aetiological diagnosis, but the usefulness of these methods was limited by the lack of general availability of specific high-potency antisera which determine the sensitivity of the procedure. These screening tests do not replace standard analysis of CSF but provide useful ancillary evidence of meningitis. Negative results obtained from screening tests should not exclude a diagnosis of bacterial meningitis and a decision to withhold treatment should only be made after all available CSF results are evaluated in conjunction with the clinical features.

Quantitative RT-PCR ELISA to determine the amount and ratio of positive- and negative strand viral RNA synthesis and the effect of guanidine in poliovirus infected cells.

Quantitative reverse transcription-polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR ELISA) is the method of choice to study positive- and negative strand viral RNA synthesis during poliovirus replication. In comparison with other methods used for this purpose, it fulfils all necessary requirements to accurately determine RNA of different polarity. It combines high specificity, high sensitivity, safety, speed, and the ability to perform quantitative analysis. The enterovirus specific RT-PCR ELISA method described in this work, was used to determine quantitatively the amount of de novo poliovirus positive- and negative strand RNA synthesis at different time-points in the viral replication cycle, both in presence and absence of the viral RNA synthesis inhibitor guanidine hydrochloride.

Screening of an enterovirus specific RT-PCR ELISA method for the quantification of enterovirus genomes in human body fluids by means of a three-level experimental design.

In order to obtain a detection limit as low as possible for a quantitative enterovirus specific RT-PCR ELISA assay, optimal reaction conditions, which give rise to the highest response, need to be determined. This was done by investigating the influence of 13 factors, selected from RT and PCR, in a multivariate approach by means of a well-balanced three-level screening design, derived from a three-level Plackett--Burman design. Optimal reaction conditions could be determined by calculation and evaluation of the effects of the different factors on the response, i.e. the measured absorbance of the ELISA detection. The method will be used to study a possible longitudinal relationship between enteroviruses and the development of multiple sclerosis and juvenile diabetes.

Screening and optimisation of an ELISA method for the quantitative detection of enterovirus specific RT-PCR products by means of a two-level experimental design.

In a previous paper, optimal reaction conditions were determined for the RT-PCR part of a quantitative enterovirus specific RT-PCR ELISA method (J. Pharm. Biomed. Anal., 25 (2001) 131-142). In order to obtain a detection limit as low as possible, the ELISA part of the procedure was optimised as well. This was done by investigating the influence of seven factors at three levels in a multivariate approach. A reflected two-level screening design, derived from a Plackett-Burman design, was used. Optimal reaction conditions were established by calculation and by evaluation of the effects of the factors on the measured absorbance of the ELISA detection. Under these conditions, the linear range and detection limit of the test were determined and compared with the ELISA conditions before optimisation. The optimised RT-PCR ELISA will be used to study a possible longitudinal relationship between enteroviruses and the development of multiple sclerosis and juvenile diabetes.

Temocillin in the treatment of pyelonephritis in children.

Temocillin is a beta-lactamase-stable penicillin with a selective. Gram-negative spectrum of activity and a long half-life. Previous studies in adult patients have demonstrated its efficacy and safety in the treatment of Gram-negative infections. The aim of the study was to evaluate the clinical and bacteriological efficacy and safety of temocillin in children with complicated urinary tract infections. Twenty-two children, aged 3 months to 13 years (mean 5.8 years) were treated with temocillin i.v. at a dose of 25 mg/kg 12 hourly for a mean period of 5.9 days (range 3-12 days). Acute pyelonephritis was diagnosed in 21 patients (one case associated with septicaemia); one patient presented recurrent bacteriuria due to a multiresistant pathogen. Some 20/22 children presented an underlying condition complicating the urinary tract infection (UTl). The causative pathogens, isolated from the urine, were: E. coli (17), Proteus mirabilis (3), Enterobacter cloacae (1), enterococcus (1). The enterococcus and one Proteus mirabilis were found to be resistant to temocillin. Clinical improvement was obtained after 24-36 h in all children with temocillin-sensitive organisms. Bacteriological cure was obtained in all patients with temocillin-sensitive organisms. The two patients with temocillin-resistant pathogens were treated with another antibiotic. Follow-up treatment was given per os during +/- 2 weeks. No adverse reactions or abnormal laboratory values were noted. In the authors' limited experience temocillin proved to be effective and safe in the treatment of pyelonephritis often due to ampicillin-resistant strains in children.