Contaminations occurring in fungal PCR assays.
Successful in vitro amplification of fungal DNA in clinical specimens has been reported recently. In a collaboration among five European centers, the frequency and risk of contamination due to airborne spore inoculation or carryover contamination in fungal PCR were analyzed. The identities of all contaminants were specified by cycle sequencing and GenBank analysis. Twelve of 150 PCR assays that together included over 2,800 samples were found to be contaminated (3.3% of the negative controls were contaminated during the DNA extraction, and 4.7% of the PCR mixtures were contaminated during the amplification process). Contaminants were specified as Aspergillus fumigatus, Saccharomyces cerevisiae, and Acremonium spp. Further analysis showed that commercially available products like zymolyase powder or 10x PCR buffer may contain fungal DNA. In conclusion, the risk of contamination is not higher in fungal PCR assays than in other diagnostic PCR-based assays if general precautions are taken. (+info)
Variation of hepatitis C virus following serial transmission: multiple mechanisms of diversification of the hypervariable region and evidence for convergent genome evolution.
We have studied the evolution of hepatitis C virus (HCV) from a common source following serial transmission from contaminated batches of anti-D immunoglobulin. Six secondary recipients were each infected with virus from identifiable primary recipients of HCV-contaminated anti-D immunoglobulin. Phylogenetic analysis of virus E1/E2 gene sequences [including the hypervariable region (HVR)] and part of NS5B confirmed their common origin, but failed to reproduce the known epidemiological relationships between pairs of viruses, probably because of the frequent occurrence of convergent substitutions at both synonymous and nonsynonymous sites. There was no evidence that the rate at which the HCV genome evolves is affected by transmission events. Three different mechanisms appear to have been involved in generating variation of the hypervariable region; nucleotide substitution, insertion/deletion of nucleotide triplets at the E1/E2 boundary and insertion of a duplicated segment replacing almost the entire HVR. These observations have important implications for the phylogenetic analysis of HCV sequences from epidemiologically linked isolates. (+info)
Postsurgical Candida albicans infections associated with an extrinsically contaminated intravenous anesthetic agent.
From 16 to 30 April 1990, four of 364 (1%) postsurgical patients at one hospital developed Candida albicans fungemia or endophthalmitis. The case patients' surgeries were clustered on two days. To identify risk factors for C. albicans infections, we conducted a cohort study comparing these 4 patients with 67 control patients who had surgeries on the same days but did not acquire C. albicans infections. The participation of anesthesiologist 9 (relative risk [RR], undefined; P < 0.001) and receipt of intravenous propofol, an anesthetic agent without preservative, which was administered by an infusion pump (RR, 8.8; P = 0.048) were identified as risk factors for C. albicans infections. The anesthetic had been recently introduced in the hospital. Hand cultures of 8 of 14 (57%) anesthesiologists were positive for Candida species; one yielded C. albicans. Anesthesiologist 9 was the only one to use stored syringes of propofol in the infusion pump and to reuse propofol syringes. DNA fingerprinting with a digoxigenin-labeled C. albicans repetitive element 2 probe and electrophoretic karyotyping showed two distinct banding patterns among patient isolates. We hypothesize that extrinsic contamination of propofol by anesthesiologist 9 likely resulted in C. albicans infections. These data suggest that strict aseptic techniques must be used when preparing and administering propofol. (+info)
GB virus C/hepatitis G virus infection in HIV infected patients with haemophilia despite treatment with virus inactivated clotting factor concentrates.
AIM: To determine the frequency of GB virus C (GBV-C)/hepatitis G virus (HGV) infection before and after switch to the use of virus inactivated concentrates in haemophiliac patients infected with human immunodeficiency virus (HIV). PATIENTS AND METHODS: Initial and follow up sera from 49 children with haemophilia were analysed for the presence of GBV-C/HGV RNA and antibodies to HGV (anti-HGV). All patients had been infected with HIV while receiving concentrates without virus inactivation before 1984 and were subsequently treated with virus inactivated concentrates. RESULTS: In the first available serum sample (1987 or later), two of 49 patients were GBV-C/HGV RNA positive and two further patients were anti-HGV positive. During follow up (mean, 6 years), 14 patients developed markers of GBV-C/HGV infection. Eleven of these had received no blood products except clotting factor concentrates that had been prepared with virus inactivation. CONCLUSIONS: Despite being treated with virus inactivated clotting factor concentrates, HIV positive patients with haemophilia are at an increased risk of manifesting GBV-C/HGV infection. We hypothesise that GBV-C/HGV is transmitted by these clotting factor concentrates. However, we cannot rule out the emergence of markers of GBV-C/HGV infection as a result of the progression of immune impairment in the course of HIV infection. (+info)
Clinical outcomes after hepatitis C infection from contaminated anti-D immune globulin. Irish Hepatology Research Group.
BACKGROUND AND METHODS: In February 1994, batches of anti-D immune globulin used in Ireland during 1977 and 1978 to prevent Rh isoimmunization were found to be contaminated with hepatitis C virus (HCV) from a single infected donor. In March 1994, a national screening program was initiated for all women who had received anti-D immune globulin between 1970 and 1994. Of the 62,667 women who had been screened when this study began, 704 (1.1 percent) had evidence of past or current HCV infection, and 390 of those 704 (55 percent) had positive tests for serum HCV RNA on reverse-transcription-polymerase-chain-reaction analysis. All 390 were offered a referral for clinical assessment and therapy. We evaluated 376 of these 390 women (96 percent); the other 14 were not seen at one of the designated treatment centers. RESULTS: The mean (+/-SD) age of the 376 women was 45+/-6 years at the time of screening. They had been infected with hepatitis C for about 17 years. A total of 304 women (81 percent) reported symptoms, most commonly fatigue (248 [66 percent]). Serum alanine aminotransferase concentrations were slightly elevated (40 to 99 U per liter) in 176 of 371 women (47 percent), and the concentrations were 100 U per liter or higher in 31 (8 percent). Liver biopsies showed inflammation in 356 of 363 women (98 percent); in most cases the inflammation was slight (41 percent) or moderate (52 percent). Although the biopsy samples from 186 of the 363 women (51 percent) showed evidence of fibrosis, only 7 women (2 percent) had probable or definite cirrhosis. Two of the seven reported excessive alcohol consumption. CONCLUSIONS: Most of the women with HCV infection 17 years after receiving HCV-contaminated anti-D immune globulin had evidence of slight or moderate hepatic inflammation on liver biopsy, about half had fibrosis, and 2 percent had probable or definite cirrhosis. (+info)
Cluster of postinjection abscesses related to corticosteroid injections and use of benzalkonium chloride.
Benzalkonium chloride (BC) is an unreliable disinfectant. A matched case-control study and environmental investigation were conducted to determine the cause of and risk factors for a cluster of postinjection abscesses at a private medical clinic where BC was used as a disinfectant. Twenty-eight case-patients who had an abscess at the injection site were matched with 126 control patients who had received an intramuscular injection at the clinic on the same day. Risk factors for abscess development in a multivariable logistic model were corticosteroid injection and being female. All case-patients had received a corticosteroid injection from a multidose vial. Cultures of abscesses from 20 of 23 case-patients grew Pseudomonas aeruginosa. Cultures of BC prepared at the clinic also grew P aeruginosa, suggesting that BC was the source of infection. Injection site cleaning with BC did not appear to be the route of infection since use of BC at the time of injection was not associated with abscess development. A more likely route of infection was injection of contaminated corticosteroid from multidose vials that could have been inoculated with pseudomonads via needle puncture after vial septa were wiped with contaminated BC. Benzalkonium chloride should not be used to clean injection vial septa or injection sites. (+info)
Peripheral blood stem cell contamination in mantle cell non-Hodgkin lymphoma: the case for purging?
Intensification using peripheral blood stem cells collected after chemotherapy followed by growth factors is being increasingly investigated as an alternative to conventional chemotherapy for mantle cell non-Hodgkin lymphoma. We investigated 14 grades III-IV, t(11;14)-positive cases for contamination of PBSC collected after a polychemotherapy regimen followed by G-CSF. Patients were first treated with a polychemotherapy regimen. There were four CR, seven PR, two refractory and one early death. Seven patients have been transplanted, in whom PBSC were mobilized, using either cyclophosphamide/VP16 or Dexa-BEAM followed by G-CSF. For all patients, whether actually autografted or not, PB cells were tested at the time of regeneration on G-CSF after the first polychemotherapy or after the mobilizing regimen. PCR evaluation of contamination was performed first by a semi-quantitative approach, using serial dilutions of initial DNA, then confirmed using a limiting-dilution analysis. Two patients were not informative (one early death and one without an available molecular marker). PB cells collected at regeneration contained at least one log more lymphoma cells than steady-state blood or marrow, apart from in two cases. Moreover, where a mobilizing treatment diminished tumor burden in the patient, at the same time it increased PB contamination in most cases. We conclude that advanced mantle cell NHL appears to be largely resistant to significant in vivo purging by conventional chemotherapy. Where treatment brings benefits by reducing tumor load, it may at the same time negate it by mobilizing malignant cells into the collections used to intensify. Although the clonogenic potential of this massive infiltration is unknown (only gene marking studies could provide a definitive answer regarding the source of relapses), strategies aimed at reducing the level of contamination in the graft should be considered when designing future protocols. (+info)
Inhibition of P-glycoprotein-mediated transport by a hydrophobic contaminant in commercial gluconate salts.
The substitution of gluconate for Cl- is commonly used to characterize Cl- transport or Cl--dependent transport mechanisms. We evaluated the effects of substituting gluconate for Cl- on the transport of the P-glycoprotein substrate rhodamine 123 (R123). The replacement of Ringer solution containing Cl- (Cl--Ringer) with gluconate-Ringer inhibited R123 efflux, whereas the replacement of Cl- by other anions (sulfate or cyclamate) had no effect. The inhibition of R123 efflux by gluconate-Ringer was absent after chloroform extraction of the sodium gluconate salt. The readdition of the sodium gluconate-chloroform extract to the extracted gluconate-Ringer or to cyclamate-Ringer inhibited R123 efflux, whereas its addition to Cl--Ringer had no effect. These observations indicate that the inhibition of P-glycoprotein-mediated R123 transport by gluconate is due to one or more chloroform-soluble contaminants and that the inhibition is absent in the presence of Cl-. The results are consistent with the fact that P-glycoprotein substrates are hydrophobic. Care should be taken when replacing ions to evaluate membrane transport mechanisms because highly pure commercial preparations may still contain potent contaminants that affect transport. (+info)