Comparison of an in house and a commercial real-time polymerase chain reaction targeting Toxoplasma gondii RE gene using various samples collected from patients in Turkey.

BACKGROUND: Toxoplasma gondii is an opportunistic protozoan parasite that can infect all warm-blooded animals including humans and cause serious clinical manifestations. Toxoplasmosis can be diagnosed using histological, serological, and molecular methods. In this study, we aimed to detect T. gondii RE gene in various human samples by in house and commercial real time polymerase chain reactions. METHODS: A total of 38 suspected cases of toxoplasmosis [peripheral blood (n:12), amnion fluid (n:11), tissue (n:9), cerebrospinal fluid (n:5), and intraocular fluid (n:1)] were included to the study. An in house and a commercial RT-PCR were applied to investigate the T. gondii RE gene in these samples. RESULTS: The compatibility rate of the two tests was 94.7% (37/38). When the commercial RT-PCR kit was taken as reference, the sensitivity and specificity of in house RT-PCR test was 87.5 and 100%. When the in house RT-PCR test was taken as reference, the commercial RT-PCR kit has 100% sensitivity and 96.8% specificity. Incompatibility was detected in only in a buffy coat sample with high protein content. CONCLUSIONS: Both the commercial and in house RT-PCR tests can be used to investigate T. gondii RE gene in various clinical specimens with their high sensitivity and specificity. In house RT-PCR assay can be favorable due to cost savings compared to using the commercial test.

A comparison of two molecular methods for diagnosing leptospirosis from three different sample types in patients presenting with fever in Laos.

OBJECTIVES: To compare two molecular assays (rrs quantitative PCR (qPCR) versus a combined 16SrRNA and LipL32 qPCR) on different sample types for diagnosing leptospirosis in febrile patients presenting to Mahosot Hospital, Vientiane, Laos. METHODS: Serum, buffy coat and urine samples were collected on admission, and follow-up serum ∼10 days later. Leptospira spp. culture and microscopic agglutination tests (MAT) were performed as reference standards. Bayesian latent class modelling was performed to estimate sensitivity and specificity of each diagnostic test. RESULTS: In all, 787 patients were included in the analysis: 4/787 (0.5%) were Leptospira culture positive, 30/787 (3.8%) were MAT positive, 76/787 (9.7%) were rrs qPCR positive and 20/787 (2.5%) were 16SrRNA/LipL32 qPCR positive for pathogenic Leptospira spp. in at least one sample. Estimated sensitivity and specificity (with 95% CI) of 16SrRNA/LipL32 qPCR on serum (53.9% (33.3%-81.8%); 99.6% (99.2%-100%)), buffy coat (58.8% (34.4%-90.9%); 99.9% (99.6%-100%)) and urine samples (45.0% (27.0%-66.7%); 99.6% (99.3%-100%)) were comparable with those of rrs qPCR, except specificity of 16SrRNA/LipL32 qPCR on urine samples was significantly higher (99.6% (99.3%-100%) vs. 92.5% (92.3%-92.8%), p <0.001). Sensitivities of MAT (16% (95% CI 6.3%-29.4%)) and culture (25% (95% CI 13.3%-44.4%)) were low. Mean positive Cq values showed that buffy coat samples were more frequently inhibitory to qPCR than either serum or urine (p <0.001). CONCLUSIONS: Serum and urine are better samples for qPCR than buffy coat, and 16SrRNA/LipL32 qPCR performs better than rrs qPCR on urine. Quantitative PCR on admission is a reliable rapid diagnostic tool, performing better than MAT or culture, with significant implications for clinical and epidemiological investigations of this global neglected disease.

Efficiency of riboflavin and ultraviolet light treatment against high levels of biofilm-derived Staphylococcus epidermidis in buffy coat platelet concentrates.

BACKGROUND AND OBJECTIVES: Staphylococcus epidermidis forms surface-attached aggregates (biofilms) in platelet concentrates (PCs), which are linked to missed detection during PC screening. This study was aimed at evaluating the efficacy of riboflavin-UV treatment to inactivate S. epidermidis biofilms in buffy coat (BC) PCs. MATERIALS AND METHODS: Biofilm and non-biofilm cells from S. epidermidis ST-10002 and S. epidermidis AZ-66 were individually inoculated into whole blood (WB) units (~106 colony-forming units (CFU)/ml) (N = 4-5). One spiked and three unspiked WB units were processed to produce a BC-PC pool. Riboflavin was added to the pool which was then split into two bags: one for UV treatment and the second was untreated. Bacterial counts were determined before and after treatment. In vitro PC quality was assessed by flow cytometry and dynamic light scattering. RESULTS: Bacterial counts were reduced during BC-PC production from ~106 CFU/ml in WB to 103 -104 CFU/ml in PCs (P < 0·0001). Riboflavin-UV treatment resulted in significantly higher reduction of S. epidermidis AZ-66 than strain ST-10002 (≥3·5 log reduction and 2·6-2·8 log reduction, respectively, P < 0·0001). Remaining bacteria post-treatment were able to proliferate in PCs. No differences in S. epidermidis inactivation were observed in PCs produced from WB inoculated with biofilm or non-biofilm cells (P > 0·05). Platelet activation was enhanced in PCs produced with WB inoculated with biofilms compared to non-biofilm cells (P < 0·05). CONCLUSION: Riboflavin-UV treatment was similarly efficacious in PCs produced from WB inoculated with S. epidermidis biofilm or non-biofilm cells. Levels of biofilm-derived S. epidermidis ≥103 CFU/ml were not completely inactivated; however, further testing is necessary with lower (real-life) bacterial levels.

Bacterial survival and distribution during buffy coat platelet production.

BACKGROUND AND OBJECTIVES: At Canadian Blood Services, buffy coat (BC) platelet concentrates (BC-PCs) show a generally lower bacterial contamination rate than apheresis PCs. This study investigated whether the PC production method contributes to this observation. MATERIALS AND METHODS: Whole blood (WB) inoculated with eight bacterial strains was processed using the BC method. Bacteria were enumerated throughout BC-PC production and subsequent PC storage. Endotoxin production and bacterial adhesion to PC bags were evaluated during PC storage. PC quality was monitored by CD62P expression (flow cytometry) and changes in dynamic light scattering (ThromboLUX® ). RESULTS: During overnight WB hold, Staphylococcus epidermidis titres remained unchanged, commercial Escherichia coli and Klebsiella pneumoniae were eliminated and the remaining organisms proliferated to high concentrations. Through BC-PC production, bacteria segregated preferentially towards the cellular fractions compared to plasma (P < 0·05). During PC storage, most bacteria adhered to the PC bags and Gram negatives produced clinically significant endotoxin levels. Changes in CD62P expression or ThromboLUX scoring did not consistently reflect bacterial contamination in BC-PCs. CONCLUSION: WB hold during BC-PC production does not have a broad-spectrum bactericidal effect, and therefore, other factors contribute to low rates of contamination in BC-PCs.

Usefulness of direct fluorescent in buffy coat in the diagnosis of Candida sepsis in neonates.

OBJECTIVE: The objective of this study is to assess the clinical utility of direct fluorescent assay in buffy coat in the diagnosis of Candida sepsis (CS) in neonates. STUDY DESIGN: A cross-sectional study was conducted in a Neonatal Intensive Care Unit and 22 neonates with suspected CS were enrolled. Fungus isolation from blood cultures and direct fluorescent tests in buffy coat were performed and validity parameters were estimated. RESULTS: Candida was isolated in 13/22 (59%) blood cultures. The direct fluorescent test was positive in 12/13 and 1/9 cases with positive and negative blood culture as corresponding. Estimated sensitivity, specificity, positive predictive value, negative predictive value, positive likehood ratio and negative likehood ratio were 92%, 89%, 92%, 89%, 8.31 and 0.09, respectively. CONCLUSION: The direct fluorescent assay in buffy coat might be useful to support early and accurate diagnosis of CS in neonates.

Validation of sterility testing of cord blood: challenges and results.

BACKGROUND: Sterility testing for cord blood (CB) products is mandatory to prevent transplantation-transmitted microbial infections. Here, the automated BacT/ALERT (bioMérieux) culture system was validated to detect microbial contamination in CB units processed at the Canadian National Public Cord Blood Bank. STUDY DESIGN AND METHODS: A three-phase validation was developed. CB units were prepared with pentastarch (Phases 1 and 2) or hetastarch (Phase 3). In Phase 1, CB was spiked with approximately 100 colony-forming units/mL of Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Bacteroides fragilis, and Candida albicans. Plasma (8 mL) and buffy coat (BC; 0.5 and 8 mL) were inoculated into culture bottles. In Phases 2 and 3, a mix of red blood cells (RBCs) and plasma (4 mL each) was used as the inoculant. In Phase 3, Aspergillus brasiliensis was added as a test organism and microbial concentrations in the by-product RBCs and plasma were determined. The BC fractions were cryopreserved and tested 3 months later. RESULTS: In Phase 1, bacteria failed to grow in CB units containing antibiotics. Thus, antibiotic-free units were used for the other phases. C. albicans was not always captured in plasma, but using a mix of RBCs and plasma, all organisms were detected. The use of pentastarch or hetastarch did not affect microbial recovery. C. albicans and A. brasiliensis were preferentially recovered in RBCs and BC. Cryopreservation did not affect microbial survival during CB processing. CONCLUSIONS: A mix of plasma and RBCs is appropriate for CB sterility testing. Interestingly, fungi preferentially segregate to cellular fractions. The clinical significance of the bactericidal /or bacteriostatic effect of antibiotics in CB merits further investigation.

Preserved functional and biochemical characteristics of platelet components prepared with amotosalen and ultraviolet A for pathogen inactivation.

BACKGROUND: Platelet concentrate (PC) functionality decreases during storage. This is referred to as the storage lesion. Pathogen inactivation may accelerate or induce lesions, potentially accounting for reduced viability. Our aim was to characterize functional and biochemical properties of platelets (PLTs) from photochemically treated buffy-coat PCs (PCT-PCs) compared to those from conventional PCs. STUDY DESIGN AND METHODS: Four PCT-PCs and four conventional PCs were stored for 6.5 days and PLT function and proteomic profiles were examined at various time points during storage. To evaluate their intrinsic properties, samples of stored PLTs were taken, washed, and suspended in Tyrode's buffer before testing. RESULTS: PLT counts and morphology were conserved although a slight increase in the PLT volume was observed after PCT. Glycoprotein (GP) IIbIIIa, IaIIa, and VI expression remained stable while GPIbα declined similarly in both types of PCs. A steep decrease (50%) in GPV occurred on Day 1.5 in PCT-PCs and Day 2.5 in control PCs. For both PCT- and control PCs, P-selectin expression and activated GPIIbIIIa remained low during storage. PCT- and control PCs were fully responsive to aggregation agonists up to Day 4.5 and exhibited similar perfusion functionality. Mitochondrial membrane potential and annexin A5 binding of PCT-PCs and control PCs were comparable. Two-dimensional differential in-gel electrophoresis and mass spectrometry profiles for 1882 protein spots revealed only three proteins selectively changed in PCT-PCs compared to control-PCs. CONCLUSION: Washed treated and untreated PCs have similar functional, morphologic, and proteomic characteristics provided that PLTs are suspended in an appropriate medium during testing.

Diagnostic accuracy of buffy coat culture compared to total blood culture in late-onset sepsis of the newborn.

OBJECTIVES: To study the potential of buffy coat culture as a diagnostic tool for neonatal late-onset sepsis. METHODS: This was a study of diagnostic accuracy in newborn infants born at 28-41 weeks of gestation, weighing >800g, with ≥8 points on the NOSEP-1 scale. Paired samples for total blood culture (TBC) and buffy coat culture were drawn. We established the positivity rate, sensitivity, specificity, predictive values, and likelihood ratios, and compared time to positivity and contamination rates. RESULTS: Fifty-two newborns were included in the study. Twenty-one TBC and 22 buffy coat cultures were positive. The positivity rate for TBC was 40.4% and for buffy coat culture was 42.3% (p=not significant). Three TBC were positive with negative buffy coat culture. Four buffy coat cultures were positive with negative TBC; Kappa agreement was 0.723, p <0.001. Buffy coat culture sensitivity was 86% (95% confidence interval (CI) 68.5-95.4%), specificity 87% (75.4-93.7%), positive predictive value 82% (65.4-91.1%), negative predictive value 90% (77.9-96.8%), positive likelihood ratio 6.64 (2.79-15.05), and negative likelihood ratio 0.16 (0.05-0.42). We found no difference in time to positivity in hours; Wilcoxon Z=1224, p=0.22. The contamination rate was 1.9% for both methods. CONCLUSIONS: Buffy coat culture is as good as TBC for the microbiological diagnosis of late-onset sepsis of the newborn. Buffy coat culture allows the use of remaining plasma for further analysis.

Proteomic analysis of Intercept-treated platelets.

In the past decades, transfusion medicine has been driven by the quest for increased safety against transfusion-transmitted infections, mainly by better donor selection and by the development of improved serological and nucleic-acid-based screening assays. Recently, pathogen reduction technologies became available and started to be implemented in several countries, with the primary goal to fight against bacterial contamination of blood products, a rare but dramatic event against which there was no definitive measure. Though pathogen reduction technologies represent a quantum leap in transfusion safety, the biomedical efficacy of platelet concentrates (PCs) treated with various pathogen reduction techniques has been recently questioned by clinical studies. Here, a gel-based proteomic analysis of PCs (n=5), Intercept-treated or untreated, from pooled buffy-coat (10 donors per PC) at Days 1, 2 and 8, shows that the Intercept process that is the most widespread pathogen reduction technique to date, has relatively low impact on the proteome of treated platelets: the process induces modifications of DJ-1 protein, glutaredoxin 5, and G(i)alpha 2 protein. As for the impact of storage, chloride intracellular channel protein 4 (CLIC4) and actin increased independently of Intercept treatment during storage. Whereas alteration of the DJ-1 protein and glutaredoxin 5 points out an oxidative stress-associated lesion, modification of G(i)alpha2 directly connects a possible Intercept-associated lesion to haemostatic properties of Intercept-treated platelets. This article is part of a Special Issue entitled: Integrated omics.

Bacterial screening of outdated buffy coat platelet pools using a culture system and a rapid immunoassay.

BACKGROUND: Canadian Blood Services performs bacterial screening of buffy coat platelet pools (BCPs) using aerobic BacT/ALERT cultures. This study aimed to determine the rate of detection failures during initial platelet (PLT) screening and evaluate the introduction of anaerobic cultures and immunoassay testing to assess the safety of extending PLT storage beyond 5 days. STUDY DESIGN AND METHODS: Outdated (7- to 10-day-old) BCPs that tested negative during initial screening were assayed with BacT/ALERT and the Verax PLT Pan Genera detection (PGD) test, an immunoassay that detects Gram-positive (GP) and Gram-negative (GN) bacteria. BacT/ALERT aerobic and anaerobic culture bottles were inoculated with 8 to 10 mL of BCP and incubated for up to 6 days. The PGD test was performed following manufacturer's instructions. Positive results were confirmed using the BacT/ALERT and PGD tests, blood agar culture, and Gram staining. Invalid PGD results were investigated. RESULTS: A total of 4002 BCPs were tested with one (0.025%) true positive (Staphylococcus epidermidis) found by both the BacT/ALERT and the PGD assays. Fifty-four (1.35%) false-positive BacT/ALERT cultures were obtained mainly due to instrument errors involving anaerobic cultures. Eleven (0.27%) false-positive PGD tests were observed in the GP window of the strip. Forty-nine (1.2%) invalid PGD results were obtained mostly before implementation of a humidity chamber. CONCLUSION: Testing of outdated BCPs suggests that introducing anaerobic cultures would result in significant PLT wastage due to a high rate of false positives. Contaminated BCPs still escape detection during initial testing; therefore, extension of PLT storage may be possible if repeat screening is performed before transfusion.