Transfusion-transmission of hepatitis E virus through red blood cell transfusion but not through platelet concentrates: A case report from Spain.

BACKGROUND: Few cases of transfusion-transmitted hepatitis E virus (HEV) have been published in Spain. Here, we describe a well-characterized lookback investigation of a transfusion-transmitted HEV case at the Community Centre for Blood and Tissues of Asturias (Spain). CASE REPORT: A female patient with chronic myeloid leukemia underwent an allogeneic bone marrow transplant in March 2019 and showed alterations in liver function shortly afterwards. This patient received blood components from 30 different donors in the 3 months before the transplant. Frozen plasma samples from these donations were investigated for the presence of HEV-RNA. One frequent donor was identified as asymptomatic HEV RNA-positive at the time of his whole blood donation. The investigation revealed that this donor's plasma unit, originally intended for the fractionation industry, had a viral RNA concentration of 1.9 × 104 copies/mL. HEV RNA was detected initially in the index patient who received the red cell concentrate from this donor 25 days after the transfusion. HEV RNA isolated from both donor and recipient were identified as subtype 3f. The recipient of platelet concentrate (PC), treated with a riboflavin-based pathogen reduction technology (PRT) was not infected, being negative for the presence of HEV IgM, IgG, and HEV RNA before and after the transfusion. CONCLUSION: This case study shows that HEV was transmitted through red cell transfusion to a recipient, while the patient who received riboflavin/UV light treated PC did not develop signs of infection. A causal relationship between PRT treatment of the PC and the non-transmission of HEV remains to be established.

Serological screening of Chagas disease in an immigrant population in Asturias, Spain proceeding from Chagas-endemic areas.

Population movements from Chagas disease-endemic areas to non-endemic countries due to immigration make the occurrence of this disease in these latter areas possible. We describe the results of a screening programme conducted in an immigrant population from endemic areas, attending the Tropical Medicine Unit of the Hospital Central de Asturias between June 2006 and June 2008. The ID-Chagas antibody test (particle gel immunoassay (PaGIA); DiaMed-ID) was used as a screening assay. We analysed 64 patients, 9 of whom (14%) tested positive for Chagas disease antibodies, a diagnosis that was confirmed in all cases. Six patients came from Bolivia, 2 from Paraguay and 1 from Brazil. Chagas disease is of increasing importance, even in areas with low migratory flows; hence screening programmes for this population group are especially important.

Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state.

Changes in the outer membrane subproteome of Escherichia coli along the transition to the viable but nonculturable state (VBNC) were studied. The VBNC state was triggered by exposure of E. coli cells to adverse conditions such as aquatic systems, starvation, suboptimal temperature, visible light irradiation and seawater. The subproteome, obtained according to Molloy et al., was analysed at the beginning of exposure (inoculum, phase 1), after a variable exposure time (95% of population culturable, phase 2) and when populations were mainly in the VBNC state (95% of cells VBNC, phase 3). Proteome changes were dependent on adverse conditions inducing the transition and were detected mainly in phase 2. The permanence of E. coli cells in seawater under illumination conditions entailed a dramatic rearrangement of the outer membrane subproteome involving 106 new spots, some of which could be identified by peptide fingerprinting. However, proteins exclusive to the VBNC state were not detected.

Inability of Escherichia coli to resuscitate from the viable but nonculturable state.

After induction of the viable but nonculturable (VBNC) state in Escherichia coli populations, we analysed abiotic and biotic factors suggested to promote the resuscitation process. The response to the stressing conditions implied the formation of three subpopulations, culturable, VBNC and nonviable. In most adverse situations studied, the VBNC subpopulation did not represent the dominant fraction, decreasing with time. This suggests that, in most cases, the VBNC is not a successful phenotype. Combining methods of dilution and inhibition of remaining culturable cells, we designed a working protocol in order to distinguish unequivocally between regrowth and resuscitation. Reversion of abiotic factors inducing nonculturability as well as prevention of additional oxidative stress did not provoke resuscitation. Participation of biotic factors was studied by addition of supernatants from different origin without positive results. These results indicate that the E. coli strain used is not able to resuscitate from the VBNC state. VBNC cells release into the surrounding medium, and could thus aid in the survival of persisting culturable cells. The formation of a VBNC subpopulation could thus be considered as an adaptive process, designed for the benefit of the population as a whole.

Effect of temperature and starvation upon survival strategies of Pseudomonas fluorescens CHA0: comparison with Escherichia coli.

Microorganisms in aquatic systems are exposed to continuous modifications in their environmental conditions. In these systems, both autochthonous and allochthonous bacteria respond to adverse conditions by expressing viable but nonculturable phenotype. On the basis of this common response, the behaviour of a few species is extrapolated to others. We compared the survival strategies of Escherichia coli (allochthonous, mesophile bacterium) and Pseudomonas fluorescens CHA0 (ubiquitous, psychrotrophic bacteria) under nonoptimal temperature and nutrient deprivation. In the absence of nutrients, the effect of temperature on the loss of culturability did not show a common pattern. Whereas the survival of E. coli had an inverse relationship with temperature, whereas for P. fluorescens a direct relationship between temperature and T90 values was only established in the range 5-15°C, with an inverse relationship at higher temperatures. When the subproteome of the outer membrane of P. fluorescens was comparatively analysed, starvation was not the main source of change. The most relevant modifications were due to variations in temperature. OprF, the major surface protein of the genus Pseudomonas, showed a high expression in nonculturable as well as culturable populations under all the adverse situations analysed. We therefore propose OprF as a suitable marker for Pseudomonas detection in the environment.

Is Urografin density gradient centrifugation suitable to separate nonculturable cells from Escherichia coli populations?

The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the different origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heterogeneous population, but working conditions should be carefully chosen to avoid Urografin toxicity.

Relationships between Escherichia coli cells and the surrounding medium during survival processes.

In Escherichia coli, during survival under adverse conditions, namely starvation and luminous radiation, two things occur. On the one hand organic substances are released into the surrounding medium and on the other there is a transition from the culturable state to viable but non-culturable (VBNC). An analysis of organic molecules released into the surrounding medium showed the presence of proteins, dissolved free amino acids, and dissolved monomeric carbohydrates. The concentration of these substances in the medium changed with exposure time, type of stress and type of molecule. The proteins accumulated in the medium and in some cases their identification revealed the presence of components of the outer membrane. Variations in the concentration of amino acids and carbohydrates point to a twofold process of excretion and uptake. Indeed, cell free supernatants supported the growth of several generations of a population of 10(4) cells ml(-1). The survival of E. coli in supernatants previously colonized by cells in the VBNC state was greater than that observed in the control experiments, with a short delay in the loss of culturability. It was thus clear that organic molecules released into the medium play a role in the transition from culturable to VBNC state.