Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria

Background Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion‐Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion‐Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. Material and Methods Four Bacteria References (Staphylococcus epidermidis PEI‐B‐06, Streptococcus pyogenes PEI‐B‐20, Klebsiella pneumoniae PEI‐B‐08 and Escherichia coli PEI‐B‐19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. Results Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19–1·32 × 10⁷ CFU/ml, S. pyogenes: 0·58–0·69 × 10⁷ CFU/ml, K. pneumoniae: 18·71–20·26 × 10⁷ CFU/ml and E. coli: 1·78–2·10 × 10⁷ CFU/ml. Conclusion The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low‐titre spiking of blood components, (ii) the property of donor‐independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion‐Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.     

Evaluation of a universal point‐of‐issue assay for bacterial detection in buffy coat platelet components

Bacterial contamination of platelet concentrates poses a major post‐transfusion infectious risk. This study was aimed at evaluating the efficacy of the BacTx ^® assay (Immunetics Inc.) for bacterial detection in leucocyte‐reduced buffy coat platelet pools and for its sensitivity in detecting clinical isolates, including bacteria that form surface‐attached aggregates (biofilm positives). Platelet pools were inoculated at bacterial concentrations of 0·8–13 CFU/ml. The BacTx ^® assay detected all species at concentrations ≥10³ CFU/ml within 20–69 h of platelet incubation. Detection of slow‐growing and biofilm‐forming strains was delayed in comparison with the other strains. This assay could be used as a point‐of‐issue method to increase the safety of the platelet supply.     

Serum CXC ligand 5 is a new marker of subclinical atherosclerosis in type 2 diabetes

Objective To assess the relationship between serum chemokine CXC ligand 5 (CXCL5) and intima‐media thickness (IMT) of the common carotid artery, a marker of preclinical atherosclerosis. Design, patients and measurements We measured the IMT (mean of three segments of both carotid arteries by ultrasonography), serum CXCL5, fasting plasma glucose, serum insulin, serum urea, serum uric acid, HbA1c, serum CRP, adiponectin and lipid profile of 730 Chinese type 2 diabetic participants older than 30 years in a cross‐sectional community‐based study performed in downtown Shanghai. Results Serum CXCL5 correlated with carotid IMT (r = 0·112, P = 0·019) after adjustment for age, gender, serum CRP and HbA1c. Multivariate linear regression analysis demonstrated that age (P = 2·30 × 10⁻⁷), gender (P = 2·70 × 10⁻²), systolic blood pressure (P = 9·26 × 10⁻³), serum CRP (P = 2·12 × 10⁻²), low‐density lipoprotein cholesterol (P = 2·40 × 10⁻²), CXCL5 (P = 5·27 × 10⁻³) and high‐density lipoprotein cholesterol (P = 3·80 × 10⁻³) were independently associated with carotid IMT. Conclusions Serum CXCL5 is an important determinant of carotid artery IMT in patients with type 2 diabetes. The correlation is independent of inflammation and glucose control.     

The utility of ADAMTS13 in differentiating TTP from other acute thrombotic microangiopathies: results from the UK TTP Registry

Thrombotic microangiopathies (TMAs) are frequently difficult to differentiate clinically, and measurement of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) remains vital in thrombotic thrombocytopenic purpura (TTP) diagnosis. We retrospectively reviewed cases referred for ADAMTS13 testing, using UK TTP Registry screening data. Of a total 810 cases, 350 were confirmed as TTP. The 460 non‐TTP cases comprised secondary TMAs (24·57%) and haemolytic uraemic syndrome (HUS) (27·17% aHUS, 2·83% Shiga‐like toxin‐producing E. coli [STEC]‐HUS); the remainder were TMAs with no clear association, not TMAs, or had no confirmed diagnosis. ADAMTS13 levels were significantly lower in TTP than STEC‐HUS, aHUS and other TMAs. TTP patients had significantly lower platelet count (15 × 10⁹/l; range 0–96) than aHUS (57 × 10⁹/l; range 13–145, P < 0·0001) or STEC‐HUS (35 × 10⁹/l; range 14–106, P < 0·0001); they also had lower creatinine levels (92 μmol/l; range 43–374) than aHUS (255 μmol/l; range 23–941, P < 0·0001) and STEC‐HUS (324 μmol/l; range 117–639, P < 0·0001). However, 12/34 (35·3%) aHUS patients had a platelet count <30 × 10⁹/l and 26/150 (17·3%) of TTP patients had a platelet count >30 × 10⁹/l; 23/150 (15·3%) of TTP patients had a creatinine level >150 μmol/l. This study highlights the wide variety of TMA presentations, and confirms the utility of ADAMTS13 testing in TTP diagnosis.     

Novel X-linked inhibitor of apoptosis inhibiting compound as sensitizer for TRAIL-mediated apoptosis in chronic lymphocytic leukaemia with poor prognosis

Given that aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukaemia (CLL), alternative therapeutic approaches are needed. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is able to induce tumour‐specific apoptosis. However, apoptosis might be inhibited by elevated levels of X‐linked inhibitor of apoptosis (XIAP). Use of XIAP‐inhibiting compounds might sensitize primary CLL cells towards TRAIL‐mediated apoptosis. A novel small molecule, compound A (CA), an inhibitor of XIAP, was used in combination with TRAIL to induce apoptosis in primary CLL cells (n = 48). XIAP was significantly more highly expressed in primary CLL cells (n = 28) compared to healthy B cells (n = 16) (P = 0·02). Our data obtained by specific knock‐down of XIAP by siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Combined treatment with CA/TRAIL significantly increased apoptosis compared to untreated (P = 8·5 × 10^?10), solely CA (P = 4·1 × 10^?12) or TRAIL treated (P = 4·8 × 10^?10) CLL cells. CA rendered 40 of 48 (83·3%) primary CLL samples susceptible to TRAIL‐mediated apoptosis. In particular, cells derived from patients with poor prognosis CLL (ZAP‐70⁺, IGHV unmutated, 17p‐) were highly responsive to this drug combination. Our highly‐effective XIAP inhibitor CA, in concert with TRAIL, shows potential for the treatment of CLL cases with poor prognosis and therefore warrants further clinical investigation.     

Saccharomyces boulardii Inhibits Water and Electrolytes Changes Induced by Castor Oil in the Rat Colon

The biotherapeutic agent Saccharomyces boulardii has been shown to inhibit castor oil-induced diarrhoea in rats. The present study investigated the mechanism(s) of this antidiarrhoeal effect in terms of water and electrolyte (sodium, potassium and chloride) changes using two rat models. A single oral dose of S. boulardii of up to 12 × 10¹⁰ CFU/kg of viable cells did not inhibit castor oil-induced fluid secretion in the enteropooling model. However, the yeast dose dependently reduced castor oil induced fluid secretion into the colon, with a significant protection at 12 × 10¹⁰ CFU/kg. In this model, castor oil reversed net sodium and chloride absorption into net secretion, and increased net potassium secretion into the lumen. Single pre-treatment with S. boulardii at 4 and 12 × 10¹⁰ CFU/kg dose dependently decreased these electrolyte changes. In conclusion, S. boulardii possesses potent anti-secretory properties versus water and electrolyte secretion induced by castor oil in the rat colon.     

Minimal residual disease before and after transplantation for childhood acute lymphoblastic leukaemia: is there any room for intervention?

Eighty‐two children and adolescents who underwent allogeneic transplantation for acute lymphoblastic leukaemia in remission (period 2001–2011, median follow‐up 4·9 years) had been assessed for minimal residual disease (MRD) by real‐time quantitative polymerase chain reaction before and at 1, 3, 6, 9 and 12 months after transplantation. Five‐year event‐free survival (EFS) and cumulative incidence of relapse were 77·7% [standard error (SE) 5·7] and 11·4% (SE 4·4), respectively, for patients with pre‐transplant MRD <1 × 10^?4 (68%), versus 30·8% (SE 9·1; P < 0·001) and 61·5% (SE 9·5; P < 0·001), respectively, for those with MRD ≥1 × 10^?4 (32%). Pre‐transplant MRD ≥1 × 10^?4 was associated with a 9·2‐fold risk of relapse [95% confidence interval (CI) 3·54–23·88; P < 0·001] compared with patients with MRD <1 × 10^?4. Patients who received additional chemotherapy pre‐transplant to reduce MRD had a fivefold reduction of risk of failure (hazard ratio 0·19, CI 0·05–0·70, P = 0·01). Patients who experienced MRD positivity post‐transplant did not necessarily relapse (5‐year EFS 40·3%, SE 9·3), but had a 2·5‐fold risk of failure (CI 1·05–5·75; P = 0·04) if any MRD was detected in the first 100 d, which increased to 7·8‐fold (CI 2·2–27·78; P = 0·002) if detected after 6 months. Anticipated immunosuppression‐tapering according to MRD may have improved outcome, nevertheless all patients with post‐transplant MRD ≥1 × 10^?3 ultimately relapsed, regardless of immunosuppression discontinuation or donor‐lymphocyte‐infusion. In conclusion, MRD before transplantation had the strongest impact on relapse and MRD positivity after transplantation, mostly if detected early and at low levels, did not necessarily imply relapse. Additional intensified chemotherapy and modulation of immunosuppression may reduce relapse risk and improve ultimate outcome.     

Lymphocyte subset reconstitution after unrelated cord blood or bone marrow transplantation in children

We report the post‐transplant lymphocyte subset recovery of 226 children treated with Unrelated Cord Blood transplant (UCBT) (n = 112) or Unrelated Bone Marrow Transplant (UBMT) (n = 114) for malignant or non‐malignant diseases. Absolute numbers of natural killer (NK), B and T cells were monitored by flow cytometry up to 5 years post‐transplant. Immunological endpoints were: time to achieve a CD3⁺ cell count >0·5 and 1·5 × 10⁹/l, CD4⁺ > 0·2 and 0·5 × 10⁹/l, CD8⁺ > 0·25 × 10⁹/l, CD19⁺ > 0·2 × 10⁹/l, NK > 0·1 × 10⁹/l. These endpoints were analysed through the use of cumulative incidence curves in the context of competing risks. CD8⁺ T cell recovery was delayed after UCBT with a median time to reach CD8⁺ T cells > 0·25 × 10⁹/l of 7·7 months whereas it was 2·8 months in UBMT (P < 0·001). B cell recovery was better in UCBT, with a median time to reach CD19⁺ cells > 0·2 × 10⁹/l of 3·2 months in UCBT and 6·4 months in UBMT (P = 0·03). Median time for CD4⁺ T cell and NK cell recovery was similar in UCBT and UBMT. CD4⁺ T cells recovery was negatively correlated to age (better reconstitution in younger patients, P = 0·002). CD8⁺ T cells recovery was shorter in recipients with a positive cytomegalovirus serology (P = 0·001).     

Beyond the platelet count: immature platelet fraction and thromboelastometry correlate with bleeding in patients with immune thrombocytopenia

Platelet counts (PC) estimate bleeding risk in Immune Thrombocytopenia (ITP). We investigated whether measures of thromboelastometry and absolute immature platelet fraction (A‐IPF) would correlate better with acute bleeding score (ABS) than PC or mean platelet volume (MPV). Simultaneous determination of ABS, complete blood count and thromboelastometry was performed in 141 ITP patients; 112 underwent A‐IPF testing. Subgroup analyses were performed for paediatric subjects, PC <60 × 10⁹/l and <30 × 10⁹/l. PC significantly inversely correlated with ABS in all subjects, PC <30 × 10⁹/l and total paediatric cohort. MPV did not correlate with ABS in any subgroup. Thromboelastometry measures of clot firmness, but not PC, significantly correlated with ABS in all subjects with PC <60 × 10⁹/l, and children with PC <60 × 10⁹/l and <30 × 10⁹/l. A‐IPF demonstrated stronger correlation with ABS than did PC among all subjects, those with PC <60 × 10⁹/l, all children and children with PC <30 × 10⁹/l (r = ?0·37; r = ?0·34; r = ?0·44; r = ?0·60) versus ABS with PC (r = ?0·36; ns; r = ?0·32; ns). Stronger correlations of both thromboelastometry measures of clot firmness and A‐IPF than PC with ABS suggest factors beyond PC, i.e. related to platelet function, contribute to ITP bleeding pathophysiology. Thromboelastometry, A‐IPF and ABS can be incorporated into routine or acute visits.     

Application of the ADVIA cerebrospinal fluid assay to count residual red blood cells in blood components

Background and Objectives There is no automated, accurate assay for the enumeration of residual red blood cells (rRBCs) in non‐RBC components for transfusion, despite the potential risk of allo‐immunization when mismatched components are transfused. Materials and Methods The automated ADVIA 120 cerebrospinal fluid (CSF) assay, which is approved to count RBCs and WBCs in CSF samples, was optimized and tested to measure rRBC in platelet concentrate (PC) and plasma components. Results Sample dilution, incubation time and reagent volume were optimized for use with non‐RBC blood products. The assay was linear (R² = 0·99), even at low rRBCs counts. Intra‐ and inter‐assay variation gave coefficients of variance (CV) between 2·2 and 9·4% and 2·6 and 14·9%, respectively, depending on rRBC levels. Good correlation (r = 0·995) was found between the automated assay and manual counting, which is considered the gold standard. Using the automated assay, the range of rRBCs (count/unit) in buffy‐coat platelet concentrate (PCs) was 27–5505 × 10⁶ and in apheresis PCs was 1–361 × 10⁶. Conclusion The ADVIA CSF assay is a sensitive, precise and accurate means to assess rRBC counts in non‐RBC components.     

Non‐D Rh antibodies appearing after apheresis platelet transfusion: stimulation by red cells or microparticles?

Background Apheresis platelets (APs) have gained favour over whole blood‐derived platelets on the presumption that they are less likely to provoke alloimmunization to red‐blood‐cell antigens. Case Reports Non‐D Rh antibodies appeared in three patients after apheresis platelet transfusion. Anti‐C and anti‐E arose in two female patients with previous antigen exposure. Both anti‐c and anti‐E arose in a male recipient with no prior transfusion history. Materials and Methods Fifty APs were analysed for residual RBCs and RBC‐derived microparticles, using samples obtained from a local blood centre. Cells and microparticles were quantified with a flow cytometry gating scheme, using PE‐labelled anti‐CD235a (glycophorin A) and FITC‐labelled anti‐CD41a (platelet gp IIb/IIIa) to distinguish lineage. Results Apheresis platelets were found to contain a mean of 7·5 × 10⁶ (95% C.I. [6·3–8·5 × 10⁶]) RBCs on one manufacturer’s device and 5·2 × 10⁶ (95% C.I. [4·0–6·3 × 10⁶]) RBCs on another’s. RBC‐derived microparticles averaged 210·7 × 10⁶ (95% C.I. [166·2–254·2 × 10⁶]) on one manufacturer’s device and 232·3 × 10⁶ (95% C.I. [194·3–272·9 × 10⁶]) on another’s. These counts all correspond to volumes of < 1 μl. Conclusion Despite RBC contamination of APs below commonly accepted thresholds for Rh immunogenicity, AP transfusion can provoke non‐D Rh antibody formation. RBC‐derived microparticles, smaller but more numerous than RBCs, are volumetrically comparable and may be a hitherto underappreciated antibody stimulus. Further microparticle research will guide considerations of extended phenotypic matching of platelet components.     

Fatal outcome of a hepatitis B virus transfusion‐transmitted infection

Background and Objectives In 2008, hepatitis B virus (HBV) DNA testing was not yet mandatory for the screening of blood donations in Switzerland. At that time, HBsAg was the only specific mandatory marker for HBV. The importance of high sensitivity for HBV NAT screening is shown. Materials and Methods Donor and recipient of a transfusion‐transmitted HBV infection were followed up. Multiple samples were tested for HBV serological and molecular markers. Results At donation, the donor appeared healthy, HBsAg was negative and had a normal ALAT level. Ten weeks later, clinical symptoms suggested acute HBV infection as was confirmed with positive HBsAg, HBeAg, anti‐HBc IgG, anti‐HBc IgM and anti‐HBe. The archived sample from the original donation was negative for anti‐HBc, but positive for HBV DNA (17 IU/ml). A recipient transfused with the red cell concentrate was HBV DNA positive (3100 IU/ml) 3 months post‐transfusion. After five months, HBsAg, HBeAg, anti‐HBc and HBV DNA (1·1 × 10¹¹ IU/ml) were positive. Two weeks later, the patient died from complications associated with HBV infection and his underlying bone marrow disease. Conclusions The present case illustrates the importance of introducing highly sensitive HBV NAT screening strategy to prevent possible HBV transfusion‐transmitted infections from donors with low viral load.     

Thyroid status in a large cohort of patients with mental retardation: the TOP-R (Thyroid Origin of Psychomotor Retardation) study

Objective Abnormalities in thyroid state may affect development and function of the brain and result in mental retardation (MR). Thyroid parameters have not been systematically investigated in institutionalized MR subjects. The objective is to measure thyroid parameters in a novel cohort of 946 institutionalized subjects. Design The TOP‐R (Thyroid Origin of Psychomotor Retardation) study is a cross‐sectional nation‐wide multicentre study. Patients Subjects with unexplained MR. Results The majority of the MR subjects had thyroid parameters within the reference range used in our laboratory. Antiepileptic drugs (AEDs) use affected thyroid hormones (T4: 102·1 ± 1·2 vs 83·9 ± 1·2 nmol/l, P < 1 × 10^?24; FT4: 18·0 ± 0·2 vs 16·1 ± 0·2 pmol/l, P < 1 × 10^?9; T3: 1·72 ± 0·02 vs 1·57 ± 0·02 nmol/l, P < 1 × 10^?9; and rT3: 0·37 ± 0·01 vs 0·27 ± 0·01 nmol/l, P < 1 × 10^?28 in subjects without vs with AEDs). The prevalence of unrecognized primary hypothyroidism and hyperthyroidism was 5·2% and 2·8%, respectively. Conclusions We report thyroid parameters in a cohort of institutionalized subjects with MR. Our findings substantiate the fact that AEDs affect thyroid hormone levels. Future studies will be employed to investigate genetic causes of MR related to abnormalities in thyroid hormone homeostasis.     

Similar clinical and laboratory findings in patients with symptomatic autosomal dominant and sporadic pseudohypoparathyroidism type Ib despite different epigenetic changes at the GNAS locus

Objective Most patients with autosomal dominant pseudohypoparathyroidism type Ib (AD‐PHP‐Ib) carry an identical maternally inherited 3‐kb microdeletion up‐stream of GNAS (STX16del4‐6^mat), which is associated with a methylation loss restricted to exon A/B. STX16del4‐6^mat is not found in sporadic PHP‐Ib (sporPHP‐Ib) patients, who show broad GNAS methylation changes. Because of the epigenetic differences between both groups, we searched for clinical and/or laboratory differences. Patients and methods Age at diagnosis, calcium, phosphorus and PTH were analysed in 43 patients with AD‐PHP‐Ib due to STX16del4‐6^mat and in 22 patients with sporPHP‐Ib. Results All AD‐PHP‐Ib patients with STX16del4‐6^mat showed only loss of exon A/B methylation. Of the 43 individuals, 26 were symptomatic when diagnosis was established at age 12·1 ± 1·34 years (mean ± SEM); laboratory findings at presentation were calcium 1·69 ± 0·06 mmol/l, phosphorus 2·25 ± 0·12 mmol/l and PTH 442 ± 54·1 pg/ml. The remaining 17 individuals with STX16del4‐6^mat were asymptomatic when diagnosed at age 23·5 ± 3·93 years (calcium 2·18 ± 0·05 mmol/l, phosphorus 1·63 ± 0·10 mmol/l, PTH 222 ± 40·3 pg/ml). Patients with sporPHP‐Ib showed methylation changes at two or more GNAS exons, presented at age 10·0 ± 1·01 years and had, as a group, similar laboratory findings as patients with symptomatic AD‐PHP‐Ib (calcium 1·51 ± 0·06 mmol/l, phosphorus 2·65 ± 0·10 mmol/l, PTH 634 ± 162·1 pg/ml). However, sporPHP‐Ib females appeared to be more severely affected. Conclusions Patients with symptomatic AD‐PHP‐Ib due to STX16del4‐6^mat and sporPHP‐Ib have similar changes in calcium, phosphate and PTH. STX16del4‐6^mat often leads to asymptomatic disease and screening of all siblings of affected individuals is therefore advised. The cause of the apparent sexual dimorphism in patients with sporPHP‐Ib remains uncertain.     

Distinct strains of Leishmania major induce different cytokine mRNA expression in draining lymph node of BALB/c mice

Four genotypically distinct strains of L. major collected from persons residing in different endemic areas of cutaneous leishmaniasis in Iran were evaluated in BALB/c mice. Parasite virulence was evaluated by measuring the parasite burden in the lymph nodes. Immunogenicity of the strains was assessed by analysis of cytokines mRNA expression levels in popliteal lymph nodes of the mice in early (3, 16, 40 h) and late (week 1, W3, W5 and W8) time periods after infection. The expression of cytokines mRNA, namely Ifng, Il2,Il4,Il10 and Il12, was quantitated by real‐time PCR. The lowest and the highest parasite loads were induced by Damghan (2·15 ×  10⁷) and Shiraz (9·59 ×  10⁹) strains, respectively. Moreover, Damghan strain elicited higher expression levels of Ifng and Il2 mRNA and the highest ratio of Ifng/Il4 mRNA expression compared with the other strains at 40 h and 8 weeks post‐infection. The results indicate that the inoculation of BALB/c mice with different strains induced high diversity in parasite burden and cytokines gene expression. Amongst the four strains, Damghan strain showed the lowest parasite load and the highest tendency to induce expression of Th1 cytokines gene and might be considered as a safe and immunogenic strain.     

Detection of HHV‐8 (human herpesvirus‐8) genomes in induced peripheral blood mononuclear cells (PBMCs) from US blood donors

Background Human herpesvirus‐8 (HHV‐8) causes Kaposi’s sarcoma and can be detected and induced in peripheral blood mononuclear cells (PBMCs) from infected individuals. The prevalence of viral genomes in induced/cultured PBMCs from healthy blood donors has not been systematically studied. Materials and Methods PBMCs from 164 donors were purified and stored as two equal aliquots in liquid nitrogen. One aliquot was used for CD19⁺ B‐cell purification with a fraction reserved for DNA extraction. The second aliquot was cultured for 2 or 4 days in culture media containing n‐butyrate and tetradecanoyl phorbol acetate. DNA was extracted from all four cell sources: PBMCs, purified B cells, induced PBMCs harvested at days 2 and 4 of culture. A sensitive real‐time PCR with a DNA equivalent of 3 × 10⁵ cells per reaction was run in duplicate for all samples along with a quantitative HHV‐8 DNA standard ranging from 1·6 to 200 copies. Results For all 164 donors, HHV‐8 genomes were not detected in the DNA equivalent of 3–6 × 10⁵ of PBMCs and induced/cultured PBMCs with a real‐time PCR assay (95% CI: 0–3·5/164). HHV‐8 DNA was not detected from DNA equivalent of 1·5 (0·5–5·6) × 10⁵ CD19⁺ B cells from 139/164 donors. HHV‐8 antibodies were detected in 7 of the 164 donors (4·3%). Conclusions HHV‐8 genomes were not detected from PBMCs, induced/cultured PBMCs and CD19⁺ B cells from 164 blood donors. The level of detectable HHV‐8 genomes in blood donors seems to be extremely low, if they exist.     

Bacteriological safety of plastic–bagged sachet drinking water sold in Amassoma,Nigeria

ObjectiveTo evaluate the bacteriological safety of sachet water sold in Amassoma, a rural community in Bayelsa State, Nigeria.MethodsSix samples of each of the different sachet drinking water brands were bought at random from shop shelves, markets and street vendors and were studies for microbial indicators of safety and quality. Bacterial counts were analyzed by one-way Analysis of Variance (ANOVA) and significance of differences was tested at 5% probability.ResultsMinimum and maximum counts with regard to the sachet water samples investigated were (4.3±1.1)×10⁶ CFU mL^?1 and (8.2±1.0) ×10⁶ CFU mL^?1 for heterotrophic plate counts; (0.9± 0.3) ×10⁶ CFU mL^?1 and (1.2± 0.4) ×10⁶ CFU mL^?1 for aerobic spore-former counts; (1.3±0.5) ×10³ CFU mL^?1 and (2.5± 0.8)×10³ CFU mL^?1 for total coliforms; (1.6±0.9) ×10³ CFU mL^?1 and (9.5±11.2)×10³ CFU mL^?1 for thermotolerant coliforms. Klebsiella spp but not Escherichia coli was present in all samples of the brands; non-coliform bacteria detected in some samples were Staphylococcus, Pseudomonas and Bacillus species.ConclusionsThe brands of sachet water sold (at the time of this study) in Amassoma did not meet the minimum acceptable standard for microbiologically safe drinking water as recommended by the World Health Organization.     

Pre-emptive rituximab based on viraemia and T cell reconstitution: a highly effective strategy for the prevention of Epstein-Barr virus-associated lymphoproliferative disease following stem cell transplantation

This study investigated the efficacy of a pre‐emptive strategy based on the combination of Epstein–Barr virus (EBV) viraemia and poor T cell reconstitution in preventing post‐transplant lymphoproliferative disease (PTLD) following T cell depleted stem cell transplant (SCT). EBV viral load and immune reconstitution were prospectively monitored in 70 consecutive children undergoing SCT following reduced intensity conditioning with alemtuzumab. Patients who developed significant EBV viraemia (>40 000 copies/ml blood) were treated pre‐emptively with rituximab if they were within 3 months of SCT or their CD3 count was <0·3 × 10⁹/l. Of 20/70 patients who developed significant EBV viraemia, 13 received pre‐emptive rituximab. The incidence of PTLD was significantly reduced in the pre‐emptive cohort compared to historical controls (1·4% vs. 21·7%, P = 0·003). This difference was more marked among viraemic patients (2·7% vs. 62·5%P < 0·0001). Patients treated with rituximab demonstrated significantly delayed B cell reconstitution at 1 year post‐SCT but this was not associated with an increase in infectious mortality. In 6/6 patients >3 months post‐SCT who had a CD3 count >0·3 × 10⁹/l, reduced immunosuppression only resulted in successful resolution of EBV viraemia without PTLD. This strategy is safe and highly effective in preventing PTLD following T cell depleted SCT, and directs rituximab therapy to patients at highest risk of this complication.     

Multicentre,randomised phase III study of the efficacy and safety of eltrombopag in Chinese patients with chronic immune thrombocytopenia

Eltrombopag, a thrombopoietin receptor agonist, raises platelet counts and reduces bleeding in patients with immune thrombocytopenia (ITP ). In Chinese patients, eltrombopag was evaluated at an initial dose of 25 mg, vs. 50 mg for non‐Asians, because the plasma exposure of eltrombopag is higher in East Asians. A multicentre, double‐blind, randomised, placebo‐controlled, 8‐week, phase III study enrolled 155 patients with chronic, previously treated ITP . Dosage could be adjusted (25–75 mg/day) to maintain platelet counts 50–250 × 10⁹/l. The primary efficacy endpoint was the proportion of patients with a platelet count ≥50 × 10⁹/l after Day 42. Pharmacokinetics and pharmacodynamics of eltrombopag were analysed in an open‐label extension. After Day 42, 57·7% of eltrombopag‐treated and 6·0% of placebo‐treated patients achieved platelet counts ≥50 × 10⁹/l. Odds of achieving a platelet count ≥50 × 10⁹/l were 26·08 times greater with eltrombopag than placebo (P  <  0·001). Compared with placebo, time to response and duration of response were better with eltrombopag (P  <  0·001) and the odds of any bleeding were reduced by 72% (P  =  0·001). Tolerability, pharmacokinetics, and pharmacokinetics/pharmacodynamics were similar to previous findings in East Asian patients. In conclusion, in Chinese patients with chronic ITP , eltrombopag 25 mg once daily, elevated platelet counts to a safe range and reduced bleeding.