Blood Titers Research Articles

Performance of parasitological and molecular techniques for the diagnosis and surveillance of gambiense sleeping sickness.

ObjectivesRecently, improvements have been made to diagnostics for gambiense sleeping sickness control but their performance remains poorly documented and may depend on specimen processing prior to examination. In a prospective study in the Democratic Republic of the Congo, we compared the diagnostic performance of several parasite detection techniques, immune trypanolysis and of m18S PCR on whole blood stored in a stabilisation buffer or dried on filter paper.MethodsIndividuals with CATT whole blood (WB) titer ≥1∶4 or with clinical signs indicative for sleeping sickness were examined for presence of trypanosomes in lymph node aspirate (LNA) and/or in blood. Blood was examined with Capillary Centrifugation Technique (CTC), mini-Anion Exchange Centrifugation Technique (mAECT) and mAECT on buffy coat (BC). PCR was performed on whole blood (i) stored in guanidine hydrochloride EDTA (GE) stabilisation buffer and (ii) dried on filter paper, and repeatability and reproducibility were assessed. Immune trypanolysis (TL) was performed on plasma.ResultsA total of 237 persons were included. Among 143 parasitologically confirmed cases, 85.3% had a CATT-WB titre of ≥1/8, 39.2% were positive in LNA, 47.5% in CTC, 80.4% in mAECT-WB, 90.9% in mAECT-BC, 95.1% in TL and up to 89.5% in PCR on GE-stabilised blood. PCR on GE-stabilised blood showed highest repeatability (87.8%) and inter-laboratory reproducibility (86.9%). Of the 94 non-confirmed suspects, respectively 39.4% and 23.4% were TL or PCR positive. Suboptimal specificity of PCR and TL was also suggested by latent class analysis.ConclusionThe combination of LNA examination with mAECT-BC offered excellent diagnostic sensitivity. For PCR, storage of blood in stabilisation buffer is to be preferred over filter paper. TL as well as PCR are useful for remote diagnosis but are not more sensitive than mAECT-BC. For TL and PCR, the specificity, and thus usefulness for management of non-confirmed suspects remain to be determined.

Comparative quantitative monitoring of rabbit haemorrhagic disease viruses in rabbit kittens.

BackgroundOnly one strain (the Czech CAPM-v351) of rabbit haemorrhagic disease virus (RHDV) has been released in Australia and New Zealand to control pest populations of the European rabbit O. cuniculus. Antigenic variants of RHDV known as RHDVa strains are reportedly replacing RHDV strains in other parts of the world, and Australia is currently investigating the usefulness of RHDVa to complement rabbit biocontrol efforts in Australia and New Zealand. RHDV efficiently kills adult rabbits but not rabbit kittens, which are more resistant to RHD the younger they are and which may carry the virus without signs of disease for prolonged periods. These different infection patterns in young rabbits may significantly influence RHDV epidemiology in the field and hence attempts to control rabbit numbers.MethodsWe quantified RHDV replication and shedding in 4–5 week old rabbits using quantitative real time PCR to assess their potential to shape RHDV epidemiology by shedding and transmitting virus. We further compared RHDV-v351 with an antigenic variant strain of RHDVa in kittens that is currently being considered as a potential RHDV strain for future release to improve rabbit biocontrol in Australia.ResultsKittens were susceptible to infection with virus doses as low as 10 ID50. Virus growth, shedding and transmission after RHDVa infection was found to be comparable or non-significantly lower compared to RHDV. Virus replication and shedding was observed in all kittens infected, but was low in comparison to adult rabbits. Both viruses were shed and transmitted to bystander rabbits. While blood titres indicated that 4–5 week old kittens mostly clear the infection even in the absence of maternal antibodies, virus titres in liver, spleen and mesenteric lymph node were still high on day 5 post infection.ConclusionsRabbit kittens are susceptible to infection with very low doses of RHDV, and can transmit virus before they seroconvert. They may therefore play an important role in RHDV field epidemiology, in particular for virus transmission within social groups during virus outbreaks.

Evaluation of antiviral efficacy of ribavirin, arbidol, and T-705 (favipiravir) in a mouse model for Crimean-Congo hemorrhagic fever.

BackgroundMice lacking the type I interferon receptor (IFNAR−/− mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR−/− mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus.Methodology/Principal FindingsCCHF virus-infected IFNAR−/− mice died 2–6 days post infection with elevated aminotransferase levels and high virus titers in blood and organs. Main pathological alteration was acute hepatitis with extensive bridging necrosis, reactive hepatocyte proliferation, and mild to moderate inflammatory response with monocyte/macrophage activation. Virus-infected and apoptotic hepatocytes clustered in the necrotic areas. Ribavirin, arbidol, and T-705 suppressed virus replication in vitro by ≥3 log units (IC50 0.6–2.8 µg/ml; IC90 1.2–4.7 µg/ml). Ribavirin [100 mg/(kg×d)] did not increase the survival rate of IFNAR−/− mice, but prolonged the time to death (p<0.001) and reduced the aminotransferase levels and the virus titers. Arbidol [150 mg/(kg×d)] had no efficacy in vivo. Animals treated with T-705 at 1 h [15, 30, and 300 mg/(kg×d)] or up to 2 days [300 mg/(kg×d)] post infection survived, showed no signs of disease, and had no virus in blood and organs. Co-administration of ribavirin and T-705 yielded beneficial rather than adverse effects.Conclusions/SignificanceActivated hepatic macrophages and monocyte-derived cells may play a role in the proinflammatory cytokine response in CCHF. Clustering of infected hepatocytes in necrotic areas without marked inflammation suggests viral cytopathic effects. T-705 is highly potent against CCHF virus in vitro and in vivo. Its in vivo efficacy exceeds that of the current standard drug for treatment of CCHF, ribavirin.

Complement Profile in Neonates of Different Gestational Ages

Blood levels of regulators of the complement system in preterm babies were reported in few studies only. The aim of this study was to set up a complement profile in premature and term babies focusing on the development of blood levels of MBL, key regulatory proteins and on classical pathway activity, which may allow an estimation of potential susceptibility to infection. Complement activity (CH50), levels of mannan-binding lectin (MBL), complement regulators (factors H and I, C1 inhibitor, properdin) and C3a as marker of complement activation were assessed in three groups of healthy newborns: (1) prematures (≤34 weeks); (2) late prematures (>34-<37 weeks) and (3) term neonates (≥37 weeks). CH50 increased with gestational age with lower titres in cord blood than in day 5 post-delivery venous blood. MBL concentrations were not significantly different among groups. Quantitative and functional C1 inhibitor were below adult normal range in prematures <34 weeks and lower in cord blood as compared to day 5. Factor I, factor H and properdin remained below adult values in all groups. Low C3a levels excluded that low complement titres were due to activation-induced consumption. These results demonstrate the relative immaturity of the complement system and its regulation, especially in premature infants.

Antibodies to islet cell autoantigens, rotaviruses and/or enteroviruses in cord blood and healthy mothers in relation to the 2010–2011 winter viral seasons in Israel: a pilot study

To determine whether antivirus and/or islet cell antibodies can be detected in healthy pregnant mothers without diabetes and/or their offspring at birth in two winter viral seasons. Maternal and cord blood sera from 107 healthy pregnant women were tested for islet cell autoantibodies using radioligand binding assays and for anti-rotavirus and anti-CoxB3 antibody using an enzyme-linked immunosorbent assay. Glutamic acid decarboxylase (GAD)65 autoantibodies and rotavirus antibodies, present in both maternal and cord blood sera, correlated with an odds ratio of 6.89 (95% CI: 1.01-46.78). For five, 22 and 17 pregnancies, antibodies to GAD65, rotavirus and CoxB3, respectively, were detected in cord blood only and not in the corresponding maternal serum. In 10 pregnancies, rotavirus antibody titres in the cord blood exceeded those in the corresponding maternal serum by 2.5-5-fold. Increased antibody titres after the 20(th) week of gestation suggested CoxB3 infection in one of the 20 pregnancies and rotavirus in another. The concurrent presence of GAD65 antibodies in cord blood and their mothers may indicate autoimmune damage to islet cells during gestation, possibly caused by cross-placental transmission of viral infections and/or antivirus antibodies. Cord blood antibody titres that exceed those of the corresponding maternal sample by >2.5-fold, or antibody-positive cord blood samples with antibody-negative maternal samples, may imply an active in utero immune response by the fetus.

Dynamics of perinatal bovine leukemia virus infection

BackgroundBovine leukemia virus (BLV) is highly endemic in many countries, including Argentina. As prevention of the spread from infected animals is of primary importance in breaking the cycle of BLV transmission, it is important to know the pathophysiology of BLV infection in young animals, as they are the main source of animal movement. In this work, we determined the proviral load and antibody titers of infected newborn calves from birth to first parturition (36 months).ResultsAll calves under study were born to infected dams with high proviral load (PVL) in blood and high antibody titers and detectable provirus in the colostrum. The PVL for five out of seven calves was low at birth. All animals reached PVLs of more than 1% infected peripheral blood mononuclear cells (PBMCs), three at 3 months, one at 6 months, and one at 12 months. High PVLs persisted until the end of the study, and, in two animals, exceeded one BLV copy per cell. Two other calves maintained a high PVL from birth until the end of the study. Antibody titers were 32 or higher in the first sample from six out of seven calves. These decayed at 3–6 months to 16 or lower, and then increased again after this point.ConclusionsCalves infected during the first week of life could play an active role in early propagation of BLV to susceptible animals, since their PVL raised up during the first 12 months and persist as high for years. Early elimination could help to prevent transmission to young susceptible animals and to their own offspring. To our knowledge, this is the first study of the kinetics of BLV proviral load and antibody titers in newborn infected calves.

Influenza Vaccination Given at Least 2 Weeks Before Delivery to Pregnant Women Facilitates Transmission of Seroprotective Influenza-specific Antibodies to the Newborn

Pregnant women and infants are at higher risk of complications secondary to influenza infection. Immunization during pregnancy facilitates protection of the neonates through passive transfer of maternal antibodies. This was a cross-sectional study performed during the post-H1N1 pandemic winter season of 2010/2011 in Geneva, Switzerland. We measured antibody titers against the seasonal influenza A H1N1, H3N2 and B 2010/2011 strains by hemagglutination inhibition in the umbilical cord blood of newborns born to vaccinated and nonvaccinated mothers. Seroprotection was defined as a hemagglutination inhibition titer ≥ 40. A total of 188 women were enrolled, 101 of whom had been vaccinated with a nonadjuvanted influenza vaccine (all during the second or third trimester) and the other 87 had not. Among newborns of vaccinated women, 84-86% showed seroprotective levels depending on the strain. In comparison, seroprotection rates were significantly lower in babies of nonvaccinated women (29-33%, P < 0.001). Adjusting for various confounding factors and applying multivariate regression analysis, vaccination during pregnancy ≥ 2 weeks before delivery increased geometric mean titers in umbilical cord blood 5-17 times and seroprotection rates 5.8-34.4 times, depending on the strain and the interval between vaccination and delivery. Vaccinating pregnant women only 2-4 weeks before delivery was still more effective than no vaccination at all (geometric mean titers increased 6.8-11.1 times and seroprotection rates increased 5.8-34.4 times compared with nonvaccinated women). Influenza vaccination at any time during the second and third trimester of pregnancy, but at least 15 days before delivery, confers seroprotection to many neonates.

ACIP tackles range of vaccine topics at meeting

The following article is part one of a two-part series covering vaccine topics addressed at the October 2013 meeting of the Advisory Committee on Immunization Practice (ACIP). More topics will be covered in next month's Immunization Update. The recent publication of the 2013–14 ACIP influenza recommendations included new vaccine strains, licensure of six new influenza vaccines, and clarification of egg allergies. Since the publication of the report, Flulaval Quadrivalent (ID Biomedical) was licensed and Flulaval Trivalent was approved for a new age indication (patients >3 years). As of October 12, influenza activity was low, which is normal for this early in the influenza season. About 90% of the strains reported so far are type A. Influenza-like illness (ILI) activity remains low. Vaccination coverage rates for the 2012–13 season were reviewed. A total of 134.9 million doses were distributed last season, with coverage rates of 57% in children, 41.5% in adults older than 18 years, and 66% in individuals 65 years or older. Rates were higher in all age groups. A summary of the Fluzone HighDose (Sanofi Pasteur) vaccine efficacy trial results was presented. The prelicensure trials demonstrated that the vaccine induced a statistically significantly higher antibody response against all three strains compared with regulardose Fluzone vaccine. Fluzone HighDose is indicated for adults 65 years or older, while regular-dose Fluzone is recommended for patients 6 months or older. A study comparing the clinical efficacy of Fluzone High-Dose with Fluzone trivalent vaccine was presented. The study enrolled approximately 32,000 participants 65 years or older in 128 study sites over two influenza seasons. The study was designed to compare the efficacy with respect to laboratory-confirmed influenza caused by any influenza strain with the occurrence of protocol-defined ILI. Relative efficacy was 24.2% higher for Fluzone High-Dose (1.43% vs. 1.89% ILI). Slight improvement also was seen for pneumonia, hospitalizations, and cardiorespiratory episodes. Additional analysis and studies are under way.■ACIP's 2013–14 influenza recommendations include new vaccine strains, licensure of six new influenza vaccines, and clarification of egg allergies.■ACIP needs more evidence before it can recommend reducing the number of doses in the pneumococcal childhood series from three to four doses. ■ACIP's 2013–14 influenza recommendations include new vaccine strains, licensure of six new influenza vaccines, and clarification of egg allergies.■ACIP needs more evidence before it can recommend reducing the number of doses in the pneumococcal childhood series from three to four doses. Three conjugated meningococcal vaccines have been approved in the United States. MenACYW-135-CRM (Menveo—Novartis) recently received an indication for age as early as 2 months (2 months to 55 years). The studies submitted for FDA approval were presented. The vaccine demonstrated effective blood titers and did not interfere with other vaccines given in the infant series. The GRADE analysis was similar to the other meningococcal vaccines. The working group decided that Menveo should be added as an option for vaccinating high-risk infants. Many countries have implemented pneumococcal conjugate vaccine (PVC) in a three-dose schedule (compared with the four-dose schedule in the United States). CDC has been evaluating the incidence of invasive pneumococcal dis-ease (IPD) in children since 2010 (when 13-valent PCV [PCV13] was licensed) and is observing a decline. Also, as a result of herd effect, IPD appears to be declining in adults. The vaccine effectiveness in one study against all IPD in children was 65%. However, if vaccine effectiveness is measured against the PCV13 serotypes in the vaccine, it climbs to 89%. Because the World Health Organization recognizes a three-dose schedule as acceptable, the working group is discussing a reduction in the number of doses in the pneumococcal childhood series. We also are seeing a large decline in disease burden in the United States caused by serotypes in the vaccine. Looking at studies from other countries, the vaccine effectiveness ranged from 70% to 100% against vaccine-type IPD. The vaccines used in these studies included PCV7, −9, and −13. The four-dose series studies reported a vaccine effectiveness of 81% to 100%. No studies comparing three- and four-dose schedules have emerged. Differing types of studies (e.g., randomized controlled versus observational) and differing diseases (e.g., IPD, pneumonia, acute otitis media) are under way. In conclusion, the three-dose PCV studies demonstrated effectiveness against IPD, pneumonia, and otitis media. The immunogenicity studies demonstrated that after the primary series, three doses are more effective than two, but this difference does not continue after the second year of life. Also, little difference was noted after the booster dose for the two-plus-one versus three-plus-one series. The same pattern was seen with nasopharyngeal carriage. However, the differences may not be meaningful because PCV7 serotypes rarely cause disease and the PVC13 serotype disease is disappearing. The herd effects are similar, so changes at the population level are not expected.

Hepatitis B Related Serological Events In Hematopoietic Stem Cell Transplant Patients and Efficacy Of Lamivudine Prophylaxis Against Reactivation

IntroductionRemote Hepatitis B Infection (RHBI) is an immunological stage in the life cycle of Hepatitis B virus (HBV). It is characterized by the persistence of HBV DNA at low level within the liver or blood of people infected in past. As a result HBsAg, HBeAg and HBV DNA in serum are negative while Anti-HBs and/or Anti-HBc IgG are positive with normal liver enzymes. In Hematopoietic stem cell transplant (HSCT) patients, reactivation of RHBI leads to increased HBV replication and increased HBV DNA titres in blood and/or HBsAg or HBeAg positivity. There may be loss or gain of Anti-HBs and/or Anti-HBc IgG. The importance of recognizing this stage with a potential for reactivation is highlighted by the fact that: 1) HBV reactivation can lead to fulminant hepatitis & 2) Anti-HBV therapy such as lamivudine can prevent HBV reactivation. In this study we have aimed to determine the incidence of RHBI and HBV reactivation in our patients, the role of lamivudine in preventing HBV reactivation and the pattern of change in serological markers after reactivation. MethodsAll patients undergoing HSCT from March 2010 to May 2013 were included. Pre transplant, all patients were tested by ELISA for HBsAg, Anti-HBs, Anti-HBc IgM and Anti-HBc IgG. Post transplant, at the time of deranged liver function, all patients were tested for HBsAg, Anti-HBs, Anti-HBc IgM, Anti-HBc IgG, Anti-HCV, Anti-HEV and Anti-HAV. HBV DNA and HCV RNA were tested by polymerase chain reaction. RHBI was defined as positive Anti-HBc IgG and/or Anti-HBs (without history of HBV vaccination) with negative HBsAg and Anti-HBc IgM and normal liver function. HBV reactivation was defined as increase in transaminases to more than 3 times the upper limit of normal with HBV DNA and/or Anti-HBs and/or Anti-HBc IgG positivity (HBsAg and Anti-Hbc IgM being negative) at the time of deranged liver function. Lamivudine at a dose of 100 mg/d or 3 mg/kg/d (children <12 years) was given to all but one patient with Anti-HBc IgG positivity pre transplant irrespective of Anti-HBs positivity (Prophylaxis Group). Patients who had only Anti-HBs positivity did not receive prophylaxis (No Prophylaxis Group). Data was analyzed to explore the role of prophylactic lamivudine in preventing HBV reactivation and to determine the pattern of change of HBV serological markers at the time of reactivation. The time to respond to lamivudine was calculated as the interval from start of prophylaxis to normalization of liver enzymes. ResultsTwo hundred five patients underwent HSCT (autologous - 103 and allogeneic - 102) from March 2010 to May 2013. Twenty-eight (14%) patients were diagnosed to have RHBI (autologous-9, allogeneic-19). Thirteen patients did not receive prophylactic lamivudine (No-Prophylaxis Group) while 15 patients received prophylactic lamivudine (Prophylaxis Group). Twelve (92%) patients in No-Prophylaxis Group developed HBV reactivation while none (0%) in the Prophylaxis Group (P<0.001). The incidence of HBV reactivation was 10% (21 of 205 patients) which included 11 (52%) with only Anti-HBs positive, 1 with only Anti-HBc IgG positive and nine (43%) with all HBV serological markers negative (i.e. no evidence of RHBI) pre transplant. Six of these 9 patients sero-converted (5 - Anti-HBc IgG and 1 - Anti-HBs positive) at the time of reactivation. Seven (33%) developed HBV viremia at the time of HBV reactivation, of which 6 were Anti-HBs negative pre transplant. In contrast, of the patients with undetectable HBV DNA at the time of reactivation (14 patients), only 4(29%) were Anti-HBs negative pre transplant (P=0.0237). Seven (33%) patients were on steroids and 9(43%) were on cyclosporine at the time of reactivation. All patients responded to lamivudine within a median time of 17 days. ConclusionsIncidence of RHBI in patients undergoing HSCT is high in our setting.Lamivudine prophylaxis protects against HBV reactivation post transplant. We would recommend lamivudine prophylaxis in patients with RHBI with isolated Anti-HBs positivity in addition to those with Anti-HBc IgG positivity given the high rate of HBV reactivation in these patients.All serological markers for Hepatitis B being negative pre transplant does not preclude HBV reactivation post transplant. Therefore, all serological markers for HBV should be repeated at the time of deranged liver functions post transplant.Anti-HBs positivity pre transplant decreases the risk of HBV viremia at the time of reactivation. Disclosures:No relevant conflicts of interest to declare.

Tissue Factor On The Herpes Simplex Virus Type 1 Surface Enhances Infection In Vivo

Tissue factor (TF) is a multifunctional transmembrane receptor. As the pivotal initiator of the coagulant response to vascular damage, it accelerates factor (F) VIIa-dependent generation of FXa. However, TF also functions in FVIIa/FXa-dependent cell signaling via protease activated receptors (PARs), and has consequently been implicated in a wide variety of physiological and pathological conditions. Our previous work adds to this repertoire by revealing that TF, originating from the host cell membrane, is on the surface of enveloped viruses. Using herpes simplex virus type 1 (HSV1) as a model for enveloped viruses, we recently produced TF +/- HSV1 variants, which showed that viral TF enhances infection of cultured cells through a mechanism involving FXa/FVIIa-mediated activation of PAR-2. In the current project we extended these studies in vivo, hypothesizing that TF on the HSV1 surface would enhance infection by influencing the activation of coagulation proteases and/or their role in cell signaling. As before, HSV1 NS strain (with restored glycoprotein C) was propagated in TF- or genetically engineered TF+ human melanoma A7 cells, then purified and quantified. HSV1 TF+ or HSV1 TF- virus (5 x 105 virus plaque forming units of similar particle number) was injected slowly in 100 µL into the tail vein of female, eight-week old balb/c mice. On the third day, organs and blood were harvested. Live virus and total HSV1 genome were evaluated by plaque assays and rtPCR, respectively. Strikingly, production of live HSV1 TF+ (n=13) in the lung, heart, spinal cord, liver and brain was greater by an order of magnitude compared to HSV1 TF- (n=13), which was not detected in some tissues. The presence of TF on HSV1 furthermore increased virus titers in spleen and blood by several fold. Viral replication in organs was corroborated by rtPCR. The procoagulant and signaling functions of TF can be dissected using epitope-specific monoclonal antibodies (mAbs) selective for human TF. These mAbs only recognize HSV1 surface TF and not the endogenous mouse TF. These mAbs or an irrelevant mAb (TIB115, n=8) were administered by intraperitoneal injection 4 hours pre-inoculation (1 mg/mouse). When TF-dependent coagulation and the related effects on cell signaling due to attenuated protease activation was blocked by a potent combination of three mAbs to non-overlapping epitopes (5G9/6B4/9C3; 0.33 mg/mouse each, n=10), reduced levels of live HSV1 TF+ were seen in lung, heart, liver, brain and blood, with concordantly reduced viral genome levels measured by rtPCR. Similar results were obtained with mAb 5G9 alone (n=6), which selective blocks FXa generation. Interestingly, titers of HSV1 TF+ in blood and spleen were not affected by mAB 10H10 (n=6) that has no effect on coagulation, but is an effective direct inhibitor of TF signaling. Despite normal blood titers, mAb 10H10 (n=6) reduced virus titers in heart and brain, but not in other organs. These data demonstrate that virus surface TF is important for HSV1 infection in vivo and indicate that distinct functions of TF contribute to organ-specific tropism. Disclosures:No relevant conflicts of interest to declare.

Infectious Complications After Unrelated Donor Allogeneic Hematopoietic Cell Transplantation With Or Without Alemtuzumab Based In Vivo Lymphocyte Depletion

The addition of alemtuzumab to conditioning may decrease the incidence of graft versus host disease (GVHD) after unrelated donor (URD) allogeneic hematopoietic cell transplantation (alloHCT) but may also contribute to impaired pathogen immunity due to donor lymphocyte depletion. We identified and graded infections occurring within 6 months after URD alloHCT in 78 individuals treated either with (N=40) or without (N=38) alemtuzumab during alloHCT for hematologic cancers. Conditioning included fludarabine (120 mg/m2 total) and cyclophosphamide (4,800 mg/m2 total) over 4 days with either alemtuzumab (100 mg total days -8 to -4) followed by cyclosporine (AC) or with tacrolimus, methotrexate, and sirolimus (TMS) based GVHD prophylaxis. Both groups received a filgrastim mobilized peripheral blood graft from an 8/8 or 7/8 HLA-matched unrelated donor and received GVHD prophylaxis for 180 days. Filgrastim was administered post HCT until neutrophil recovery. Infections were graded according to Cordonnier et al. (Transplantation 2006). Only proven/probable pulmonary fungal episodes by EORT/MSG criteria were included. Infection prophylaxis during the study period included trimethoprim/sulfamethoxazole BID thrice weekly after HCT engraftment and daily acyclovir or valacyclovir. Fungal prophylaxis was fluconazole until day 100 or either micafungin or voriconazole in persons receiving 1+ week of &gt;1 mg/kg/d prednisone or equivalent. Ceftazidime was administered to persons with neutrophil count &lt;500/μL until recovery. Persons were monitored for CMV, adenovirus, toxoplasmosis (in seropositive persons), EBV (after 2009), and HHV-6 with at least weekly blood PCR. CMV was treated with pre-emptive therapy according to published guidelines. Blood aspergillus glactomannin and beta D-glucan assays were obtained when clinically indicated. The use of AC compared to TMS resulted in greater total infections per patient (4.4 versus 2.8, P&lt;0.0001) but similar rates of grade 3 (most serious) infections (0.5 versus 0.6, P=0.49). CMV reactivation in recipient seropositive persons was more common in AC (24/24 AC arm versus 11/24 TMS arm, P&lt;0.0001). The median duration of CMV viremia was 6 weeks in AC versus 2 weeks in TMS (P=0.001). CMV colitis or pneumonitis occurred in 3 persons treated with AC and in 1 person treated with TMS at a mean of 47 days post alloHCT (range 15-121). Bacterial infections per patient in AC and TMS were similar (1.6 versus 1.1, P=0.08). Clostridium difficile colitis was relatively common (10 episodes AC; 11 episodes TMS). HHV-6 infections were similar (24/40 AC versus 16/38 TMS, P=0.16). HHV-6 encephalitis developed in five individuals (3 TMS, 2 AC). The median blood titer at the time CNS involvement was identified was 15,950 copies/mL (0-40,000 copies/mL) and was not predictive on onset of encephalopathy (P=0.6). EBV infection developed in 9 persons in AC and 6 persons in TMS. Three cases of EBV lymphoproliferative disorder occurred in persons treated with AC. An additional three persons in each AC and TMS were treated pre-emptively with rituximab for EBV viremia. Adenovirus infection developed in 4 persons in each TMS and AC. One person treated with AC developed adenoviral colitis. There was one episode of invasive fungal infection in TMS (Candida krusei blood stream infection) and 4 episodes in AC (3 episodes Candida blood stream infection [2 parapsilosis, 1 albicans], one pulmonary aspergillosis). In summary, the use of AC versus TMS resulted greater total infections and CMV infections but with similar bacterial and fungal infection incidence. Weekly monitoring of HHV-6 by blood PCR was ineffective at predicting development of HHV-6 CNS disease. The rate of invasive fungal infections in this cohort was low. Disclosures: No relevant conflicts of interest to declare.

Infection of Type I Interferon Receptor-Deficient Mice with Various Old World Arenaviruses: A Model for Studying Virulence and Host Species Barriers

Lassa virus causes hemorrhagic Lassa fever in humans, while the related Old World arenaviruses Mopeia, Morogoro, and Mobala are supposedly apathogenic to humans and cause only inapparent infection in non-human primates. Here, we studied whether the virulence of Old World arenaviruses in humans and non-human primates is reflected in type I interferon receptor deficient (IFNAR-/-) mice by testing several strains of Lassa virus vs. the apathogenic viruses Mopeia, Morogoro, and Mobala. All Lassa virus strains tested—Josiah, AV, BA366, and Nig04-10—replicated to high titers in blood, lung, kidney, heart, spleen, brain, and liver and caused disease as evidenced by weight loss and elevation of aspartate and alanine aminotransferase (AST and ALT) levels with a high AST/ALT ratio. Lassa fever-like pathology included acute hepatitis, interstitial pneumonia, and pronounced disturbance of splenic cytoarchitecture. Infiltrations of activated monocytes/macrophages expressing inducible nitric oxide synthase and T cells were found in liver and lung. In contrast, Mopeia, Morogoro, and Mobala virus replicated poorly in the animals and acute inflammatory alterations were not noted. Depletion of CD4+ and CD8+ T cells strongly enhanced susceptibility of IFNAR-/- mice to the apathogenic viruses. In conclusion, the virulence of Old World arenaviruses in IFNAR-/- mice correlates with their virulence in humans and non-human primates. In addition to the type I interferon system, T cells seem to regulate whether or not an arenavirus can productively infect non-host rodent species. The observation that Lassa virus overcomes the species barrier without artificial depletion of T cells suggests it is able to impair T cell functionality in a way that corresponds to depletion.

High Circulatory Titer of Platelet-Associated Autoantibodies in Childhood Onset Schizophrenia and Its Diagnostic Implications

Background: It has been suggested that the etiology of schizophrenia, in a distinct group of patients, originates from an autoimmune reaction against platelets. Previous studies have demonstrated significantly higher blood titers of platelet-associated autoantibodies (PAA) in adult schizophrenia patients as compared to normal healthy subjects. In addition, young adult schizophrenia patients at their early stages of the disorder displayed higher PAA titers than older patients with longer duration of the disorder. Aim: To assess the blood titers of PAA in children with schizophrenia as compared to matched control subjects without psychotic disorders, as a possible diagnostic parameter. Methods: Twenty-nine children with DSM-IV schizophrenia in the active psychotic state, with an age range of 6-12 years (mean ± SD: 9.6 ± 1.5 years), with average Positive and Negative Syndrome Scale scores of 108 ± 19.2, were assessed. The control group consisted of 25 children with DSM-IV conduct disorder in a similar age range of 5-12 years (mean ± SD: 9.5 ± 1.6 years). The blood titers of PAA were evaluated using an optimized ELISA test, expressed by a linear optical density (OD) scale. The blood samples of all participants were tested anonymously and were scored under a code number. A test recording above 1.4 OD units was predefined as positive. Results: The titers of PAA of children with schizophrenia (1.9 ± 0.5 OD units, range: 0.7-2.44 units) were significantly (p < 0.00001) higher than those of the control group (1.0 ± 0.4 OD units, range: 0.45-2.28 units). In 83% of the children with schizophrenia (24 out of the 29 patients) a positive test, i.e. OD >1.4, was detected. In contrast, in the control group, only 12% (3 of the 25 subjects) displayed a positive test, p < 0.00001. Conclusions: High titers of PAA in children with schizophrenia as compared with nonpsychotic controls may indicate an active autoimmune process in the early onset of schizophrenia. The PAA level may therefore provide a supportive diagnostic biomarker for childhood schizophrenia.

The role of the RNA chaperone Hfq in Haemophilus influenzae pathogenesis

BackgroundThe RNA binding protein Hfq of Haemophilus influenzae is highly homologous to Hfq from other bacterial species. In many of these other bacteria, Hfq affects the expression of a broad range of genes and enhances the ability to respond to stressful environments. However, the role of Hfq in H. influenzae is unknown.ResultsDeletion mutants of hfq were generated in the nontypeable H. influenzae strains R2866 and 86-028NP to assess the role of Hfq in these well characterized but genotypically and phenotypically divergent clinical isolates. A deletion mutation of hfq had no effect on growth of H. influenzae in nutrient rich media and had no effect on survival in several stressful conditions in vitro. However, the mutation resulted in a reduced ability to utilize heme from hemoglobin. The mutant and wild type strains were assessed for virulence and competitive fitness in models of invasive disease and otitis media. In the chinchilla model of otitis media, the hfq mutant of 86-028NP exhibited impaired competitive fitness when compared to its wild type progenitor but exhibited no apparent defect in virulence. In the infant rat model, deletion of hfq in R2866 resulted in reduced bacterial titers in blood and a shorter duration of infection when compared to the wild type strain in the competitive fitness study.ConclusionWe conclude that Hfq is involved in the utilization of essential nutrients and facilitates infection by H. influenzae.

Prevalence of Neutralizing Factors Against Adeno-Associated Virus Types 2 in Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy

Adeno-associated virus type 2 (AAV2) mediated gene therapy providing a potential treatment in the eye. However, immune responses can limit virally mediated gene transfer and therapy. To assess preexisting AAV2 neutralizing factors (NF) titers in peripheral blood and the vitreous in patients with age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). 130 subjects were enrolled: 50 with neovascular AMD, 30 with PCV, and 50 controls. The serum and the vitreous were obtained for AAV2 NF assay. We found AAV2 NF are present in all of AMD, PCV patients and controls we tested. There were no significant differences in prevalence of NAb in serum between AMD, PCV and controls (P=0.999). There was no correlation between NF in serum and in vitreous (P>0.05), and NF in vitreous was significantly less than in serum. Our results for the first time showed in Chinese population, NF against AAV2 was present in serum of all the patients with AMD or PCV and controls, and there were no significant differences among these groups. Therefore, it demonstrated there were no correlations between AAV2 NF titer and these diseases. We found NF in vitreous was considerably less than in serum in all groups. We also found no direct correlation between NF in vitreous and in serum suggesting serum antibody levels may not be used to predict their counterparts in the vitreous. Our results will provide crucial information for future clinical studies in the development of new therapies based on AAV2 mediated gene delivery in the eye.

Comparative investigations of immune response of calves at different intervals between primary and secondary immunization using inactivated bovine herpes virus 1 vaccine

Bovine herpesvirus 1 (BHV-1) is one of the most siginificant causes of infections of the respiratory tract of cattle and immunoprophylaxis has a key role in curbing this infection. The intensity of the immune response against BHV-1 following immunization using inactivated commercial vaccines varies depending on the type of vaccine, but it is generally believed that they provide good protection from the development of the clinical form of the infection, and that they are safe. The paper present the development of the humoral immune response in fattening calves that were immunized against bovine herpesvirus 1 (BHV-1) at different time intervals between the primary and the secondary immunization. Calves were administered a commercial vaccine, and then they were divided into two groups which were revaccinated on days 14 or 21. Over a course of the 120 days of the duration of the experiment, blood and nasal mucus were sampled 11 times. The blood serum samples were examined for antibodies to BHV-1 using the virus neutralization (VN) test, and the nasal mucus samples were analyzed using the VN test and the ELISA method. Following revaccination, it was established that there was an increase in the antibody titer in blood of all experimental animals, and it was maintained at a high level up until the very end of the experiment (day 120). In the blood serums, maximum mean values for the antibody titer were determined on day 30 in the group that was revaccinated on day 14, and on day 45 in the group of calves revaccinated on day 21. In nasal mucus, antibodies were established at the earliest, using the virus neutralization test, on day 14 following vaccination, and using the ELISA method only after revaccination. The highest antibody titer in nasal mucus was established on day 45 in the group revaccinated on day 21, and on day 120 in the group revaccinated on day 14. Based on the established antibody titer values, calves can be revaccinated using the inactivated BHV-1 vaccine already on day 14.

NK cells from an AML patient have recovered in remission and reached comparable cytolytic activity to that of a healthy monozygotic twin mediated by the single-chain triplebody SPM-2

BackgroundThe capacity of patient’s Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling’s NKs were compared with the patient’s drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient’s autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared.MethodsPatient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling’s NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody RituximabTM were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays.ResultsPatient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient’s autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission.ConclusionsPeripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for cells drawn from an AML patient in remission and a healthy twin. If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML.

Concentrations of nitric oxide (NO) metabolites in the liver of Neisseria meningitidis-infected mice: The effect of bacterial nitric oxide detoxification

Rationale and hypothesis Meningococcal septicaemia is characterised by high bacterial titres in blood and an increased production of circulating nitric oxide metabolites, including nitrates NO 3 - , nitrites NO 2 - and S-nitrosothiol [SNO]. The formation of SNO, termed S-nitrosylation, is an important form of post-translational protein modification akin to phosphorylation. Previously, bacterial NO detoxification was demonstrated to reduce the concentration of host-cell SNO. We hypothesise that bacterial NO detoxification during sepsis interferes with host NO homeostasis within the liver, a vital organ in physiological response to infection, and contributes toward pathogenesis. Objectives 1. To establish an in vivo murine model of acute fulminant meningococcal sepsis. 2. To study the link between bacterial NO detoxification and circulating NO metabolites in murine liver. Methodology Female C57 Bl/6 mice were infected by intraperitoneal injection of wild type Neisseria meningitidis (serogroup B strain MC58) or an isogenic mutant unable to detoxify NO (ΔnorB). Eight hours after infection, livers were extracted and lysates were subjected to ozone-based chemiluminescence-based measurement of multiple NO metabolites. We obtained reproducibly high bacterial titres both in blood and liver lysates. Findings 1. Increased bacterial burden in liver lysates is positively and significantly correlated with hepatic nitrite concentrations NO 2 - (Spearman’s rank correlation, r = 0.5682, p = 0.0020, n = 9), negatively and significantly correlated with the hepatic concentration of all NO [NOx] deriviatives (r = −0.4390, p = 0.0220, n = 9) 2. In an acute fulminant meningococcal sepsis model, the production of NO metabolites was found to be unperturbed by the presence of bacterial NO detoxification machinery (p = 0.2704, n = 9).

Highly Efficient Prion Transmission by Blood Transfusion

It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 103ID50 as measured by intracerebral inoculation of tg338 mice (ID50 IC in tg338). This was consistent with a whole blood titer greater than 103.6 ID50 IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation.

Infection regulates pro-resolving mediators that lower antibiotic requirements

Underlying mechanisms for how bacterial infections contribute to active resolution of acute inflammation are unknown1-4. Here, we performed exudate leukocyte trafficking and mediator-metabololipidomics of murine peritoneal Escherichia coli (E. coli) infections with temporal identification of pro-inflammatory (prostaglandins and leukotrienes) and specialized pro-resolving mediators (SPM). In self-resolving E. coli exudates (105 CFU), the dominant SPM identified were resolvin (Rv) D5 and protectin D1 (PD1), which at 12 h were significantly greater than levels in exudates from higher titer E. coli (107 CFU) challenged mice. Germ-free mice displayed endogenous RvD1 and PD1 levels higher than in conventional mice. RvD1 and RvD5 (ng/mouse) each reduced bacterial titers in blood and exudates, E. coli-induced hypothermia and increased survival, demonstrating the first actions of RvD5. With human polymorphonuclear neutrophils (PMN) and macrophages, RvD1, RvD5, and PD1 each directly enhanced phagocytosis of E. coli, and RvD5 counter-regulated a panel of pro-inflammatory genes, including NF-κB and TNF-α. RvD5 activated the RvD1 receptor, GPR32, to enhance phagocytosis. With self-limited E. coli infections, RvD1 and the antibiotic ciprofloxacin accelerated resolution, each shortening resolution intervals (Ri). Host-directed RvD1 actions enhanced ciprofloxacin’s therapeutic actions. In 107 CFU E. coli infections, SPM (RvD1, RvD5, PD1) together with ciprofloxacin also heightened host antimicrobial responses. In skin infections, SPM enhanced vancomycin clearance of Staphylococcus aureus. These results demonstrate that specific SPM are temporally and differentially regulated during infections and that they are anti-phlogistic, enhance containment and lower antibiotic requirements for bacterial clearance.