Molecular characteristics and successful management of a respiratory syncytial virus outbreak among pediatric patients with hemato-oncological disease.

Background: Respiratory syncytial virus (RSV) is responsible for upper and lower respiratory tract infection in adults and children. Especially immunocompromised patients are at high risk for a severe course of infection, and mortality is increased. Moreover RSV can spread in healthcare settings and can cause outbreaks. Herein we demonstrate the successful control and characteristics of a RSV outbreak that included 8 patients in our Department of Pediatric Hematology and Oncology. Methods: We performed an epidemiologic investigation and a molecular analysis of the outbreak strains. Moreover we present the outbreak control bundle and our concept for RSV screening in the winter season. Results: RSV A and B strains caused the outbreak. RSV B strains affected 3 patients, 2 of whom were co-infected with RSV A. Exactly this RSV A strain was detected in another 5 patients. Our multimodal infection control bundle including prophylactic RSV screening was able to rapidly stop the outbreak. Conclusion: An infection control bundle in RSV outbreaks should address all potential transmission pathways. In pediatric settings the restriction of social activities might have a temporal negative impact on quality of life but helps to limit transmission opportunities. Molecular analysis allows better understanding of RSV outbreaks and, if done in a timely manner, might be helpful for guidance of infection control measures.

Fulminant anaplastic large cell lymphoma (ALCL) concomitant with primary cytomegalovirus (CMV) infection, and human herpes virus 8 (HHV-8) infection together with Epstein-Barr-virus (EBV) reactivation in a patient with asymptomatic HIV-infection.

BACKGROUND: Most malignant lymphomas in HIV-patients are caused by reactivation of EBV-infection. Some lymphomas have a very rapid fulminant course. HHV-8 has also been reported to be a cause of lymphoma. The role of CMV in the development of lymphoma is not clear, though both CMV and HHV-8 have been reported in tissues adjacent to the tumour in Burkitt lymphoma patients. Here we present a patient with asymptomatic HIV infection, that contracted a primary cytomegalovirus (CMV) infection and human herpes virus 8 (HHV-8) infection. Three weeks before onset of symptoms the patient had unprotected sex which could be possible source of his CMV and also HHV-8 infection He deteriorated rapidly and died with a generalized anaplastic large cell lymphoma (ALCL). METHODS: A Caucasian homosexual male with asymptomatic human immunodeficiency virus (HIV) infection contracted a primary cytomegalovirus (CMV) infection and human herpes virus 8 (HHV-8) infection. He deteriorated rapidly and died with a generalized anaplastic large cell lymphoma (ALCL). Clinical and laboratory records were compiled. Immunohistochemistry was performed on lymphoid tissues, a liver biopsy, a bone marrow aspirate and the spleen during the illness and at autopsy. Serology and PCR for HIV, CMV, EBV, HHV-1-3 and 6-8 was performed on blood drawn during the course of disease. RESULTS: The patient presented with an acute primary CMV infection. Biopsies taken 2 weeks before death showed a small focus of ALCL in one lymph node of the neck. Autopsy demonstrated a massive infiltration of ALCL in lymph nodes, liver, spleen and bone marrow. Blood samples confirmed primary CMV- infection, a HHV-8 infection together with reactivation of Epstein- Barr-virus (EBV). CONCLUSION: Primary CMV-infection and concomitant HHV-8 infection correlated with reactivation of EBV. We propose that these two viruses influenced the development and progression of the lymphoma. Quantitative PCR blood analysis for EBV, CMV and HHV-8 could be valuable in diagnosis and treatment of this type of very rapidly developing lymphoma. It is also a reminder of the importance of prevention and prophylaxis of several infections by having protected sex.

Viral Origin, Clinical Course, and Renal Outcomes in Patients With BK Virus Infection After Living-Donor Renal Transplantation.

BACKGROUND: BK virus (BKV) nephropathy remains the main cause of renal graft loss after living-donor renal transplantation. The aim of the study was to investigate the source and factors influencing the course of BKV infection. METHODS: We investigated 214 living donor-recipient pairs. Urine and blood of donors and recipients were tested by qPCR for the presence of BKV DNA before and after transplantation; genotyping of BKV subtypes was performed. RESULTS: Eighty-five recipients (40%) had posttransplant BK viruria including 61 with additional viremia and 22 with nephropathy. Pretransplant urinary BKV shedding of donor or recipient was a significant risk factor for posttransplant viruria and viremia (OR, 4.52; CI, 2.33-8.77; P < 0.0001) and nephropathy (OR, 3.03; CI, 1.16-7.9; P = 0.02). In the BKV nephropathy group, urine and blood became BKV positive earlier than in the group with viruria and viremia. Renal function was worse in BKV-nephropathy compared with BKV-negative patients beginning at transplantation. Comparing BKV subtypes of donor and recipient before with the subtype of the infected recipient after transplantation, donor-derived transmission was identified in 24 of 28 corresponding pairs. BKV subtype IV had a higher prevalence in recipients with BKV nephropathy than in those with viruria and viremia (P = 0.045). CONCLUSIONS: Pretransplant urinary BKV shedding of donor and recipient is a risk for posttransplant infection. Donor-derived BKV transmission is an important mode of infection. BKV subtype IV may be one of the viral determinants. Early BKV positivity of urine and blood indicates later BKV nephropathy. Decreased renal function may favor BKV infection.

Amino Acid Variation in HLA Class II Proteins Is a Major Determinant of Humoral Response to Common Viruses.

The magnitude of the human antibody response to viral antigens is highly variable. To explore the human genetic contribution to this variability, we performed genome-wide association studies of the immunoglobulin G response to 14 pathogenic viruses in 2,363 immunocompetent adults. Significant associations were observed in the major histocompatibility complex region on chromosome 6 for influenza A virus, Epstein-Barr virus, JC polyomavirus, and Merkel cell polyomavirus. Using local imputation and fine mapping, we identified specific amino acid residues in human leucocyte antigen (HLA) class II proteins as the most probable causal variants underlying these association signals. Common HLA-DRß1 haplotypes showed virus-specific patterns of humoral-response regulation. We observed an overlap between variants affecting the humoral response to influenza A and EBV and variants previously associated with autoimmune diseases related to these viruses. The results of this study emphasize the central and pathogen-specific role of HLA class II variation in the modulation of humoral immune response to viral antigens in humans.

A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages, Is Associated with Borrelia Seropositivity.

In a first genome-wide association study (GWAS) approach to anti-Borrelia seropositivity, we identified two significant single nucleotide polymorphisms (SNPs) (rs17850869, P = 4.17E-09; rs41289586, P = 7.18E-08). Both markers, located on chromosomes 16 and 3, respectively, are within or close to genes previously connected to spinocerebellar ataxia. The risk SNP rs41289586 represents a missense variant (R263H) of anoctamin 10 (ANO10), a member of a protein family encoding Cl(-) channels and phospholipid scramblases. ANO10 augments volume-regulated Cl(-) currents (IHypo) in Xenopus oocytes, HEK293 cells, lymphocytes and macrophages and controls volume regulation by enhancing regulatory volume decrease (RVD). ANO10 supports migration of macrophages and phagocytosis of spirochetes. The R263H variant is inhibitory on IHypo, RVD and intracellular Ca(2+) signals, which may delay spirochete clearance, thereby sensitizing adaptive immunity. Our data demonstrate for the first time that ANO10 has a central role in innate immune defense against Borrelia infection.

Donor origin of BKV replication after kidney transplantation.

BACKGROUND: BK virus associated nephropathy (BKVN) leads to renal allograft dysfunction and loss. However, it is still unclear whether BKV replication in the transplant recipient is a result of reactivation in the recipient's native kidneys or whether BKV originates from the donor kidney. STUDY DESIGN: Urine of 249 donor/recipient pairs was investigated for the presence of BKV-DNA by qPCR before living transplantation (Tx) and consecutively after Tx. In BKV positive samples, the VP1 typing region (TR) and, in case of the presence of sufficient amount of DNA, the complete VP1 gene, the NCCR and a fragment of the Large T-antigen were sequenced and compared between donors and corresponding recipients before and after Tx. RESULTS: In 20 pairs, sequencing of the BKV TR succeeded in donors and corresponding recipients after Tx. The derived sequences were completely identical in donor and post-Tx recipient samples. For comparison, identical TR sequences were detected in only 24% of 1068 randomly assembled pairs. This difference was statistically highly significant (p<0.0001, Fisher's exact test). Furthermore, all VP1, Large T-antigen and NCCR BKV sequences were also identical between donors and corresponding post-Tx recipients. In two of the 20 donor/recipient pairs, VP1 TR sequencing was also successful from the recipient before Tx. In both cases the sequence differed from the sequence detected in donor and recipient after Tx giving further evidence that recipient BKV was replaced by donor BKV after Tx. CONCLUSIONS: Our study for the first time provides evidence of BKV donor origin in renal transplant recipients.

Prevalence dependent calibration of a predictive model for nasal carriage of methicillin-resistant Staphylococcus aureus.

BACKGROUND: Published models predicting nasal colonization with Methicillin-resistant Staphylococcus aureus among hospital admissions predominantly focus on separation of carriers from non-carriers and are frequently evaluated using measures of discrimination. In contrast, accurate estimation of carriage probability, which may inform decisions regarding treatment and infection control, is rarely assessed. Furthermore, no published models adjust for MRSA prevalence. METHODS: Using logistic regression, a scoring system (values from 0 to 200) predicting nasal carriage of MRSA was created using a derivation cohort of 3091 individuals admitted to a European tertiary referral center between July 2007 and March 2008. The expected positive predictive value of a rapid diagnostic test (GeneOhm, Becton & Dickinson Co.) was modeled using non-linear regression according to score. Models were validated on a second cohort from the same hospital consisting of 2043 patients admitted between August 2008 and January 2012. Our suggested correction score for prevalence was proportional to the log-transformed odds ratio between cohorts. Calibration before and after correction, i.e. accurate classification into arbitrary strata, was assessed with the Hosmer-Lemeshow-Test. RESULTS: Treating culture as reference, the rapid diagnostic test had positive predictive values of 64.8% and 54.0% in derivation and internal validation corhorts with prevalences of 2.3% and 1.7%, respectively. In addition to low prevalence, low positive predictive values were due to high proportion (> 66%) of mecA-negative Staphylococcus aureus among false positive results. Age, nursing home residence, admission through the medical emergency department, and ICD-10-GM admission diagnoses starting with "A" or "J" were associated with MRSA carriage and were thus included in the scoring system, which showed good calibration in predicting probability of carriage and the rapid diagnostic test's expected positive predictive value. Calibration for both probability of carriage and expected positive predictive value in the internal validation cohort was improved by applying the correction score. CONCLUSIONS: Given a set of patient parameters, the presented models accurately predict a) probability of nasal carriage of MRSA and b) a rapid diagnostic test's expected positive predictive value. While the former can inform decisions regarding empiric antibiotic treatment and infection control, the latter can influence choice of screening method.

Prolonged detection of herpes simplex virus type 2 (HSV-2) DNA in cerebrospinal fluid despite antiviral therapy in a patient with HSV-2-associated radiculitis.

Herpes simplex virus type 2 (HSV-2) can cause radiculo-myelitis as a neurological manifestation. We report a case of ongoing HSV-2 DNA positivity in the cerebrospinal fluid (CSF) of at least eight weeks under antiviral therapy with acyclovir in a highly immunocompromised hemato-oncologic patient with HSV-2-associated radiculitis. Upon admission, the patient presented with pain, leg paresis, and urinary incontinence, as well as pleocytosis in the CSF. Quantitative real-time PCR of the CSF at day 3 after admission revealed HSV-2 with a concentration of 2.0×10(5) copies/ml and treatment with acyclovir intravenously and prednisolone by mouth was started. Clinical symptoms resolved almost completely after approximately 3 weeks of antiviral therapy. However, CSF samples of day 12, 19, 26, 33, 39, 48 and 54 after admission showed a slow decline of HSV-2 DNA concentrations. HSV-2 DNA was still detectable (1.6×10(4) copies/ml) at day 54 after admission. Genotypic resistance testing showed, as far as available, no mutations indicative for acyclovir resistance. Since an increasing specific antibody index for HSV was observed, we speculate that the prolonged detectability of HSV-2 DNA in the CSF might not necessarily indicate ongoing viral replication but neutralized virus. Other hypotheses and the consequences on treatment are discussed. To our knowledge this is the first report about the long-term viral load kinetics of HSV-2 in the CSF of a patient with radiculitis under antiviral therapy, highlighting the need for further studies on HSV DNA kinetics in the CSF and their significance for an appropriate antiviral treatment.

Pathogen-specific DNA enrichment does not increase sensitivity of PCR for diagnosis of invasive aspergillosis in neutropenic patients.

PCR assays designed for the diagnosis of invasive aspergillosis (IA) in high-risk patients have to detect minute amounts of target DNA to reach sufficient analytical sensitivity to be of clinical use. This prospective study assessed the use of a novel strategy for selective pathogen DNA enrichment for enhancing the performance of diagnostic PCR in a direct comparison with a highly sensitive in-house quantitative PCR (qPCR) assay and the galactomannan enzyme-linked immunosorbent assay (ELISA). Surprisingly, and in contrast to experience with other patient groups, the novel protocol for selective pathogen DNA enrichment did not enhance but instead significantly impaired sensitivity. This could be explained by the small amounts of host DNA in the specimens, which were derived mostly from severely neutropenic patients. In the qPCR assay, positive samples required an average of 43.5 amplification cycles (range, 39.2 to 50) for detection in the in-house PCR. Repetitive testing of selected samples showed test positivity to be variable, most likely due to the small amounts of target DNA. Despite this, the in-house protocol proved helpful in the diagnosis of IA, detecting 2 out of 3 patients with probable IA and 10 out of 19 patients with possible IA. Our results underline the necessity for diagnostic PCR protocols that help diagnose IA to be highly sensitive and show that selective pathogen DNA enrichment using affinity purification may not be useful in severely neutropenic patients.

The transcriptional repressor FarR is not involved in meningococcal fatty acid resistance mediated by the FarAB efflux pump and dependent on lipopolysaccharide structure.

Free fatty acids are important antimicrobial substances regulating the homeostasis of colonizing bacteria on epithelial surfaces. Here, we show that meningococci express a functional farAB efflux pump, which is indispensable for fatty acid resistance. However, other than in Neisseria gonorrhoeae, the transcriptional regulator FarR is not involved in regulation of this operon in Neisseria meningitidis. We tested the susceptibility of 23 meningococcal isolates against saturated and unsaturated long-chain fatty acids, proving that meningococci are generally highly resistant, with the exception of serogroup Y strains belonging to sequence type 23. Using genetically determined lipopolysaccharide (LPS)-truncated mutant strains, we show that addition of the LPS core oligosaccharide and hexa-acylation of its membrane anchor lipid A are imperative for fatty acid resistance of meningococci. The sensitivity of the serogroup Y strains is due to naturally occurring mutations within the lpxL1 gene, which is responsible for addition of the sixth acyl chain on the LPS membrane anchor lipid A. Therefore, fatty acid resistance in meningococci is provided by both the active efflux pump FarAB and by the natural permeability barrier of the Gram-negative outer membrane. The transcriptional regulator FarR is not implicated in fatty acid resistance in meningococci, possibly giving rise to a constitutively active FarAB efflux pump system and thus revealing diverse mechanisms of niche adaptation in the two closely related Neisseria species.

Recognition of meningococcal molecular patterns by innate immune receptors.

The innate immune system plays an important role in mounting a protective immune response against invading pathogens. By presenting antigens to the acquired immune system and chaperoning the activation of B- and T-lymphocytes it determines the course of immune activation. Protective immunity against Neisseria meningitidis is rather well characterized and based on the presence of anti-meningococcal antibodies capable of activating the complement system. However, the activation patterns in innate immune cells that lead to the induction of these antibodies are only partially understood. In this review, bacterial factors and cellular receptors mediating the initial interaction of meningococci with cells of the innate immune system, such as dendritic cells, macrophages and neutrophils, are discussed.

Recognition via the class A scavenger receptor modulates cytokine secretion by human dendritic cells after contact with Neisseria meningitidis.

Proinflammatory cytokines play a major role in the pathogenesis of meningococcal disease and their serum levels in patients are correlated with the outcome of infection. Dendritic cells initiate immunity against Neisseria meningitidis and are a major source of proinflammatory cytokines. Here we show that physical interaction of human DC with N. meningitidis via the class A scavenger receptor (SRA) modulates cytokine release by DC. Phagocytosis and uptake via SRA were shown to increase release of TNF-alpha, IL-1 beta and IL-6. In contrast, secretion of IL-8 is enhanced after recognition of N. meningitidis via SRA and not dependent on phagocytosis. Binding of N. meningitidis results in dephosphorylation of SRA but not in upregulation of SRA transcription. Unlike previously thought, not all meningococcal strains are recognized via SRA. A constitutively unencapsulated carriage isolate could be shown not to bind to SRA and induce proinflammatory cytokines independent of this receptor. In conclusion, recognition via SRA by dendritic cells is likely to play a central role in the immune response to N. meningitidis.

Whole-genome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis.

Neisseria meningitidis is a leading cause of infectious childhood mortality worldwide. Most research efforts have hitherto focused on disease isolates belonging to only a few hypervirulent clonal lineages. However, up to 10% of the healthy human population is temporarily colonized by genetically diverse strains mostly with little or no pathogenic potential. Currently, little is known about the biology of carriage strains and their evolutionary relationship with disease isolates. The expression of a polysaccharide capsule is the only trait that has been convincingly linked to the pathogenic potential of N. meningitidis. To gain insight into the evolution of virulence traits in this species, whole-genome sequences of three meningococcal carriage isolates were obtained. Gene content comparisons with the available genome sequences from three disease isolates indicate that there is no core pathogenome in N. meningitidis. A comparison of the chromosome structure suggests that a filamentous prophage has mediated large chromosomal rearrangements and the translocation of some candidate virulence genes. Interspecific comparison of the available Neisseria genome sequences and dot blot hybridizations further indicate that the insertion sequence IS1655 is restricted only to N. meningitidis; its low sequence diversity is an indicator of an evolutionarily recent population bottleneck. A genome-based phylogenetic reconstruction provides evidence that N. meningitidis has emerged as an unencapsulated human commensal from a common ancestor with Neisseria gonorrhoeae and Neisseria lactamica and consecutively acquired the genes responsible for capsule synthesis via horizontal gene transfer.

Capsule acetylation does not impair recognition of serogroup C, W-135 and Y meningococci by human dendritic cells.

The capsule polysaccharide of several Neisseria meningitidis serogroups can be modified by O-acetylation in vivo. As capsule expression is a major determinant of the interaction between N. meningitidis and human dendritic cells (DCs), the influence of the capsule polysaccharide acetylation status on activation of DCs was investigated. For serogroup C, W-135 and Y, mutations resulting in a lack of capsule acetylation did not interfere with recognition and phagocytosis of N. meningitidis, induction of DC maturation or triggering of cytokine release. Therefore, acetylation of the meningococcal capsule does not modify the activation of dendritic cells by this pathogen.

Induction of ERK-kinase signalling triggers morphotype-specific killing of Candida albicans filaments by human neutrophils.

Candida albicans is among the most important fungal pathogens in humans. Morphological plasticity has been linked to its pathogenic potential as filamentous forms are associated with tissue invasion and infection. Here we show that human neutrophils discriminate between yeasts and filaments of C. albicans. Whereas filaments induced targeted motility, resulting in the establishment of close contact between neutrophils and fungal cells, yeast forms were largely ignored during coincubation. In transwell assays, C. albicans filaments induced significantly higher migratory activity in neutrophils than yeasts. Neutrophil motility based on actin rearrangement was essential for killing of C. albicans filaments but not involved in killing of yeast forms. Using inhibitors for MAP-kinase cascades, it was shown that recognition of C. albicans filaments by neutrophils is mediated via the MEK/ERK cascade and independent of JNK or p38 activation. Inhibition of the ERK signalling pathway abolished neutrophil migration induced by C. albicans filaments and selectively impaired the ability to kill this morphotype. These data show that invasive filamentous forms of C. albicans trigger a morphotype-specific activation of neutrophils, which is strongly dependent on neutrophil motility. Therefore, human neutrophils are capable of sensing C. albicans invasion and initiating an appropriate early immune response.

A functional two-partner secretion system contributes to adhesion of Neisseria meningitidis to epithelial cells.

Neisseria meningitidis is a frequent commensal of the human nasopharynx causing severe invasive infections in rare cases. A functional two-partner secretion (TPS) system in N. meningitidis, composed of the secreted effector protein HrpA and its cognate transporter HrpB, is identified and characterized in this study. Although all meningococcal strains harbor at least one TPS system, the hrpA genes display significant C-terminal sequence variation. Meningococcal genes encoding the TPS effector proteins and their transporters are closely associated and transcribed into a single mRNA. HrpA proteins are translocated across the meningococcal outer membrane by their cognate transporters HrpB and mainly released into the environment. During this process, HrpA is proteolytically processed to a mature 180-kDa form. In contrast to other known TPS systems, immature HrpA proteins are stable in the absence of HrpB and accumulate within the bacterial cell. A small percentage of mature HrpA remains associated with the bacteria and contributes to the interaction of meningococci with epithelial cells.

Carbohydrate composition of meningococcal lipopolysaccharide modulates the interaction of Neisseria meningitidis with human dendritic cells.

Meningococcal lipopolysaccharide (LPS) is of crucial importance for the pathogenesis of invasive infection. We show that sialylation and elongation of the alpha-chain effectively shields viable unencapsulated Neisseria meningitidis from recognition by human dendritic cells (DC). In contrast, beta- and gamma- chain of the LPS carbohydrate moiety play only a minor role in the interaction with DC. The protective function of the LPS for the bacteria can be counteracted in vivo by phase variation of the lgtA gene encoding LPS glycosyltransferase A. Capsule expression protects N. meningitidis efficiently from recognition and phagocytosis by DC independent of the LPS structure. Despite the significant impact of LPS composition on the adhesion and phagocytosis of N. meningitidis no differences were found in terms of cytokine levels secreted by DC for IL1-beta, IL-6, IL-8, TNF-alpha, IFN-gamma and GM-CSF. However, significantly lower levels of the regulatory mediator IL-10 were induced by encapsulated strains in comparison to isogenic unencapsulated derivatives. IL-10 secretion was shown to depend on phagocytosis because poly alpha-2,8 sialic acid did not influence IL-10 secretion. The use of truncated LPS isoforms in vaccine preparations can therefore not only result in attenuation but also in more efficient targeting of DC.

Polymorphism of Candida albicans is a major factor in the interaction with human dendritic cells.

Morphological plasticity of Candida albicans is a major virulence factor. Using pH-dependent dimorphism we show, that human dendritic cells (DC) recognize filamentous forms and blastoconidia of a virulent C. albicans isolate (strain SC5314). Heat inactivated and viable blastoconidia are rapidly phagocytosed by human DC. However, viable yeast cells start to filament inside the DC at later stages of infection, leading to penetration and loss of cellular integrity. The cytokine burst of human DC induced upon contact with Candida is dominated by the granulocyte-activating, chemotactic factor IL-8 and the proinflammatory mediator TNF-alpha. Blastoconidia induce markedly lower cytokine levels than filamentous forms. Whereas IL-8 secretion is mainly cell mass dependent, release of TNF-alpha, a major proinflammatory cytokine, is clearly dependent on the morphology of Candida.