A pilot study to understand feasibility and acceptability of stool and cord blood sample collection for a large-scale longitudinal birth cohort.

BACKGROUND: Few data are available to guide biological sample collection around the time of birth for large-scale birth cohorts. We are designing a large UK birth cohort to investigate the role of infection and the developing immune system in determining future health and disease. We undertook a pilot to develop methodology for the main study, gain practical experience of collecting samples, and understand the acceptability of sample collection to women in late pregnancy. METHODS: Between February-July 2014, we piloted the feasibility and acceptability of collecting maternal stool, baby stool and cord blood samples from participants recruited at prolonged pregnancy and planned pre-labour caesarean section clinics at University College London Hospital. Participating women were asked to complete acceptability questionnaires. RESULTS: Overall, 265 women were approached and 171 (65%) participated, with ≥1 sample collected from 113 women or their baby (66%). Women had a mean age of 34 years, were primarily of white ethnicity (130/166, 78%), and half were nulliparous (86/169, 51%). Women undergoing planned pre-labour caesarean section were more likely than those who delivered vaginally to provide ≥1 sample (98% vs 54%), but less likely to provide maternal stool (10% vs 43%). Pre-sample questionnaires were completed by 110/171 women (64%). Most women reported feeling comfortable with samples being collected from their baby (<10% uncomfortable), but were less comfortable about their own stool (19% uncomfortable) or a vaginal swab (24% uncomfortable). CONCLUSIONS: It is possible to collect a range of biological samples from women around the time of delivery, and this was acceptable for most women. These data inform study design and protocol development for large-scale birth cohorts.

Prognostic value of C-reactive protein in HIV-infected patients with Pneumocystis jirovecii pneumonia.

C-reactive protein (CRP) is a sensitive marker of inflammation and tissue damage. We aimed to describe CRP responses in HIV-infected patients presenting with Pneumocystis pneumonia (PCP), bacterial pneumonia (BP) and pulmonary tuberculosis (TB) and, in patients with PCP, to identify if elevated CRP has prognostic significance. Data obtained by case-note review of consecutive HIV-infected adults with acute respiratory episodes included admission CRP (elevated >5 mg/L), haemoglobin, white blood count, CD4 count and partial pressure of oxygen in the blood (PaO(2)), presence of pulmonary co-pathology/intercurrent infection and outcome (survival). Median (range) CRP in patients with BP = 120 mg/L (<5-620 mg/L), TB = 44 mg/L (<5-256.3 mg/L) and PCP = 35 mg/L (<5-254 mg/L). CRP was elevated in 93/103 (90.3%) patients with PCP; six patients died; and all had an elevated CRP. PaO(2) and CRP values were associated as follows: average CRP levels declined by 10% (95% confidence interval [CI] 0.20%) per kPa increase in PaO(2) = 0.002. Factors associated with death were higher CRP, odds ratio (OR) (95% CI) = 5.30 (1.61 to 17.51) per 100 mg/L increase, P = 0.006 and haemoglobin, OR (95% CI) = 0.52 (0.29 to 0.93) per g/dL, P = 0.033. CRP is elevated in the majority of HIV-infected patients with PCP, BP and TB. Admission CRP measurement lacks specificity, but in PCP elevations of CRP are associated with disease severity (PaO(2)) and poor outcome and might be used prognostically, together with other mortality risk factors; further prospective evaluation is needed.

Mismatch between suspected pyelonephritis and microbiological diagnosis: a cohort study from a UK teaching hospital.

Urinary tract infections are a common reason for prescribing empirical antibiotics in the emergency department. This study investigated the role of microbiological culture and urinalysis in the diagnosis of pyelonephritis by extracting data on 105 patients with a clinical diagnosis of pyelonephritis at a London teaching hospital. In total, 99 of 102 patients were treated empirically with intravenous antibiotics, but only 55 of 100 patients who were sampled had microbiological evidence of infection in urine and/or blood. Almost half (10/21) of the patients with a negative urine dipstick test had a positive urine culture. Diagnostic uncertainty in this context undoubtedly drives inappropriate antibiotic use.

18F-Fluorodeoxyglucose positron emission tomography-computed tomography imaging in HIV-infected patients with lymphadenopathy, with or without fever and/or splenomegaly.

We audited whether 18F-Fluorodeoxyglucose positron emission tomography-computed tomography (18FDG PET-CT) imaging could discriminate between different diagnoses in HIV-infected patients presenting with lymphadenopathy, with or without fever and/or splenomegaly. Maximum standardised uptake (SUVmax) values were similar in lymphoma and mycobacterial and fungal infections and were lower but similar in those with human herpesvirus (HHV) 8-associated disease and HIV-associated reactive lymphadenopathy. Nodal 18FDG avidity, with SUVmax ≥10, excluded diagnoses of HHV 8-associated disease and miscellaneous conditions, and HIV-associated reactive lymphadenopathy was additionally excluded in those who had undetectable plasma HIV viral loads. This audit suggests 18FDG PET-CT imaging did not permit discrimination between specific diagnoses but has utility in identifying lymph nodes with increased avidity that could be targeted for biopsy and in ruling out significant pathology.

A Four-Biomarker Blood Signature Discriminates Systemic Inflammation Due to Viral Infection Versus Other Etiologies.

The innate immune system of humans and other mammals responds to pathogen-associated molecular patterns (PAMPs) that are conserved across broad classes of infectious agents such as bacteria and viruses. We hypothesized that a blood-based transcriptional signature could be discovered indicating a host systemic response to viral infection. Previous work identified host transcriptional signatures to individual viruses including influenza, respiratory syncytial virus and dengue, but the generality of these signatures across all viral infection types has not been established. Based on 44 publicly available datasets and two clinical studies of our own design, we discovered and validated a four-gene expression signature in whole blood, indicative of a general host systemic response to many types of viral infection. The signature's genes are: Interferon Stimulated Gene 15 (ISG15), Interleukin 16 (IL16), 2',5'-Oligoadenylate Synthetase Like (OASL), and Adhesion G Protein Coupled Receptor E5 (ADGRE5). In each of 13 validation datasets encompassing human, macaque, chimpanzee, pig, mouse, rat and all seven Baltimore virus classification groups, the signature provides statistically significant (p < 0.05) discrimination between viral and non-viral conditions. The signature may have clinical utility for differentiating host systemic inflammation (SI) due to viral versus bacterial or non-infectious causes.

Trans-activation of the murine dystrophin gene in human-mouse hybrid myotubes.

Myotube cultures of the myogenic cell line, C2, produce significantly lower levels of dystrophin than primary mouse cultures. We demonstrate that expression of the C2 dystrophin gene increases 10-fold in hybrid myotubes formed by fusion of C2 and dystrophin-deficient human myoblasts from a Duchenne muscular dystrophy patient. These results indicate that C2 cells are deficient in endogenous gene regulatory factors which enhance dystrophin expression, and that the C2 cell line may therefore be used to identify putative trans-acting factors involved in the regulation of dystrophin gene expression.

An unusual cause of fever in an HIV-positive patient.

Patients with HIV frequently present with fever. However, the spectrum of diseases affecting patients with HIV has changed in the era of antiretroviral therapy (ART). Here we present a patient who has an unusual cause for his fever with an eventual diagnosis of adult-onset Still's disease.

Can plasma HHV8 viral load be used to differentiate multicentric Castleman disease from Kaposi sarcoma?

We measured plasma human herpesvirus 8 (HHV8) DNA load in consecutive patients presenting with HIV-associated multicentric Castleman disease (MCD) and in contemporaneous patients who had Kaposi sarcoma (KS), lymphoma or other diagnoses. All 11 patients with MCD had detectable plasma HHV8 DNA compared with 18 (72%) of 25 patients with KS, none with lymphoma and one of 38 patients with other diagnoses. Detectable plasma HHV8 DNA levels were higher among MCD patients, median (interquartile range [IQR]) = 43,500 (5200-150,000) copies/mL, when compared with those with KS, median (IQR) = 320 (167-822) copies/mL and those with lymphoma and other diagnoses (one-way analysis of variance; P = 0.0303). Using receiver operating characteristic analysis, a cut-off of >1000 copies HHV8 DNA/mL of plasma helped to discriminate between MCD and other diagnoses, with a specificity of 94.7% and a negative predictive value of 97.3%. The level of HHV8 viraemia, while not diagnostic, may aid discrimination between patients with MCD and those with KS and other systemic illnesses.

Monocyte and macrophage dysfunction as a cause of HIV-1 induced dysfunction of innate immunity.

HIV-1 can establish both long lived and productive infection of macrophages (Mϕ) but circulating monocytes are less permissive to infection. Multiple studies have identified extensive changes to monocyte and Mϕ phenotype, differentiation or function. These include alterations in Toll-like receptor signaling and resultant changes to cytokine responses, specific defects in phagocytosis and microbial killing and modulation of apoptotic responses, all of which may perturb the important role of these cells in innate immunity. Interpretation of contradictory data however, is complicated by the use of different experimental models and many of the reported effects may be an indirect consequence of HIV 1 infection that result from exposure to viral products or from disruption of cellular and cytokine networks in the immune system, rather than the direct consequence of productive HIV 1 infection. Future research should focus on refining experimental models and on elucidating the physiological mechanisms of monocyte/ Mϕ dysfunction during HIV 1 infection.

Relationship of granulocyte colony stimulating factor with other acute phase reactants in man.

The non-specific acute phase response in mice is associated with increased resistance to bacterial infection, which is critically mediated by granulocyte colony stimulating factor (G-CSF), but the behaviour of G-CSF in the human acute phase response is not known. Cardiothoracic surgery is a powerful acute phase stimulus and we show here that this procedure caused increased production of G-CSF, in addition to increases in the circulating concentrations of the proinflammatory cytokine interleukin (IL)-6 and the acute phase plasma proteins C-reactive protein (CRP) and serum amyloid A protein (SAA). Values of G-CSF correlated positively with IL-6 concentrations and circulating neutrophil counts, but not with CRP values. These results confirm that G-CSF is a physiological component of the acute phase response in humans that shares some of the same regulatory controls as IL-6, but its downstream effects are on neutrophils, not hepatic acute phase protein synthesis. Our observations are compatible with a protective role against bacterial infection for G-CSF in the human acute phase response, and support investigation of the prophylactic use of G-CSF in at-risk patients.

Successful liposomal amphotericin B treatment of Leishmania braziliensis cutaneous leishmaniasis.

Existing systemic treatments for New World cutaneous leishmaniasis (CL) caused by Leishmania (vianna) braziliensis are unsatisfactory. Liposomal amphotericin B has been used extensively for the treatment of visceral leishmaniasis, but in few cases of CL, and an appropriate regimen for CL has not been described. We successfully treated a patient with multiple L. braziliensis CL lesions acquired in Belize. Liposomal amphotericin B (AmBisome) was given to our patient as an inpatient for seven daily doses of 3 mg kg(-1) day(-1) and then as an outpatient at 3 mg kg(-1) twice weekly for a further three weeks, a total of 40 mg kg(-1). Liposomal amphotericin offers a well-tolerated alternative to pentavalent antimony or amphotericin B deoxycholate for the systemic treatment of New World CL.

Co-localization and molecular association of dystrophin with laminin at the surface of mouse and human myotubes.

In Duchenne muscular dystrophy (DMD), deficiency of the protein dystrophin results in necrosis of muscle myofibres, associated with lesions in the sarcolemma and surrounding basal lamina. Dystrophin has been proposed to be a major component of the sub-sarcolemmal cytoskeleton involved in maintaining the integrity of the myofibre plasma membrane, and is known to associate with a group of sarcolemmal glycoproteins, one of which exhibits high affinity binding to the basal lamina component laminin. However, a direct or indirect transmembrane association of dystrophin in muscle cells with the myofibre basal lamina has not been demonstrated. To address this question we have examined dystrophin immunostaining and immunoprecipitation patterns in cultured mouse and human myotubes in comparison with that of the basal lamina component, laminin. Dual-immunolabelling revealed virtually complete co-localization of dystrophin on the inside surface of the muscle cell sarcolemma with plaques and veined arrays of laminin accumulating on the extracellular face. This pattern of laminin and dystrophin distribution was distinct from that of other cell surface molecules expressed in myotubes such as the neural cell adhesion molecule, NCAM, and the beta 1 integrin receptor, and immunoprecipitation of dystrophin from solubilized myotube extracts resulted in co-purification of laminin B1 chain confirming an association between these two components. The results thus provide the first direct cellular evidence of a transmembrane linkage between dystrophin in the sarcolemmal cytoskeleton with laminin in the overlying basal lamina. While the immunocytochemical distribution of laminin was apparently normal in dystrophin-deficient muscle cells, elevated levels of soluble laminin were present in extracts of mdx compared with normal mouse skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo.

We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA(-) mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo.

Role of serum amyloid P component in bacterial infection: protection of the host or protection of the pathogen.

Serum amyloid P component (SAP) binds to Streptococcus pyogenes, and we show here that it also binds to Neisseria meningitidis, including a lipopolysaccharide (LPS)-negative mutant, and to rough variants of Escherichia coli. Surprisingly, this binding had a powerful antiopsonic effect both in vitro and in vivo, reducing phagocytosis and killing of bacteria. Furthermore, SAP knockout mice survived lethal infection with S. pyogenes and rough E. coli J5, organisms to which SAP binds. The susceptibility of SAP(-/-) mice was fully restored by injection of isolated human SAP. However, SAP(-/-) mice were more susceptible than wild-type animals to lethal infection with E. coli O111:B4, a smooth strain to which SAP does not bind, suggesting that SAP also has some host defense function. Although SAP binds to LPS in vitro, SAP(-/-) mice were only marginally more susceptible to lethal LPS challenge, and injection of large amounts of human SAP into wild-type mice did not affect sensitivity to LPS, indicating that SAP is not a significant modulator of LPS toxicity in vivo. In contrast, the binding of SAP to pathogenic bacteria enabled them to evade neutrophil phagocytosis and display enhanced virulence. Abrogation of this molecular camouflage is thus potentially a novel therapeutic approach, and we show here that administration to wild-type mice of (R)-1-[6-(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine -2- carboxylic acid, a drug that inhibits SAP binding, significantly prolonged survival during lethal infection with E. coli J5.

Generalized chorea in two patients harboring the Friedreich's ataxia gene trinucleotide repeat expansion.

Recently, a trinucleotide repeat expansion in intron 1 of the frataxin gene on chromosome 9p13 has been identified as the genetic defect in Friedreich's ataxia (FA). We have identified two patients exhibiting generalized chorea in the absence of cerebellar signs who were homozygous for this intron 1 expansion. Chorea as a rare manifestation of FA has previously been controversial. This is the first report of chorea in patients confirmed to have the FA genetic abnormality and broadens further the clinical phenotype associated with the FA genotype.