Genetic diversity and host associations in Campylobacter jejuni from human cases and broilers in 2000 and 2008.

Campylobacter jejuni is an important food-borne pathogen, with a global distribution. It can colonize numerous host species, including both domestic and wild animals, but is particularly associated with birds (poultry and wild birds). For human campylobacteriosis, poultry products are deemed the most significant risk factor for acquiring infection. We conducted a genotyping and host attribution study of a large representative collection of C. jejuni isolated from humans and broilers in Sweden in the years 2000 and 2008. In total 673 broiler and human isolates from 10 different abattoirs and 6 different hospitals were genotyped with multilocus sequence typing. Source attribution analyses confirmed the strong linkage between broiler C. jejuni and domestic human cases, but also indicated a significant association to genotypes more commonly found in wild birds. Genotype distributions did not change dramatically between the two study years, suggesting a stable population of infecting bacteria.

The risk of HCV RNA contamination in serology screening instruments with a fixed needle for sample transfer.

BACKGROUND: Hepatitis C diagnostics involve antibody screening and confirmation of current infection by detection of HCV RNA positivity. In screening instruments with fixed pipetting needle, there is a risk of sample carry-over contamination. OBJECTIVES: The aim of this study was to evaluate the risk of such contamination in a proposed clinical setting. STUDY DESIGN: In the present study, known HCV RNA positive (n=149) and negative (n=149) samples were analysed by anti-HCV Abbott in an Architect instrument in an alternating fashion in order to test for contamination. RESULTS: In subsequent retesting of the previously HCV RNA-negative samples, six samples (4%) were positive by the Cobas Taqman assay with a maximum level of 33 IU/mL. The results show that there is a risk for transfer of HCV in the Architect instrument but they also show that the levels of HCV RNA observed are low. CONCLUSIONS: We conclude that complementary HCV RNA testing on samples identified as anti-HCV positive by screening can be recommended because the complementary results are reliable in the majority of cases when either HCV RNA is negative or HCV RNA is positive with a level >1000 IU/mL. In a minority of cases, with low HCV RNA after anti-HCV antibody screening, cross-contamination should be suspected and a new sample requested for HCV RNA testing. This strategy would reduce the need for obtaining a new sample from the vast majority of patients with a newly discovered HCV antibody positivity.

Endocine™, N3OA and N3OASq; three mucosal adjuvants that enhance the immune response to nasal influenza vaccination.

Annual outbreaks of seasonal influenza are controlled or prevented through vaccination in many countries. The seasonal vaccines used are either inactivated, currently administered parenterally, or live-attenuated given intranasally. In this study three mucosal adjuvants were examined for the influence on the humoral (mucosal and systemic) and cellular influenza A-specific immune responses induced by a nasally administered vaccine. We investigated in detail how the anionic Endocine™ and the cationic adjuvants N3OA and N3OASq mixed with a split inactivated influenza vaccine induced influenza A-specific immune responses as compared to the vaccine alone after intranasal immunization. The study showed that nasal administration of a split virus vaccine together with Endocine™ or N3OA induced significantly higher humoral and cell-mediated immune responses than the non-adjuvanted vaccine. N3OASq only significantly increased the cell-mediated immune response. Furthermore, nasal administration of the influenza vaccine in combination with any of the adjuvants; Endocine™, N3OA or N3OASq, significantly enhanced the mucosal immunity against influenza HA protein. Thus the addition of these mucosal adjuvants leads to enhanced immunity in the most relevant tissues, the upper respiratory tract and the systemic circulation. Nasal influenza vaccination with an inactivated split vaccine can therefore provide an important mucosal immune response, which is often low or absent after traditional parenteral vaccination.

Real-time PCR detection of human herpesvirus 1-5 in patients lacking clinical signs of a viral CNS infection.

BACKGROUND: Infections of the central nervous system (CNS) with herpes- or enterovirus can be self-limiting and benign, but occasionally result in severe and fatal disease. The polymerase chain reaction (PCR) has revolutionized the diagnostics of viral pathogens, and by multiple displacement amplification (MDA) prior to real-time PCR the sensitivity might be further enhanced. The aim of this study was to investigate if herpes- or enterovirus can be detected in cerebrospinal fluid (CSF) from patients without symptoms. METHODS: Cerebrospinal fluid (CSF) samples from 373 patients lacking typical symptoms of viral CNS infection were analysed by real-time PCR targeting herpesviruses or enteroviruses with or without prior MDA. RESULTS: In total, virus was detected in 17 patients (4%). Epstein-Barr virus (EBV) was most commonly detected, in general from patients with other conditions (e.g. infections, cerebral hemorrhage). MDA satisfactorily amplified viral DNA in the absence of human nucleic acids, but showed poor amplification capacity for viral DNA in CSF samples, and did not increase the sensitivity for herpes virus-detection with our methodology. CONCLUSIONS: Viral pathogens are rarely detected in CSF from patients without signs of CNS infection, supporting the view that real-time PCR is a highly specific method to detect symptomatic CNS-infection caused by these viruses. However, EBV may be subclinically reactivated due to other pathological conditions in the CNS.

Innate immunity proteins and a new truncated form of SPLUNC1 in nasopharyngeal aspirates from infants with respiratory syncytial virus infection.

PURPOSE: Respiratory syncytial virus (RSV) is the most common cause of severe respiratory tract infection in infants. The aim was to identify host defence components in nasopharyngeal aspirate (NPA) from infants with RSV infection and to study the expression of the novel 25 kDa innate immunity protein SPLUNC1. EXPERIMENTAL DESIGN: NPAs from infants were analyzed with 2-DE and MS in a pilot study. The levels of SPLUNC1 were analyzed with immunoblotting in 47 NPAs, admitted for RSV diagnosis. RESULTS: Totally, 35 proteins were identified in NPA, including several innate immunity proteins such as group X phospholipase A(2) , different S100 proteins and SPLUNC1. In addition, a new truncated 15 kDa form of SPLUNC1 was identified that was detected in about 50% of the aspirates admitted for RSV diagnosis. RSV-positive boys had significantly less 25 kDa SPLUNC1 than RSV-negative boys while there were no significant differences among girls. CONCLUSIONS AND CLINICAL RELEVANCE: Several important innate immunity proteins were identified in NPA. Notably, a new truncated form of the newly suggested anti-bacterial protein SPLUNC1 was found. It is possible that a decrease in SPLUNC1 in the upper airways may increase the risk for severe pneumonia in boys.

The cost-effectiveness of introducing nucleic acid testing to test for hepatitis B, hepatitis C, and human immunodeficiency virus among blood donors in Sweden.

BACKGROUND: The purpose of this study was to estimate the cost-effectiveness of using individual-donor nucleic acid testing (ID-NAT) in addition to serologic tests compared with the sole use of serologic tests for the identification of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) among blood donors in Sweden. STUDY DESIGN AND METHODS: The two strategies analyzed were serologic tests and ID-NAT plus serologic tests. A health-economic model was used to estimate the lifetime costs and effects. The effects were measured as infections avoided and quality-adjusted life-years (QALYs) gained. A societal perspective was used. RESULTS: The largest number of viral transmissions occurred with serologic testing only. However, the risks for viral transmissions were very low with both strategies. The total cost was mainly influenced by the cost of the test carried out. The cost of using ID-NAT plus serologic tests compared to serologic tests alone was estimated at Swedish Krona (SEK) 101 million (USD 12.7 million) per avoided viral transmission. The cost per QALY gained was SEK 22 million (USD 2.7 million). CONCLUSION: Using ID-NAT for testing against HBV, HCV, and HIV among blood donors leads to cost-effectiveness ratios that are far beyond what is usually considered cost-effective. The main reason for this is that with current methods, the risks for virus transmission are very low in Sweden.

Monitoring hepatitis C infection in a major Swedish nephrology unit and molecular resolution of a new case of nosocomial transmission.

Hepatitis C virus (HCV) infection is a frequent problem in hemodialysis units. The prevalence and incidence of HCV infection over a decade were studied in a nephrology unit affected by previous nosocomial HCV transmission. The HCV non-structural 5B protein gene was sequenced to achieve phylogenetic analysis of a new (incident) case of infection. Proportions of patients who were and were not infected with HCV remained similar over the period, as did the inflow and outflow of patients infected previously. In 1997, 12/157 (8%) of patients at the unit (treatment: hemodialysis, peritoneal dialysis, and renal transplant recipients) were positive in HCV RNA, whereas in 2007 the overall number was 9/239 (4%). One patient acquired an HCV infection, and the NS5B sequence in that case clustered with genotype 2b sequences found in patients from an earlier outbreak. Comparing the HCV from the incident patient with several stored longitudinal samples and cloned PCR products from the most likely source patient revealed close phylogenetic relationship with an HCV quasispecies member from the possible source. The source patient and the incident newly infected patient were not scheduled on the same dialysis shift, although the records showed that simultaneous treatment occurred on two occasions during the months preceding transmission. In conclusion, over the 10-year period, the proportion of HCV-infected patients at the unit was unchanged. Only one new infection occurred, which originated from a fellow patient's quasispecies. This establishes phylogenetic analysis as a valuable tool for tracing patient sources of HCV transmission.

Excellent response rate to a double dose of the combined hepatitis A and B vaccine in previous nonresponders to hepatitis B vaccine.

BACKGROUND: Hepatitis B vaccine has been shown to be highly efficient in preventing hepatitis B. However, 5%-10% of individuals fail to develop protective levels (>or=10 mIU/mL) of antibodies to hepatitis B surface antigen (anti-HBs) and are considered to be nonresponders. METHODS: A total of 48 nonresponders and 20 subjects naive to the HBV vaccine received a double dose of combined hepatitis A and B vaccine (Twinrix) at 0, 1, and 6 months. The levels of anti-HBs and antibodies to hepatitis A virus (anti-HAV) were determined before vaccination and 1 month after each dose. RESULTS: Among 44 nonresponders, protective anti-HBs levels were found in 26 (59%) after the first dose and in 42 (95%) after the third dose. Among the control subjects, the corresponding figures were 10% and 100%, respectively. All subjects seroconverted to anti-HAV. The titers of both anti-HBs and anti-HAV were lower in the previously nonresponsive subjects (P< .01). CONCLUSION: Revaccination of nonresponders to the standard hepatitis B vaccine regimen with a double dose of the combined hepatitis A and B vaccine was highly effective. This is most likely explained by the increased dose, a positive bystander effect conferred by the hepatitis A vaccine, or both.

A deletion in the chemokine receptor 5 (CCR5) gene is associated with tickborne encephalitis.

Tickborne encephalitis (TBE) virus infections can be asymptomatic or cause moderate to severe injuries of the central nervous system. Why some individuals develop severe disease is unknown, but a role for host genetic factors has been suggested. To investigate whether chemokine receptor CCR5 is associated with TBE, CCR5Delta32 genotyping was performed among Lithuanian patients with TBE (n=129) or with aseptic meningoencephalitis (n=76) as well as among control subjects (n=134). We found individuals homozygous for CCR5Delta32 (P= .026) only among patients with TBE and a higher allele prevalence among patients with TBE compared with the other groups studied. CCR5Delta32 allele prevalence also increased with the clinical severity of disease.

Host genetic resistance to symptomatic norovirus (GGII.4) infections in Denmark.

A total of 61 individuals involved in five norovirus outbreaks in Denmark were genotyped at nucleotides 428 and 571 of the FUT2 gene, determining secretor status, i.e., the presence of ABH antigens in secretions and on mucosa. A strong correlation (P = 0.003) was found between the secretor phenotype and symptomatic disease, extending previous knowledge and confirming that nonsense mutations in the FUT2 gene provide protection against symptomatic norovirus (GGII.4) infections.

Antibody prevalence and titer to norovirus (genogroup II) correlate with secretor (FUT2) but not with ABO phenotype or Lewis (FUT3) genotype.

BACKGROUND: Histo-blood group antigens and secretor status have been associated with susceptibility to Norovirus infections, which suggests that antibody prevalence and titer might correlate with these phenotypes. METHODS: Plasma samples (n = 105) from Swedish blood donors that had been genotyped for secretor (FUT2) and Lewis (Le; FUT3) genotypes and phenotyped for ABO and Le blood groups were analyzed for immunoglobulin G antibody prevalence and titers to norovirus genogroup (GG) II.4. RESULTS: The results showed that nonsecretors (se4128se428) and Lea+b- individuals not only had significantly lower antibody titers than did secretors (P < .0001) and Lea-b+ individuals (P < .0002) but were also significantly more often antibody negative (P < .05). Antibody titers in secretors were not significantly different between individuals of different Le (FUT3) genotypes or different ABO phenotypes. CONCLUSIONS: Nonsecretors and Lea+b- individuals are significantly less prone to be infected with GGII noroviruses. This new information extends previous knowledge and supports the hypothesis that nonsecretors are relatively but not absolutely resistant to norovirus infections.

Inner ear and facial nerve complications of acute otitis media with focus on bacteriology and virology.

CONCLUSION: Among 20 patients with inner ear complications and/or peripheral facial palsy secondary to acute otitis media (AOM) a proven or probable bacteriological cause was found in 13 (65%). In seven patients (35%), a proven or probable viral cause was found. Only two of the patients (10%), with a proven bacterial AOM and a clinical picture of a purulent labyrinthitis in both, together with a facial palsy in one, had a substantial degree of dysfunction. Although the number of patients in this study is relatively low our findings show that inner ear complications and facial palsy due to AOM can be of both bacterial and viral origin. Severe sequelae were found only where a bacterial origin was proven. OBJECTIVES: Inner ear complications and/or peripheral facial palsy secondary to AOM are rare. The general understanding is that they are due to bacterial infections. However, in some of these patients there are no clinical or laboratory signs of bacterial infections and they have negative bacterial cultures. During recent years different viruses have been isolated from the middle ear or serologically proven in AOM patients and are thought to play a pathogenetic role. We suggest that in some cases of AOM complications from the inner ear and the facial nerve can be caused by viruses. The purpose of our study was to analyze infectious agents present in patients with inner ear complications and/or facial palsy arising from AOM. PATIENTS AND METHODS: The medical records of 20 patients who had inner ear complications and/or facial palsy following AOM ( unilateral in 18, bilateral in 2) between January 1989 and March 2003 were evaluated. Bacterial cultures were carried out for all patients. Sera from 12 of the patients were stored and tested for a battery of specific viral antibodies. In three patients, investigated between November 2002 and March 2003, viral cultures were also performed on samples from the middle ear and nasopharynx. RESULTS: Nineteen patients had inner ear symptoms. Eight of them had a unilateral sensorineural hearing loss and vertigo, three had vertigo as an isolated symptom and one, with bilateral AOM, had bilateral sensorineural hearing loss. Seven patients had a combination of facial palsy and inner ear symptoms (unilateral sensorineural hearing loss in three, unilateral sensorineural hearing loss and vertigo in two, bilateral sensorineural hearing loss and vertigo in one, with bilateral AOM, and vertigo alone in one). One patient had an isolated facial palsy. Healing was complete in 11 of the 20 patients. In seven patients a minor defect remained at follow-up (a sensorineural hearing loss at higher frequencies in all). Only two patients had obvious defects (a pronounced hearing loss in combination with a moderate to severe facial palsy (House-Brackman grade 4) in one, distinct vestibular symptoms and a total caloric loss in combination with a high-frequency loss in the other. Eight patients had positive bacteriological cultures from middle ear contents: Streptococcus pneumoniae in two, beta-hemolytic Streptococcus group A in two, beta-hemolytic Streptococcus group A together with Staphylococcus aureus in one, Staph. aureus alone in one and coagulase-negative staphylococci (interpreted as pathogens) in two. In the 12 patients with negative cultures, there was a probable bacteriological cause due to the outcome in SR/CRP and leukocyte count in five. In four patients serological testing showed a concomitant viral infection that was interpreted to be the cause (varicella zoster virus in two, herpes simplex virus in one and adenovirus in one.) In three there was a probable viral cause despite negative viral antibody test due to normal outcome in SR/CRP, normal leukocyte count, serous fluid at myringotomy and a relatively short pre-complication antibiotic treatment period.

Hepatocyte growth factor (HGF) in fecal samples: rapid detection by surface plasmon resonance.

BACKGROUND: The development of biosensors, based on surface plasmon resonance (SPR) technology, enables monitoring of a variety of biospecific interactions without the need for chemical-, biological- or radiological-labelled reagents. METHOD: We utilised SPR to detect hepatocyte growth factor (HGF) in reconstituted faecal samples and studied samples from patients with infectious gastroenteritis (n = 20) and normal controls (n = 10). Mouse anti-human HGF monoclonal antibodies and recombinant human HGF receptor (c-Met)/Fc chimera were immobilised in flow cells of a CM5 biosensor chip. RESULTS: We found that infectious gastroenteritis produced a higher signal response compared to controls, due to binding of HGF to monoclonal anti-HGF antibody as well as binding of HGF to c-Met receptor (p < 0.01). The SPR signal response correlated with results from ELISA (r = 72%, p > 0.001). The signal response decreased significantly (p < 0.05) when samples were diluted with dextran, because of reduction in both specific as well as unspecific binding of HGF to dextran. The decrease in the specific response might imply that the dextran- binding site for HGF overlaps with the antibody binding epitope, or that dextran binding induces a conformational change of the HGF molecule. Bands corresponding to HGF were found by gel electrophoresis of purified faeces in an affinity chromatography column immobilised by HGF ligands. CONCLUSION: Determination of HGF by SPR might be beneficial in diagnosis of acute situations that present with symptoms of gastroenteritis and may, possibly, guide appropriate medical treatments. This is to our knowledge the first report on the use of SPR for detection of HGF in faeces samples.

High hepatocyte growth factor levels in faeces during acute infectious gastroenteritis.

Hepatocyte growth factor (HGF) is a potent mitogen of mature epithelial cells which is produced after organ injuries and acts as a trigger for regeneration in the impaired organ. The aim of the present study was to investigate local production of HGF during infectious gastroenteritis. We measured the concentration of HGF in serum and faeces in 49 patients with acute infectious gastroenteritis (bacterium = 30, virus = 10, amoebae = 1, and probable infection = 8) at the time of referral to hospital and at convalescence (n = 31). The values were compared with normal healthy vaccination volunteers (n = 11) as well as patients with acute non-infectious diarrhoea (n = 10). The presence of HGF in faeces was confirmed by ELISA and Western immunoblot. HGF concentrations in faeces was significantly higher in the patients with infectious gastroenteritis compared to the control groups (p < 0.0001). Using a cut-off concentration of 20 ng/g, the overall sensitivity of faeces HGF to distinguish infectious gastroenteritis (bacterial, viral, probable infection) was 98% with a specificity of 100%. At convalescence all patients had normal values. There was no significant correlation between HGF concentrations in faeces and serum. Determination of faeces HGF may identify cases of transmittable diarrhoea requiring isolation at an early stage.