Correction to: Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene.

After publication of our article [1] it came to our notice that the source of the sequence for the control plasmid, pNeo (Materials and methods: Controls) was incorrectly stated as AB094461. The correct accession number is AB074461. The authors apologize for any confusion this may have caused.

Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene.

BACKGROUND: Candidatus Neoehrlichia mikurensis is an emerging tick-borne pathogen. It is widely distributed in Ixodes ricinus ticks in Europe, but knowledge of its distribution in Norway, where I. ricinus reaches its northern limit, is limited. In this study we have developed a real time PCR test for Ca. N. mikurensis and used it to investigate the distribution of Ca. N. mikurensis in Norway. RESULTS: Real time PCR targeting the groEL gene was developed and shown to be highly sensitive. It was used to detect Ca. N. mikurensis in 1651 I. ricinus nymphs and adults collected from twelve locations in Norway, from the eastern Oslo Fjord in the south to near the Arctic Circle in the north. The overall prevalence was 6.5% and varied locally between 0 and 16%. Prevalence in adults and nymphs was similar, suggesting that ticks acquire Ca. N. mikurensis predominantly during their first blood meal. In addition, 123 larvae were investigated; Ca. N. mikurensis was not found in larvae, suggesting that transovarial transmission is rare or absent. Sequence analysis suggests that a single variant dominates in Norway. CONCLUSIONS: Ca. N. mikurensis is widespread and common in ticks in Norway and reaches up to their northern limit near the Arctic Circle. Ticks appear to acquire Ca. N. mikurensis during their first blood meal. No evidence for transovarial transmission was found.

"Candidatus Neoehrlichia mikurensis" in Ixodes ricinus ticks collected near the Arctic Circle in Norway.

BACKGROUND: "Candidatus Neoehrlichia mikurensis" is a gram-negative bacterium belonging to the family Anaplasmataceae that, in Europe, is transmitted by Ixodes ricinus ticks. "Candidatus N. mikurensis" can cause a severe systemic inflammatory syndrome, neoehrlichiosis, mostly in persons with other underlying diseases. To date, "Ca. N. mikurensis" has been found in ticks in different countries in Asia and Europe, but never as far north as at the Arctic Circle. METHODS: A total of 1104 I. ricinus ticks collected from vegetation and from animals in northern Norway (64-68°N) were analysed for the prevalence of "Ca. N. mikurensis". Of them, 495 ticks were collected from vegetation by flagging and 609 ticks were collected from dogs and cats. Total nucleic acid extracted from the ticks were converted to cDNA and analyzed with real-time PCR targeting the 16S rRNA gene of "Ca. N. mikurensis". Positive samples were further analysed by nested PCR and sequencing. RESULTS: "Candidatus N. mikurensis" was detected in 11.2% of all collected I. ricinus ticks in northern Norway. The prevalence differed between ticks collected from vegetation (18.2%; 90/495) compared to ticks collected from dogs and cats (5.6%; 34/609). The ticks from dogs and cats were collected in Brønnøy area and seven additional districts further north. The prevalence of "Ca. N. mikurensis" in these ticks differed between geographical localities, with the highest prevalence in the Brønnøy area. CONCLUSIONS: The detection of "Ca. N. mikurensis" in I. ricinus ticks from the Arctic Circle in northern Norway indicates potential risk for tick-bitten humans at this latitude to be infected with "Ca. N. mikurensis".

Blood donor Borrelia burgdorferi sensu lato seroprevalence and history of tick bites at a northern limit of the vector distribution.

In order to study the antibody seroprevalence of the causal agent of Lyme borreliosis, Borrelia burgdorferi sensu lato (s.l.), and the history of tick bites at a geographical distribution limit of Ixodes ricinus, we compared healthy blood donors in geographically extreme regions: the borreliosis-endemic Vestfold County (59°N) and the region of northern Norway. Blood samples were screened using IgG/VlsE ELISA, and positive/borderline samples were confirmed using C6 ELISA and immunoblot assays. Also, donors completed a questionnaire consisting of several items including the places they have lived, and whether they owned any pets. The seroprevalence was 0.48% (5/1048) in northern Norway and 9.25% (48/519) in Vestfold County. Seven donors (of 1048) had experienced a single tick bite in the southern part of Nordland County (65°N) in northern Norway. This first study on B. burgdorferi s.l. antibody seroprevalence and tick bites on humans and pets in northern Norway showed that the seroprevalence of B. burgdorferi s.l. infection and the risk of tick bite in northern Norway are insignificant; the fact that only five positive IgG samples were detected underscores the very low background seroprevalence. These results suggest that so far I. ricinus has not expanded north of the previously established geographical distribution limit.

Detection of Anaplasma phagocytophilum in Ixodes ricinus ticks from Norway using a realtime PCR assay targeting the Anaplasma citrate synthase gene gltA.

BACKGROUND: A TaqMan real-time PCR assay targeting the Anaplasma citrate synthase gene, gltA, was developed and used for detection of Anaplasma phagocytophilum in 765 Ixodes ricinus ticks collected from dogs and cats in northern Norway (n = 669) and Telemark county in southern Norway (n = 96). RESULTS: Among the ticks from northern Norway the prevalence of A. phagocytophilum was 3.0 %, while the prevalence in southern Norway was 2.1 % (p = 0.63). The gltA PCR assay showed a high analytical sensitivity (30 genomic units) and efficiency (98.5 %), and its utility in clinical diagnostics should be evaluated in future studies. CONCLUSION: This is the first report of A. phagocytophilum occurrence in ticks collected north of the Arctic Circle in Norway. The prevalence is comparable to that found in Telemark county in southern Norway.

Borrelia burgdorferi sensu lato-infected Ixodes ricinus collected from vegetation near the Arctic Circle.

This is the first study to determine the density of questing Ixodes ricinus in northern Norway. It was performed at two sites in Brønnøy, which has been known for its tick permissive habitats for decades and is one of the northernmost habitats with an abundant I. ricinus population in the world. From April to November 2011, all stages of host-seeking I. ricinus were collected from the two sites. The overall prevalence of nymphs infected with Borrelia burgdorferi sensu lato was 21% and that of adult ticks 46%. The rates of the genospecies Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana were similar to findings in most other studies in Scandinavia, with B. afzelii by far the most prevalent at 76%. The high Borrelia-infection prevalence in ticks from Brønnøy may explain the high incidence rate of reported Lyme borreliosis in the municipality.

Ixodes ricinus and Borrelia prevalence at the Arctic Circle in Norway.

The distribution limit of Ixodes ricinus ticks in northwestern Europe (Brønnøy, Norway, 1° south of the Arctic Circle), has been known since the 1930s. To reconfirm this finding and extend studies in the areas adjacent to the Arctic Circle (66°33' N), ticks were collected from dogs and cats in 8 districts in northern Norway from 64°56' N to 68°48' N. We detected 549 I. ricinus, 244 (44%) of them in Brønnøy district, and 305 (range 6-87 ticks) in 7 districts in the northern part of the study area. The prevalence of Borrelia in these ticks was determined by real-time PCR. In the Brønnøy district (65°28' N, 12°12' E), 29% of the I. ricinus were Borrelia spp.-positive, and the species B. afzelii was nearly twice as prevalent as B. garinii and/or B. valaisiana. In the study area north of Brønnøy district, only 12 (4%) of the collected ticks contained Borrelia spp. In conclusion, tick occurrence and Borrelia prevalence are high in the Brønnøy district. In contrast, I. ricinus occurrence and Borrelia prevalence are low further north across the Arctic Circle in Norway.

Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Norway: evaluation of a PCR test targeting the chromosomal flaB gene.

A consensus TaqMan real-time PCR test targeting the chromosomal flaB gene of Borrelia burgdorferi sensu lato was constructed. The test was compared with a recently published generic Light Upon eXtension (LUX) 16S rRNA real-time PCR test (Wilhelmsson et al. in J Clin Microbiol 48:4169-4176, 2010) on material consisting of 242 Ixodes ricinus ticks collected from dogs and cats in Northern Norway (n = 139) and Telemark County in Southern Norway (n = 103). Ticks positive in either test were further tested by nested PCR amplification of the 5S-23S rRNA intergenic-spacer region followed by sequencing for species identification. A tick was defined as Borrelia positive if two of three tests were positive. Thirty-four of the 242 (14 %) ticks satisfied this definition of positivity. Of these ticks 32 were positive both in the rRNA and flaB test, while two were positive only in the rRNA test. One tick was positive only in the rRNA test and was considered false positive since PCR for sequencing failed. The sensitivity of the flaB test was 94 % and the specificity 100 %. It was possible to determine the species present using Tm analysis. Among ticks from Northern Norway the prevalence of Borrelia was 13 %, whereas the prevalence in Telemark was 16 %. Among identified species (n = 33) B. afzelii was found in 16 (47 %), B. garinii in 15 (44 %) and B. valaisiana in 2 (6 %) ticks, respectively. The flaB test is a rapid, sensitive and specific test for detection and quantification of Borrelia burgdorferi s.l. in I. ricinus ticks. This is the first report on Borrelia prevalence in I. ricinus in Northern Norway.

Respiratory viruses in nepalese children with and without pneumonia: a case-control study.

BACKGROUND: The causative role of respiratory viruses detected in upper airway secretions in childhood pneumonia needs further investigation. OBJECTIVE: To measure the association between infection with respiratory RNA viruses and pneumonia in children. METHODS: From March 2006 to July 2007, we conducted a case-control study of 680 pneumonia cases (WHO criteria) and 680 randomly selected, concurrently sampled age-matched controls among children aged 2-35 months in Bhaktapur, Nepal. A nasopharyngeal aspirate from each child was examined for 7 respiratory viruses using reverse transcription polymerase chain reaction. We calculated the matched odds ratios (MORs) for the detection of the individual viruses from a case compared with a control as measures of pathogenicity using conditional logistic regression. RESULTS: At least 1 virus was recovered in 248 (36.5%) cases and 48 (7.1%) controls. The MOR varied from 2.0 to 13.0; the highest associations were observed for parainfluenza virus type 3 (MOR 13.0; 95% confidence interval [CI] 6.0-28.0), respiratory syncytial virus (MOR 10.7; CI 4.6-24.6), and influenza A (MOR 6.3; CI 1.9-21.4). We observed that the association was lower for children age 2-5 months compared with older children for parainfluenza virus type 3 (P value for interaction 0.002). CONCLUSIONS: All of the 7 respiratory viruses were associated with pneumonia, but their pathogenicity varied. Parainfluenza type 3, RSV, and influenza A were most strongly associated with pneumonia.

Clinical presentation and severity of viral community-acquired pneumonia in young Nepalese children.

BACKGROUND: Most deaths from pneumonia in children <5 years of age occur in developing countries, where information about the clinical impact and severity of viral causes of respiratory infections is limited. METHODS: From June 29, 2004 to June 30, 2007 we evaluated 2230 cases of pneumonia (World Health Organization criteria) in children aged 2 to 35 months in Bhaktapur, Nepal. A nasopharyngeal aspirate from each case was examined for 7 respiratory viruses using reverse-transcription polymerase chain reaction. We compared illness duration, severity, and treatment failure between cases positive and negative for the individual viruses in multiple regression models. RESULTS: A total of 2219 cases had a valid polymerase chain reaction result and were included in the analyses. Overall, 46.1% of cases were 2 to 11 months of age. Being infected with respiratory syncytial virus (RSV) was associated with lower chest indrawing (odds ratio [OR] 2.17; 95% confidence interval [CI] 1.42-3.30) and, among infants, oxygen saturation <93% (OR: 1.88; CI: 1.32-2.69). Among the 2088 nonsevere pneumonia cases, those positive for RSV had a longer time to recovery (hazard ratio 0.82; CI 0.75-0.90; P < 0.001) and an increased risk of treatment failure (OR: 1.75; CI: 1.34-2.28; P < 0.001) than the RSV negative cases. CONCLUSIONS: Being infected with RSV was associated with a more severe clinical presentation of pneumonia, longer illness duration, and increased risk of treatment failure. The severity of RSV infection was age related, infants being more severely affected.

RNA viruses in community-acquired childhood pneumonia in semi-urban Nepal; a cross-sectional study.

BACKGROUND: Pneumonia is among the main causes of illness and death in children <5 years of age. There is a need to better describe the epidemiology of viral community-acquired pneumonia (CAP) in developing countries. METHODS: From July 2004 to June 2007, we examined nasopharyngeal aspirates (NPA) from 2,230 cases of pneumonia (World Health Organization criteria) in children 2 to 35 months old recruited in a randomized trial of zinc supplementation at a field clinic in Bhaktapur, Nepal. The specimens were examined for respiratory syncytial virus (RSV), influenza virus type A (InfA) and B (InfB), parainfluenza virus types 1, 2 and 3 (PIV1, PIV2, and PIV3), and human metapneumovirus (hMPV) using a multiplex reverse transcriptase polymerase chain reaction (PCR) assay. RESULTS: We identified 919 virus isolates in 887 (40.0%) of the 2,219 NPA specimens with a valid PCR result, of which 334 (15.1%) yielded RSV, 164 (7.4%) InfA, 129 (5.8%) PIV3, 98 (4.4%) PIV1, 93 (4.2%) hMPV, 84 (3.8%) InfB, and 17 (0.8%) PIV2. CAP occurred in an epidemic pattern with substantial temporal variation during the three years of study. The largest peaks of pneumonia occurrence coincided with peaks of RSV infection, which occurred in epidemics during the rainy season and in winter. The monthly number of RSV infections was positively correlated with relative humidity (rs = 0.40, P = 0.01), but not with temperature or rainfall. An hMPV epidemic occurred during one of the three winter seasons and the monthly number of hMPV cases was also associated with relative humidity (rs = 0.55, P = 0.0005). CONCLUSION: Respiratory RNA viruses were detected from NPA in 40% of CAP cases in our study. The most commonly isolated viruses were RSV, InfA, and PIV3. RSV infections contributed substantially to the observed CAP epidemics. The occurrence of viral CAP in this community seemed to reflect more or less overlapping micro-epidemics with several respiratory viruses, highlighting the challenges of developing and implementing effective public health control measures.

The course of C-reactive protein response in untreated upper respiratory tract infection.

BACKGROUND: High C-reactive protein (CRP) values are frequently found in patients with bacterial respiratory infection, and CRP testing has been shown to be useful in differentiating pneumonia from other respiratory infections. Raised CRP values may also be found in viral respiratory infection, and as a result there is a risk that antibiotics may be wrongly prescribed. AIMS: To describe the course of the CRP response during untreated upper respiratory tract infections and associations between the development of CRP values, erythrocyte sedimentation rate (ESR) and respiratory symptoms. DESIGN OF STUDY: Prospective study. SETTING: Seven general practices in northern Norway. METHOD: Patients with upper respiratory tract infection aged 16 years or over, who were not treated with antibiotics and who had been ill for no more than 3 days, were recruited. Microbiological examinations were undertaken, together with measurements of CRP, ESR and recording of symptoms daily during the first week of illness and on days 10, 14 and 21. RESULTS: An aetiological agent was established in 23 of the 41 included subjects. These were: influenza A, influenza B, rhinovirus, and other agents. Among the 15 patients examined on both the second and the third day of illness, the median CRP value increased from 7-10 mg/l, and the mean value was from 19-24 mg/l between day 2 and day 3. Peak CRP values were reached on days 2 to 4. Higher CRP values were found in those infected with influenza A and B than in the other subjects (P <0.001). A CRP value >10 mg/l was found in 26 subjects during the first 7 days, compared to five subjects after 1 week. Evidence of a secondary infection with group A streptococci was found in two of these five subjects. The development of the symptoms of sore throat, fatigue, clamminess, and pain from muscles and joints followed a similar course as the CRP response, while stuffy nose, cough, sputum production, and dyspnoea tended to persist after the CRP values had approached the normal range. CONCLUSION: A moderately elevated CRP value (10-60 mg/l) is a common finding in viral upper respiratory tract infection, with a peak during days 2-4 of illness. Moderately elevated CRP values cannot support a diagnosis of bacterial infection when the illness has lasted less than 7 days, but may indicate a complication of viral infection after a week.

[Kingella kingae and osteoarticular infections in children]. / Kingella kingae og skjelettinfeksjon hos barn.

BACKGROUND: In septic arthritis and osteomyelitis in children, the infectious agent is frequently not identified because of failure in obtaining bacterial growth from blood culture or osteoarticular aspirations. MATERIAL AND METHODS: We report a case of septic arthritis caused by Kingella kingae in a two-year-old boy. On the basis of a PubMed literature search, we present an overview of osteoarticular infections with K kingae in childhood. RESULTS AND INTERPRETATION: Over the last ten years there has been an increasing number of reports on osteoarticular infection in children caused by K kingae. The clinical course is usually benign. Direct inoculation of osteoarticular specimens into blood culture bottles is important in order to enhance isolation of K kingae, as this organism will often fail to grow when plated directly on solid media. Improved culture methods and increased awareness are important in order to identify the organism and thus enable targeted antibiotic therapy.