Using amplicon sequencing of rpoB for identification of inoculant rhizobia from peanut nodules.

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.

European all-cause excess and influenza-attributable mortality in the 2017/18 season: should the burden of influenza B be reconsidered?

OBJECTIVES: Weekly monitoring of European all-cause excess mortality, the EuroMOMO network, observed high excess mortality during the influenza B/Yamagata dominated 2017/18 winter season, especially among elderly. We describe all-cause excess and influenza-attributable mortality during the season 2017/18 in Europe. METHODS: Based on weekly reporting of mortality from 24 European countries or sub-national regions, representing 60% of the European population excluding the Russian and Turkish parts of Europe, we estimated age stratified all-cause excess morality using the EuroMOMO model. In addition, age stratified all-cause influenza-attributable mortality was estimated using the FluMOMO algorithm, incorporating influenza activity based on clinical and virological surveillance data, and adjusting for extreme temperatures. RESULTS: Excess mortality was mainly attributable to influenza activity from December 2017 to April 2018, but also due to exceptionally low temperatures in February-March 2018. The pattern and extent of mortality excess was similar to the previous A(H3N2) dominated seasons, 2014/15 and 2016/17. The 2017/18 overall all-cause influenza-attributable mortality was estimated to be 25.4 (95%CI 25.0-25.8) per 100,000 population; 118.2 (116.4-119.9) for persons aged 65. Extending to the European population this translates into over-all 152,000 deaths. CONCLUSIONS: The high mortality among elderly was unexpected in an influenza B dominated season, which commonly are considered to cause mild illness, mainly among children. Even though A(H3N2) also circulated in the 2017/18 season and may have contributed to the excess mortality among the elderly, the common perception of influenza B only having a modest impact on excess mortality in the older population may need to be reconsidered.

Are we missing respiratory viral infections in infants and children? Comparison of a hospital-based quality management system with standard of care.

OBJECTIVES: Hospital-based surveillance of influenza and acute respiratory infections relies on International Classification of Diseases (ICD) codes and hospital laboratory reports (Standard-of-Care). It is unclear how many cases are missed with either method, i.e. remain undiagnosed/coded as influenza and other respiratory virus infections. Various influenza-like illness (ILI) definitions co-exist with little guidance on how to use them. We compared the diagnostic accuracy of standard surveillance methods with a prospective quality management (QM) programme at a Berlin children's hospital with the Robert Koch Institute. METHODS: Independent from routine care, all patients fulfilling pre-defined ILI-criteria (QM-ILI) participated in the QM programme. A separate QM team conducted standardized clinical assessments and collected nasopharyngeal specimens for blinded real-time quantitative PCR for influenza A/B viruses, respiratory syncytial virus, adenovirus, rhinovirus and human metapneumovirus. RESULTS: Among 6073 individuals with ILI qualifying for the QM programme, only 8.7% (528/6073) would have undergone virus diagnostics during Standard-of-Care. Surveillance based on ICD codes would have missed 61% (359/587) of influenza diagnoses. Of baseline ICD codes, 53.2% (2811/5282) were non-specific, most commonly J06 ('acute upper respiratory infection'). Comparison of stakeholder case definitions revealed that QM-ILI and the WHO ILI case definition showed the highest overall sensitivities (84%-97% and 45%-68%, respectively) and the CDC ILI definition had the highest sensitivity for influenza infections (36%, 95% CI 31.4-40.8 for influenza A and 48%, 95% CI 40.5-54.7 for influenza B). CONCLUSIONS: Disease-burden estimates and surveillance should account for the underreporting of cases in routine care. Future studies should explore the effect of ILI screening and surveillance in various age groups and settings. Diagnostic algorithms should be based on the WHO ILI case definition combined with targeted testing.

Human-Dromedary Camel Interactions and the Risk of Acquiring Zoonotic Middle East Respiratory Syndrome Coronavirus Infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) cases without documented contact with another human MERS-CoV case make up 61% (517/853) of all reported cases. These primary cases are of particular interest for understanding the source(s) and route(s) of transmission and for designing long-term disease control measures. Dromedary camels are the only animal species for which there is convincing evidence that it is a host species for MERS-CoV and hence a potential source of human infections. However, only a small proportion of the primary cases have reported contact with camels. Other possible sources and vehicles of infection include food-borne transmission through consumption of unpasteurized camel milk and raw meat, medicinal use of camel urine and zoonotic transmission from other species. There are critical knowledge gaps around this new disease which can only be closed through traditional field epidemiological investigations and studies designed to test hypothesis regarding sources of infection and risk factors for disease. Since the 1960s, there has been a radical change in dromedary camel farming practices in the Arabian Peninsula with an intensification of the production and a concentration of the production around cities. It is possible that the recent intensification of camel herding in the Arabian Peninsula has increased the virus' reproductive number and attack rate in camel herds while the 'urbanization' of camel herding increased the frequency of zoonotic 'spillover' infections from camels to humans. It is reasonable to assume, although difficult to measure, that the sensitivity of public health surveillance to detect previously unknown diseases is lower in East Africa than in Saudi Arabia and that sporadic human cases may have gone undetected there.

Comparing introduction to Europe of highly pathogenic avian influenza viruses A(H5N8) in 2014 and A(H5N1) in 2005.

Since the beginning of November 2014, nine outbreaks of highly pathogenic avian influenza virus (HPAIV) A(H5N8) in poultry have been detected in four European countries. In this report, similarities and differences between the modes of introduction of HPAIV A(H5N1) and A(H5N8) into Europe are described. Experiences from outbreaks of A(H5N1) in Europe demonstrated that early detection to control HPAIV in poultry has proven pivotal to minimise the risk of zoonotic transmission and prevention of human cases.

Taking stock of the first 133 MERS coronavirus cases globally--Is the epidemic changing?

Since June 2012, 133 Middle East respiratory syndrome coronavirus (MERS-CoV) cases have been identified in nine countries. Two time periods in 2013 were compared to identify changes in the epidemiology. The case-fatality risk (CFR) is 45% and is decreasing. Men have a higher CFR (52%) and are over-represented among cases. Thirteen out of 14 known primary cases died. The sex-ratio is more balanced in the latter period. Nosocomial transmission was implied in 26% of the cases.

Influenza-related deaths--available methods for estimating numbers and detecting patterns for seasonal and pandemic influenza in Europe.

Two methodologies are used for describing and estimating influenza-related mortality: Individual-based methods, which use death certification and laboratory diagnosis and predominately determine patterns and risk factors for mortality, and population-based methods, which use statistical and modelling techniques to estimate numbers of premature deaths. The total numbers of deaths generated from the two methods cannot be compared. The former are prone to underestimation, especially when identifying influenza-related deaths in older people. The latter are cruder and have to allow for confounding factors, notably other seasonal infections and climate effects. There is no routine system estimating overall European influenza-related premature mortality, apart from a pilot system EuroMOMO. It is not possible at present to estimate the overall influenza mortality due to the 2009 influenza pandemic in Europe, and the totals based on individual deaths are a minimum estimate. However, the pattern of mortality differed considerably between the 2009 pandemic in Europe and the interpandemic period 1970 to 2008, with pandemic deaths in 2009 occurring in younger and healthier persons. Common methods should be agreed to estimate influenza-related mortality at national level in Europe, and individual surveillance should be instituted for influenza-related deaths in key groups such as pregnant women and children.

Associations of dietary polyunsaturated fatty acids with bone mineral density in elderly women.

BACKGROUND/OBJECTIVES: Significance of dietary fatty acids on bone health is not clear, and the evidence is controversial. This study aimed to investigate the relationship between dietary polyunsaturated fatty acids (PUFAs) and bone mineral density (BMD) among elderly women. SUBJECTS/METHODS: Subjects (n=554) were drawn from the Kuopio OSTPRE Fracture Prevention Study. At baseline they filled a 3-day food record and a questionnaire on lifestyle factors, diseases and medications. BMD was measured at lumbar spine (L2-L4), femoral neck and total body by dual energy X-ray absorptiometry at baseline and after 3 years. The associations between dietary fatty acids and BMD were analyzed by a linear mixed model adjusting for potential dietary and non-dietary confounders. RESULTS: Our findings suggested a positive relationship between the dietary PUFAs and BMD at lumbar spine and in total body but not at femoral neck. Further analyses revealed that these results were due to associations among the women without hormone therapy (HT) at baseline. Among them, the intake of total PUFAs as well the intakes of linoleic and linolenic acids and total n-3 and n-6 fatty acids were significantly associated with BMD at lumbar spine; P for trend over the quartiles ranged between 0.013 and 0.001. Similarly, significant associations were demonstrated for total body BMD and fatty acids with an exception of total PUFA. No significant associations were found among women with HT at baseline. CONCLUSIONS: Our findings among elderly women without HT support the suggested beneficial effect of dietary PUFAs on bone health.

Initial surveillance of 2009 influenza A(H1N1) pandemic in the European Union and European Economic Area, April-September 2009.

European Union (EU) and European Economic Area (EEA) countries reported surveillance data on 2009 pandemic influenza A(H1N1) cases to the European Centre for Disease Prevention and Control (ECDC) through the Early Warning and Response System (EWRS) during the early phase of the 2009 pandemic. We describe the main epidemiological findings and their implications in respect to the second wave of the 2009 influenza pandemic. Two reporting systems were in place (aggregate and case-based) from June to September 2009 to monitor the evolution of the pandemic. The notification rate was assessed through aggregate reports. Individual data were analysed retrospectively to describe the population affected. The reporting peak of the first wave of the 2009 pandemic influenza was reached in the first week of August. Transmission was travel-related in the early stage and community transmission within EU/EEA countries was reported from June 2009. Seventy eight per cent of affected individuals were less than 30 years old. The proportions of cases with complications and underlying conditions were 3% and 7%, respectively. The most frequent underlying medical conditions were chronic lung (37%) and cardio-vascular diseases (15%). Complication and hospitalisation were both associated with underlying conditions regardless of age. The information from the first wave of the pandemic produced a basis to determine risk groups and vaccination strategies before the start of the winter wave. Public health recommendations should be guided by early capture of profiles of affected populations through monitoring of infectious diseases.

In vivo and in vitro efficacy of amodiaquine monotherapy for treatment of infection by chloroquine-resistant Plasmodium vivax.

Amodiaquine retains efficacy against infection by chloroquine-resistant Plasmodium falciparum; however, little information is available on its efficacy against infection by chloroquine-resistant Plasmodium vivax. Patients presenting to a rural clinic with a pure P. vivax infection that recurred after recent antimalarial treatment were retreated, this time with amodiaquine monotherapy, and the risk of further recurrence within 4 weeks was assessed. Of the 87 patients with pure P. vivax infection, 15 patients did not complete a full course of treatment, 4 of whom were intolerant to treatment. In the 72 patients completing treatment, 91% (63 of 69) had cleared their parasitemia within 48 h with no early treatment failure. Follow-up to day 28 or recurrent parasitemia was achieved for 56 patients (78%). The cumulative incidence of treatment failure by day 28 was 22.8% (95% confidence interval, 7.3 to 38%). The in vitro sensitivity profile was determined for a separate set of isolates from outpatients with pure P. vivax infection. The median 50% inhibitory concentration of amodiaquine was 11.3 nM (range, 0.37 to 95.8) and was correlated significantly with that of chloroquine (Spearman rank correlation coefficient, 0.602; P < 0.001). Although amodiaquine results in a rapid clinical response, the risk of recurrence by day 28 is unacceptably high, reducing its suitability as an alternative treatment of infection by chloroquine-resistant P. vivax in this region.

A public-private partnership for TB control in Timika, Papua Province, Indonesia.

SETTING: A district-level tuberculosis (TB) control programme in Papua Province, Indonesia. OBJECTIVE: To describe a successful partnership between the District Health Department, a private company and non-governmental health care providers. METHODS: Routinely collected surveillance data were analysed. A conceptual model was constructed to describe TB control in the district. Data were compared with the National TB Control Programme (NTP) performance indicators. RESULTS: Funding for the programme's TB clinic is provided by a private company (PT Freeport Indonesia). The NTP provides the policy framework, treatment guidelines and some supplies. TB clinic staff are included in training programmes and the TB laboratory in the provincial quality assurance system. TB clinic staff are responsible for diagnosis, treatment, default tracing, recording and reporting, health education and community mobilisation. The largest proportion of TB patient referrals came from the community hospital (41%). The TB notification rate (311/100000), TB-HIV (human immunodeficiency virus) co-infection (12%) and multidrug-resistant (MDR) TB (2%) are significantly higher in Mimika, but the treatment success rate for smear-positive patients (91%) is similar to Indonesian national figures. CONCLUSIONS: For true progress in attaining the United Nations Millennium Development Goals for TB in Indonesia, innovative local solutions utilising public-private partnerships are essential. The Mimika model is one such solution that should be tested elsewhere.

Clinical and pharmacological determinants of the therapeutic response to dihydroartemisinin-piperaquine for drug-resistant malaria.

Dihydroartemisinin-piperaquine (DHP) is an important new treatment for drug-resistant malaria, although pharmacokinetic studies on the combination are limited. In Papua, Indonesia, we assessed determinants of the therapeutic efficacy of DHP for uncomplicated malaria. Plasma piperaquine concentrations were measured on day 7 and day 28, and the cumulative risk of parasitological failure at day 42 was calculated using survival analysis. Of the 598 patients in the evaluable population 342 had infections with Plasmodium falciparum, 83 with Plasmodium vivax, and 173 with a mixture of both species. The unadjusted cumulative risks of recurrence were 7.0% (95% confidence interval [CI]: 4.6 to 9.4%) for P. falciparum and 8.9% (95% CI: 6.0 to 12%) for P. vivax. After correcting for reinfections the risk of recrudescence with P. falciparum was 1.1% (95% CI: 0.1 to 2.1%). The major determinant of parasitological failure was the plasma piperaquine concentration. A concentration below 30 ng/ml on day 7 was observed in 38% (21/56) of children less than 15 years old and 22% (31/140) of adults (P = 0.04), even though the overall dose (mg per kg of body weight) in children was 9% higher than that in adults (P < 0.001). Patients with piperaquine levels below 30 ng/ml were more likely to have a recurrence with P. falciparum (hazard ratio [HR] = 6.6 [95% CI: 1.9 to 23]; P = 0.003) or P. vivax (HR = 9.0 [95% CI: 2.3 to 35]; P = 0.001). The plasma concentration of piperaquine on day 7 was the major determinant of the therapeutic response to DHP. Lower plasma piperaquine concentrations and higher failure rates in children suggest that dose revision may be warranted in this age group.

Dihydroartemisinin-piperaquine versus artesunate-amodiaquine: superior efficacy and posttreatment prophylaxis against multidrug-resistant Plasmodium falciparum and Plasmodium vivax malaria.

BACKGROUND: Antimalarial drug resistance is now well established in both Plasmodium falciparum and Plasmodium vivax. In southern Papua, Indonesia, where both strains of plasmodia coexist, we have been conducting a series of studies to optimize treatment strategies. METHODS: We conducted a randomized trial that compared the efficacy and safety of dihydroartemisinin-piperaquine (DHP) with artesunate-amodiaquine (AAQ). The primary end point was the overall cumulative parasitological failure rate at day 42. RESULTS: Of the 334 patients in the evaluable patient population, 185 were infected with P. falciparum, 80 were infected with P. vivax, and 69 were infected with both species. The overall parasitological failure rate at day 42 was 45% (95% confidence interval [CI], 36%-53%) for AAQ and 13% (95% CI, 7.2%-19%) for DHP (hazard ratio [HR], 4.3; 95% CI, 2.5-7.2; P<.001). Rates of both recrudescence of P. falciparum infection and recurrence of P. vivax infection were significantly higher after receipt of AAQ than after receipt of DHP (HR, 3.4 [95% CI, 1.2-9.4] and 4.3 [95% CI, 2.2-8.2], respectively; P<.001). By the end of the study, AAQ recipients were 2.95-fold (95% CI, 1.2- to 4.9-fold) more likely to be anemic and 14.5-fold (95% CI, 3.4- to 61-fold) more likely to have carried P. vivax gametocytes. CONCLUSIONS: DHP was more effective and better tolerated than AAQ against multidrug-resistant P. falciparum and P. vivax infections. The prolonged therapeutic effect of piperaquine delayed the time to P. falciparum reinfection, decreased the rate of recurrence of P. vivax infection, and reduced the risk of P. vivax gametocyte carriage and anemia.

A case-cohort study to investigate concomitant waterborne outbreaks of Campylobacter and gastroenteritis in Söderhamn, Sweden, 2002-3.

Increased domestic, laboratory confirmed, Campylobacter notifications were reported in Siderhamn municipality, December 2002 and January 2003. Concurrently, during preliminary investigations a large outbreak of acute gastroenteritis was detected. Simultaneously, two studies were completed to identify risk factors for infection with Campylobacter and acute gastrointestinal infection (AGI): (1) a case-cohort study using Campylobacter cases (N = 101) with a large random sample from the municipal population as referents (N = 1000) and (2) a retrospective cohort study for the outcome AGI using the same sample. A postal questionnaire was used to collect demographic, clinical, water and food consumption data. Measures of association (risk ratio (RR), odds ratio (OR)) and 95% confidence intervals (CI) were calculated. Stool, environmental and water samples were tested by standard methods at Gävle Hospital and SMI laboratories respectively. In the case-cohort study, Camplylobacter cases were more likely than referents to consume communal water (OR = 12.6 (95% CI 1.7-92.3)). In the cohort study, risk of gastroenteritis was 2.3 times higher in those who consumed water (AR = 27.3%) than others (AR = 12%). Risk of illness was associated with the amount of water consumed in both studies. Campylobacter was detected in stools and Escherichia coli (E. coli) from routine communal water (CW) samples. Results suggest both Söderhamn outbreaks of Campylobacter and AGI were associated with consumption of CW. The method used strengthened epidemiological evidence and was efficient in the use of time and resources.