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Serologic studies are crucial for clarifying dynamics of the coronavirus disease pandemic. Past work on serologic studies (e.g., during influenza pandemics) has made relevant contributions, but specific conditions of the current situation require adaptation. Although detection of antibodies to measure exposure, immunity, or both seems straightforward conceptually, numerous challenges exist in terms of sample collection, what the presence of antibodies actually means, and appropriate analysis and interpretation to account for test accuracy and sampling biases. Successful deployment of serologic studies depends on type and performance of serologic tests, population studied, use of adequate study designs, and appropriate analysis and interpretation of data. We highlight key questions that serologic studies can help answer at different times, review strengths and limitations of different assay types and study designs, and discuss methods for rapid sharing and analysis of serologic data to determine global transmission of severe acute respiratory syndrome coronavirus 2.

Background: Tuberculosis (TB) is a major global health burden, with an estimated quarter of the world's population being infected. The World Health Organization (WHO) launched the "End TB Strategy" in 2014 emphasising knowing the epidemic. WHO ranks Vietnam 12th in the world of high burden countries. TB spatial and temporal patterns have been observed globally with evidence of Vitamin D playing a role in seasonality. We explored the presence of temporal and spatial clustering of TB in Vietnam and their determinants to aid public health measures., Methods: Data were collected by the National TB program of Vietnam from 2010 to 2015 and linked to the following datasets: socio-demographic characteristics; climatic variables; influenza-like-illness (ILI) incidence; geospatial data. The TB dataset was aggregated by province and quarter. Descriptive time series analyses using LOESS regression were completed per province to determine seasonality and trend. Harmonic regression was used to determine the amplitude of seasonality by province. A mixed-effect linear model was used with province and year as random effects and all other variables as fixed effects., Results: There were 610,676 cases of TB notified between 2010 and 2015 in Vietnam. Heat maps of TB incidence per quarter per province showed substantial temporal and geospatial variation. Time series analysis demonstrated seasonality throughout the country, with peaks in spring/summer and troughs in autumn/winter. Incidence was consistently higher in the south, the three provinces with the highest incidence per 100,000 population were Tay Ninh, An Giang and Ho Chi Minh City. However, relative seasonal amplitude was more pronounced in the north. Mixed-effect linear model confirmed that TB incidence was associated with time and latitude. Of the demographic, socio-economic and health related variables, population density, percentage of those under 15 years of age, and HIV infection prevalence per province were associated with TB incidence. Of the climate variables, absolute humidity, average temperature and sunlight were associated with TB incidence., Conclusion: Preventative public health measures should be focused in the south of Viet Nam where incidence is highest. Vitamin D is unlikely to be a strong driver of seasonality but supplementation may play a role in a package of interventions.

In the Mekong Delta region of Vietnam, small-scale chicken farming is common. However, high levels of disease or mortality in such flocks impair economic development and challenge the livelihoods of many rural households. We investigated 61 diseased small-scale flocks (122 chickens) for evidence of infection with 5 bacteria, 4 viruses, and helminths. Serological profiles (ELISA) were also determined against 6 of these pathogens. The aims of this study were the following: (1) to investigate the prevalence of different pathogens and to compare the probability of detection of bacterial pathogens using PCR and culture; (2) to investigate the relationship between detection of organisms in birds' tissues and the observed morbidity and mortality, as well as their antibody profile; and (3) to characterize risk factors for infection with specific viral or bacterial pathogens. We used PCR to test for viral (viruses causing infectious bronchitis [IB], highly pathogenic avian influenza [HPAI], Newcastle disease, and infectious bursal disease [IBD]) and bacterial pathogens (Mycoplasma gallisepticum, Pasteurella multocida, Avibacterium paragallinarum, and Ornithobacterium rhinotracheale [ORT]). The latter two were also investigated in respiratory tissues by conventional culture. Colisepticemic Escherichia coli was investigated by liver or spleen culture. In 49 of 61 (80.3%) flocks, at least one bacterial or viral pathogen was detected, and in 29 (47.5%) flocks, more than one pathogen was detected. A. paragallinarum was detected in 62.3% flocks, followed by M. gallisepticum (26.2%), viruses causing IBD (24.6%) and IB (21.3%), septicemic E. coli (14.8%), ORT (13.1%), and HPAI viruses (4.9%). Of all flocks, 67.2% flocks were colonized by helminths. Mortality was highest among flocks infected with HPAI (100%, interquartile range [IQR]: 81.6-100%) and lowest with flocks infected with ORT (5.3%, IQR: 1.1-9.0%). The results indicated slight agreement (kappa ≤ 0.167) between detection by PCR and culture for both A. paragallinarum and ORT, as well as between the presence of cestodes and ORT infection (kappa = 0.317). Control of A. paragallinarum, viruses causing HPAI, IBD, and IB, M. gallisepticum, and gastrointestinal helminths should be a priority in small-scale flocks., (Copyright © 2019. Published by Elsevier Inc.)

The accurate assessment of antimicrobial use (AMU) requires relating quantities of active ingredients (AAIs) with population denominators. These data can be used to prioritize potential sources of selective pressure for antimicrobial resistance and to establish reduction targets. Here, we estimated AMU in Vietnam (human population 93.4 M in 2015), and compared it with European Union (EU) data (population 511.5 M in 2014). We extrapolated AMU data on each key animal species and humans from different published sources to calculate overall AMU (in tonnes) in Vietnam. We then compared these data with published statistics on AMU in the European Union (EU). A total of 3838 t of antimicrobials were used in Vietnam, of which 2751 (71.7%) corresponded to animal use, and the remainder (1086 t; 28.3%) to human AMU. This equates to 261.7 mg and 247.3 mg per kg of human and animal biomass, compared with 122.0 mg and 151.5 mg in the EU. The greatest quantities of antimicrobials (in decreasing order) were used in pigs (41.7% of total use), humans (28.3%), aquaculture (21.9%) and chickens (4.8%). Combined AMU in other species accounted for < 1.5%. These results are approximate and highlight the need to conduct targeted surveys to improve country-level estimates of AMU., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2020.)

In recent years, serosurveillance has gained momentum as a way of determining disease transmission and immunity in populations, particularly with respect to vaccine-preventable diseases. At the end of 2017, the Oxford University Clinical Research Unit and the National Institute of Hygiene and Epidemiology held a meeting in Vietnam with national policy makers, researchers, and international experts to discuss current seroepidemiologic projects in Vietnam and future needs and plans for nationwide serosurveillance. This report summarizes the meeting and the plans that were discussed to set up nationwide serosurveillance in Vietnam.

Chicken is, among farmed species, the target of the highest levels of antimicrobial use (AMU). There are considerable knowledge gaps on how and when antimicrobials are used in commercial small-scale chicken farms. These shortcomings arise from cross-sectional study designs and poor record keeping practiced by many such farmers. Furthermore, there is a large diversity of AMU metrics, and it is not clear how these metrics relate to each other. We performed a longitudinal study on a cohort of small-scale chicken farms ( n = 102) in the Mekong Delta (Vietnam), an area regarded as a hotspot of AMU, from October 2016 to May 2018. We collected data on all medicine products administered to 203 flocks with the following aims: (1) to describe types and quantities of antimicrobial active ingredients (AAIs) used; (2) to describe critical time points of AMU; and (3) to compare AMU using three quantitative metrics: (a) weight of AAIs related to bird weight at the time of treatment (mg/kg at treatment); (b) weight of AAIs related to weight of birds sold (mg/kg sold); and (c) "treatment incidence" (TI), or the number of daily doses per kilogram of live chicken [Vietnamese animal daily dose (ADDvetVN)] per 1,000 days. Antimicrobials contained in commercial feed, administered by injection ( n = N = 6), or antimicrobials for human medicine ( n = N = 16) were excluded. A total of 236 products were identified, containing 42 different AAIs. A total of 76.2% products contained AAIs of "critical importance" according to the World Health Organization (WHO). On average, chickens consumed 791.8 (SEM ±16.7) mg/kg at treatment, 323.4 (SEM ±11.3) mg/kg sold, and the TI was 382.6 (SEM ±5.5) per 1,000 days. AMU was more common early in the production cycle and was highly skewed, with the upper 25% quantile of flocks accounting for 60.7% of total AMU. The observed discrepancies between weight- and dose-based metrics were explained by differences in the strength of AAIs, mortality levels, and the timing of administration. Results suggest that in small-scale chicken production, AMU reduction efforts should preferentially target the early (brooding) period, which is when birds are most likely to be exposed to antimicrobials, whilst restricting access to antimicrobials of critical importance for human medicine.

Raising chickens in small-scale flocks following all-in-all-out management is common in the Mekong Delta of Vietnam. These flocks represent an intermediate category between backyard and intensive (industrial) farming systems. However, little is known about the occurrence and burden of disease and/or mortality in such flocks, and their potential association with antimicrobial usage (AMU). We investigated mortality, disease and weekly antimicrobial use (AMU) in 124 cycles of meat chicken flocks raised in 88 farms in the Mekong Delta of Vietnam (with a median cycle duration of 18 weeks [inter-quartile range IQR 17-20]). We visited each farm 4 times per cycle to review data collected weekly by the farmers on clinical signs, mortality, and AMU. The overall probability of disease and AMU were 0.31 (95% CI 0.29-0.32) and 0.26 (95% CI 0.24-0.28), respectively. The average weekly incidence of mortality was 2.6 (95% CI 2.2-3.0) per 100 birds. Both the probabilities of a flock experiencing disease and mortality, as well as of using antimicrobials decreased with the flock's age. However, mortality peaked at the 5-10 week period. The only significant explanatory factors associated with presence of disease was the stage of production ≥5 weeks (protective) (OR ≤ 0.51). Factors independently associated with AMU (p < 0.05) were: (1) Number of chickens (log) (OR=1.46), (2) Stage of production ≥5 weeks (OR≤0.67) (protective), (3) Cao Lanh district (OR=2.23), (4) Density of veterinary drug shops at commune level (log) (OR=1.58), and (5) Disease in flocks (OR=1.80). Factors independently associated with overall increased weekly incidence of mortality (p < 0.05) were: (1) High level of education attainment (secondary education or higher) (Hazard rate Ratio [HR]=1.70), (2) number of chickens (log) (HR=1.39), and (3) Stage of production >5 weeks (HR≤2.14). In flocks reporting disease, AMU significantly reduced the incidence of mortality (HR=0.90). These results confirm an exceptionally high mortality in chicken flocks in the area, jeopardizing the profitability and sustainability of these small-scale farming systems. The data also suggest an association between nearby access to antimicrobials and AMU, and a high correlation of AMU over consecutive cycles. The atomized farming landscape of the Mekong Delta, the high incidence of disease and mortality, and the unrestricted and easy access to antimicrobials present major challenges to the implementation of policies aimed at AMU reductions., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Rickettsioses are endemic to Vietnam; however, only a limited number of clinical studies have been performed on these vectorborne bacteria. We conducted a prospective hospital-based study at 2 national referral hospitals in Hanoi to describe the clinical characteristics of scrub typhus and murine typhus in northern Vietnam and to assess the diagnostic applicability of quantitative real-time PCR assays to diagnose rickettsial diseases. We enrolled 302 patients with acute undifferentiated fever and clinically suspected rickettsiosis during March 2015-March 2017. We used a standardized case report form to collect clinical information and laboratory results at the time of admission and during treatment. We confirmed scrub typhus in 103 (34.1%) patients and murine typhus in 12 (3.3%) patients. These results highlight the need for increased emphasis on training for healthcare providers for earlier recognition, prevention, and treatment of rickettsial diseases in Vietnam.

Due to worldwide increased human mobility, air-transportation data and mathematical models have been widely used to measure risks of global dispersal of pathogens. However, the seasonal and interannual risks of pathogens importation and onward transmission from endemic countries have rarely been quantified and validated. We constructed a modelling framework, integrating air travel, epidemiological, demographical, entomological and meteorological data, to measure the seasonal probability of dengue introduction from endemic countries. This framework has been applied retrospectively to elucidate spatiotemporal patterns and increasing seasonal risk of dengue importation from South-East Asia into China via air travel in multiple populations, Chinese travelers and local residents, over a decade of 2005-15. We found that the volume of airline travelers from South-East Asia into China has quadrupled from 2005 to 2015 with Chinese travelers increased rapidly. Following the growth of air traffic, the probability of dengue importation from South-East Asia into China has increased dramatically from 2005 to 2015. This study also revealed seasonal asymmetries of transmission routes: Sri Lanka and Maldives have emerged as origins; neglected cities at central and coastal China have been increasingly vulnerable to dengue importation and onward transmission. Compared to the monthly occurrence of dengue reported in China, our model performed robustly for importation and onward transmission risk estimates. The approach and evidence could facilitate to understand and mitigate the changing seasonal threat of arbovirus from endemic regions., Competing Interests: The authors have declared that no competing interests exist.

Multi-species interactions can often have non-intuitive consequences. However, the study of parasite interactions has rarely gone beyond the effects of pairwise combinations of species, and the outcomes of multi-parasite interactions are poorly understood. We investigated the effects of co-infection by four gastrointestinal helminth species on the development of cerebral malaria among Plasmodium falciparum-infected patients. We characterized associations among the helminth parasite infra-community, and then tested for independent (direct) and co-infection dependent (indirect) effects of helminths on cerebral malaria risk. We found that infection by Ascaris lumbricoides and Trichuris trichiura were both associated with direct reductions in cerebral malaria risk. However, the benefit of T. trichiura infection was halved in the presence of hookworm, revealing a strong indirect effect. Our study suggests that the outcome of interactions between two parasite species can be significantly modified by a third, emphasizing the critical role that parasite community interactions play in shaping infection outcomes., Competing Interests: The authors have declared that no competing interests exist.

Background: Acute respiratory tract infections (ARIs) are the leading cause of morbidity and mortality in young children in low/middle-income countries. Using routine hospital data, we aimed to examine the spatial distribution, temporal trends and climatic risk factors of paediatric ARIs in Vietnam., Methods: Data from hospitalised paediatric (<16 years) patients with ARIs residing in Ho Chi Minh City (HCMC) between 2005 and 2010 were retrieved from the two main Children's Hospitals and the Hospital for Tropical Diseases in HCMC. Spatial mapping and time series analysis were performed after disaggregating data into upper respiratory tract infections (URIs) and lower respiratory tract infections (LRIs)., Results: Over the study period, there were 155 999 paediatric patients admitted with ARIs (33% of all hospital admissions). There were 68 120 URIs (14%) and 87 879 LRIs (19%). The most common diagnoses were acute pharyngitis (28% of all ARI), pneumonia (21%), bronchitis (18%) and bronchiolitis (16%). A significant increasing trend over time was found for both URIs (mean weekly incidence per 1000 population, I=3.12), incidence rate ratio for 1-week increase in time (RR 1.0, 95% CI 1.02 to 1.17) for URI and (I=4.02, RR 1.08 (95% CI 1.006 to 1.16)) for LRI. The weekly URI incidence peaked in May-June and was significantly associated with lags in weekly URI incidence and the average humidity, rainfall and water level. The weekly LRI incidence exhibited significant seasonality (P<0.0001), with an annual peak in September-October and was significantly associated with lags in weekly LRI incidence and lags in weekly average temperature, rainfall and water level., Conclusions: ARIs are a leading cause of childhood hospitalisation in HCMC, Vietnam. The incidence of ARIs was higher in the wet season and in specific HCMC districts. These results may guide health authorities in where and when to effectively allocate resources for the prevention and control of ARIs., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Evaluating the risk of emergence and transmission of vector-borne diseases requires knowledge of the genetic and environmental contributions to pathogen transmission traits. Compared to the significant effort devoted to understanding the biology of malaria transmission from vertebrate hosts to mosquito vectors, the strategies that malaria parasites have evolved to maximize transmission from vectors to vertebrate hosts have been largely overlooked. While determinants of infection success within the mosquito host have recently received attention, the causes of variability for other key transmission traits of malaria, namely the duration of parasite development and its virulence within the vector, as well as its ability to alter mosquito behavior, remain largely unknown. This important gap in our knowledge needs to be bridged in order to obtain an integrative view of the ecology and evolution of malaria transmission strategies. Associations between transmission traits also need to be characterized, as they trade-offs and constraints could have important implications for understanding the evolution of parasite transmission. Finally, theoretical studies are required to evaluate how genetic and environmental influences on parasite transmission traits can shape malaria dynamics and evolution in response to disease control.

While climate is often presented as a key factor influencing the seasonality of diseases, the importance of anthropogenic factors is less commonly evaluated. Using a combination of methods - wavelet analysis, economic analysis, statistical and disease transmission modelling - we aimed to explore the influence of climatic and economic factors on the seasonality of H5N1 Highly Pathogenic Avian Influenza in the domestic poultry population of Vietnam. We found that while climatic variables are associated with seasonal variation in the incidence of avian influenza outbreaks in the North of the country, this is not the case in the Centre and the South. In contrast, temporal patterns of H5N1 incidence are similar across these 3 regions: periods of high H5N1 incidence coincide with Lunar New Year festival, occurring in January-February, in the 3 climatic regions for 5 out of the 8 study years. Yet, daily poultry meat consumption drastically increases during Lunar New Year festival throughout the country. To meet this rise in demand, poultry production and trade are expected to peak around the festival period, promoting viral spread, which we demonstrated using a stochastic disease transmission model. This study illustrates the way in which economic factors may influence the dynamics of livestock pathogens.

The Lao People's Democratic Republic (PDR) is still considered a highly endemic country for hepatitis B, mainly due to perinatal transmission of hepatitis B virus (HBV), despite efforts made since 2004 for universal immunization of newborns. The prevalence of HBV surface antigen (HBsAg) carriage in pregnant women is a relevant marker for the risk of mother-to-child HBV transmission. This study aimed to assess the changes in prevalence of HBV infection among pregnant women attending the Mahosot Prenatal Clinic (Vientiane Capital). Methods . A retrospective study was performed in the Mahosot Hospital Laboratory to collect and analyze all the results of HBsAg testing in pregnant women from 2008 to 2014. Results . Of a total of 13,238 tested women of mean age of 26 years, 720 women (5,44% [95 CI: 5.1-5.8%]) were found HBsAg positive, the annual prevalence ranging from 4.6% to 6.2%. A slight but steady and significant decrease in prevalence over the 7 years of the study could be documented. Conclusion . Although below the 8% hyperendemic threshold, the HBsAg prevalence observed in pregnant women in Vientiane reflects a high risk of HBV perinatal transmission and call for a widespread infant immunization with an HBV vaccine birth dose.

Background: The global burden of diarrhea is a leading cause of morbidity and mortality worldwide. In montane areas of South-East Asia such as northern Laos, recent changes in land use have induced increased runoff, soil erosion and in-stream suspended sediment loads, and potential pathogen dissemination. To our knowledge, few studies have related diarrhea incidences to catchment scale hydrological factors such as river discharge, and loads of suspended sediment and of Fecal Indicator Bacteria (FIB) such as Escherichia coli, together with sociological factors such as hygiene practices. We hypothesized that climate factors combined with human behavior control diarrhea incidence, either because higher rainfall, leading to higher stream discharges, suspended sediment loads and FIB counts, are associated with higher numbers of reported diarrhea cases during the rainy season, or because water shortage leads to the use of less safe water sources during the dry season. Using E. coli as a FIB, the objectives of this study were thus (1) to characterize the epidemiological dynamics of diarrhea in Northern Laos, and (2) to identify which hydro-meteorological and sociological risk factors were associated with diarrhea epidemics., Methods: Considering two unconnected river catchments of 22 and 7,448 km2, respectively, we conducted a retrospective time series analysis of meteorological variables (rainfall, air temperature), hydrological variables (discharge, suspended sediments, FIB counts, water temperature), and the number of diarrheal disease cases reported at 6 health centers located in the 5 southern districts of the Luang Prabang Province, Lao PDR. We also examined the socio-demographic factors potentially affecting vulnerability to the effect of the climate factors, such as drinking water sources, hygiene habits, and recreational water exposure., Results: Using thus a mixed methods approach, we found E. coli to be present all year long (100-1,000 Most Probable Number or MPN 100 mL-1) indicating that fecal contamination is ubiquitous and constant. We found that populations switch their water supply from wells to surface water during drought periods, the latter of which appear to be at higher risk of bacterial contamination than municipal water fountains. We thus found that water shortage in the Luang Prabang area triggers diarrhea peaks during the dry and hot season and that rainfall and aquifer refill ends the epidemic during the wet season. The temporal trends of reported daily diarrhea cases were generally bimodal with hospital admissions peaking in February-March and later in May-July. Annual incidence rates were higher in more densely populated areas and mostly concerned the 0-4 age group and male patients., Conclusions: We found that anthropogenic drivers, such as hygiene practices, were at least as important as environmental drivers in determining the seasonal pattern of a diarrhea epidemic. For diarrheal disease risk monitoring, discharge or groundwater level can be considered as relevant proxies. These variables should be monitored in the framework of an early warning system provided that a tradeoff is found between the size of the monitored catchment and the frequency of the measurement., Competing Interests: The authors have declared that no competing interests exist.

Despite their classification as low pathogenicity avian influenza viruses (LPAIV), A/H9N2 viruses cause significant losses in poultry in many countries throughout Asia, the Middle East and North Africa. To date, poultry surveillance in Vietnam has focused on detection of influenza H5 viruses, and there is limited understanding of influenza H9 epidemiology and transmission dynamics. We determined prevalence and diversity of influenza A viruses in chickens from live bird markets (LBM) of 7 northern Vietnamese provinces, using pooled oropharyngeal swabs collected from October to December 2014. Screening by real time RT-PCR revealed 1207/4900 (24.6%) of pooled swabs to be influenza A virus positive; overall prevalence estimates after accounting for pooling (5 swabs/pools) were 5.8% (CI 5.4-6.0). Subtyping was performed on 468 pooled swabs with M gene Ct<26. No influenza H7 was detected; 422 (90.1%) were H9 positive; and 22 (4.7%) were H5 positive. There was no evidence was of interaction between H9 and H5 virus detection rates. We sequenced 17 whole genomes of A/H9N2, 2 of A/H5N6, and 11 partial genomes. All H9N2 viruses had internal genes that clustered with genotype 57 and were closely related to Chinese human isolates of A/H7N9 and A/H10N8. Using a nucleotide divergence cutoff of 98%, we identified 9 distinct H9 genotypes. Phylogenetic analysis suggested multiple introductions of H9 viruses to northern Vietnam rather than in-situ transmission. Further investigations of H9 prevalence and diversity in other regions of Vietnam are warranted to assess H9 endemicity elsewhere in the country., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Mycobacterium tuberculosis, the tuberculosis (TB) pathogen, despite a low level of genetic diversity, has revealed a high variety of biological and epidemiological characteristics linked to their lineages, such as transmissibility, fitness and propensity to acquire drug resistance. This has important implications for the epidemiology of TB. We conducted this first countrywide cross-sectional study to identify the prevalent M. tuberculosis lineages and to assess their epidemiological associations and their relation to drug resistance. The study was conducted among isolates acquired in reference hospitals across Vietnam. Isolates with drug susceptibility testing profiles were identified for their lineages by spoligotyping. Logistic regression was used to investigate the association of M. tuberculosis lineages with location, age and sex of the patients and drug resistance levels., Results: Results showed that the most prevalent lineage was Beijing (55.4 %), followed by EAI (27.5 %), T (6.4 %), LAM (1.3 %), Haarlem (1 %) and Zero type (0.3 %). The proportion of Beijing isolates in the North (70.4 %) and the South (68 %) was higher than in the Centre (28 %) (OR = 1.7 [95 % CI: 1.4-2.0], p < 0.0001), whereas the proportion of EAI isolates in the North (7.1 %) and the South (17 %) was much lower compared with the Centre (59 %) (OR = 0.5 [95 % CI: 0.4-0.6], p < 0.0001). Overall, Beijing isolates were the most likely to be drug-resistant and EAI isolates were the least likely to be drug-resistant, except in the South of Vietnam where EAI is also highly drug-resistant. The proportion of Beijing isolates was significantly higher (p < 0.01), and the proportion of EAI isolates was significantly lower (p < 0.05) in younger patients. The proportion of drug-resistance was higher in isolates collected from male patients and from patients in the middle age groups., Conclusions: The findings suggest ongoing replacement of EAI lineage, which is mainly more drug-susceptible with highly drug-resistant Beijing lineage in all studied regions of Vietnam. Male patients of working ages should be the focus for better control to prevent the emergence of drug-resistant TB.

Background: The agar dilution method is currently considered as the reference method for Mycobacterium marinum drug susceptibility testing (DST). As it is time-consuming, alternative methods, such as the E-test, were evaluated for M. marinum DST, but without success. The SLOMYCO Sensititre(®) panel, recently commercialized by TREK Diagnostic Systems (Cleveland, OH), can be used for DST in slow-growing mycobacteria and for antimicrobial agents recommended by the Clinical and Laboratory Standards Institute (CLSI) for M. marinum DST. The main goal of this work was to evaluate the SLOMYCO Sensititre(®) panel method for DST in M. marinum isolates from human patients and fish relative to the reference agar dilution method., Methods/results: The reproducibility of the minimum inhibitory concentration (MIC) determination (±1 log2 dilution) was very good for both the agar dilution method and SLOMYCO Sensititre(®) panel (>90 % agreement). The percentage essential agreement between methods varied, depending on the drug: between 97 and 75 % for ciprofloxacin, moxifloxacin, linezolid, isoniazid, clarithromycin, amikacin, rifabutin and rifampin, 74 % for trimethoprim, 72 % for doxycycline, 70 % for sulfamethoxazole, 59 % for streptomycin, 33 % for ethambutol and only 2.2 % for ethionamide. When the agar dilution and SLOMYCO Sensititre(®) panel results were converted into interpretive criteria, the category agreement was 100 % for amikacin, ciprofloxacin, clarithromycin, moxifloxacin, rifabutin, sulfamethoxazole and trimethoprim, 98 % for ethambutol and 96 % for rifampin and no agreement for doxycycline., Conclusions: The SLOMYCO Sensititre(®) panel method could provide a potential alternative to the reference agar dilution method, when DST in M. marinum is required, except for doxycycline.

The immunity profile against H5N1 highly pathogenic avian influenza (HPAI) in the commercial poultry value chain network in Egypt was modeled with the use of different vaccination scenarios. The model estimated the vaccination coverage, the protective seroconversion level, and the duration of immunity for each node of the network and vaccination scenario. Partial budget analysis was used to compare the benefit-cost of the different vaccination scenarios. The model predicted that targeting day-old chick avian influenza (AI) vaccination in industrial and large hatcheries would increase immunity levels in the overall poultry population in Egypt and especially in small commercial poultry farms (from <30% to >60%). This strategy was shown to be more efficient than the current strategy of using inactivated vaccines. Improving HPAI control in the commercial poultry sector in Egypt would have a positive impact to improve disease control.

Background: Experimental and ecological studies have shown the role of climatic factors in driving the epidemiology of influenza. In particular, low absolute humidity (AH) has been shown to increase influenza virus transmissibility and has been identified to explain the onset of epidemics in temperate regions. Here, we aim to study the potential climatic drivers of influenza-like illness (ILI) epidemiology in Vietnam, a tropical country characterized by a high diversity of climates. We specifically focus on quantifying and explaining the seasonality of ILI., Methods: We used 18 years (1993-2010) of monthly ILI notifications aggregated by province (52) and monthly climatic variables (minimum, mean, maximum temperatures, absolute and relative humidities, rainfall and hours of sunshine) from 67 weather stations across Vietnam. Seasonalities were quantified from global wavelet spectra, using the value of the power at the period of 1 year as a measure of the intensity of seasonality. The 7 climatic time series were characterized by 534 summary statistics which were entered into a regression tree to identify factors associated with the seasonality of AH. Results were extrapolated to the global scale using simulated climatic times series from the NCEP/NCAR project., Results: The intensity of ILI seasonality in Vietnam is best explained by the intensity of AH seasonality. We find that ILI seasonality is weak in provinces experiencing weak seasonal fluctuations in AH (annual power <17.6), whereas ILI seasonality is strongest in provinces with pronounced AH seasonality (power >17.6). In Vietnam, AH and ILI are positively correlated., Conclusions: Our results identify a role for AH in driving the epidemiology of ILI in a tropical setting. However, in contrast to temperate regions, high rather than low AH is associated with increased ILI activity. Fluctuation in AH may be the climate factor that underlies and unifies the seasonality of ILI in both temperate and tropical regions. Alternatively, the mechanism of action of AH on disease transmission may be different in cold-dry versus hot-humid settings., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Dengue is a mosquito-transmitted virus infection that causes epidemics of febrile illness and hemorrhagic fever across the tropics and subtropics worldwide. Annual epidemics are commonly observed, but there is substantial spatiotemporal heterogeneity in intensity. A better understanding of this heterogeneity in dengue transmission could lead to improved epidemic prediction and disease control. Time series decomposition methods enable the isolation and study of temporal epidemic dynamics with a specific periodicity (e.g., annual cycles related to climatic drivers and multiannual cycles caused by dynamics in population immunity). We collected and analyzed up to 18 y of monthly dengue surveillance reports on a total of 3.5 million reported dengue cases from 273 provinces in eight countries in Southeast Asia, covering ∼ 10(7) km(2). We detected strong patterns of synchronous dengue transmission across the entire region, most markedly during a period of high incidence in 1997-1998, which was followed by a period of extremely low incidence in 2001-2002. This synchrony in dengue incidence coincided with elevated temperatures throughout the region in 1997-1998 and the strongest El Niño episode of the century. Multiannual dengue cycles (2-5 y) were highly coherent with the Oceanic Niño Index, and synchrony of these cycles increased with temperature. We also detected localized traveling waves of multiannual dengue epidemic cycles in Thailand, Laos, and the Philippines that were dependent on temperature. This study reveals forcing mechanisms that drive synchronization of dengue epidemics on a continental scale across Southeast Asia.

Background: A dengue outbreak in an ecotourism destination spot in Vietnam, from September to November 2013, impacted a floating village of fishermen on the coastal island of Cat Ba. The outbreak raises questions about how tourism may impact disease spread in rural areas., Methods: Epidemiological data were obtained from the Hai Phong Preventive Medical Center (PMC), including case histories and residential location from all notified dengue cases from this outbreak. All household addresses were geo-located. Knox test, a spatio-temporal analysis that enables inference dengue clustering constrained by space and time, was performed on the geocoded locations. From the plasma available from two patients, positive for Dengue serotype 3 virus (DENV3), the Envelope (E) gene was sequenced, and their genetic relationships compared to other E sequences in the region., Results: Of 192 dengue cases, the odds ratio of contracting dengue infections for people living in the floating villages compared to those living on the island was 4.9 (95 % CI: 3.6-6.7). The space-time analyses on 111 geocoded dengue residences found the risk of dengue infection to be the highest within 4 days and a radius of 20 m of a given case. Of the total of ten detected clusters with an excess risk greater than 2, the cluster with the highest number of cases was in the floating village area (24 patients for a total duration of 31 days). Phylogenetic analysis revealed a high homology of the two DENV3 strains (genotype III) from Cat Ba with DENV3 viruses circulating in Hanoi in the same year (99.1 %)., Conclusions: Our study showed that dengue transmission is unlikely to be sustained on Cat Ba Island and that the 2013 epidemic likely originated through introduction of viruses from the mainland, potentially Hanoi. These findings suggest that prevention efforts should be focused on mainland rather than on the island.

Background: Dengue fever (DF) has been emerging in Hanoi over the last decade. Both DF epidemiology and climate in Hanoi are strongly seasonal. This study aims at characterizing the seasonality of DF in Hanoi and its links to climatic variables as DF incidence increases from year to year., Methods: Clinical suspected cases of DF from the 14 central districts of Hanoi were obtained from the Ministry of Health over a 8-year period (2002-2009). Wavelet decompositions were used to characterize the main periodic cycles of DF and climatic variables as well as the mean phase angles of these cycles. Cross-wavelet spectra between DF and each climatic variables were also computed. DF reproductive ratio was calculated from Soper's formula and smoothed to highlight both its long-term trend and seasonality., Results: Temperature, rainfall, and vapor pressure show strong seasonality. DF and relative humidity show both strong seasonality and a sub-annual periodicity. DF reproductive ratio is increasing through time and displays two clear peaks per year, reflecting the sub-annual periodicity of DF incidence. Temperature, rainfall and vapor pressure lead DF incidence by a lag of 8-10 weeks, constant through time. Relative humidity leads DF by a constant lag of 18 weeks for the annual cycle and a lag decreasing from 14 to 5 weeks for the sub-annual cycle., Conclusion: Results are interpreted in terms of mosquito population dynamics and immunological interactions between the different dengue serotypes in the human compartment. Given its important population size, its strong seasonality and its dengue emergence, Hanoi offers an ideal natural experiment to test hypotheses on dengue serotypes interactions, knowledge of prime importance for vaccine development.

Animal medication against parasites can occur either as a genetically fixed (constitutive) or phenotypically plastic (induced) behavior. Taking the tritrophic interaction between the monarch butterfly Danaus plexippus, its protozoan parasite Ophryocystis elektroscirrha, and its food plant Asclepias spp. as a test case, we develop a game-theory model to identify the epidemiological (parasite prevalence and virulence) and environmental (plant toxicity and abundance) conditions that predict the evolution of genetically fixed versus phenotypically plastic forms of medication. Our model shows that the relative benefits (the antiparasitic properties of medicinal food) and costs (side effects of medicine, the costs of searching for medicine, and the costs of plasticity itself) crucially determine whether medication is genetically fixed or phenotypically plastic. Our model suggests that animals evolve phenotypic plasticity when parasite risk (a combination of virulence and prevalence and thus a measure of the strength of parasite-mediated selection) is relatively low to moderately high and genetically fixed medication when parasite risk becomes very high. The latter occurs because at high parasite risk, the costs of plasticity are outweighed by the benefits of medication. Our model provides a simple and general framework to study the conditions that drive the evolution of alternative forms of animal medication.

Background: During the last century, WHO led public health interventions that resulted in spectacular achievements such as the worldwide eradication of smallpox and the elimination of malaria from the Western world. However, besides major successes achieved worldwide in infectious diseases control, most elimination/control programs remain frustrating in many tropical countries where specific biological and socio-economical features prevented implementation of disease control over broad spatial and temporal scales. Emblematic examples include malaria, yellow fever, measles and HIV. There is consequently an urgent need to develop affordable and sustainable disease control strategies that can target the core of infectious diseases transmission in highly endemic areas., Discussion: Meanwhile, although most pathogens appear so difficult to eradicate, it is surprising to realize that human activities are major drivers of the current high rate of extinction among upper organisms through alteration of their ecology and evolution, i.e., their "niche". During the last decades, the accumulation of ecological and evolutionary studies focused on infectious diseases has shown that the niche of a pathogen holds more dimensions than just the immune system targeted by vaccination and treatment. Indeed, it is situated at various intra- and inter- host levels involved on very different spatial and temporal scales. After developing a precise definition of the niche of a pathogen, we detail how major advances in the field of ecology and evolutionary biology of infectious diseases can enlighten the planning and implementation of infectious diseases control in tropical countries with challenging economic constraints., Summary: We develop how the approach could translate into applied cases, explore its expected benefits and constraints, and we conclude on the necessity of such approach for pathogen control in low-income countries.

Theory predicts that selection for pathogen virulence and horizontal transmission is highest at the onset of an epidemic but decreases thereafter, as the epidemic depletes the pool of susceptible hosts. We tested this prediction by tracking the competition between the latent bacteriophage λ and its virulent mutant λcI857 throughout experimental epidemics taking place in continuous cultures of Escherichia coli. As expected, the virulent λcI857 is strongly favored in the early stage of the epidemic, but loses competition with the latent virus as prevalence increases. We show that the observed transient selection for virulence and horizontal transmission can be fully explained within the framework of evolutionary epidemiology theory. This experimental validation of our predictions is a key step towards a predictive theory for the evolution of virulence in emerging infectious diseases.

The environmental conditions experienced by hosts are known to affect their mean parasite transmission potential. How different conditions may affect the variance of transmission potential has received less attention, but is an important question for disease management, especially if specific ecological contexts are more likely to foster a few extremely infectious hosts. Using the obligate-killing bacterium Pasteuria ramosa and its crustacean host Daphnia magna, we analysed how host nutrition affected the variance of individual parasite loads, and, therefore, transmission potential. Under low food, individual parasite loads showed similar mean and variance, following a Poisson distribution. By contrast, among well-nourished hosts, parasite loads were right-skewed and overdispersed, following a negative binomial distribution. Abundant food may, therefore, yield individuals causing potentially more transmission than the population average. Measuring both the mean and variance of individual parasite loads in controlled experimental infections may offer a useful way of revealing risk factors for potential highly infectious hosts.

Although bubonic plague is an endemic zoonosis in many countries around the world, the factors responsible for the persistence of this highly virulent disease remain poorly known. Classically, the endemic persistence of plague is suspected to be due to the coexistence of plague resistant and plague susceptible rodents in natural foci, and/or to a metapopulation structure of reservoirs. Here, we test separately the effect of each of these factors on the long-term persistence of plague. We analyse the dynamics and equilibria of a model of plague propagation, consistent with plague ecology in Madagascar, a major focus where this disease is endemic since the 1920s in central highlands. By combining deterministic and stochastic analyses of this model, and including sensitivity analyses, we show that (i) endemicity is favoured by intermediate host population sizes, (ii) in large host populations, the presence of resistant rats is sufficient to explain long-term persistence of plague, and (iii) the metapopulation structure of susceptible host populations alone can also account for plague endemicity, thanks to both subdivision and the subsequent reduction in the size of subpopulations, and extinction-recolonization dynamics of the disease. In the light of these results, we suggest scenarios to explain the localized presence of plague in Madagascar.

Prediction and control of the geographical spread of emerging pathogens has become a central public health issue. Because these infectious diseases are by definition novel, there are few data to characterize their dynamics. One possible solution to this problem is to apply lessons learnt from analyses of historical data on familiar and epidemiologically similar pathogens. However, the portability of the spatial ecology of an infectious disease in a different epoch to other infections remains unexamined. Here, we study this issue by taking advantage of the recent re-emergence of pertussis in the United States to compare its spatial transmission dynamics throughout the 1950s with the past decade. We report 4-year waves, sweeping across the continent in the 1950s. These waves are shown to emanate from highly synchronous foci in the northwest and northeast coasts. In contrast, the recent resurgence of the disease is characterized by 5.5-year epidemics with no particular spatial structure. We interpret this to be the result of dramatic changes in patterns of human movement over the second half of the last century, together with changing age distribution of pertussis. We conclude that extrapolation regarding the spatial spread of contemporaneous pathogens based on analyses of historical incidence may be potentially very misleading.

Mycobacterium marinum causes a systemic tuberculosis-like disease in fish and skin infections in humans that can spread to deeper structures, resulting in tenosynovitis, arthritis, and osteomyelitis. However, little information is available concerning (i) the intraspecific genetic diversity of M. marinum isolated from humans and animals; (ii) M. marinum genotype circulation in the different ecosystems, and (iii) the link between M. marinum genetic diversity and hosts (humans and fish). Here, we conducted a genetic study on 89 M. marinum isolates from humans (n = 68) and fish (n = 21) by using mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. The results show that the M. marinum population is genetically structured not only according to the host but also according to the ecosystem as well as to tissue tropism in humans. This suggests the existence of different genetic pools in the function of the biological and ecological compartments. Moreover, the presence of only certain M. marinum genotypes in humans suggests a different zoonotic potential of the M. marinum genotypes. Considering that the infection is linked to aquarium activity, a significant genetic difference was also detected when the human tissue tropism of M. marinum was taken into consideration, with a higher genetic polymorphism in strains isolated from patients with cutaneous forms than from individuals with deeper-structure infection. It appears that only few genotypes can produce deeper infections in humans, suggesting that the immune system might play a filtering role.

Adaptation depends greatly on the distribution of mutation fitness effects (DMFE), but the phenotypic expression of mutations is often environment dependent. The environments faced by multihost pathogens are mostly governed by their hosts and therefore measuring the DMFE on multiple hosts can inform on the likelihood of short-term establishment and longer term adaptation of emerging pathogens. We explored this by measuring the growth rate of 36 mutants of the lytic bacteriophage φX174 on two host backgrounds, Escherichia coli (EcC) and Salmonella typhimurium (StGal). The DMFE showed higher mean and variance on EcC than on StGal. Most mutations were either deleterious or neutral on both hosts, but a greater proportion of mutations were deleterious on StGal. We identified two mutations with beneficial fitness effects on EcC that were neutral on StGal. Host-specific differences in fitness were associated with particular functional classes of genes involved in the initial stages of infection in accordance with previous studies of host specificity. Overall, there was a positive correlation between the effects of mutations on each host, suggesting that most new mutations will have general, rather than host-specific fitness effects. We consider these results in light of simple fitness landscape models of adaptation and discuss the relevance of context-dependent DMFE for multihost pathogens., (© 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.)

A total of 221 isolates of M. tuberculosis were sampled from hospitals and the general population in the northern plain of Vietnam, one of the most populated region of the country. Genotypic composition and diversity were characterized, and we investigated how they are affected by sampling (hospital vs. general population), correcting for potential confounding effects (location, age and gender of the patients). Spoligotyping and 12 MIRU-VNTR typing were used as first line. Then 15 MIRU-VNTR standard set was used, making 21 MIRU-VNTR typing for the clustered isolates. Result showed that 8 lineages and 13 sub-lineages were circulating in the region. The most predominant lineages were Beijing (38.5%) and EAI (38.5%). Others appeared with small proportions H (1.4%), LAM (1.8%), T (8.1%), X (0.9%), MANU (2.3%), and Zero (0.4%). Higher clustering rate was found in the hospital samples (17.9% in urban and 19.2% in rural areas) compared to the population ones (0%). The typical Vietnamese EAI4-VNM sub-lineage of EAI lineage accounted for 67% of EAI strains and was associated with older ages. Beijing genotypes were associated with younger, urban population and were characterized by high clustering rates. These characteristics strongly suggest that Beijing strains are invading the population, replacing the local EAI-VNM4, thus predicting a more serious tuberculosis situation in the future in the absence of more effective control strategies.

Mixed-genotype parasite infections are common in nature. Theoretical studies analyze the effects of such infections over evolutionary time and predict an increase in virulence due to the competitive advantage of virulent parasites. In contrast, experimental studies compare the overall virulence of mixed and single infections within one generation. Although these within-generation comparisons have limited relevance to existing theory, they demonstrate that within-host parasite interactions are not restricted to competition for resources, as envisaged by theory. Instead, mixed infections may result in phenotypic changes in growth rate or impaired immune clearance. Developing and using a two-parasite epidemiological model with recovery, we confirm that within-host competition for resources selects for higher virulence. However, parasite phenotypic plasticity and impaired host immunity can select for lower virulence. Because these latter two mechanisms would be detected by experimentalists as an increase in pathology, our results warn against the temptation to draw inferences on virulence evolution on the basis of single-generation experiments.

clonality V.0.4 is a program for testing heterozygosity-genet size relationships in clonal organisms using a randomization procedure. The software has been developed under the Borland Delphi developing environment and a Windows-executable version is freely downloadable from http://gemi.mpl.ird.fr/SiteSGASS/Prugnolle/ClonalityPage.html. The program compares the observed F(IS) of the population with the F(IS) expected if genets (multilocus genotypes present in multiple copies within the population) were chosen randomly from the set of different multilocus genotypes. The randomization procedure is performed with the same number of genets and the same number of repetitions per genet as what is observed in the original data set., (© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.)

Background: Social scientists have suggested that cultural diversity in a nation leads to societal instability. However, societal instability may be affected not only by within-nation or alpha diversity, but also diversity between a nation and its neighbours or beta diversity. It is also necessary to distinguish different domains of diversity, namely linguistic, ethnic and religious, and to distinguish between the direct effects of diversity on societal instability, and effects that are mediated by economic conditions., Methodology/principal Findings: We assembled a large cross-national dataset with information on alpha and beta cultural diversity, economic conditions, and indices of societal instability. Structural equation modeling was used to evaluate the direct and indirect effects of cultural diversity on economics and societal stability. Results show that different types and domains of diversity have interacting effects. As previously documented, linguistic alpha diversity has a negative effect on economic performance, and we show that it is largely through this economic mechanism that it affects societal instability. For beta diversity, the higher the linguistic diversity among nations in a region, the less stable the nation. But, religious beta diversity has the opposite effect, reducing instability, particularly in the presence of high linguistic diversity., Conclusions: Within-nation linguistic diversity is associated with reduced economic performance, which, in turn, increases societal instability. Nations which differ linguistically from their neighbors are also less stable. However, religious diversity between neighboring nations has the opposite effect, decreasing societal instability.

Theoretical studies of wildlife population dynamics have proved insightful for sustainable management, where the principal aim is to maximize short-term yield, without risking population extinction. Surprisingly, infectious diseases have not been accounted for in harvest models, which is a major oversight because the consequences of parasites for host population dynamics are well-established. Here, we present a simple general model for a host species subject to density dependent reproduction and seasonal demography. We assume this host species is subject to infection by a strongly immunizing, directly transmitted pathogen. In this context, we show that the interaction between density dependent effects and harvesting can substantially increase both disease prevalence and the absolute number of infectious individuals. This effect clearly increases the risk of cross-species disease transmission into domestic and livestock populations. In addition, if the disease is associated with a risk of mortality, then the synergistic interaction between hunting and disease-induced death can increase the probability of host population extinction.

The mode of reproduction (sexual and/or asexual) and the mating system determine the patterns of gene transmission and genotype formation across generations. Schistosoma mansoni is a dioecious trematode that necessarily alternates sexual and asexual reproduction during its life cycle. In a previous study of the distribution of S. mansoni genetic variability within and between definitive host individuals, we noticed that deleting multilocus genotypes from each infrapopulation so as to keep only one copy of each multilocus genotype, seemed to have a substantial effect on FIS values. More precisely, female FIS increased when repeated genotypes were removed whereas no effect was observed on male FIS. This suggested that multilocus genotypes at high frequency tended to be more heterozygous. The aim of the present study is specifically to test and analyse this phenomenon. We demonstrate that the number of repetitions per clone correlates with individual heterozygosity. This effect is however, sex-specific: only female clone size correlates with heterozygosity. We discuss this phenomenon in relation to the heterozygosity-fitness relationship and the sex-specific response to inbreeding depression.

Fourteen isolates of Rice yellow mottle virus (RYMV) were selected as representative of the genetic variability of the virus in Africa from a total set of 320 isolates serologically typed or partially sequenced. The 14 isolates were fully sequenced and analyzed together with two previously reported sequences. RYMV had a genomic organization similar to that of Cocksfoot mottle sobemovirus. The average nucleotide diversity among the 16 isolates of RYMV was 7%, and the maximum diversity between any two isolates was 10%. A strong conservative selection was apparent on both synonymous and nonsynonymous substitutions, through the amino acid replacement pattern, on the genome size, and through the limited number of indel events. Furthermore, there was a lack of positive selection on single amino acid sites and no evidence of recombination events. RYMV diversity had a pronounced and characteristic geographic structure. The branching order of the clades correlated with the geographic origin of the isolates along an east-to-west transect across Africa, and there was a marked decrease in nucleotide diversity moving westward across the continent. The insertion-deletion polymorphism was related to virus phylogeny. There was a partial phylogenetic incongruence between the coat protein gene and the rest of the genome. Overall, our results support the hypothesis that RYMV originated in East Africa and then dispersed and differentiated gradually from the east to the west of the continent.

Molecular adaptation, as characterized by the detection of positive selection, was quantified in a number of genes from different human immunodeficiency virus type 1 (HIV-1) group M subtypes, group O, and an HIV-2 subtype using the codon-based maximum-likelihood method of Yang and coworkers (Z. H. Yang, R. Nielsen, N. Goldman, and A. M. K. Pedersen, Genetics 155:431-449, 2000). The env gene was investigated further since it exhibited the strongest signal for positive selection compared to those of the other two major HIV genes (gag and pol). In order to investigate the pattern of adaptive evolution across env, the location and strength of positive selection in different HIV-1 sequence alignments was compared. The number of sites having a significant probability of being positively selected varied among these different alignment data sets, ranging from 25 in HIV-1 group M subtype A to 40 in HIV-1 group O. Strikingly, there was a significant tendency for positively selected sites to be located at the same position in different HIV-1 alignments, ranging from 10 to 16 shared sites for the group M intersubtype comparisons and from 6 to 8 for the group O to M comparisons, suggesting that all HIV-1 variants are subject to similar selective forces. As the host immune response is believed to be the dominant driving force of adaptive evolution in HIV, this result would suggest that the same sites are contributing to viral persistence in diverse HIV infections. Thus, the positions of the positively selected sites were investigated in reference to the inferred locations of different epitope types (antibody, T helper, and cytotoxic T lymphocytes) and the positions of N and O glycosylation sites. We found a significant tendency for positively selected sites to fall outside T-helper epitopes and for positively selected sites to be strongly associated with N glycosylation sites.

Although multihost complex life cycles (CLCs) are common in several distantly related groups of parasites, their evolution remains poorly understood. In this article, we argue that under particular circumstances, adding a second host to a single-host life cycle is likely to enhance transmission (i.e., reaching the target host). For instance, in several situations, the propagules of a parasite exploiting a predator species will achieve a higher host-finding success by encysting in a prey of the target predator than by other dispersal modes. In such a case, selection should favor the transition from a single- to a two-host life cycle that includes the prey species as an intermediate host. We use an optimality model to explore this idea, and we discuss it in relation to dispersal strategies known among free-living species, especially animal dispersal. The model found that selection favored a complex life cycle only if intermediate hosts were more abundant than definitive hosts. The selective value of a complex life cycle increased with predation rates by definitive hosts on intermediate hosts. In exploring trade-offs between transmission strategies, we found that more costly trade-offs made it more difficult to evolve a CLC while less costly trade-offs between traits could favor a mixed strategy.