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Background Intramuscular antitoxin is recommended in tetanus treatment, but there are few data comparing human and equine preparations. Tetanus toxin acts within the CNS, where there is limited penetration of peripherally administered antitoxin; thus, intrathecal antitoxin administration might improve clinical outcomes compared with intramuscular injection. Methods In a 2 × 2 factorial trial, all patients aged 16 years or older with a clinical diagnosis of generalised tetanus admitted to the intensive care unit of the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam, were eligible for study entry. Participants were randomly assigned first to 3000 IU human or 21 000 U equine intramuscular antitoxin, then to either 500 IU intrathecal human antitoxin or sham procedure. Interventions were delivered by independent clinicians, with attending clinicians and study staff masked to treatment allocations. The primary outcome was requirement for mechanical ventilation. The analysis was done in the intention-to-treat population. The study is registered at ClinicalTrials.gov, NCT02999815; recruitment is completed. Findings 272 adults were randomly assigned to interventions between Jan 8, 2017, and Sept 29, 2019, and followed up until May, 2020. In the intrathecal allocation, 136 individuals were randomly assigned to sham procedure and 136 to antitoxin; in the intramuscular allocation, 109 individuals were randomly assigned to equine antitoxin and 109 to human antitoxin. 54 patients received antitoxin at a previous hospital, excluding them from the intramuscular antitoxin groups. Mechanical ventilation was given to 56 (43%) of 130 patients allocated to intrathecal antitoxin and 65 (50%) of 131 allocated to sham procedure (relative risk [RR] 0·87, 95% CI 0·66–1·13; p=0·29). For the intramuscular allocation, 48 (45%) of 107 patients allocated to human antitoxin received mechanical ventilation compared with 48 (44%) of 108 patients allocated to equine antitoxin (RR 1·01, 95% CI 0·75–1·36, p=0·95). No clinically relevant difference in adverse events was reported. 22 (16%) of 136 individuals allocated to the intrathecal group and 22 (11%) of 136 allocated to the sham procedure experienced adverse events related or possibly related to the intervention. 16 (15%) of 108 individuals allocated to equine intramuscular antitoxin and 17 (16%) of 109 allocated to human antitoxin experienced adverse events related or possibly related to the intervention. There were no intervention-related deaths. Interpretation We found no advantage of intramuscular human antitoxin over intramuscular equine antitoxin in tetanus treatment. Intrathecal antitoxin administration was safe, but did not provide overall benefit in addition to intramuscular antitoxin administration.

IntroductionAntimicrobial stewardship (AMS) is an important strategy to control antimicrobial resistance. Resources are available to provide guidance for design and implementation of AMS programmes, however these may have limited applicability in resource-limited settings including those in Asia. This scoping review aims to identify context-specific domains and items for the development of a healthcare facility (HCF)-level tool to guide AMS implementation in Asia.Methods and analysisThis review is the first step in a larger project to assess AMS implementation, needs and gaps in Asia. We will employ a deductive qualitative approach to identify locally appropriate domains and items of AMS implementation guided by Nilsen and Bernhardsson’s contextual dimensions. This process is also informed by discussions from a technical advisory group coordinated by the US Centers for Disease Control and Prevention to develop an AMS HCF-level assessment tool for low-income and middle-income countries. We will review English-language documents that discuss HCF-level implementation, including those describing frameworks, components/elements or recommendations for design, implementation or assessment globally and specific to Asia. We have performed the search in August–September 2021 including general electronic databases (MEDLINE, Embase, Web of Science and Google Scholar), region-specific databases, national action plans, grey literature sources and reference lists to identify eligible documents. Country-specific documents will be restricted to countries in three subregions: South Asia, East Asia and Southeast Asia. Codes and themes will be derived through a content analysis, classified following the predefined context dimensions and used for developing domains and items of the assessment tool.Ethics and disseminationResults from this review will feed into our stepwise process for developing a context-specific HCF-level assessment tool for AMS programmes to assess the implementation status, identify intervention opportunities and monitor progress over time. The process will be done in consultation with local stakeholders, the end-users of the generated knowledge.

Enterovirus A71 (EV-A71)–related hand, foot, and mouth disease (HFMD) imposes a substantial clinical burden in the Asia Pacific region. To inform policy on the introduction of the EV-A71 vaccine into the National Immunization Programme, we investigated the seroepidemiological characteristics of EV-A71 in two prospective cohorts of children in southern China conducted between 2013 and 2018. Our results show that maternal antibody titres declined rapidly in neonates, with over half becoming susceptible to EV-A71 at 1 month of age. Between 6 months and 2 years of age, over 80% of study participants were susceptible, while one third remained susceptible at 5 years old. The highest incidence of EV-A71 infections was observed in children aged 5-6 months. Our findings support EV-A71 vaccination before 6 months for birth cohorts in southern China, potentially with a one-time catch-up vaccination for children 6 months-5 years old. More regionally representative longitudinal seroepidemiological studies are needed to further validate these findings.

Background: Intramuscular antitoxin is recommended in tetanus treatment, but there are few data comparing human and equine preparations. As tetanus toxin acts within the central nervous system, where there is limited penetration of peripherally-administered antitoxin, intrathecal antitoxin administration may improve clinical outcomes compared to intramuscular injection. Methods: In a 2x2 factorial trial, adults with tetanus in a single-centre in Vietnam were randomized first to 3,000 IU human or 21,000 U equine intramuscular antitoxin, then to either 500 IU intrathecal human antitoxin or sham procedure. Interventions were delivered by independent clinicians, with attending clinicians and study staff blind to treatment allocations. The primary outcome was requirement for mechanical ventilation. Secondary outcomes included in-hospital mortality, death and disability at 240-days, duration of intensive care unit (ICU) stay, and adverse events. The study was registered at ClinicalTrials.gov, NCT 02999815 (status: recruitment completed). Findings: 272 adults were randomized. Mechanical ventilation was given to 56/130 (43%) of patients allocated to intrathecal antitoxin and 65/131 (50%) allocated to sham procedure (RR 0.87; 95% CI 0.66 to 1.13; p=0.29). For the intramuscular allocation 48/107 (45%), patients allocated to human antitoxin received mechanical ventilation compared to 48/108 (44%) patients allocated to equine antitoxin (relative risk (RR) 1.01, 95% confidence interval (CI) 0.75, 1.36, p=0.95). No clinically-relevant differences in secondary outcomes or adverse events were seen except for shorter length of ICU stay in those treated with intrathecal antitoxin compared to sham. Interpretation: We found no advantage of intramuscular human antitoxin over intramuscular equine antitoxin in tetanus treatment. Intrathecal antitoxin administration was safe but did not provide overall benefit in addition to intramuscular antitoxin administration. Funding: The Wellcome Trust, grant number 107367/Z/15/Z.

Contains fulltext : 248581.pdf (Publisher’s version ) (Open Access) Colistin is widely used in agriculture and aquaculture as prophylaxis, particularly in Asia. Recently, mcr-1 and other mobilizable genes conferring colistin resistance have spread globally in community and hospital populations. Characterizing mcr-1 mobile genetic elements and host genetic background is important to understand the transmission of this resistance mechanism. We conducted whole-genome sequencing of 94 mcr-1-positive Escherichia coli isolates (Mcr1-Ec isolates) from human and animal feces, food, and water in a community cohort (N = 87) and from clinical specimens from a referral hospital (N = 7) in northern Vietnam. mcr-1 was plasmid-borne in 71 and chromosomally carried in 25 (2 isolates contain one copy on chromosome and one copy on a plasmid) of 94 E. coli isolates from the community and hospital settings. All seven clinical isolates carried mcr-1 on plasmids. Replicon types of mcr-1-carrying plasmids included IncI2, IncP, IncX4, and IncFIA single replicons and combinations of IncHI2, IncN, and IncX1 multireplicons. Alignment of a long-read sequence of an IncI2 plasmid from animal feces with short-read sequences of IncI2 plasmids from a healthy human, water, and hospitalized patients showed highly similar structures (query cover from 90% to 98%, overall identity of >81%). We detected the potential existence of multireplicon plasmids harboring mcr-1 regardless of sample setting, confirming 10/71 with long-read sequencing. An intact/conserved Tn6330 transposon sequence or its genetic context variants were found in 6/25 Mcr1-Ec isolates with chromosomally carried mcr-1. The dissemination of mcr-1 is facilitated by a high diversity of plasmid replicon types and a high prevalence of the chromosomal Tn6330 transposon. IMPORTANCE The article presented advances our understanding of genetic elements carrying mcr-1 in Escherichia coli in both community and hospital settings. We provide evidence to suggest that diverse plasmid types, including multireplicon plasmids, have facilitated the successful transmission of mcr-1 in different reservoirs. The widespread use of colistin in agriculture, where a high diversity of bacteria are exposed, has allowed the selection and evolution of various transmission mechanisms that will make it a challenge to get rid of. Colocalization of mcr-1 and other antibiotic resistance genes (ARGs) on multireplicon plasmids adds another layer of complexity to the rapid dissemination of mcr-1 genes among community and hospital bacterial populations and to the slow pandemic of antimicrobial resistance (AMR) in general.

ObjectivesAntibiotic overuse is one of the main drivers of antimicrobial resistance (AMR), especially in low-income and middle-income countries. This study aimed to understand the perceptions and views towards AMR, antibiotic prescribing practice and antimicrobial stewardship (AMS) among hospital physicians in Jakarta, Indonesia.DesignCross-sectional, self-administered questionnaire-based survey, with descriptive statistics, exploratory factor analysis (EFA) to identify distinct underlying constructs in the dataset, and multivariable linear regression of factor scores to analyse physician subgroups.SettingSix public and private acute-care hospitals in Jakarta in 2019.Participants1007 of 1896 (53.1% response rate) antibiotic prescribing physicians.ResultsPhysicians acknowledged the significance of AMR and contributing factors, rational antibiotic prescribing, and purpose and usefulness of AMS. However, this conflicted with reported suboptimal local hospital practices, such as room cleaning, hand hygiene and staff education, and views regarding antibiotic decision making. These included insufficiently applying AMS principles and utilising microbiology, lack of confidence in prescribing decisions and defensive prescribing due to pervasive diagnostic uncertainty, fear of patient deterioration or because patients insisted. EFA identified six latent factors (overall Crohnbach’s α=0.85): awareness of AMS activities; awareness of AMS purpose; views regarding rational antibiotic prescribing; confidence in antibiotic prescribing decisions; perception of AMR as a significant problem; and immediate actions to contain AMR. Factor scores differed across hospitals, departments, work experience and medical hierarchy.ConclusionsAMS implementation in Indonesian hospitals is challenged by institutional, contextual and diagnostic vulnerabilities, resulting in externalising AMR instead of recognising it as a local problem. Appropriate recognition of the contextual determinants of antibiotic prescribing decision making will be critical to change physicians’ attitudes and develop context-specific AMS interventions.

Highlights • MALDITOF MS provided more rapid identification of invasive bacterial and fungal pathogens than conventional microbiology. • MALDITOF MS did not increase the proportion of patients on optimal therapy at 24 or 48 hours after positive culture. • MALDITOF MS did not increase the proportion of patients receiving adequate therapy at 24 hours after positive culture. • The most common reason for therapy being sub-optimal was use of overly broad spectrum or unnecessary multiple antibiotics., Summary Objectives We assessed the impact of MALDITOF-MS on the timeliness of optimal antimicrobial therapy through a parallel-arm randomised controlled trial in two hospitals in Vietnam. Methods We recruited patients with a pathogen (bacterial or fungal) cultured from a normally sterile sample. Samples were randomly assigned (1:1) to identification by MALDITOF-MS or conventional diagnostics. The primary outcome was the proportion on optimal antimicrobial therapy within 24 h of positive culture, determined by a blinded independent review committee. Trial registered at ClinicalTrials.gov (NCT02306330). Results Among 1005 randomised patients, pathogens were isolated from 628 (326 intervention, 302 control), with 377 excluded as likely contaminants or discharged/died before positive culture. Most isolates were cultured from blood (421/628, 67.0%). The proportion receiving optimal antimicrobial therapy within 24 h (the primary outcome) or 48 h of growth was not significantly different between MALDITOF-MS and control arms (135/326, 41.4% vs 120/302, 39.7%; Adjusted Odds ration (AOR) 1.17, p = 0.40 and 151/326, 46.3% vs 141/302, 46.7%; AOR 1.05 p = 0.79, respectively). Conclusions MALDITOF-MS, in the absence of an antimicrobial stewardship programme, did not improve the proportion on optimal antimicrobial therapy at 24 or 48 h after first growth in a lower-middle income setting with high rates of antibiotic resistance.

Community-acquired (CA) sepsis is a major public health problem worldwide, yet the etiology remains unknown for >50% of the patients. Here we applied metagenomic next-generation sequencing (mNGS) to characterize the human virome in 492 clinical samples (384 sera, 92 pooled nasal and throat swabs, 10 stools, and 6 cerebrospinal fluid samples) from 386 patients (213 adults and 173 children) presenting with CA sepsis who were recruited from 6 hospitals across Vietnam between 2013 and 2015., Community-acquired (CA) sepsis is a major public health problem worldwide, yet the etiology remains unknown for >50% of the patients. Here we applied metagenomic next-generation sequencing (mNGS) to characterize the human virome in 492 clinical samples (384 sera, 92 pooled nasal and throat swabs, 10 stools, and 6 cerebrospinal fluid samples) from 386 patients (213 adults and 173 children) presenting with CA sepsis who were recruited from 6 hospitals across Vietnam between 2013 and 2015. Specific monoplex PCRs were used subsequently to confirm the presence of viral sequences detected by mNGS. We found sequences related to 47 viral species belonging to 21 families in 358 of 386 (93%) patients, including viruses known to cause human infections. After PCR confirmation, human viruses were found in 52 of 386 patients (13.4%); picornavirus (enteroviruses [n = 14], rhinovirus [n = 5], and parechovirus [n = 2]), hepatitis B virus (n = 10), cytomegalovirus (n = 9), Epstein-Barr virus (n = 5), and rotavirus A (n = 3) were the most common viruses detected. Recently discovered viruses were also found (gemycircularvirus [n = 5] and WU polyomavirus, Saffold virus, salivirus, cyclovirus-VN, and human pegivirus 2 [HPgV2] [n, 1 each]), adding to the growing literature about the geographic distribution of these novel viruses. Notably, sequences related to numerous viruses not previously reported in human tissues were also detected. To summarize, we identified 21 viral species known to be infectious to humans in 52 of 386 (13.4%) patients presenting with CA sepsis of unknown cause. The study, however, cannot directly impute sepsis causation to the viruses identified. The results highlight the fact that it remains a challenge to establish the causative agents in CA sepsis patients, especially in tropical settings such as Vietnam.

Background Community acquired bloodstream infection (CABSI) in low- and middle income countries is associated with a high mortality. This study describes the clinical manifestations, laboratory findings and correlation of SOFA and qSOFA with mortality in patients with CABSI in northern Vietnam. Methods This was a retrospective study of 393 patients with at least one positive blood culture with not more than one bacterium taken within 48 h of hospitalisation. Clinical characteristic and laboratory results from the first 24 h in hospital were collected. SOFA and qSOFA scores were calculated and their validity in this setting was evaluated. Results Among 393 patients with bacterial CABSI, approximately 80% (307/393) of patients had dysfunction of one or more organ on admission to the study hospital with the most common being that of coagulation (57.1% or 226/393). SOFA performed well in prediction of mortality in those patients initially admitted to the critical care unit (AUC 0.858, 95%CI 0.793–0.922) but poor in those admitted to medical wards (AUC 0.667, 95%CI 0.577–0.758). In contrast qSOFA had poor predictive validity in both settings (AUC 0.692, 95%CI 0.605–0.780 and AUC 0.527, 95%CI 0.424–0.630, respectively). The overall case fatality rate was 28%. HIV infection (HR = 3.145, p = 0.001), neutropenia (HR = 2.442, p = 0.002), SOFA score 1-point increment (HR = 1.19, p

Improved understanding of pathogens that cause sepsis would aid management and antimicrobial selection. In this study, we aimed to identify the causative pathogens of sepsis in southeast Asia.In this multinational multicentre cross-sectional study of community-acquired sepsis and severe sepsis, we prospectively recruited children (age ≥30 days and18 years) and adults (age ≥18 years) at 13 public hospitals in Indonesia (n=3), Thailand (n=4), and Vietnam (n=6). Hospitalised patients with suspected or documented community-acquired infection, with at least three diagnostic criteria for sepsis according to the Surviving Sepsis Campaign 2012, and within 24 h of admission were enrolled. Blood from every patient, and nasopharyngeal swab, urine, stool, and cerebrospinal fluid, if indicated, were collected for reference diagnostic tests to identify causative pathogens. We report causative pathogens of sepsis and 28-day mortality. We also estimate mortality associated with enrolment with severe sepsis. This study was registered with ClinicalTrials.gov, number NCT02157259.From Dec 16, 2013, to Dec 14, 2015, 4736 patients were screened and 1578 patients (763 children and 815 adults) were enrolled. Dengue viruses (n=122 [8%]), Leptospira spp (n=95 [6%]), rickettsial pathogens (n=96 [6%]), Escherichia coli (n=76 [5%]), and influenza viruses (n=65 [4%]) were commonly identified in both age groups; whereas Plasmodium spp (n=12 [1%]) and Salmonella enterica serovar Typhi (n=3 [0·2%]) were rarely observed. Emerging pathogens identified included hantaviruses (n=28 [2%]), non-typhoidal Salmonella spp (n=21 [1%]), Streptococcus suis (n=18 [1%]), Acinetobacter spp (n=12 [1%]), and Burkholderia pseudomallei (n=5 [1%]). 28-day mortality occurred in 14 (2%) of 731 children with known statuses and 108 (13%) of 804 adults. Severe sepsis was identified on enrolment in 194 (28%) of 731 children and 546 (68%) of 804 adults, and was associated with increased mortality (adjusted odds ratio 5·3, 95% CI 2·7-10·4; p0·001).Sepsis in southeast Asia is caused by a wide range of known and emerging pathogens, and is associated with substantial mortality.National Cancer Institute, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA, and Wellcome Trust, UK.

Rogier van Doorn and colleagues analyze the initial outbreak, attempts at containment, and establishment of community transmission of pandemic H1N1 influenza in Ho Chi Minh City, Vietnam., Background To date, little is known about the initial spread and response to the 2009 pandemic of novel influenza A (“2009 H1N1”) in tropical countries. Here, we analyse the early progression of the epidemic from 26 May 2009 until the establishment of community transmission in the second half of July 2009 in Ho Chi Minh City (HCMC), Vietnam. In addition, we present detailed systematic viral clearance data on 292 isolated and treated patients and the first three cases of selection of resistant virus during treatment in Vietnam. Methods and Findings Data sources included all available health reports from the Ministry of Health and relevant health authorities as well as clinical and laboratory data from the first confirmed cases isolated at the Hospital for Tropical Diseases in HCMC. Extensive reverse transcription (RT)-PCR diagnostics on serial samples, viral culture, neuraminidase-inhibition testing, and sequencing were performed on a subset of 2009 H1N1 confirmed cases. Virological (PCR status, shedding) and epidemiological (incidence, isolation, discharge) data were combined to reconstruct the initial outbreak and the establishment of community transmission. From 27 April to 24 July 2009, approximately 760,000 passengers who entered HCMC on international flights were screened at the airport by a body temperature scan and symptom questionnaire. Approximately 0.15% of incoming passengers were intercepted, 200 of whom tested positive for 2009 H1N1 by RT-PCR. An additional 121 out of 169 nontravelers tested positive after self-reporting or contact tracing. These 321 patients spent 79% of their PCR-positive days in isolation; 60% of PCR-positive days were spent treated and in isolation. Influenza-like illness was noted in 61% of patients and no patients experienced pneumonia or severe outcomes. Viral clearance times were similar among patient groups with differing time intervals from illness onset to treatment, with estimated median clearance times between 2.6 and 2.8 d post-treatment for illness-to-treatment intervals of 1–4 d, and 2.0 d (95% confidence interval 1.5–2.5) when treatment was started on the first day of illness. Conclusions The patients described here represent a cross-section of infected individuals that were identified by temperature screening and symptom questionnaires at the airport, as well as mildly symptomatic to moderately ill patients who self-reported to hospitals. Data are observational and, although they are suggestive, it is not possible to be certain whether the containment efforts delayed community transmission in Vietnam. Viral clearance data assessed by RT-PCR showed a rapid therapeutic response to oseltamivir. Please see later in the article for the Editors' Summary, Editors' Summary Background Every year, millions of people catch influenza—a viral infection of the airways—and about half a million people die as a result. These yearly seasonal epidemics occur because small but frequent changes in the influenza virus mean that the immune response produced by infection with one year's virus provides only partial protection against the next year's virus. Sometimes, however, a very different influenza virus emerges to which people have virtually no immunity. Such viruses can start global epidemics (pandemics) and can kill millions of people. Consequently, when the first case of influenza caused by a new virus called pandemic A/H1N1 2009 (2009 H1N1, swine flu) occurred in March 2009 in Mexico, alarm bells rang. National and international public health agencies quickly issued advice about how the public could help to control the spread of the virus and, as the virus spread, some countries banned flights from affected regions and instigated screening for influenza-like illness at airports. However, despite everyone's efforts, the virus spread rapidly and on June 11, 2009 the World Health Organization (WHO) declared that an influenza pandemic was underway. Why Was This Study Done? To date, little is known about the spread of and response to 2009 H1N1 in tropical countries. In this study, therefore, the researchers investigate the early progression of the 2009 H1N1 pandemic in Ho Chi Minh City, Vietnam, and the treatment of infected patients. On April 27, 2009, when WHO announced that human-to-human transmission of 2009 H1N1 was occurring, the Vietnamese Ministry of Health mandated airport body temperature scans and symptom questionnaire screening of travelers arriving in Vietnam's international airports. Suspected cases were immediately transferred to in-hospital isolation, screened for virus using a sensitive test called PCR, and treated with the anti-influenza drug oseltamivir if positive. The first case of 2009 H1N1 infection in Vietnam was reported on May 31, 2009 in a student who had returned from the US on May 26, 2009, and, despite these efforts to contain the infection, by the second half of July the virus was circulating in Ho Chi Minh City (community transmission). What Did the Researchers Do and Find? The researchers used reports from the Ministry of Health and relevant health authorities and clinical and laboratory data for people infected with 2009 H1N1 and isolated in hospital to reconstruct the initial outbreak and the establishment of community transmission in Ho Chi Minh City. Between April 27 and July 24 2009, three-quarters of a million passengers arriving in the city on international flights were screened at the airport. 200 passenger tested positive for 2009 H1N1 as did 121 nontravelers who were identified during this period after self-reporting illness or through contact tracing. The infected individuals spent 79% of the days when they tested positive for 2009 H1N1 by PCR (days when they were infectious) in isolation; 60% of their PCR-positive days were spent in isolation and treatment. Importantly, travelers and nontravelers spent 10% and 42.2%, respectively, of their potentially infectious time in the community. None of the patients became severely ill but 61% experienced an influenza-like illness. Finally, the average time from starting treatment to clearance of the virus was between 2.6 and 2.8 days for patients who began treatment 1 to 4 days after becoming ill; for those who started treatment on the first day of illness, the average virus clearance time was 2.0 days. What Do These Findings Mean? These findings, although limited by missing data, suggest that the strict containment measures introduced early in the 2009 H1N1 pandemic in Ho Chi Minh City may have reduced the circulation of infected people in the community. This reduction in circulation might have delayed the onset of community transmission, suggest the researchers, but because the study was observational, this possibility cannot be proven. However, importantly, these findings show that the containment measures were unable to prevent the eventual establishment of pandemic influenza in Vietnam, presumably because many imported cases were not detected by airport screening. Finally, these findings suggest that in Vietnam, as in other countries, 2009 H1N1 causes a mild disease and that this disease responds quickly to treatment with oseltamivir whenever treatment is started in relation to the onset of illness. Additional Information Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000277. The US Centers for Disease Control and Prevention provides information about influenza for patients and professionals, including specific information on H1N1 influenza and how to prevent its spread Flu.gov, a US government website, provides information on H1N1, avian, and pandemic influenza The World Health Organization provides information on seasonal influenza and has detailed information on H1N1 influenza (in several languages); the WHO Representative Office in Vietnam provides an overview of the current 2009 H1N1 situation in Vietnam The UK Health Protection Agency provides information on pandemic influenza and on H1N1 influenza Wikipedia has a timeline of the 2009 H1N1 pandemic (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)

Background The family Flaviviridae, genus Flavivirus, holds many of the world’s most prevalent arboviral diseases that are also considered the most important travel related arboviral infections. In most cases, flavivirus diagnosis in travelers is primarily based on serology as viremia is often low and typically has already been reduced to undetectable levels when symptoms set in and patients seek medical attention. Serological differentiation between flaviviruses and the false-positive results caused by vaccination and cross-reactivity among the different species, are problematic for surveillance and diagnostics of flaviviruses. Their partially overlapping geographic distribution and symptoms, combined with increase in travel, and preexisting antibodies due to flavivirus vaccinations, expand the need for rapid and reliable multiplex diagnostic tests to supplement currently used methods. Goal We describe the development of a multiplex serological protein microarray using recombinant NS1 proteins for detection of medically important viruses within the genus Flavivirus. Sera from clinical flavivirus patients were used for primary development of the protein microarray. Results Results show a high IgG and IgM sensitivity and specificity for individual NS1 antigens, and limited cross reactivity, even within serocomplexes. In addition, the serology based on this array allows for discrimination between infection and vaccination response for JEV vaccine, and no cross-reactivity with TBEV and YFV vaccine induced antibodies when testing for antibodies to other flaviviruses. Conclusion Based on these data, multiplex NS1-based protein microarray is a promising tool for surveillance and diagnosis of flaviviruses., Author Summary The number of international travelers has increased dramatically in recent decades. This has contributed to the increase in infectious diseases in travelers which are not present in their countries of origin and so may cause a threat to the public health. Viruses transmitted by biting insects (vector-borne viruses) are an important group within these travel-related diseases. They are found across the world and can cause debilitating and life-threatening symptoms, like inflammation of the brain or excessive bleeding. Many of these diseases are difficult to distinguish from each other. They cause comparable symptoms and are genetically closely related. Testing for long lists of diseases is time consuming and expensive. Here we develop a novel testing tool that allows doctors and researchers to test for multiple viruses with just one test. The method, which uses a specific part of the virus that makes distinguishing between infections with these closely related viruses possible, requires only one drop of blood. This allows us to test for multiple viruses simultaneously with the same amount of blood previously used to test for only one virus, while distinguishing between genetically closely related viruses.