Genotyping Hepatitis B virus by Next-Generation Sequencing: Detection of Mixed Infections and Analysis of Sequence Conservation.

Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5' end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A-H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies.

The imperative for quality control programs in Monkeypox virus DNA testing by PCR: CIBERINFEC quality control.

To evaluate molecular assays for Mpox diagnosis available in various clinical microbiology services in Spain through a quality control (QC) approach. A total of 14 centers from across Spain participated in the study. The Reference Laboratory dispatched eight serum samples and eight nucleic acid extracts to each participating center. Some samples were spiked with Mpox or Vaccinia virus to mimic positive samples for Mpox or other orthopox viruses. Participating centers provided information on the results obtained, as well as the laboratory methods used. Among the 14 participating centers seven different commercial assays were employed, with the most commonly used kit being LightMix Modular Orthopox/Monkeypox (Mpox) Virus (Roche®). Of the 12 centers conducting Mpox determinations, concordance ranged from 62.5% (n = 1) to 100% (n = 11) for eluates and from 75.0% (n = 1) to 100% (n = 10) for serum. Among the 10 centers performing Orthopoxvirus determinations, a 100% concordance was observed for eluates, while for serum, concordance ranged from 87.5% (n = 6) to 100% (n = 4). Repeatedly, 6 different centers reported a false negative in serum samples for Orthopoxvirus diagnosis, particularly in a sample with borderline Ct = 39. Conversely, one center, using the TaqMan™ Mpox Virus Microbe Detection Assay (Thermo Fisher), reported false positives in Mpox diagnosis for samples spiked with vaccinia virus due to cross-reactions. We observed a positive correlation of various diagnostic assays for Mpox used by the participating centers with the reference values. Our results highlight the significance of standardization, validation, and ongoing QC in the microbiological diagnosis of infectious diseases, which might be particularly relevant for emerging viruses.

Impact of SARS-CoV-2 viral load and duration of symptoms before hospital admission on the mortality of hospitalized COVID-19 patients.

PURPOSE: Assess the impact of viral load estimated by cycle threshold (Ct) of reverse transcription real time-polymerase chain reaction (rRT-PCR) and the days from symptoms onset on mortality in hospitalized patients with COVID19. METHODS: Retrospective observational study of 782 patients with a positive rRT-PCR from a nasopharyngeal swab was performed within the first 24 h from admission. Demographic data, clinical manifestations and laboratory parameters were collected. Uni- and multivariate analyses were performed to identify factors associated with mortality at 60 days. RESULTS: Ct was divided into three groups and the mortality rate decreased from 27.3 to 20.7% and 9.8% for Ct values of ≤ 20, 21-25 and > 25, respectively (P = 0.0001). The multivariate analysis identified as predictors of mortality, a Ct value < 20 (OR 3.13, CI 95% 1.38-7.10), between 21-25 (OR 2.47, CI 95% 1.32-4.64) with respect to a Ct value > 25. Days from symptoms onset is a variable associated with mortality as well (DSOA) ≤ 6 (OR 1.86, CI 95% 1.00-3.46), among other factors. Patients requiring hospital admission within 6 DSOA with a Ct value ≤ 25 had the highest mortality rate (28%). CONCLUSIONS: The inclusion of Ct values and DSOA in the characterization of study populations could be a useful tool to evaluate the efficacy of antivirals.

Frequent detection of monkeypox virus DNA in saliva, semen, and other clinical samples from 12 patients, Barcelona, Spain, May to June 2022.

A monkeypox (MPX) outbreak has expanded worldwide since May 2022. We tested 147 clinical samples collected at different time points from 12 patients by real-time PCR. MPX DNA was detected in saliva from all cases, sometimes with high viral loads. Other samples were frequently positive: rectal swab (11/12 cases), nasopharyngeal swab (10/12 cases), semen (7/9 cases), urine (9/12 cases) and faeces (8/12 cases). These results improve knowledge on virus shedding and the possible role of bodily fluids in disease transmission.

Minimally Invasive Tissue Sampling as an Alternative to Complete Diagnostic Autopsies in the Context of Epidemic Outbreaks and Pandemics: The Example of Coronavirus Disease 2019 (COVID-19).

BACKGROUND: Infectious diseases' outbreak investigation requires, by definition, conducting a thorough epidemiological assessment while simultaneously obtaining biological samples for an adequate screening of potential responsible pathogens. Complete autopsies remain the gold-standard approach for cause-of-death evaluation and characterization of emerging diseases. However, for highly transmissible infections with a significant associated lethality, such as COVID-19, complete autopsies are seldom performed due to biosafety challenges, especially in low-resource settings. Minimally invasive tissue sampling (MITS) is a validated new approach based on obtaining postmortem samples from key organs and body fluids, a procedure that does not require advanced biosafety measures or a special autopsy room. METHODS: We aimed to review the use of MITS or similar procedures for outbreak investigation up to 27 March 2021 and their performance for evaluating COVID-19 deaths. RESULTS: After a literature review, we analyzed in detail the results of 20 studies conducted at international sites, whereby 216 COVID-19-related deaths were investigated. MITS provided a general and more granular understanding of the pathophysiological changes secondary to the infection and high-quality samples where the extent and degree of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related damage could be evaluated. CONCLUSIONS: MITS is a useful addition in the investigation and surveillance of infections occurring in outbreaks or epidemics. Its less invasive nature makes the tool more acceptable and feasible and reduces the risk of procedure-associated contagion, using basic biosafety measures. Standardized approaches protocolizing which samples should be collected-and under which exact biosafety measures-are necessary to facilitate and expand its use globally.

Minimally Invasive Tissue Sampling: A Tool to Guide Efforts to Reduce AIDS-Related Mortality in Resource-Limited Settings.

BACKGROUND: Available information on the causes of death among people living with human immunodeficiency virus (PLHIV) in low- and middle-income countries (LMICs) remains scarce. We aimed to provide data on causes of death in PLHIV from two LMICs, Brazil and Mozambique, to assess the impact of clinical misdiagnosis on mortality rates and to evaluate the accuracy of minimally invasive tissue sampling (MITS) in determining the cause of death in PLHIV. METHODS: We performed coupled MITS and complete autopsy on 164 deceased PLHIV (18 children, 36 maternal deaths, and 110 adults). HIV antibody levels and HIV RNA viral loads were determined from postmortem serum samples. RESULTS: Tuberculosis (22.7%), toxoplasmosis (13.9%), bacterial infections (13.9%), and cryptococcosis (10.9%) were the leading causes of death in adults. In maternal deaths, tuberculosis (13.9%), bacterial infections (13.9%), cryptococcosis (11.1%), and cerebral malaria (8.3%) were the most frequent infections, whereas viral infections, particularly cytomegalovirus (38.9%), bacterial infections (27.8%), pneumocystosis (11.1%), and HIV-associated malignant neoplasms (11.1%) were the leading cause among children. Agreement between the MITS and the complete autopsy was 100% in children, 91% in adults, and 78% in maternal deaths. The MITS correctly identified the microorganism causing death in 89% of cases. CONCLUSIONS: Postmortem studies provide highly granular data on the causes of death in PLHIV. The inaccuracy of clinical diagnosis may play a significant role in the high mortality rates observed among PLHIV in LMICs. MITS might be helpful in monitoring the causes of death in PLHIV and in highlighting the gaps in the management of the infections.

Minimally Invasive Tissue Sampling Findings in 12 Patients With Coronavirus Disease 2019.

BACKGROUND: Minimally invasive tissue sampling (MITS), a postmortem procedure that uses core needle biopsy samples and does not require opening the body, may be a valid alternative to complete autopsy (CA) in highly infectious diseases such as coronavirus disease-19 (COVID-19). This study aimed to (1) compare the performance of MITS and CA in a series of COVID-19 deaths and (2) evaluate the safety of the procedure. METHODS: From October 2020 to February 2021, MITS was conducted in 12 adults who tested positive before death for COVID-19, in a standard, well-ventilated autopsy room, where personnel used reinforced personal protective equipment. In 9 cases, a CA was performed after MITS. A thorough histological evaluation was conducted, and the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was evaluated by real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: The diagnoses provided by MITS and CA matched almost perfectly. In 9 patients, COVID-19 was in the chain of events leading to death, being responsible for diffuse alveolar damage and mononuclear T-cell inflammatory response in the lungs. No specific COVID-19 features were identified. Three deaths were not related to COVID-19. All personnel involved in MITS repeatedly tested negative for COVID-19. SARS-CoV-2 was identified by RT-PCR and immunohistochemistry in the MITS samples, particularly in the lungs. CONCLUSIONS: MITS is useful for evaluating COVID-19-related deaths in settings where a CA is not feasible. The results of this simplified and safer technique are comparable to those of CA.

Microbiology in minimally invasive autopsy: best techniques to detect infection. ESGFOR (ESCMID study group of forensic and post-mortem microbiology) guidelines.

This manuscript aims to: 1) provide specific guidelines on PMM techniques in the setting of minimally invasive autopsy (MIA), both for pathologists collecting samples and for microbiologists advising pathologists and interpreting the results and 2) introduce standardization in PMM sampling at MIA. Post-mortem microbiology (PMM) is crucial to identify the causative organism in deaths due to infection. MIA including the use of post-mortem (PM) computed tomography (CT) and PM magnetic resonance imaging (MRI), is increasingly carried out as a complement or replacement for the traditional PM. In this setting, mirroring the traditional autopsy, PMM aims to: detect infectious organisms causing sudden unexpected deaths; confirm clinically suspected but unproven infection; evaluate the efficacy of antimicrobial therapy; identify emergent pathogens; and recognize medical diagnostic errors. Meaningful interpretation of PMM results requires careful evaluation in the context of the clinical history, macroscopic and microscopic findings. These guidelines were developed by a multidisciplinary team with experts in various fields of microbiology and pathology on behalf of the ESGFOR (ESCMID - European Society of Clinical Microbiology and Infectious Diseases - Study Group of Forensic and Post-mortem Microbiology, in collaboration with the ESP -European Society of Pathology-) based on a literature search and the author's expertise. Microbiological sampling methods for MIA are presented for various scenarios: adults, children, developed and developing countries. Concordance between MIA and conventional invasive autopsy is substantial for children and adults and moderate for neonates and maternal deaths. Networking and closer collaboration among microbiologists and pathologists is vital to maximize the yield of PMM in MIA.

Hantavirus Pulmonary Syndrome in Traveler Returning from Nepal to Spain.

Most human hantavirus infections occur in Asia, but some cases have been described in Europe in travelers returning from Asia. We describe a case of hantavirus pulmonary syndrome in a previously healthy traveler occurring shortly after he returned to Spain from Nepal. Serologic tests suggested a Puumala virus-like infection.

Specialist laboratory networks as preparedness and response tool - the Emerging Viral Diseases-Expert Laboratory Network and the Chikungunya outbreak, Thailand, 2019.

We illustrate the potential for specialist laboratory networks to be used as preparedness and response tool through rapid collection and sharing of data. Here, the Emerging Viral Diseases-Expert Laboratory Network (EVD-LabNet) and a laboratory assessment of chikungunya virus (CHIKV) in returning European travellers related to an ongoing outbreak in Thailand was used for this purpose. EVD-LabNet rapidly collected data on laboratory requests, diagnosed CHIKV imported cases and sequences generated, and shared among its members and with the European Centre for Disease Prevention and Control. Data across the network showed an increase in CHIKV imported cases during 1 October 2018-30 April 2019 vs the same period in 2018 (172 vs 50), particularly an increase in cases known to be related to travel to Thailand (72 vs 1). Moreover, EVD-LabNet showed that strains were imported from Thailand that cluster with strains of the ECSA-IOL E1 A226 variant emerging in Pakistan in 2016 and involved in the 2017 outbreaks in Italy. CHIKV diagnostic requests increased by 23.6% between the two periods. The impact of using EVD-LabNet or similar networks as preparedness and response tool could be improved by standardisation of the collection, quality and mining of data in routine laboratory management systems.

Unmasking the hidden tuberculosis mortality burden in a large post mortem study in Maputo Central Hospital, Mozambique.

Sensitive tools are needed to accurately establish the diagnosis of tuberculosis (TB) at death, especially in low-income countries. The objective of this study was to evaluate the burden of TB in a series of patients who died in a tertiary referral hospital in sub-Saharan Africa using an in-house real time PCR (TB-PCR) and the Xpert MTB/RIF Ultra (Xpert Ultra) assay.Complete diagnostic autopsies were performed in a series of 223 deaths (56.5% being HIV-positive), including 54 children, 57 maternal deaths and 112 other adults occurring at the Maputo Central Hospital, Mozambique. TB-PCR was performed in all lung, cerebrospinal fluid and central nervous system samples in HIV-positive patients. All samples positive for TB-PCR or showing histological findings suggestive of TB were analysed with the Xpert Ultra assay.TB was identified as the cause of death in 31 patients: three out of 54 (6%) children, five out of 57 (9%)maternal deaths and 23 out of 112 (21%) other adults. The sensitivity of the main clinical diagnosis to detect TB as the cause of death was 19.4% (95% CI 7.5-37.5) and the specificity was 97.4% (94.0-99.1) compared to autopsy findings. Concomitant TB (TB disease in a patient dying of other causes) was found in 31 additional cases. Xpert Ultra helped to identify 15 cases of concomitant TB. In 18 patients, Mycobacterium tuberculosis DNA was identified by TB-PCR and Xpert Ultra in the absence of histological TB lesions. Overall, 62 (27.8%) cases had TB disease at death and 80 (35.9%) had TB findings.The use of highly sensitive, easy to perform molecular tests in complete diagnostic autopsies may contribute to identifying TB cases at death that would have otherwise been missed. Routine use of these tools in certain diagnostic algorithms for hospitalised patients needs to be considered. Clinical diagnosis showed poor sensitivity for the diagnosis of TB at death.

Spontaneous Abortion Associated with Zika Virus Infection and Persistent Viremia.

We report a case of spontaneous abortion associated with Zika virus infection in a pregnant woman who traveled from Spain to the Dominican Republic and developed a rash. Maternal Zika viremia persisted at least 31 days after onset of symptoms and 21 days after uterine evacuation.

Arbovirus surveillance: first dengue virus detection in local Aedes albopictus mosquitoes in Europe, Catalonia, Spain, 2015.

Dengue has emerged as the most important viral mosquito-borne disease globally. The current risk of dengue outbreaks in Europe appeared with the introduction of the vector Aedes albopictus mosquito in Mediterranean countries. Considering the increasing frequency of dengue epidemics worldwide and the movement of viraemic hosts, it is expected that new autochthonous cases will occur in the future in Europe. Arbovirus surveillance started in Catalonia in 2015 to monitor imported cases and detect possible local arboviral transmission. During 2015, 131 patients with a recent travel history to endemic countries were tested for dengue virus (DENV) and 65 dengue cases were detected. Twenty-eight patients with a febrile illness were viraemic, as demonstrated by a positive real-time RT-PCR test for DENV in serum samples. Entomological investigations around the viraemic cases led to the detection of DENV in a pool of local Ae. albopictus captured in the residency of one case. The sequence of the DENV envelope gene detected in the mosquito pool was identical to that detected in the patient. Our results show how entomological surveillance conducted around viraemic travellers can be effective for early detection of DENV in mosquitoes and thus might help to prevent possible autochthonous transmission.

Zika Virus Screening among Spanish Team Members After 2016 Rio de Janeiro, Brazil, Olympic Games.

We evaluated the risk for the Spanish Olympic Team acquiring Zika virus in Rio de Janeiro, Brazil, during 2016. We recruited 117 team members, and all tested negative for Zika virus. Lack of cases in this cohort supports the minimum risk estimates made before the Games.

Validity of a minimally invasive autopsy for cause of death determination in maternal deaths in Mozambique: An observational study.

BACKGROUND: Despite global health efforts to reduce maternal mortality, rates continue to be unacceptably high in large parts of the world. Feasible, acceptable, and accurate postmortem sampling methods could provide the necessary evidence to improve the understanding of the real causes of maternal mortality, guiding the design of interventions to reduce this burden. METHODS AND FINDINGS: The validity of a minimally invasive autopsy (MIA) method in determining the cause of death was assessed in an observational study in 57 maternal deaths by comparing the results of the MIA with those of the gold standard (complete diagnostic autopsy [CDA], which includes any available clinical information). Concordance between the MIA and the gold standard diagnostic categories was assessed by the kappa statistic, and the sensitivity, specificity, positive and negative predictive values and their 95% confidence intervals (95% CI) to identify the categories of diagnoses were estimated. The main limitation of the study is that both the MIA and the CDA include some degree of subjective interpretation in the attribution of cause of death. A cause of death was identified in the CDA in 98% (56/57) of cases, with indirect obstetric conditions accounting for 32 (56%) deaths and direct obstetric complications for 24 (42%) deaths. Nonobstetric infectious diseases (22/32, 69%) and obstetric hemorrhage (13/24, 54%) were the most common causes of death among indirect and direct obstetric conditions, respectively. Thirty-six (63%) women were HIV positive, and HIV-related conditions accounted for 16 (28%) of all deaths. Cerebral malaria caused 4 (7%) deaths. The MIA identified a cause of death in 86% of women. The overall concordance of the MIA with the CDA was moderate (kappa = 0.48, 95% CI: 0.31-0.66). Both methods agreed in 68% of the diagnostic categories and the agreement was higher for indirect (91%) than for direct obstetric causes (38%). All HIV infections and cerebral malaria cases were identified in the MIA. The main limitation of the technique is its relatively low performance for identifying obstetric causes of death in the absence of clinical information. CONCLUSIONS: The MIA procedure could be a valuable tool to determine the causes of maternal death, especially for indirect obstetric conditions, most of which are infectious diseases. The information provided by the MIA could help to prioritize interventions to reduce maternal mortality and to monitor progress towards achieving global health targets.

Validity of a minimally invasive autopsy for cause of death determination in stillborn babies and neonates in Mozambique: An observational study.

BACKGROUND: Over 5 million stillbirths and neonatal deaths occur annually. Limited and imprecise information on the cause of these deaths hampers progress in achieving global health targets. Complete diagnostic autopsies (CDAs)-the gold standard for cause of death determination-are difficult to perform in most high-burden settings. Therefore, validation of simpler and more feasible methods is needed. METHODS AND FINDINGS: In this observational study, the validity of a minimally invasive autopsy (MIA) method in determining the cause of death was assessed in 18 stillbirths and 41 neonatal deaths by comparing the results of the MIA with those of the CDA. Concordance between the categories of diseases obtained by the 2 methods was assessed by the Kappa statistic, and the sensitivity, specificity, positive, and negative predictive values of the MIA diagnoses were calculated. A cause of death was identified in 16/18 (89%) and 15/18 (83%) stillborn babies in the CDA and the MIA, respectively. Fetal growth restriction accounted for 39%, infectious diseases for 22%, intrapartum hypoxia for 17%, and intrauterine hypoxia for 11% of stillborn babies. Overall, the MIA showed in this group a substantial concordance with the CDA (Kappa = 0.78, 95% CI [0.56-0.99]). A cause of death was identified in all (100%) and 35/41 (85%) neonatal deaths in the CDA and the MIA, respectively. In this group, the majority of deaths were due to infectious diseases (66%). The overall concordance of the MIA with the CDA in neonates was moderate (Kappa = 0.40, 95% CI [0.18-0.63]). A high percentage of accuracy was observed for the MIA in all the diagnostic categories in both stillbirths and neonates (>75%). The main limitation of this study is that some degree of subjective interpretation is inherent to cause-of-death attribution in both the MIA and the CDA; this is especially so in stillbirths and in relation to fetal growth restriction. CONCLUSIONS: The MIA could be a useful tool for cause-of-death determination in stillbirths and neonatal deaths. These findings may help to accelerate progress towards meeting global health targets by obtaining more accurate information on the causes of death in these age groups, which is essential in guiding the design of new interventions and increasing the effectiveness of those already implemented.

Validity of a minimally invasive autopsy tool for cause of death determination in pediatric deaths in Mozambique: An observational study.

BACKGROUND: In recent decades, the world has witnessed unprecedented progress in child survival. However, our knowledge of what is killing nearly 6 million children annually in low- and middle-income countries remains poor, partly because of the inadequacy and reduced precision of the methods currently utilized in these settings to investigate causes of death (CoDs). The study objective was to validate the use of a minimally invasive autopsy (MIA) approach as an adequate and more acceptable substitute for the complete diagnostic autopsy (CDA) for pediatric CoD investigation in a poor setting. METHODS AND FINDINGS: In this observational study, the validity of the MIA approach in determining the CoD was assessed in 54 post-neonatal pediatric deaths (age range: ≥1 mo to 15 y) in a referral hospital of Mozambique by comparing the results of the MIA with those of the CDA. Concordance in the category of disease obtained by the two methods was evaluated by the Kappa statistic, and the sensitivity, specificity, and positive and negative predictive values of the MIA diagnoses were calculated. A CoD was identified in all cases in the CDA and in 52/54 (96%) of the cases in the MIA, with infections and malignant tumors accounting for the majority of diagnoses. The MIA categorization of disease showed a substantial concordance with the CDA categorization (Kappa = 0.70, 95% CI 0.49-0.92), and sensitivity, specificity, and overall accuracy were high. The ICD-10 diagnoses were coincident in up to 75% (36/48) of the cases. The MIA allowed the identification of the specific pathogen deemed responsible for the death in two-thirds (21/32; 66%) of all deaths of infectious origin. Discrepancies between the MIA and the CDA in individual diagnoses could be minimized with the addition of some basic clinical information such as those ascertainable through a verbal autopsy or clinical record. The main limitation of the analysis is that both the MIA and the CDA include some degree of expert subjective interpretation. CONCLUSIONS: The MIA showed substantial concordance with CDA for CoD identification in this series of pediatric deaths in Mozambique. This minimally invasive approach, simpler and more readily acceptable than the more invasive CDA, could provide robust data for CoD surveillance, especially in resource-limited settings, which could be helpful for guiding child survival strategies in the future.

Validity of a Minimally Invasive Autopsy for Cause of Death Determination in Adults in Mozambique: An Observational Study.

BACKGROUND: There is an urgent need to identify tools able to provide reliable information on the cause of death in low-income regions, since current methods (verbal autopsy, clinical records, and complete autopsies) are either inaccurate, not feasible, or poorly accepted. We aimed to compare the performance of a standardized minimally invasive autopsy (MIA) approach with that of the gold standard, the complete diagnostic autopsy (CDA), in a series of adults who died at Maputo Central Hospital in Mozambique. METHODS AND FINDINGS: In this observational study, coupled MIAs and CDAs were performed in 112 deceased patients. The MIA analyses were done blindly, without knowledge of the clinical data or the results of the CDA. We compared the MIA diagnosis with the CDA diagnosis of cause of death. CDA diagnoses comprised infectious diseases (80; 71.4%), malignant tumors (16; 14.3%), and other diseases, including non-infectious cardiovascular, gastrointestinal, kidney, and lung diseases (16; 14.3%). A MIA diagnosis was obtained in 100/112 (89.2%) cases. The overall concordance between the MIA diagnosis and CDA diagnosis was 75.9% (85/112). The concordance was higher for infectious diseases and malignant tumors (63/80 [78.8%] and 13/16 [81.3%], respectively) than for other diseases (9/16; 56.2%). The specific microorganisms causing death were identified in the MIA in 62/74 (83.8%) of the infectious disease deaths with a recognized cause. The main limitation of the analysis is that both the MIA and the CDA include some degree of expert subjective interpretation. CONCLUSIONS: A simple MIA procedure can identify the cause of death in many adult deaths in Mozambique. This tool could have a major role in improving the understanding and surveillance of causes of death in areas where infectious diseases are a common cause of mortality.

Assessing viral metagenomics for the diagnosis of acute undifferentiated fever in returned travellers: a multicenter cohort study.

BACKGROUND: Up to 45% of febrile returning travellers remain undiagnosed after a thorough diagnostic work-up, even at referral centres. Although metagenomic next-generation sequencing (mNGS) has emerged as a promising tool, evidence of its usefulness in imported fever is very limited. METHODS: Travellers returning with fever were prospectively recruited in three referral clinics from November 2017 to November 2019. Unbiased mNGS optimised for virus detection was performed on serum samples of participants with acute undifferentiated febrile illness (AUFI), and results were compared to those obtained by reference diagnostic methods (RDM). RESULTS: Among 507 returned febrile travellers, 433(85.4%) presented with AUFI. Dengue virus (n = 86) and Plasmodium spp. (n = 83) were the most common causes of fever. 103/433(23.8%) AUFI remained undiagnosed at the end of the follow-up.Metagenomic next-generation sequencing unveiled potentially pathogenic microorganisms in 196/433(38.7%) AUFI. mNGS identifications were more common in patients with a shorter duration of fever (42.3% in ≤5 days vs 28.7% in >5 days, P = 0.005). Potential causes of fever were revealed in 25/103(24.2%) undiagnosed AUFI and 5/23(21.7%) travellers with severe undiagnosed AUFI. Missed severe aetiologies included eight bacterial identifications and one co-infection of B19 parvovirus and Aspergillus spp.Additional identifications indicating possible co-infections occurred in 29/316(9.2%) travellers with AUFI, and in 11/128(8.6%) travellers with severe AUFI, who had received a diagnosis through RDM. The most common co-infections detected in severe AUFI were caused by Gram-negative bacteria. Serum mNGS was unable to detect >50% of infectious diagnoses achieved by RDM and also yielded 607 non-pathogenic identifications. DISCUSSION: mNGS of serum can be a valuable diagnostic tool for selected travellers with undiagnosed AUFI or severe disease in addition to reference diagnostic techniques, especially during the first days of symptoms. Nevertheless, mNGS results interpretation presents a great challenge. Further studies evaluating the performance of mNGS using different sample types and protocols tailored to non-viral agents are needed.

Doxycycline responding illnesses in returning travellers with undifferentiated non-malaria fever: a European multicentre prospective cohort study.

BACKGROUND: Diagnosis of undifferentiated non-malaria fevers (NMF) in returning travellers is a great challenge. Currently, there is no consensus about the use of empirical antibiotics in returning travellers with undifferentiated NMF. Although studies in endemic areas showed that a wide range of pathogens implicated in undifferentiated NMF are treatable with doxycycline, the role of doxycycline in returning travellers with fever still has to be explored. METHODS: Prospective European multicentre cohort study of febrile international travellers (November 2017-November 2019). Immunological and molecular diagnostic techniques for doxycycline responding illnesses (DRI) agents such as Anaplasma phagocytophilum, spotted fever group Rickettsia spp., typhus group Rickettsia spp., Coxiella burnetii, Bartonella spp., Orientia tsutsugamushi, Borrelia miyamotoi, Borrelia recurrentis and Leptospira spp. were systematically performed in all patients with undifferentiated NMF. We estimated the prevalence and predictive factors of DRI in returning travellers with undifferentiated NMF. RESULTS: Among 347 travellers with undifferentiated NMF, 106 (30·5%) were finally diagnosed with DRI. Only 57 (53·8%) of the 106 DRI infections were diagnosed by the standard of care. The main causes of DRI were: 55 (51·9%) Rickettsia spp., 16 (15·1%) C. burnetii; 15 (14·2%) Bartonella spp.; 13 (12·3%) Leptospira spp. and 10 (9·5%) A. phagocytophilum. The only predictive factor associated with DRI was presenting an eschar (aOR 39·52, 95%CI 4·85-322·18). Features of dengue such as retro-orbital pain (aOR 0·40, 95%CI 0·21-0·76) and neutropenia (aOR 0·41, 95%CI 0·21-0·79) were negatively associated with DRI. CONCLUSIONS: Although DRI are responsible for 30% of undifferentiated NMF cases in travellers, those are seldom recognized during the first clinical encounter. Empirical treatment with doxycycline should be considered in returning travellers with undifferentiated fever and negative tests for malaria and dengue, particularly when presenting severe illness, predictive factors for rickettsiosis or no features of dengue.