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.

om as , F e r na n d o G on zález Candelas, SeqCOVID-SPAIN consortium, Tanja Stadler & Richard A. NeherThis is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

BackgroundThere 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 FindingsIn 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.ConclusionsA 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.

Background and AimsComplete diagnostic autopsies (CDA) remain the gold standard in the determination of cause of death (CoD). However, performing CDAs in developing countries is challenging due to limited facilities and human resources, and poor acceptability. We aimed to develop and test a simplified minimally invasive autopsy (MIA) procedure involving organ-directed sampling with microbiology and pathology analyses implementable by trained technicians in low- income settings.MethodsA standardized scheme for the MIA has been developed and tested in a series of 30 autopsies performed at the Maputo Central Hospital, Mozambique. The procedure involves the collection of 20 mL of blood and cerebrospinal fluid (CSF) and puncture of liver, lungs, heart, spleen, kidneys, bone marrow and brain in all cases plus uterus in women of childbearing age, using biopsy needles.ResultsThe sampling success ranged from 67% for the kidney to 100% for blood, CSF, lung, liver and brain. The amount of tissue obtained in the procedure varied from less than 10 mm2 for the lung, spleen and kidney, to over 35 mm2 for the liver and brain. A CoD was identified in the histological and/or the microbiological analysis in 83% of the MIAs.ConclusionsA simplified MIA technique allows obtaining adequate material from body fluids and major organs leading to accurate diagnoses. This procedure could improve the determination of CoD in developing countries.

BackgroundOver 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 findingsIn 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.ConclusionsThe 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.

BackgroundIn 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 findingsIn 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.ConclusionsThe 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.

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VP30 is a phosphoprotein essential for the initiation of Ebola virus transcription. In this work, we have studied the effect of mutations in VP30 phosphorylation sites on the ebolavirus replication cycle by using a reverse genetics system. We demonstrate that VP30 is involved in reinitiation of gene transcription and that this activity is affected by mutations at the phosphorylation sites.Ebola virus (EBOV) causes a severe hemorrhagic fever syndrome, characterized by high mortality rates, in humans and nonhuman primates (7,13,14). The EBOV replication cycle takes place in the cytoplasm of infected cells, where inclusion bodies are formed (4). The inclusions contain viral ribonucleoprotein (RNP) complexes consisting of viral RNA and four nucleocapsid proteins: the nucleoprotein (NP), VP35, the polymerase (L), and VP30 (15, 16). Using an EBOV minigenome system, it was demonstrated that NP, VP35, and L are sufficient for RNA replication, whereas the addition of VP30 is required for transcription initiation (12). Recently, it has been shown that VP30 provided in trans supports transcription of the minigenome delivered by infectious virus-like particles (VLPs) and replication of a recombinant EBOV lacking the VP30 gene (5). VP30 is phosphorylated at two serine clusters (amino acids 29 to 31 and 42 to 46), each containing three serine residues (Fig. 1A). Previously, it has been shown that mutants of VP30 with both serine clusters replaced by alanine residues supported transcription of an EBOV minigenome but did not accumulate in the NP-induced inclusions. In contrast, when all serine residues at the phosphorylation sites were replaced by aspartate residues, VP30 was unable to support transcription (10). While a Ser3Ala substitution mimics nonphosphorylated serine, a Ser3Asp substitution mimics constantly phosphorylated serine.In this study, we investigated the effect of mutations simulating a constantly phosphorylated state of VP30 during a Zaire EBOV infection (Fig. 1A). VP30 mutants with nonphosphorylated and highly phosphorylated states are represented by VP30-AA and VP30-DD, respectively. Two other constructs, VP30-AD and VP30-DA, have one of the two serine clusters replaced by an alanine cluster and the other by an aspartate cluster.In an attempt to generate recombinant viruses containing the designated mutations, we used a reverse genetics system for EBOV (17). Only the wild-type EBOV genome was rescued into infectious virus (Fig. 1B). Failure to recover a virus containing VP30-DD is likely explained by the lack of transcription initiation activity of VP30-DD (10). To assess whether the ability of VP30 to support viral transcription was altered with the other three mutants, we quantified their activities in an EBOV-specific minigenome system (8) ( Fig. 2A and B). VP30-AA was 30% more active than the VP30 wild type (VP30-WT) in supporting EBOV transcription, and both VP30-AD and VP30-DA supported transcription of the reporter gene in the same range as VP30-WT. Thus, the experiments with minigenomes did not provi...