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.

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.

Report on the first two confirmed autochthonous cases of West Nile virus encephalitis in Catalonia, Spain.

BACKGROUND: West Nile virus (WNV) is a mosquito-borne flavivirus that can cause Central Nervous System infection in humans. Previous autochthonous cases of WNV encephalitis have been described in Spain, but none in Catalonia. MATERIALS AND METHODS: We report on the first two autochthonous cases of encephalitis in humans caused by the West Nile virus (WNV) diagnosed in Catalonia (northeastern region of Spain). RESULTS: An old married couple presented with clinical and biological signs compatible with viral encephalitis. Acute and convalescent serum samples showed IgM and IgG positivity for WNV. In addition, IgM was also detected in cerebrospinal fluid in the male patient. The serological results were later confirmed by microneutralization assays. CONCLUSIONS: WNV infection must be considered in patients presenting with meningoencephalitis with viral CSF characteristics when common pathogens are excluded.

Clinical evaluation of BioFire® multiplex-PCR panel for acute undifferentiated febrile illnesses in travellers: a prospective multicentre study.

BACKGROUND: Identifying the causes of Acute Undifferentiated Febrile Illness (AUFI) is key to improve the management of returning travellers with fever. We evaluated a BioFire®FilmArray® prototype panel of multiplex nucleic acid amplification tests (NAAT) targeting different relevant pathogens in travellers returning with fever. METHODS: Prospective, multicentre study to evaluate a prototype panel in whole blood samples of adult international travellers presenting with AUFI in three European travel Clinics/Hospitals (November 2017-November 2019). We evaluated 15 target analytes: Plasmodium spp., Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, chikungunya virus, dengue virus, Zika virus, Anaplasma phagocytophilum, Borrelia spp., Leptospira spp., Orientia tsutsugamushi, Rickettsia spp. and Salmonella spp. Results were compared with composite reference standards (CRSs) for each target infection, including direct methods [smear microscopy, rapid diagnostic test (RDT), reference NAAT and blood cultures] and indirect methods (paired serology). FINDINGS: Among 455 travellers with AUFI, 229 target infections were diagnosed; the prototype panel detected 143 (overall sensitivity and specificity of 62.5 and 99.8%, respectively). The panel identified all Plasmodium infections (n = 82). Sensitivity for dengue (n = 71) was 92.9, 80.8 and 68.5% compared with RDT, NAAT and CRS, respectively. Compared with direct methods and CRS, respectively, the prototype panel detected 4/4 and 4/6 chikungunya, 2/2 and 4/29 Leptospira spp., 1/1 and 1/6 O. tsutsugamushi and 2/2 and 2/55 Rickettsia spp., but 0/2 and 0/10 Zika, 0/1 and 0/11 A. phagocytophylum and 0/3 Borrelia spp. diagnosed by serology and only 1/7 Salmonella spp. diagnosed by blood cultures. 77/86 (89.5%) infections not detected by the panel were diagnosed by serology. INTERPRETATION: The prototype panel allowed rapid and reliable diagnosis for malaria, dengue and chikungunya. Further improvements are needed to improve its sensitivity for Zika and important travel-related bacterial infections.

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.

Genetic Study of SARS-CoV-2 Non Structural Protein 12 in COVID-19 Patients Non Responders to Remdesivir.

Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (n = 36/111, 32.4%) or prolonged (5 to 20 days) (n = 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the nsp12 gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the de novo E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication. IMPORTANCE This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the nsp12 pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.

Prevalence of Hepatitis C Virus Infection, Genotypes and Subtypes in Migrants from Pakistan in Barcelona, Spain.

Background: Hepatitis C virus (HCV) is a major cause of chronic liver infection with 71 million people infected worldwide. Pakistan has the second highest prevalence of HCV infection and more than half (52%) of Pakistani living in Spain reside in Barcelona. The aim of this study was to analyse the seroprevalence and viraemic rate and determine the genotypes and subtypes of HCV among Pakistanis living in the southern metropolitan area of Barcelona. Methods: We included all Pakistani patients seeking primary healthcare in the southern metropolitan area of Barcelona from August 2011 to July 2014. Serum samples were screened for HCV antibodies. HCV viral load was determined by reverse transcription polymerase chain reaction and genotypes and subtypes were performed using Versant HCV Genotype and/or deep-sequencing. Screening for hepatitis B virus (HBV) was also carried out. Results: Among 5877 Pakistani patients, 565 (9.61%) were screened for anti-HCV antibodies, with 68 (12.04%) being positive. The viral load was determined in 65, with 31 presenting active infection and the viraemic rate was 47.69% (95% confidence interval 36.02-59.62). HCV genotyping and subtyping were performed in 24 individuals. Most infections corresponded to HCV genotype 3 (91.67%), and high resolution HCV subtyping was performed in 18 samples, 16 of which presented subtype 3a. One subject presented HBV coinfection with undetectable HBV DNA. During the study period, we identified a possible case of HCV vertical transmission followed by spontaneous viraemia clearance in a chronically infected mother with a C/T IL28B genetic polymorphism. Conclusion: These results suggest that general HCV screening protocols in patients from high prevalence countries, such as Pakistan, would be helpful to identify and treat active HCV infections. This could avoid further transmission and contribute to building targeted health policies for micro-elimination of HCV infection in specific communities.

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.

Post-Mortem Diagnosis of Pediatric Dengue Using Minimally Invasive Autopsy during the COVID-19 Pandemic in Brazil.

We report the first pediatric disease in which the use of minimally invasive autopsy (MIA) confirmed severe dengue as the cause of death. During the COVID-19 pandemic, a previously healthy 10-year-old girl living in north-eastern Brazil presented fever, headache, diffuse abdominal pain, diarrhoea, and vomiting. On the fourth day, the clinical symptoms worsened and the patient died. An MIA was performed, and cores of brain, lungs, heart, liver, kidneys, and spleen were collected with 14G biopsy needles. Microscopic examination showed diffuse oedema and congestion, pulmonary intra-alveolar haemorrhage, small foci of midzonal necrosis in the liver, and tubular cell necrosis in the kidneys. Dengue virus RNA and NS1 antigen were detected in blood and cerebrospinal fluid samples. Clinical, pathological, and laboratory findings, in combination with the absence of other lesions and microorganisms, allowed concluding that the patient had died from complications of severe dengue.

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.

Surveillance of SARS-CoV-2 virus circulation using Acute Respiratory Infections sentinel system of Catalonia (PIDIRAC) during the 2019-2020 season: A retrospective observational study.

BACKGROUND: In the context of COVID-19 pandemic in Catalonia (Spain), the present study analyses respiratory samples collected by the primary care network using Acute Respiratory Infections Sentinel Surveillance System (PIDIRAC) during the 2019-2020 season to complement the pandemic surveillance system in place to detect SARS-CoV-2. The aim of the study is to describe whether SARS-CoV-2 was circulating before the first confirmed case was detected in Catalonia, on February 25th, 2020. METHODS: The study sample was made up of all samples collected by the PIDIRAC primary care network as part of the Influenza and Acute Respiratory Infections (ARI) surveillance system activities. The study on respiratory virus included coronavirus using multiple RT-PCR assays. All positive samples for human coronavirus were subsequently typed for HKU1, OC43, NL63, 229E. Every respiratory sample was frozen at-80°C and retrospectively studied for SARS-CoV-2 detection. A descriptive study was performed, analysing significant differences among variables related to SARS-CoV- 2 cases comparing with rest of coronaviruses cases through a bivariate study with Chi-squared test and statistical significance at 95%. RESULTS: Between October 2019 and April 2020, 878 respiratory samples from patients with acute respiratory infection or influenza syndrome obtained by PIDIRAC were analysed. 51.9% tested positive for influenza virus, 48.1% for other respiratory viruses. SARS-CoV-2 was present in 6 samples. The first positive SARS-CoV-2 case had symptom onset on 2 March 2020. These 6 cases were 3 men and 3 women, aged between 25 and 50 years old. 67% had risk factors, none had previous travel history nor presented viral coinfection. All of them recovered favourably. CONCLUSION: Sentinel Surveillance PIDIRAC enhances global epidemiological surveillance by allowing confirmation of viral circulation and describes the epidemiology of generalized community respiratory viruses' transmission in Catalonia. The system can provide an alert signal when identification of a virus is not achieved in order to take adequate preparedness measures.

Causes of fever in returning travelers: a European multicenter prospective cohort study.

BACKGROUND: Etiological diagnosis of febrile illnesses in returning travelers is a great challenge, particularly when presenting with no focal symptoms [acute undifferentiated febrile illnesses (AUFI)], but is crucial to guide clinical decisions and public health policies. In this study, we describe the frequencies and predictors of the main causes of fever in travelers. METHODS: Prospective European multicenter cohort study of febrile international travelers (November 2017-November 2019). A predefined diagnostic algorithm was used ensuring a systematic evaluation of all participants. After ruling out malaria, PCRs and serologies for dengue, chikungunya and Zika viruses were performed in all patients presenting with AUFI ≤ 14 days after return. Clinical suspicion guided further microbiological investigations. RESULTS: Among 765 enrolled participants, 310/765 (40.5%) had a clear source of infection (mainly traveler's diarrhea or respiratory infections), and 455/765 (59.5%) were categorized as AUFI. AUFI presented longer duration of fever (p < 0.001), higher hospitalization (p < 0.001) and ICU admission rates (p < 0.001). Among travelers with AUFI, 132/455 (29.0%) had viral infections, including 108 arboviruses, 96/455 (21.1%) malaria and 82/455 (18.0%) bacterial infections. The majority of arboviral cases (80/108, 74.1%) was diagnosed between May and November. Dengue was the most frequent arbovirosis (92/108, 85.2%). After 1 month of follow-up, 136/455 (29.9%) patients with AUFI remained undiagnosed using standard diagnostic methods. No relevant differences in laboratory presentation were observed between undiagnosed and bacterial AUFI. CONCLUSIONS: Over 40% of returning travelers with AUFI were diagnosed with malaria or dengue, infections that can be easily diagnosed by rapid diagnostic tests. Arboviruses were the most common cause of AUFI (above malaria) and most cases were diagnosed during Aedes spp. high season. This is particularly relevant for those areas at risk of introduction of these pathogens. Empirical antibiotic regimens including doxycycline or azithromycin should be considered in patients with AUFI, after ruling out malaria and arboviruses.

Encefalitis centroeuropea. Descripción del primer caso importado en España en un paciente pediátrico / Tick-borne encephalitis. Description of the first imported case in Spain in a paediatric patient

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Lack of Prognostic Value of SARS-CoV2 RT-PCR Cycle Threshold in the Community.

The immense impact of the COVID-19 pandemic on health systems has motivated the scientific community to search for clinical prognostic factors for SARS-CoV-2 infection. Low cycle threshold values (Ct) of diagnostic real-time RT-PCR assays in hospitalized patients have been associated with a poor prognosis in several studies, whereas other studies did not find this association. We explored whether SARS-CoV-2 Ct values at diagnosis were associated with a poor outcome (admission to hospital and death) in 604 community patients diagnosed at primary health centers. Although lower Ct values were found in patients who died of COVID-19, the Ct value was not significantly associated with a worse outcome in a multivariate analysis, while age remained an independent prognostic factor. We did not find evidence to support the role of Ct values as a prognostic factor of COVID-19 in community cases.

Post-mortem neuropathologic examination of a 6-case series of CAR T-cell treated patients.

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. Adverse events are common, and neurotoxicity is one of the most important. However, the physiopathology is unknown and neuropathologic information is scarce. Materials and methods: Post-mortem examination of 6 brains from patients that underwent CAR T-cell therapy from 2017 to 2022. In all cases, polymerase chain reaction (PCR) in paraffin blocks for the detection of CAR T cells was performed. Results: Two patients died of hematologic progression, while the others died of cytokine release syndrome, lung infection, encephalomyelitis, and acute liver failure. Two out of 6 presented neurological symptoms, one with extracranial malignancy progression and the other with encephalomyelitis. The neuropathology of the latter showed severe perivascular and interstitial lymphocytic infiltration, predominantly CD8+, together with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, and a diffuse gliosis of basal ganglia, hippocampus, and brainstem. Microbiological studies were negative for neurotropic viruses, and PCR failed to detect CAR T -cells. Another case without detectable neurological signs showed cortical and subcortical gliosis due to acute hypoxic-ischemic damage. The remaining 4 cases only showed a mild patchy gliosis and microglial activation, and CAR T cells were detected by PCR only in one of them. Conclusions: In this series of patients that died after CAR T-cell therapy, we predominantly found non-specific or minimal neuropathological changes. CAR T-cell related toxicity may not be the only cause of neurological symptoms, and the autopsy could detect additional pathological findings.

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.

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 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.

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens.

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis.Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination.Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures.Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013-2018) included stool culture, microscopy and immunochromatography.Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013-2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis.Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.