Ultrasound-guided minimally invasive autopsy as a tool for rapid post-mortem diagnosis in the 2018 Sao Paulo yellow fever epidemic: Correlation with conventional autopsy

Background New strategies for collecting post-mortem tissue are necessary, particularly in areas with emerging infections. Minimally invasive autopsy (MIA) has been proposed as an alternative to conventional autopsy (CA), with promising results. Previous studies using MIA addressed the cause of death in adults and children in developing countries. However, none of these studies was conducted in areas with an undergoing infectious disease epidemic. We have recently experienced an epidemic of yellow fever (YF) in Brazil. Aiming to provide new information on low-cost post-mortem techniques that could be applied in regions at risk for infectious outbreaks, we tested the efficacy of ultrasound-guided MIA (MIA-US) in the diagnosis of patients who died during the epidemic. Methodology/principal findings In this observational study, we performed MIA-US in 20 patients with suspected or confirmed YF and compared the results with those obtained in subsequent CAs. Ultrasound-guided biopsies were used for tissue sampling of liver, kidneys, lungs, spleen, and heart. Liver samples from MIA-US and CA were submitted for RT-PCR and immunohistochemistry for detection of YF virus antigen. Of the 20 patients, 17 had YF diagnosis confirmed after autopsy by histopathological and molecular analysis. There was 100% agreement between MIA-US and CA in determining the cause of death (panlobular hepatitis with hepatic failure) and main disease (yellow fever). Further, MIA-US obtained samples with good quality for molecular studies and for the assessment of the systemic involvement of the disease. Main extrahepatic findings were pulmonary hemorrhage, pneumonia, acute tubular necrosis, and glomerulonephritis. One patient was a 24-year-old, 27-week pregnant woman; MIA-US assessed the placenta and provided adequate placental tissue for analysis. Conclusions MIA-US is a reliable tool for rapid post-mortem diagnosis of yellow fever and can be used as an alternative to conventional autopsy in regions at risk for hemorrhagic fever outbreaks with limited resources to perform complete diagnostic autopsy.


Introduction
In the last decades, we have been globally identifying an unprecedented number of emerging infections [1]. As a result, considerable effort is currently being made by several actors from governmental and non-governmental institutions for better preparedness for the next expected emerging threats to public health worldwide [2][3][4]. Therefore, there is great interest in the development of tools for the early detection of infectious outbreaks for the establishment of appropriate measures [2,5].
Autopsy has been an important and indisputable diagnostic tool for the detection of novel diseases. In the past years, our group has performed autopsy studies to describe new aspects of human pathology of emerging infectious diseases, such as measles, leptospirosis, and influenza A(H1N1)pdm09 [6][7][8]. However, the global distribution of facilities capable of performing the procedure is markedly uneven. New strategies for collecting post-mortem tissue samples are necessary, particularly in areas where outbreaks of infectious diseases are occurring and where the identification of the causative agent as well as its effects on target organs is a public health priority [4,5,9]. Minimally invasive autopsy (MIA) has been proposed as an alternative to conventional autopsy, conceived as targeting small diagnostic biopsies by needle puncture of key organs, with or without the guidance of any imaging technique. The use of computed tomography (CT) and CT-angiography, associated with needle biopsy, has been proposed as feasible for diagnosis of common causes of death [10,11]. However, these techniques can only be performed in centers of excellence, requiring substantial budgets. More recently, ultrasoundguided tissue sampling (MIA-US) has also been tested, with promising results [12][13][14][15][16][17][18]. This methodology represents a portable, rapid and low-cost post-mortem technique, which may be especially useful in countries where mortality data are largely unavailable [2,[13][14][15][16][17][18].
Viral hemorrhagic fevers (VHFs) are severe viral infections that may present as hemorrhagic disease with fatal multi-organ failure. In endemic areas, they can cause long-lasting epidemics with great impact on human morbidity and mortality. Yellow fever (YF), in particular, is a re-emerging disease, endemic in tropical regions of South America and sub-Saharan Africa. It is a mosquito-borne flavivirus-induced VHF, with a high case-fatality rate, clinically manifested as hepatic dysfunction, renal failure, coagulopathy, and shock [19]. The investigation of deaths related to VHFs should put MIA-US into perspective among other potential diagnostic strategies.
From the end of 2017 to mid-May 2018, we experienced a YF epidemic in the southern region of Brazil. During this period, 498 autochthonous confirmed YF cases were registered in the state of Sao Paulo, with 176 deaths (fatality rate: 35.4%). A substantial part of the fatalities (80 cases) was referred to the autopsy service at Sao Paulo University Medical School. Aiming to provide new information on low-cost post-mortem techniques that could be applied in atrisk regions in Brazil as well as worldwide, we tested the efficacy of MIA-US in the post-mortem diagnosis of 20 patients who died with suspected or confirmed YF during the recent epidemic in Brazil.

Ethics statement
This prospective observational study was approved by the University of Sao Paulo School of Medicine Internal Review Board (CAAE protocol number: 18781813.2.0000.0068). Informed written consent was obtained from the next of kin.
From January 23, 2018 to February 27, 2018, 20 deceased patients with suspected or confirmed YF underwent MIA using ultrasound-guided percutaneous core needle biopsy. Conventional autopsy (CA) was performed subsequently by a distinct group of pathologists, blinded to the MIA-US results. Fig 1 illustrates the sequence of the autopsy procedures. All the patients included in the study fulfilled the following criteria: 1) patients died with suspected or confirmed yellow fever; 2) an autopsy was requested by the clinician; 3) death occurred during business hours, when interventional radiologists were available to perform the MIA-US; 4) informed consent to perform the autopsy was given by a family member.
The case definition of YF employed during the epidemic period was established by the Brazilian Ministry of Health and the Health Department of the State of Sao Paulo. Suspected cases referred to those patients who had a sudden onset of high fever associated with jaundice and/ or hemorrhages, who lived or had visited areas with cases of YF, YF epizootics in non-human primates, or isolation of yellow fever virus (YFV) in vectors, regardless of the vaccine status for YF, during the preceding 15 days. Confirmed cases referred to those patients who had compatible clinical presentation and laboratory confirmation by at least one of the following methods: positive serum IgM (MAC-ELISA) (performed in 10 of 20 patients); detection of YFV-RNA by RT-PCR in blood samples (performed in 17 of 20 patients); and histopathology compatible for YF hepatitis with detectable YF antigen in tissues by immunohistochemistry (wild, SP strain, hyperimmune, IAL-SP, Brazil) [20]. In all of the cases, viral hepatitis (A, B, and C) and dengue fever were excluded by serology and/or RT-PCR.

MIA-US protocol
The procedures were performed at the "Image Platform in the Autopsy Room", a research center in the University of Sao Paulo Medical School, located next to the Autopsy Service of Sao Paulo University (https://pisa.hc.fm.usp.br/). Radiologists and pathologists were aware of the information provided by the next of kin prior to autopsy.
All US examinations were performed by interventional radiology physicians, who were trained to perform gray-scale post-mortem US during a period of 8 weeks. In total, fifteen exams were necessary in order to achieve thorough ultrasound imaging and good quality biopsy samples. We used a SonoSite M-Turbo portable ultrasound (Fujifilm, Bothell, WA, USA) with broadband and multifrequency transducers: C60x (5-2 MHz Curved) and HFL38X (13-6 MHz Linear) and DICOM medical images.
Tissue samples from the liver (at least two samples), lungs (three samples from each lung), both kidneys (one sample each), spleen (one sample), and heart (one sample) were collected under ultrasound guidance using a large core (14-gauge) needle biopsy. A final MIA-US diagnosis was provided by conjunct analysis of US and biopsy data, blinded to CA results.

Conventional autopsy protocol
The autopsies were performed following the Letulle technique, where all the organs are removed en masse, requiring dissection of each organ [21]. Tissue samples were collected from all body systems. Both biopsy and autopsy samples were submitted for routine histological examination, and stained with hematoxylin-eosin (H&E). Brown-Brenn, Ziehl-Neelsen, and Grocott stains were used to detect bacteria, acid-fast bacilli, and fungi, respectively, when required.

RT-PCR protocol
Eleven liver biopsy samples and all autopsy liver samples were submitted for reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of YFV-RNA. Liver samples measuring 1 cm 3 were stored at −70˚C. The tissue was macerated, the nucleic acid extraction was performed using the TRIzol reagent (Life Technologies, Carlsbad, CA, USA), and carried out according to the manufacturer's instructions. Molecular detection of YFV was performed using the AgPath-ID One-Step RT-PCR Reagents (Ambion, Austin, TX, USA) with specific primers/probe previously described [22]. To identify cases of adverse vaccine response (i.e., fatal cases associated with the vaccine virus) we used primers/probe specific for the vaccine virus [23]. qRT-PCR reactions consisted of a step of reverse transcription at 45˚C for 10 min, enzyme activation at 95˚C for 10 min, and 40 cycles at 95˚C for 15 s and 60˚C for 45 s for

Immunohistochemistry (IHC) protocol
All biopsy and autopsy liver samples were submitted for IHC for detection of YFV antigens as previously described [24]. Briefly, after antigen retrieval using Tris-EDTA at pH 9.0, the sections were incubated overnight with the primary antibody (goat anti-human IgM polyclonal anti-arbovirus, provided by Institut Pasteur de Dakar, Dakar, Senegal, 1:20,000) at 4˚C [25]. The slides were incubated with biotinylated secondary antibody (Reveal-Biotin-Free Polyvalent HRP DAB, Spring Bioscience, cod. SPD-125) and chromogen (Dako Liquid DAB+ Substrate Cromogen System, Dako, cod. K3460) and counterstained with Harris-hematoxylin (Merck, Darmstadt, Germany). The primary antibody was tested in liver samples from patients with virus hepatitis (A, B, and C), herpes virus, cytomegalovirus, adenovirus, dengue virus, Treponema pallidum, Leptospira, and atypical mycobacteria infections, obtained from our autopsy archive. The IHC reaction resulted negative in all these samples.

Comparison between MIA-US and conventional autopsy data
We compared the MIA-US diagnosis with the CA diagnosis of the cause of death and main disease. In addition, we performed a descriptive analysis of the concordances and discrepancies between the diagnoses of MIA-US and CA in all organs analyzed.

Results
All patients who died during the yellow fever epidemic that fulfilled the inclusion criteria were included in the study (n = 20).
Of the 20 patients who died with suspected (n = 11) or confirmed YF (n = 9), 17 had their diagnosis confirmed after autopsy by histopathological and molecular analysis. A total of nine in ten patients had positive serology. Serum YFV-RT-PCR was performed in 17 patients, and 16 patients showed positive results. However, eight patients died before their serum RT-PCR results were obtained, and their diagnoses were established after autopsy.
YF was not confirmed in three patients, as described below. Interestingly, MIA-US could determine the main disease and cause of death in these three patients.
First we present the results of the 17 patients with confirmed YF.

MIA-US
Adequate biopsy samples from the liver (100%), kidneys (94%), and lungs (88%) were obtained in majority of patients. More limited samples were obtained from the spleen (82%) and heart (76%). Liver biopsies showed panlobular hepatitis with severe steatosis and midzonal apoptotic bodies, which were characteristic of fatal YF, in all samples analyzed (100%). All liver samples were submitted for IHC and 11 liver samples were submitted for RT-PCR for detection of YFV. IHC revealed positive results in 16 samples (94%), and RT-PCR revealed positive results in all 11 samples (100%).

Conventional autopsy
The cause of death in the 17 patients was hepatic failure. Associated organ-specific hemorrhages and/or hemorrhagic shock was present in 16 patients. One patient presented with septic shock.

CA and MIA-US findings in three patients in whom the diagnosis of yellow fever was not confirmed
One patient was a 19-year-old man who died due to perforated appendicitis and sepsis. Purulent fluid leaked from the abdominal cavity when the radiologist performed US-guided liver and kidney biopsies, indicating an intra-abdominal infection. Lung biopsy in this case showed secondary acute lung injury with intense pulmonary hemorrhage. One patient was an 87-yearold woman with sepsis due to bacterial pneumonia and pyelonephritis, both observed respectively on lung and kidney US-guided biopsies. The third patient was a 14-year-old boy with acute myeloid leukemia. Liver and kidney samples from MIA-US showed infiltration of atypical myeloid CD34 positive cells (blasts).

Concordances and discrepancies between MIA-US and CA in 17 FA patients
Main disease and cause of death. MIA-US and CA determined the cause of death (hepatic failure) and main disease (YF) in all 17 (100%) patients. Liver. Panlobular hepatitis was observed in all MIA-US and CA liver samples. Liver fibrosis/cirrhosis was present in two patients at CA and was not observed in the biopsies.
Lungs. Alveolar hemorrhage and pneumonia were the main pulmonary findings both at CA and MIA-US. Alveolar hemorrhage was present in 16 (94%) of 17 autopsies and in 10 (67%) of 15 lung biopsies. MIA-US was able to detect pneumonia in all nine patients, including the patient with aspergillosis. Mucorales infection (one case) and pulmonary embolism (two cases) were not observed at lung biopsy. The lung samples in two patients were not successfully obtained using MIA-US. Both of these patients presented with alveolar hemorrhage, and one presented with pulmonary embolism at CA.
Kidneys. Acute tubular necrosis (94%) and mesangial proliferative glomerulonephritis (88%) were the main kidney alterations observed at CA. Acute tubular necrosis was observed in all MIA-US kidney samples (16/16) and mesangial proliferative glomerulonephritis in 63%. Hypertensive nephropathy was present at CA in four patients; three of them were identified by kidney biopsy. Interestingly, in one patient with pyelonephritis, the etiologic agent (Aspergillus sp) was identified at MIA-US but not at CA.
Spleen. Lymphoid hypoplasia, splenitis, and cytophagocytosis were the main findings on both CA and MIA-US.
Heart. Interstitial edema and fiber hypertrophy were the main heart changes observed at CA and MIA-US. Myocardiosclerosis and myocarditis, observed at CA in 11 and 3 patients, respectively, were not detected by heart biopsies. Figs 2 and 3 illustrate the MIA-US and CA main findings in the liver and other organs, respectively.

Discussion
The present results demonstrate the effectiveness of MIA-US in the post-mortem diagnosis of the main disease (YF) and cause of death (hepatic failure) in a group of patients evaluated during the epidemic of YF that occurred in early February 2018 in Sao Paulo, Brazil. We found a 100% concordance between MIA-US and CA for the diagnosis of the main disease and cause of death, which validated our initial proposition that this diagnostic method could be a rapid and viable alternative to CA in determining post-mortem diagnosis of a VHS.
The excellent correlation between MIA-US and CA in this group of patients may be explained by some factors. First, the acknowledged accuracy of ultrasound to guide needles into the viscera affected by this viral disease, especially the liver, kidneys, and lungs. Second, important technical aspects were applied to obtain tissue samples, involving the use of the portable ultrasound equipment and Tru-Cut 14G needles. Third, procedures were performed by interventional radiologists after a training period. The ultrasound guidance, ensuring the researchers about the organ or region that underwent needle biopsies, favored good quality samples in most patients, thus allowing precise histopathologic diagnosis to be made. Technical limitations were observed in the access to the spleen and heart of some patients, due to their anatomical locations, i.e. the heart being partially obscured by the sternum and the spleen by the rib cage and intestinal gas distension. The procedure turned out to be a simple and reliable method of postmortem tissue sampling that could be applied in remote areas after adequate training.
Our results are in concert with those of other researchers [12][13][14][15][16][17][18]26,27]. In the series by Fariña et al. [12], there was an 83% diagnostic concordance between MIA-US and CA. Castilho et al. [14] reported a success rate of 83% for the determination of cause of death by MIA-US in a study of 30 patients with a predominance of infectious diseases. Recently, Martinez et al. [15] compared MIA-US and CA in 30 patients and obtained an 89.5% concordance for the detection of an infectious agent directly involved in the cause of death. Conversely, Cox et al. [13] found a 57% concordance between MIA-US and CA for infectious major diagnoses, mainly present in the lungs (63%), liver (44%), and spleen (34%). Result differences among studies possibly relate to different patient populations and different causal mixes.
Our results further showed that MIA-US is also a reliable tool to obtain samples with sufficient quality for molecular studies and microorganism identification and to assess the systemic involvement of the disease. Besides typical hepatic changes observed on histological examination, IHC and/or RT-PCR allowed definite viral detection in 94% of the patients. These results encourage further development of local technologies in endemic regions, which might provide access to real-time research data, improving epidemic preparedness.  In addition to the liver, the main affected organs at CA were the lungs and kidneys [19]. Lung biopsies showed that pulmonary hemorrhage and suppurative pneumonia were important complications of YF infection, significantly contributing to death. Besides the more prevalent bacterial pneumonia, MIA-US was able to detect severe pulmonary aspergillosis in one patient, demonstrating an immunosuppressive state associated with fatal YF. Shock-induced acute tubular necrosis was related to kidney dysfunction and could be detected in all kidney biopsies. Interestingly, in one patient with pyelonephritis, the etiologic agent (Aspergillus spp.) could be identified at MIA-US but not at CA. YF-induced glomerulonephritis could be observed in most kidney biopsies [24]. One of the 17 patients was a 24-year-old, 27-week pregnant woman. In this specific case, MIA-US was able to assess the placenta and provided adequate placental tissue for analysis. Therefore, we believe that MIA-US can be an important tool to study maternal deaths and maternal-fetal transmission.
The limitations of MIA-US in the present study were related to changes observed at autopsy that were not assessed by MIA-US. These mainly included changes related to GI tract and CNS. Shock-induced ischemic pancreatic alterations, steatonecrosis, and gastrointestinal bleeding, as well as cerebral edema, were common and important findings that certainly contributed to death. Therefore, for diseases that primarily affect the CNS and/or GI tract, strategies to include assessment of these organs should be considered.
In the past years, we have been globally identifying an unprecedented number of emerging infections [1,4,5,18]. This phenomenon is probably associated with a complex interaction of factors such as human behavior, environmental changes and vector proliferation worldwide [1]. Precise post-mortem diagnosis during the first periods of an emerging epidemic would represent an improvement in identifying the specific etiologic agent, with substantial impact in disease monitoring. For instance, it is quite plausible that if post-mortem brain sampling had been performed in the early cases of microcephaly in Northeastern Brazil, the identification of Zika virus infection could have been made earlier [28]. Moreover, protective measures could have been taken more effectively. However, such measures are hindered by the lack of autopsies in areas prone to be affected by emerging infectious agents. In addition, because of its minimal invasiveness, MIA-US may represent a safer procedure to health authorities who investigate the emerging infectious diseases of high (Ebola, for instance) or unknown lethality.
In this scenario, our results indicate that MIA-US represents a portable, low-cost post-mortem technique that can be useful for the rapid monitoring and surveillance of infectious diseases spread and outbreaks. The adoption of consolidate protocols, associated with international partnerships to use the resources of molecular diagnosis to regions where such resources are scarce, may, theoretically, expand the horizons of surveillance of global infectious threatens.
In conclusion, our results show that MIA-US is a reliable tool for the post-mortem diagnosis of YF and can be used as an alternative to conventional autopsy in regions at risk for viral hemorrhagic fever of different etiologies.