- Email: [email protected]
Investigating infectious diseases at autopsy
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
Investigating infectious diseases at autopsy
information on appropriate vaccination schedules for APTs and pathologists. This review does not include the well-known protocols for dealing with possible infection in SUDI/SIDS deaths or with the transmissible encephalopathies. Infections encountered in the UK mortuaries in all other scenarios, and how to approach their investigation are the main topic.
Sebastian Lucas
Abstract
Before the autopsy: when to suspect an infectious disease
Autopsy has a central role in diagnosing severe infectious diseases of all types: virus, prions, bacteria, mycobacteria, fungi, protozoa and helminths. The majority can be identified using histopathology e with special stains and immunohistochemistry - which is an essential part of the autopsy. In addition, cytology, microbiology culture and molecular diagnostics, serology and urine antigen testing are important adjuncts. This review encompasses a general overview of infectious autopsies, recommends working practices, and highlights the scenarios of HIV, post-transplantation and post-vaccine deaths, and rabies.
The information presented to pathologists includes e ideally the recent and past clinical history, laboratory test data, indications of immunocompromise states, and travel history. Whilst we tend to parcel patients into the binary groups of ‘normal’ and ‘immunocompromised’, in reality there is a broad spectrum. Damaged immune systems means malfunctioning innate, antibody-mediated, and cell-mediated immunities, often in combination. Table 2 lists the causes of these states, which enhance infection intensities, and alter their gross and histomorphology. Another clinical history clue to infection is a foreign travel history. ‘Under venis?’ [where have you come from?] is the question, since many severe infections cannot be acquired in north-west Europe (including leishmania and histoplasmosis). There is much available diagnostic serology for parasitic and fungal infections, which will often have been performed before death. Gathering all the available microbiology data is critical for evaluating possible infectious deaths. However, it does not generally have to be done before commencing the autopsy; the standard practice in such autopsies is to sample all relevant organs (and blood as necessary), so the pre-mortem test data can be acquired afterwards. This is important since post-mortem autolysis is the enemy of good histo-morphology, the bedrock of infection diagnostics at autopsy: do not delay the autopsy unnecessarily. The list is not exclusive, but other scenarios of death raise the probability of significant infection: intravenous drug injection habit, chronic fibrosing and industrial lung disease, people from Africa and Asia (higher risk of tuberculosis), the homeless and the emaciated. Deaths during pregnancy and around delivery are commonly related to sepsis (E. coli during the second trimester, and Strep. pyogenes in the third) and require detailed examination.3 ‘Severe sepsis’ syndromes: systemic sepsis is common, but over-diagnosed clinically. In addition to clinical suspicion, laboratory tests such as raised CRP and low platelets are evidence for sepsis. About one third of genuine sepsis cases have negative microbiology (due to prior antibiotic administration). Pathologically, systemic sepsis manifests a systemic inflammatory response syndrome (SIRS), with characteristic haemophagocytosis (in marrow and liver Kupffer cells), lymphoid atrophy, enhanced expression of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells on histology, and sometimes disseminated intravascular coagulation (DIC).4 But so do the non-infection-related autoinflammatory syndromes which are now more widely recognised.5,6 One of the commoner, causing rapid multi-organ failure from SIRS, and mimicking sepsis, is adult onset Still’s disease (AOSD). A major role for the autopsy in modern medicine is the discrimination of the common systemic
Keywords Autopsy; Cytology; Histopathology; HIV; Infections; Sepsis; Transplantation; Vaccination
Introduction Autopsy work e whether medico-legal or consented/hospital autopsies e frequently involves examination of cadavers with infectious diseases. Most of those infections encountered pose no significant risk to mortuary staff, but some are potentially serious, and a few are so virulent and dangerous that routine autopsy is effectively banned. The categorisation of infectious hazards across all areas of Medicine is regulated by the Health and Safety Executive’s (HSE) Advisory Committee on Dangerous Pathogens (ACDP).1 The schedule is regularly reviewed and updated in the light of global epidemiological trends. All agents across the globe are considered, not just those prevalent in the UK. Since international travel is the norm, people with a potentially lethal infection can now travel from any one country to any other within 24 h, and present ill or moribund to a heathcare centre; and some people die shortly after receiving travel-related vaccination. There are four Hazard Groups of infectious biological agents, categorised along three considerations: the likelihood that it will cause disease by infection or toxicity in humans how likely it is that the infection would spread to the community the availability of any prophylaxis or treatment. Table 1 presents the definitions and examples of such infections. Applying sensible universal precautions, the risk of infection with most of the HG2 & 3 agents is small during autopsy work; for further information on safe working practice in the mortuary, consult the forthcoming RCPath guideline.2 It includes
Sebastian Lucas FRCP FRCPath Emeritus Professor of Pathology, Guy’s & St Thomas’ NHS Foundation Trust, London, UK. Conflicts of interest: none declared.
DIAGNOSTIC HISTOPATHOLOGY --:-
1
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
Hazard group (HG) definitions Group 1 e Unlikely to cause human disease. Group 2 e Can cause human disease and may be a hazard to employees; it is unlikely to spread to the community and there is usually effective prophylaxis or treatment available. HG 1 & 2 are the common infections in human medicine (virus, bacteria, fungus, protozoan, helminth), as well as the more esoteric: e.g. legionellosis, nocardiosis, amoebiasis, non-tuberculosis mycobacteria. Group 3 e Can cause severe human disease and may be a serious hazard to employees; it may spread to the community, but there is usually effective prophylaxis or treatment available. The most commonly encountered are the non-A viral hepatitides, HIVs, tuberculosis and other mycobacteria, imported fungal infections such as histoplasmosis, and transmissible encephalopathies. Group 4 e Causes severe human disease and is a serious hazard to employees; it is likely to spread to the community and there is usually no effective prophylaxis or treatment available. The imported viral infections in HG 4 e such as Ebola and Lassa fevers e are only autopsied under exceptional circumstances, and are not considered further here. Table 1
sepsis deaths from the rarer autoinflammatory syndromes; it is important to feed back these diagnoses to the treating doctors, for their audit and practice improvement. This requires close attention to the histopathology of SIRS, particularly the presence of haemophagocytosis (Figure 1). A final note on cadaveric imaging: in the realm of infectious diseases: primary diagnostic cadaveric imaging has no role to play. Pre-death imaging provides clues to undiagnosed significant infections, but there is no infection scenario where a
cadaveric CT or MRI scan can diagnose or exclude a particular infection.
Pathology encountered at autopsy This review is cannot depict in detail the pathology of all the infections encountered at autopsy. It highlights some of the gross and histo-morphological features of infections that cause diagnostic problems in the UK. External findings suggesting an infection Skin and subcutis: a generalised erythematous rash (common in bacteraemias) or petechial haemorrhages (vasculitis or
The aetiologies of immunocompromised states 1. HIV-related impairment of cell-mediated immunity (CMI) 2. Congenital immune disorders: a Chronic granulomatous disease b Hyper-IgE syndrome c Severe combined immunodeficiency d X-linked hyper-IgM syndrome e Wiskott-Aldrich syndrome f Di George syndrome g Common variable immunodeficiency h Defects in the interferon-g-interleukin -12 axis i Myeloperoxidase deficiency 3. Burns 4. Diabetes mellitus 5. Sickle cell disease (HbSS and HbSC genotypes) 6. Cystic fibrosis 7. Chronic renal failure and dialysis 8. Liver cirrhosis of any cause 9. Malignancies per se, e.g. leukaemia and lymphoma 10. Iatrogenic damage to the immune system: a Cancer chemotherapy treatments b Transplantation and anti-rejection regimes i Solid organ ii Bone marrow allograft c Specific immunosuppressive therapy (e.g. anti-TNF) d Steroid therapy e Intensive care organ support systems, including vascular catheter lines and ECMO 11. Old age, with resultant impaired CMI
Figure 1 Haemophagocytosis in bone marrow highlighted by CD68 macrophage stain e this is a constant feature of SIRS.
Table 2
DIAGNOSTIC HISTOPATHOLOGY --:-
2
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
infarction); ulceration and swelling, with or without pus or underlying osteomyelitis; necrotising fasciitis.
Miliary nodules in the lung C
Internal findings at autopsy Few naked eye organ appearances at autopsy are pathognomonic of a specific infection at autopsy: these include Candida oropharyngitis, subcutaneous mycetoma, invasive ascariasis, gas gangrene of a limb (Clostridium perfringens), and classical hydatid cyst (Echinococcus granulosus) (Figure 2). Brain: swelling and congestion without known hypoxicischaemic injury suggests viral or protozoal encephalitis; milky, granular or yellow meningeal exudate suggests meninigitis (if it turns yellow on exposure to air, it is probably pneumococcal meningitis); a dusky dark red or gray brain suggest malaria; a ‘swiss-cheese’ holey brain suggests cryptococcosis; Chest: purulent pleurisy, and pericarditis; consolidated lung lobes suggest bacterial infections; focal haemorrhagic necroses in the lung suggest aspergillosis, as do mouldy balls in a cavity; anthrax is suggested by mediastinal haemorrhage and oedema; Abdomen: purulent peritonitis indicates perforation of gut, spread from an adjacent organ or primary bacterial peritonitis; Liver: macronodular cirrhosis may indicate viral hepatitis; submassive necrosis can be from hepatitis, yellow fever (or often of uncategorisable causation); focal lesions are bacterial abscesses (including Actinomyces) or possibly worm lesions (e.g. Fasciola); Intestines: distention, discolouration, inflammation and ulceration of small or large bowel may be typhoid, salmonellosis or shigellosis; advanced amoebiasis has undermining large bowel ulcers, but early lesions are merely aphthoid mucosal erosions; Spleen and lymph nodes when enlarged may represent early HIV infection, and as do large white pulp follicles; Miliary necrotic nodules have many causes. Table 3 lists those found in the lung at autopsy.
C C
C C C C C C C
Table 3
Otherwise, political and public health confidence in vaccination programs may be lost. HG3 vaccine-related infections include tuberculosis, rabies, and yellow fever; there may also be deaths following vaccination against numerous HG2 agents such as mumps, rubella, influenza, pertussis. BCG vaccine for tuberculosis causes disseminated infection in immunocompromised subjects. Rabies vaccine has no apparent association with post-vaccine mortality. Yellow fever vaccine death has become more important as more people outside the standard tropical endemic zones are vaccinated, and has occurred in the UK. As with any infection autopsy, a clear appraisal of the questions being asked concerning the death is required. Is the person seriously considered to have died of a vaccine-related complication, specifically dissemination of the infective vaccine? What is the pattern of clinical pathology produced by that vaccine? Is the time period since vaccination and reported clinical scenario consistent with a vaccine effect? What co-morbidities might or does the patient have? What other diseases are likely to have killed the patient? A protocol for addressing such deaths depends on the nature of the vaccine and target organs. Full organ sampling is essential, and e critically e retention of frozen tissue samples and frozen spun autopsy blood, so that biological investigations can be pursued later if required. Thus for a suspected yellow fever vaccine fatality, a sample of liver must be retained frozen.
Specific infection scenarios evaluated at autopsy Post-vaccination fatality Immunisations are not 100% safe; morbidity and some deaths inevitably follow. It is important that such deaths are investigated with the utmost rigour to determine, as far as is possible the contribution of the recent vaccination to the death.
HIV infection and autopsy pathology HIV disease occupies a special place in autopsy pathology, since it is a relatively recent phenomenon and, in the early days, autopsy was a major source of clinico-pathological information about what HIV can do to people. HIV has had a profound impact on autopsy practice in areas with significant infection prevalence, through the perceptions of the risks involved. Moreover with the advent of effective combination anti-retroviral infection (after 1996), the pathology encountered at autopsy has changed significantly. The cadavers of untreated patients can be highly infectious, with millions of viral particles per millilitre of blood, and thus highly infectious. Pathologists have been known to cut themselves and become infected. Previously, most deaths in people with HIV involved opportunistic infections and tumours. Now, those on combination
Figure 2 Classical hydatid cyst (E.granulosus) in mesentery causing intestinal obstruction e this is pathognomonic.
DIAGNOSTIC HISTOPATHOLOGY --:-
Tuberculosis & other mycobacteria Nocardia Fungi e aspergillus, cryptococcus, coccidiodomycosis, pneumocystis, histoplasma Viruses e HSV, VZV Protozoa e toxoplasmosis (Figure 3) Helminths e schistosomiasis, strongyloides Tumour e adenocarcinoma, lymphoma etc Reactive lymphoid nodules in HIV (Figure 4) Sarcoidosis Intravenous injected foreign material
3
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
Figure 3 (a) Miliary toxoplasma nodules in lung in HIV disease; (b) shows immunostained zoites.
antiretroviral therapy (cART) are mostly ‘well-controlled’, with undetectable viral loads in body fluids, i.e. they are effectively non-infectious. Simultaneously the range of observed pathologies has become both easier to evaluate at autopsy and more difficult. The majority of those with HIV infection now die from nonHIV related disease: cardiovascular atheroma and stroke, adenocarcinoma of lung and liver, lymphoma, COPD, accidents, suicide and drug overdose. A minority are complicated, involving cART toxicity to organs, IRIS (the immune reconstitution inflammatory syndrome), direct HIV damage to organs, and encephalopathy such as CD8 þ T-cell encephalitis e it is recommended that such HIV deaths are referred directly to specialist pathologists, or at least are sampled extensively for later review by experts. If a cadaver at autopsy is suspected to have HIV infection, and there is no known in-life diagnosis, there are two complementary means of confirming the infection: serology, and immunohistochemistry (IHC) on tissue samples. Autopsy blood can be tested for HIV antibodies, and the results are specific and sensitive providing the delay time from death to autopsy is not prolonged. Should tissue histopathology suggest HIV infection in a case, and no blood is available for testing e either from the autopsy or retained from life in a laboratory e application of immunohistochemistry with anti-HIVp24 antibodies is effective and specific as long as the viral load in the tissues is high. Both solid tissue (Figure 4) and dab imprints can be so immunostained. Rabies Suspected rabies fatalities happen in the UK once every 5e10 years, and these usually require autopsy to confirm the diagnosis. Any APT and pathologist performing the autopsy, and reconstructing the body, must have received prior rabies vaccination. The critical organs are the heart and the brain. The heart is sampled in the standard manner, and formalinfixed blocks taken. The brain should be fixed whole, but only after fresh samples of cerebellum and temporal cortex have been taken for rapid immunodiagnosis; the rabies reference centres,
DIAGNOSTIC HISTOPATHOLOGY --:-
Figure 4 (a) Lung nodules in HIV infection; (b) shows virus using antiHIVp24 immunostain in the lymphoid nodule; the other nodule, with the red centre, is a pneumocystis granuloma. H&E stain.
related to the Public Health laboratories will collect these tissues and perform the tests. Histologically, the brain shows a variable encephalitis, and the characteristic Negri bodies in cortical and cerebellar neurones (Figure 5).
4
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
(see above). Thus the identification of infections at autopsy requires histopathology e the bedrock of all diagnostics e augmented by other methods such as cytology, serology, urine antigen testing, standard microbiology culture, and molecular techniques (PCR etc). Electron microscopy is now rarely used at autopsy. Histopathology alone e formalin-fixed and paraffinembedded (FFPE) - can identify a large proportion of the infections encountered in tissues to the genus level, and often to the species. When doing special stains, it is recommended to use a gram stain, a mycobacterial stain (Ziehl-Neelsen) and a fungal stain (Grocott silver is more sensitive than PAS) as the routine starting point. Immunohistochemistry against infectious agents is increasingly available, as is in situ hybridisation technology. If mycobacteria are present, their morphology does not indicate the species (with the exception of the large beaded Mycobacterium kansasii e Figure 7), but the host cell reaction often does.
Figure 5 Rabies encephalitis e the eosinophilic Negri bodies are seen in the hippocampal neurones’ cytoplasm. H&E stain.
Cytology Dab cytology is an underused technique in autopsy practice. It is useful for identifying tumours and infections; in general, tumours stain well with Papanicolau method on alcohol-fixed preparations, whilst for infections, the Giemsa method is best, using airdried slides. Fungi, mycobacteria and Gram þve bacteria are readily identified using special stains on air-dried slides (Figure 8). Depending on the pathologists’ skill, discrimination between common bacteria such as Strep. pneumoniae, Strep. pyogenes and Staphylococcus aureus is possible. The related technique of crush-and-drag brain tissue smear has been used for decades to identify or exclude falciparum cerebral malaria. Because it is rapid and cheap, and is not generally regarded as ‘tissue retention’ in the usual sense, it can be an alternative to histology. In cases of possible lung pneumonia or bronchopneumonia, where the sensitivity and specificity of naked eye diagnosis is lamentable, a dab preparation provides confirmation or exclusion of infection: if there are no polymorphs in the cytopreparation, there is no infection.
Post-transplant infectious deaths The infection risks for patients with bone marrow and solidorgan transplants are two fold. There is the necessary immunosuppressive therapy, which predisposes to reactivation of latent infections (viral and mycobacterial) and to acquisition of new infections (nosocomial) from the hospital staff and environment. Secondly, infections are transmitted in the donor tissue. As well as the standard common such agents such as hepatitis viruses, HIV, CMV and Epstein-Barr virus, there are uncommon potentially lethal infections, listed in Table 4. Often, it is at autopsy that organ-transmitted infections are diagnosed. It is vital that these are documented accurately, to feed the information back into the transplantation networks and so help to improve outcomes in the future.
Investigations on autopsy material Histopathology The autopsy in suspected infectious disease requires a 100% histology sampling rate. Very few invasive infections are identified at the genus or species level from naked eye appearance only
Documented unusual infections transmissible in solid organ transplantation C C
C
C
Prion: TSE/CJD Viruses C Rabies virus C West Nile virus C Lymphocytic choriomeningitis (LCM) Helminths C Strongyloides stercoralis C Halicephalobus (Figure 6) Protozoa C Microsporidiosis C Trypanosoma cruzi C Free-living amoebae - Balamuthia Figure 6 Halicephalobus nematode larvae in the brain e transmitted accidentally by an infected renal transplant. H&E stain.
Table 4
DIAGNOSTIC HISTOPATHOLOGY --:-
5
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
Figure 8 Cytological dab imprint diagnosis of cerebral Cryptococcus neoformans e Giemsa stain highlights the clear space outside the yeast cell.
Figure 7 (a) Lung necrotic zone with (b) large beaded Mycobacterium kansasii bacilli e due to chronic industrial lung disease. Ziehl-Neelsen stain. Figure 9 Small bowel mucosa with macrophages containing bacilli e PCR on the fixed tissue identified these as Shigella spp. H&E stain.
Microbiology e bacteriology, virology, mycology & molecular The standard culture methods are progressively being replaced by modern molecular techniques (PCR and variants), making identification of agents quicker and more sensitive. Autopsy samples should be taken as cleanly as possible, and sent fresh to the laboratory. In influenza outbreaks, the use of the specific nasal sampling brushes is recommended, as they are designed for direct insertion into the laboratory equipment. Obviously, the results of culture and molecular identification need to be correlated with the clinical and pathological information for consistency. If, for example, a PCR result indicates the presence of a specific infection when the histology firmly identifies a different agent, the PCR data need to be treated with caution: morphology trumps molecular. That said, the results of PCR diagnostics for fungal infections are excellent, and they are improving constantly for bacterial infections, even in FFPE (Figure 9). Blood and cerebrospinal fluid (CSF) cultures are invaluable in systemic infections. Blood must be taken from above the navel and before opening the body, to avoid faecal contamination: the neck veins and heart chambers are good sites. CSF is most easily
DIAGNOSTIC HISTOPATHOLOGY --:-
obtained from the cisterna magna (inserting the needle below the occipital bone process). Serology Whilst autopsy blood is not ideal for serodiagnostics due to haemolysis and other post-mortem changes, it does work. The local virology and immunology laboratories can help, and for possible HG4 infections or rarer imported HG3 infections, the central reference centres will assist. My personal uses of this method are to confirm or exclude HIV infection in a cadaver (as part of the diagnostic process), and to exclude a helminth infection. Urine antigen testing Microbiology laboratories perform urine antigen testing regularly to confirm or exclude certain infections in the living; the tests that are available everywhere are for pneumococcus and legionella. Others e including for fungal infections - are being developed.
6
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
Specific histopathology practice points in infection diagnosis Using a standard trio of special stains - Gram, Ziehl-Neelsen (ZN), Grocott silver e ensures that the obvious agents are covered. For example, in immunocompromised tuberculosis, there are no granulomas, only amorphous necrosis, and the diagnosis could be missed, unless a Z-N stain is done to reveal the huge numbers of bacilii present (Figure 10). Further investigation of undiagnosed necrotic lesions can include IHC for toxoplasmosis and herpes (Figure 11), both sometime causes of occult tissue necrosis. For bacteria, the most useful IHC reagent is for syphilis spirochaetes; much more sensitive and easier to prepare and read than the old Warthin-Starry stain. One reason for always using the Grocott stain (rather than PAS) is its ability to highlight Nocardia bacteria in abscesses and granulomas (Figure 12). The Grocott is also more precise in visualising fungal hyphae and yeasts and usually suffices in indicating the genus diagnosis in mycotic infections.7 The travel history is helpful: for example, people from middle and southern USA frequently have latent coccidiodomycosis or histoplasmosis, which can reactivate and cause death many years later.
Figure 12 Nocardiosis in the lung e the silver stain makes the branching bacteria easy to see. Grocott silver stain.
Viral inclusion bodies: the list includes the herpes viruses (cytomegalovirus, herpes simplex viruses), measles, JC (Figure 13) and BK viruses, adenovirus, parvo/B19 virus, and rabies (Figure 5). It is wise to apply a panel of IHC reagents to determine which is present. The standard antibody for JC is SV40, which also stains BK virus. Diagnosing encephalitis relies on this methodology. Protozoal infections include amoebiasis, malaria and a large number of other ‘small blue dot’ infectious agents. Amoebae of Entamoeba histolytica are strongly PAS positive, and the cytoplasm contains phagocytosed red cells. If these features are absent, the cells are likely to be macrophages. The ‘small blue dots’ infections of red blood cells are the malarias (always imported e Figure 14) and babesiosis (endemic in UK). The other small protozoa are Toxoplasma gondii, Leishmania spp, Isospora belli, Trypanosoma cruzi, and the microsporidia. IHC is very helpful in identifying toxoplasmosis, staining the zoites and cysts in the brain and other locations. If leishmaniasis is suspected, stain the section with Grocott silver to make sure that it is not Histoplasma capsulatum instead (and vice versa). Some worms e seen as adults, larvae and eggs e are easily recognised (see Figure 2); others require help from more experienced pathologists or a parasitologist (see Figure 6). Strongyloides stercoralis disseminated hyperinfection is a particularly
Figure 10 Non-reactive tuberculosis in advanced HIV disease e this concentrated mass of mycobacteria appeared as necrosis on H&E stains. Ziehl-Neelsen stain.
Figure 13 Viral inclusion bodies e JC virus encephalitis. The bizarre astrocytes and the purple intranuclear inclusion bodies are characteristic. H&E stain.
Figure 11 Mass necrosis of lung due to herpes simplex 1 infection e anti-HSV immunohistochemistry.
DIAGNOSTIC HISTOPATHOLOGY --:-
7
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
REFERENCES 1 HSE. The approved list of biological agents - health and safety executive. www.hse.gov.uk/pubns/misc208.pdf. 2 Royal College of Pathologists. Guidelines on autopsy practice: precautions for high-risk infectious autopsies. In preparation, for publication in 2019. 3 Lucas SB. The maternal death autopsy. In: Pignatelli M, Gallagher P, eds. Recent advances in histopathology, vol. 23. London: JP Medical Pulishers, 2014; 17e30. 4 Lucas SB. The role of the autopsy in the diagnosis of sepsis and related fatal syndromes. In: Flanagan AM, ed. Recent advances in histopathology, vol. 24. London: JP Medical Publishers, 2016; 115e29. 5 Ramos-Casals M, Brita-Zeron P, Lopez-Gjuillermo A, Khamashtra MA, Bosch X. Adult haemophagocytic syndrome. Lancet 2014; 383: 1503e16. 6 Pathak S, McDermott MF, Savic S. Autoinflammatory diseases: update on classification diagnosis and management. J Clin Pathol 2017; 70: 1e8. 7 Lucas SB. Histopathology of fungal diseases. In: Kibbler C, Barton R, Gow NAR, Howell S, MacCallum DM, Manuel RJ, eds. Oxford textbook of medical mycology. Oxford: OUP, 2018; 289e97 [chapter 40].
Figure 14 Cerebral malaria e abundant parasites in the capillary red cells. H&E stain.
lethal infection in people with T-cell lymphoma, HTLV-1 infection, or on steroid therapy; they may have been infected recently or decades ago. The characteristic larvae are seen in the gut mucosa, lung and elsewhere. An uncommon problem is finding a large eosinophilic necrotic lesion, e.g. in liver, with a possible degenerate worm within. The causes of these lesions do include worms (Fasciola, Toxocara canis); more frequently they are allergic reactions to drugs, or a lymphoma, or part of a hypereosinophilia syndrome. Serology on autopsy blood to look for worm antibodies is helpful.A
Acknowledgements Most of the information presented in this review comes from personal experience and reflection since 1976, bolstered by discussion with my departmental colleagues at St Thomas’ who have also taken up infectious disease morbid anatomy and histopathology. I gratefully acknowledge: the essential role of the many coroners who asked me and my colleagues at St Thomas’ to investigate infection-related deaths in their jurisdictions - crucially, they never placed obstacles in the path of fully investigating the deaths with histopathology and microbiology; my UK clinical infectious disease colleagues who organised consented autopsies following deaths under their care; the large-scale autopsy-based collaborative research projects undertaken in several countries in Africa; and very importantly, all the pathology colleagues in UK and elsewhere who send us case consultations on their infection autopsy cases. I have no funding sources to aknowledge.
Practice points C C
C
C
C
Sample everything for cellular pathology microscopy Take blood cultures in all cases of possible systemic sepsis; and seek out all pre-mortem microbiology test results Apply the standard trio of stains: Gram, Ziehl-Neelsen, Grocott silver to lesions In systemic ? sepsis syndromes, consider the autoinflammatory syndromes when investigations for an infectious agent are negative When you are baffled, seek expert pathology consultation help; also cultivate the local infectious disease networks for clinical and laboratory advice.
DIAGNOSTIC HISTOPATHOLOGY --:-
8
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
Investigating infectious diseases at autopsy
information on appropriate vaccination schedules for APTs and pathologists. This review does not include the well-known protocols for dealing with possible infection in SUDI/SIDS deaths or with the transmissible encephalopathies. Infections encountered in the UK mortuaries in all other scenarios, and how to approach their investigation are the main topic.
Sebastian Lucas
Abstract
Before the autopsy: when to suspect an infectious disease
Autopsy has a central role in diagnosing severe infectious diseases of all types: virus, prions, bacteria, mycobacteria, fungi, protozoa and helminths. The majority can be identified using histopathology e with special stains and immunohistochemistry - which is an essential part of the autopsy. In addition, cytology, microbiology culture and molecular diagnostics, serology and urine antigen testing are important adjuncts. This review encompasses a general overview of infectious autopsies, recommends working practices, and highlights the scenarios of HIV, post-transplantation and post-vaccine deaths, and rabies.
The information presented to pathologists includes e ideally the recent and past clinical history, laboratory test data, indications of immunocompromise states, and travel history. Whilst we tend to parcel patients into the binary groups of ‘normal’ and ‘immunocompromised’, in reality there is a broad spectrum. Damaged immune systems means malfunctioning innate, antibody-mediated, and cell-mediated immunities, often in combination. Table 2 lists the causes of these states, which enhance infection intensities, and alter their gross and histomorphology. Another clinical history clue to infection is a foreign travel history. ‘Under venis?’ [where have you come from?] is the question, since many severe infections cannot be acquired in north-west Europe (including leishmania and histoplasmosis). There is much available diagnostic serology for parasitic and fungal infections, which will often have been performed before death. Gathering all the available microbiology data is critical for evaluating possible infectious deaths. However, it does not generally have to be done before commencing the autopsy; the standard practice in such autopsies is to sample all relevant organs (and blood as necessary), so the pre-mortem test data can be acquired afterwards. This is important since post-mortem autolysis is the enemy of good histo-morphology, the bedrock of infection diagnostics at autopsy: do not delay the autopsy unnecessarily. The list is not exclusive, but other scenarios of death raise the probability of significant infection: intravenous drug injection habit, chronic fibrosing and industrial lung disease, people from Africa and Asia (higher risk of tuberculosis), the homeless and the emaciated. Deaths during pregnancy and around delivery are commonly related to sepsis (E. coli during the second trimester, and Strep. pyogenes in the third) and require detailed examination.3 ‘Severe sepsis’ syndromes: systemic sepsis is common, but over-diagnosed clinically. In addition to clinical suspicion, laboratory tests such as raised CRP and low platelets are evidence for sepsis. About one third of genuine sepsis cases have negative microbiology (due to prior antibiotic administration). Pathologically, systemic sepsis manifests a systemic inflammatory response syndrome (SIRS), with characteristic haemophagocytosis (in marrow and liver Kupffer cells), lymphoid atrophy, enhanced expression of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells on histology, and sometimes disseminated intravascular coagulation (DIC).4 But so do the non-infection-related autoinflammatory syndromes which are now more widely recognised.5,6 One of the commoner, causing rapid multi-organ failure from SIRS, and mimicking sepsis, is adult onset Still’s disease (AOSD). A major role for the autopsy in modern medicine is the discrimination of the common systemic
Keywords Autopsy; Cytology; Histopathology; HIV; Infections; Sepsis; Transplantation; Vaccination
Introduction Autopsy work e whether medico-legal or consented/hospital autopsies e frequently involves examination of cadavers with infectious diseases. Most of those infections encountered pose no significant risk to mortuary staff, but some are potentially serious, and a few are so virulent and dangerous that routine autopsy is effectively banned. The categorisation of infectious hazards across all areas of Medicine is regulated by the Health and Safety Executive’s (HSE) Advisory Committee on Dangerous Pathogens (ACDP).1 The schedule is regularly reviewed and updated in the light of global epidemiological trends. All agents across the globe are considered, not just those prevalent in the UK. Since international travel is the norm, people with a potentially lethal infection can now travel from any one country to any other within 24 h, and present ill or moribund to a heathcare centre; and some people die shortly after receiving travel-related vaccination. There are four Hazard Groups of infectious biological agents, categorised along three considerations: the likelihood that it will cause disease by infection or toxicity in humans how likely it is that the infection would spread to the community the availability of any prophylaxis or treatment. Table 1 presents the definitions and examples of such infections. Applying sensible universal precautions, the risk of infection with most of the HG2 & 3 agents is small during autopsy work; for further information on safe working practice in the mortuary, consult the forthcoming RCPath guideline.2 It includes
Sebastian Lucas FRCP FRCPath Emeritus Professor of Pathology, Guy’s & St Thomas’ NHS Foundation Trust, London, UK. Conflicts of interest: none declared.
DIAGNOSTIC HISTOPATHOLOGY --:-
1
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
Hazard group (HG) definitions Group 1 e Unlikely to cause human disease. Group 2 e Can cause human disease and may be a hazard to employees; it is unlikely to spread to the community and there is usually effective prophylaxis or treatment available. HG 1 & 2 are the common infections in human medicine (virus, bacteria, fungus, protozoan, helminth), as well as the more esoteric: e.g. legionellosis, nocardiosis, amoebiasis, non-tuberculosis mycobacteria. Group 3 e Can cause severe human disease and may be a serious hazard to employees; it may spread to the community, but there is usually effective prophylaxis or treatment available. The most commonly encountered are the non-A viral hepatitides, HIVs, tuberculosis and other mycobacteria, imported fungal infections such as histoplasmosis, and transmissible encephalopathies. Group 4 e Causes severe human disease and is a serious hazard to employees; it is likely to spread to the community and there is usually no effective prophylaxis or treatment available. The imported viral infections in HG 4 e such as Ebola and Lassa fevers e are only autopsied under exceptional circumstances, and are not considered further here. Table 1
sepsis deaths from the rarer autoinflammatory syndromes; it is important to feed back these diagnoses to the treating doctors, for their audit and practice improvement. This requires close attention to the histopathology of SIRS, particularly the presence of haemophagocytosis (Figure 1). A final note on cadaveric imaging: in the realm of infectious diseases: primary diagnostic cadaveric imaging has no role to play. Pre-death imaging provides clues to undiagnosed significant infections, but there is no infection scenario where a
cadaveric CT or MRI scan can diagnose or exclude a particular infection.
Pathology encountered at autopsy This review is cannot depict in detail the pathology of all the infections encountered at autopsy. It highlights some of the gross and histo-morphological features of infections that cause diagnostic problems in the UK. External findings suggesting an infection Skin and subcutis: a generalised erythematous rash (common in bacteraemias) or petechial haemorrhages (vasculitis or
The aetiologies of immunocompromised states 1. HIV-related impairment of cell-mediated immunity (CMI) 2. Congenital immune disorders: a Chronic granulomatous disease b Hyper-IgE syndrome c Severe combined immunodeficiency d X-linked hyper-IgM syndrome e Wiskott-Aldrich syndrome f Di George syndrome g Common variable immunodeficiency h Defects in the interferon-g-interleukin -12 axis i Myeloperoxidase deficiency 3. Burns 4. Diabetes mellitus 5. Sickle cell disease (HbSS and HbSC genotypes) 6. Cystic fibrosis 7. Chronic renal failure and dialysis 8. Liver cirrhosis of any cause 9. Malignancies per se, e.g. leukaemia and lymphoma 10. Iatrogenic damage to the immune system: a Cancer chemotherapy treatments b Transplantation and anti-rejection regimes i Solid organ ii Bone marrow allograft c Specific immunosuppressive therapy (e.g. anti-TNF) d Steroid therapy e Intensive care organ support systems, including vascular catheter lines and ECMO 11. Old age, with resultant impaired CMI
Figure 1 Haemophagocytosis in bone marrow highlighted by CD68 macrophage stain e this is a constant feature of SIRS.
Table 2
DIAGNOSTIC HISTOPATHOLOGY --:-
2
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
infarction); ulceration and swelling, with or without pus or underlying osteomyelitis; necrotising fasciitis.
Miliary nodules in the lung C
Internal findings at autopsy Few naked eye organ appearances at autopsy are pathognomonic of a specific infection at autopsy: these include Candida oropharyngitis, subcutaneous mycetoma, invasive ascariasis, gas gangrene of a limb (Clostridium perfringens), and classical hydatid cyst (Echinococcus granulosus) (Figure 2). Brain: swelling and congestion without known hypoxicischaemic injury suggests viral or protozoal encephalitis; milky, granular or yellow meningeal exudate suggests meninigitis (if it turns yellow on exposure to air, it is probably pneumococcal meningitis); a dusky dark red or gray brain suggest malaria; a ‘swiss-cheese’ holey brain suggests cryptococcosis; Chest: purulent pleurisy, and pericarditis; consolidated lung lobes suggest bacterial infections; focal haemorrhagic necroses in the lung suggest aspergillosis, as do mouldy balls in a cavity; anthrax is suggested by mediastinal haemorrhage and oedema; Abdomen: purulent peritonitis indicates perforation of gut, spread from an adjacent organ or primary bacterial peritonitis; Liver: macronodular cirrhosis may indicate viral hepatitis; submassive necrosis can be from hepatitis, yellow fever (or often of uncategorisable causation); focal lesions are bacterial abscesses (including Actinomyces) or possibly worm lesions (e.g. Fasciola); Intestines: distention, discolouration, inflammation and ulceration of small or large bowel may be typhoid, salmonellosis or shigellosis; advanced amoebiasis has undermining large bowel ulcers, but early lesions are merely aphthoid mucosal erosions; Spleen and lymph nodes when enlarged may represent early HIV infection, and as do large white pulp follicles; Miliary necrotic nodules have many causes. Table 3 lists those found in the lung at autopsy.
C C
C C C C C C C
Table 3
Otherwise, political and public health confidence in vaccination programs may be lost. HG3 vaccine-related infections include tuberculosis, rabies, and yellow fever; there may also be deaths following vaccination against numerous HG2 agents such as mumps, rubella, influenza, pertussis. BCG vaccine for tuberculosis causes disseminated infection in immunocompromised subjects. Rabies vaccine has no apparent association with post-vaccine mortality. Yellow fever vaccine death has become more important as more people outside the standard tropical endemic zones are vaccinated, and has occurred in the UK. As with any infection autopsy, a clear appraisal of the questions being asked concerning the death is required. Is the person seriously considered to have died of a vaccine-related complication, specifically dissemination of the infective vaccine? What is the pattern of clinical pathology produced by that vaccine? Is the time period since vaccination and reported clinical scenario consistent with a vaccine effect? What co-morbidities might or does the patient have? What other diseases are likely to have killed the patient? A protocol for addressing such deaths depends on the nature of the vaccine and target organs. Full organ sampling is essential, and e critically e retention of frozen tissue samples and frozen spun autopsy blood, so that biological investigations can be pursued later if required. Thus for a suspected yellow fever vaccine fatality, a sample of liver must be retained frozen.
Specific infection scenarios evaluated at autopsy Post-vaccination fatality Immunisations are not 100% safe; morbidity and some deaths inevitably follow. It is important that such deaths are investigated with the utmost rigour to determine, as far as is possible the contribution of the recent vaccination to the death.
HIV infection and autopsy pathology HIV disease occupies a special place in autopsy pathology, since it is a relatively recent phenomenon and, in the early days, autopsy was a major source of clinico-pathological information about what HIV can do to people. HIV has had a profound impact on autopsy practice in areas with significant infection prevalence, through the perceptions of the risks involved. Moreover with the advent of effective combination anti-retroviral infection (after 1996), the pathology encountered at autopsy has changed significantly. The cadavers of untreated patients can be highly infectious, with millions of viral particles per millilitre of blood, and thus highly infectious. Pathologists have been known to cut themselves and become infected. Previously, most deaths in people with HIV involved opportunistic infections and tumours. Now, those on combination
Figure 2 Classical hydatid cyst (E.granulosus) in mesentery causing intestinal obstruction e this is pathognomonic.
DIAGNOSTIC HISTOPATHOLOGY --:-
Tuberculosis & other mycobacteria Nocardia Fungi e aspergillus, cryptococcus, coccidiodomycosis, pneumocystis, histoplasma Viruses e HSV, VZV Protozoa e toxoplasmosis (Figure 3) Helminths e schistosomiasis, strongyloides Tumour e adenocarcinoma, lymphoma etc Reactive lymphoid nodules in HIV (Figure 4) Sarcoidosis Intravenous injected foreign material
3
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
Figure 3 (a) Miliary toxoplasma nodules in lung in HIV disease; (b) shows immunostained zoites.
antiretroviral therapy (cART) are mostly ‘well-controlled’, with undetectable viral loads in body fluids, i.e. they are effectively non-infectious. Simultaneously the range of observed pathologies has become both easier to evaluate at autopsy and more difficult. The majority of those with HIV infection now die from nonHIV related disease: cardiovascular atheroma and stroke, adenocarcinoma of lung and liver, lymphoma, COPD, accidents, suicide and drug overdose. A minority are complicated, involving cART toxicity to organs, IRIS (the immune reconstitution inflammatory syndrome), direct HIV damage to organs, and encephalopathy such as CD8 þ T-cell encephalitis e it is recommended that such HIV deaths are referred directly to specialist pathologists, or at least are sampled extensively for later review by experts. If a cadaver at autopsy is suspected to have HIV infection, and there is no known in-life diagnosis, there are two complementary means of confirming the infection: serology, and immunohistochemistry (IHC) on tissue samples. Autopsy blood can be tested for HIV antibodies, and the results are specific and sensitive providing the delay time from death to autopsy is not prolonged. Should tissue histopathology suggest HIV infection in a case, and no blood is available for testing e either from the autopsy or retained from life in a laboratory e application of immunohistochemistry with anti-HIVp24 antibodies is effective and specific as long as the viral load in the tissues is high. Both solid tissue (Figure 4) and dab imprints can be so immunostained. Rabies Suspected rabies fatalities happen in the UK once every 5e10 years, and these usually require autopsy to confirm the diagnosis. Any APT and pathologist performing the autopsy, and reconstructing the body, must have received prior rabies vaccination. The critical organs are the heart and the brain. The heart is sampled in the standard manner, and formalinfixed blocks taken. The brain should be fixed whole, but only after fresh samples of cerebellum and temporal cortex have been taken for rapid immunodiagnosis; the rabies reference centres,
DIAGNOSTIC HISTOPATHOLOGY --:-
Figure 4 (a) Lung nodules in HIV infection; (b) shows virus using antiHIVp24 immunostain in the lymphoid nodule; the other nodule, with the red centre, is a pneumocystis granuloma. H&E stain.
related to the Public Health laboratories will collect these tissues and perform the tests. Histologically, the brain shows a variable encephalitis, and the characteristic Negri bodies in cortical and cerebellar neurones (Figure 5).
4
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
(see above). Thus the identification of infections at autopsy requires histopathology e the bedrock of all diagnostics e augmented by other methods such as cytology, serology, urine antigen testing, standard microbiology culture, and molecular techniques (PCR etc). Electron microscopy is now rarely used at autopsy. Histopathology alone e formalin-fixed and paraffinembedded (FFPE) - can identify a large proportion of the infections encountered in tissues to the genus level, and often to the species. When doing special stains, it is recommended to use a gram stain, a mycobacterial stain (Ziehl-Neelsen) and a fungal stain (Grocott silver is more sensitive than PAS) as the routine starting point. Immunohistochemistry against infectious agents is increasingly available, as is in situ hybridisation technology. If mycobacteria are present, their morphology does not indicate the species (with the exception of the large beaded Mycobacterium kansasii e Figure 7), but the host cell reaction often does.
Figure 5 Rabies encephalitis e the eosinophilic Negri bodies are seen in the hippocampal neurones’ cytoplasm. H&E stain.
Cytology Dab cytology is an underused technique in autopsy practice. It is useful for identifying tumours and infections; in general, tumours stain well with Papanicolau method on alcohol-fixed preparations, whilst for infections, the Giemsa method is best, using airdried slides. Fungi, mycobacteria and Gram þve bacteria are readily identified using special stains on air-dried slides (Figure 8). Depending on the pathologists’ skill, discrimination between common bacteria such as Strep. pneumoniae, Strep. pyogenes and Staphylococcus aureus is possible. The related technique of crush-and-drag brain tissue smear has been used for decades to identify or exclude falciparum cerebral malaria. Because it is rapid and cheap, and is not generally regarded as ‘tissue retention’ in the usual sense, it can be an alternative to histology. In cases of possible lung pneumonia or bronchopneumonia, where the sensitivity and specificity of naked eye diagnosis is lamentable, a dab preparation provides confirmation or exclusion of infection: if there are no polymorphs in the cytopreparation, there is no infection.
Post-transplant infectious deaths The infection risks for patients with bone marrow and solidorgan transplants are two fold. There is the necessary immunosuppressive therapy, which predisposes to reactivation of latent infections (viral and mycobacterial) and to acquisition of new infections (nosocomial) from the hospital staff and environment. Secondly, infections are transmitted in the donor tissue. As well as the standard common such agents such as hepatitis viruses, HIV, CMV and Epstein-Barr virus, there are uncommon potentially lethal infections, listed in Table 4. Often, it is at autopsy that organ-transmitted infections are diagnosed. It is vital that these are documented accurately, to feed the information back into the transplantation networks and so help to improve outcomes in the future.
Investigations on autopsy material Histopathology The autopsy in suspected infectious disease requires a 100% histology sampling rate. Very few invasive infections are identified at the genus or species level from naked eye appearance only
Documented unusual infections transmissible in solid organ transplantation C C
C
C
Prion: TSE/CJD Viruses C Rabies virus C West Nile virus C Lymphocytic choriomeningitis (LCM) Helminths C Strongyloides stercoralis C Halicephalobus (Figure 6) Protozoa C Microsporidiosis C Trypanosoma cruzi C Free-living amoebae - Balamuthia Figure 6 Halicephalobus nematode larvae in the brain e transmitted accidentally by an infected renal transplant. H&E stain.
Table 4
DIAGNOSTIC HISTOPATHOLOGY --:-
5
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
Figure 8 Cytological dab imprint diagnosis of cerebral Cryptococcus neoformans e Giemsa stain highlights the clear space outside the yeast cell.
Figure 7 (a) Lung necrotic zone with (b) large beaded Mycobacterium kansasii bacilli e due to chronic industrial lung disease. Ziehl-Neelsen stain. Figure 9 Small bowel mucosa with macrophages containing bacilli e PCR on the fixed tissue identified these as Shigella spp. H&E stain.
Microbiology e bacteriology, virology, mycology & molecular The standard culture methods are progressively being replaced by modern molecular techniques (PCR and variants), making identification of agents quicker and more sensitive. Autopsy samples should be taken as cleanly as possible, and sent fresh to the laboratory. In influenza outbreaks, the use of the specific nasal sampling brushes is recommended, as they are designed for direct insertion into the laboratory equipment. Obviously, the results of culture and molecular identification need to be correlated with the clinical and pathological information for consistency. If, for example, a PCR result indicates the presence of a specific infection when the histology firmly identifies a different agent, the PCR data need to be treated with caution: morphology trumps molecular. That said, the results of PCR diagnostics for fungal infections are excellent, and they are improving constantly for bacterial infections, even in FFPE (Figure 9). Blood and cerebrospinal fluid (CSF) cultures are invaluable in systemic infections. Blood must be taken from above the navel and before opening the body, to avoid faecal contamination: the neck veins and heart chambers are good sites. CSF is most easily
DIAGNOSTIC HISTOPATHOLOGY --:-
obtained from the cisterna magna (inserting the needle below the occipital bone process). Serology Whilst autopsy blood is not ideal for serodiagnostics due to haemolysis and other post-mortem changes, it does work. The local virology and immunology laboratories can help, and for possible HG4 infections or rarer imported HG3 infections, the central reference centres will assist. My personal uses of this method are to confirm or exclude HIV infection in a cadaver (as part of the diagnostic process), and to exclude a helminth infection. Urine antigen testing Microbiology laboratories perform urine antigen testing regularly to confirm or exclude certain infections in the living; the tests that are available everywhere are for pneumococcus and legionella. Others e including for fungal infections - are being developed.
6
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
Specific histopathology practice points in infection diagnosis Using a standard trio of special stains - Gram, Ziehl-Neelsen (ZN), Grocott silver e ensures that the obvious agents are covered. For example, in immunocompromised tuberculosis, there are no granulomas, only amorphous necrosis, and the diagnosis could be missed, unless a Z-N stain is done to reveal the huge numbers of bacilii present (Figure 10). Further investigation of undiagnosed necrotic lesions can include IHC for toxoplasmosis and herpes (Figure 11), both sometime causes of occult tissue necrosis. For bacteria, the most useful IHC reagent is for syphilis spirochaetes; much more sensitive and easier to prepare and read than the old Warthin-Starry stain. One reason for always using the Grocott stain (rather than PAS) is its ability to highlight Nocardia bacteria in abscesses and granulomas (Figure 12). The Grocott is also more precise in visualising fungal hyphae and yeasts and usually suffices in indicating the genus diagnosis in mycotic infections.7 The travel history is helpful: for example, people from middle and southern USA frequently have latent coccidiodomycosis or histoplasmosis, which can reactivate and cause death many years later.
Figure 12 Nocardiosis in the lung e the silver stain makes the branching bacteria easy to see. Grocott silver stain.
Viral inclusion bodies: the list includes the herpes viruses (cytomegalovirus, herpes simplex viruses), measles, JC (Figure 13) and BK viruses, adenovirus, parvo/B19 virus, and rabies (Figure 5). It is wise to apply a panel of IHC reagents to determine which is present. The standard antibody for JC is SV40, which also stains BK virus. Diagnosing encephalitis relies on this methodology. Protozoal infections include amoebiasis, malaria and a large number of other ‘small blue dot’ infectious agents. Amoebae of Entamoeba histolytica are strongly PAS positive, and the cytoplasm contains phagocytosed red cells. If these features are absent, the cells are likely to be macrophages. The ‘small blue dots’ infections of red blood cells are the malarias (always imported e Figure 14) and babesiosis (endemic in UK). The other small protozoa are Toxoplasma gondii, Leishmania spp, Isospora belli, Trypanosoma cruzi, and the microsporidia. IHC is very helpful in identifying toxoplasmosis, staining the zoites and cysts in the brain and other locations. If leishmaniasis is suspected, stain the section with Grocott silver to make sure that it is not Histoplasma capsulatum instead (and vice versa). Some worms e seen as adults, larvae and eggs e are easily recognised (see Figure 2); others require help from more experienced pathologists or a parasitologist (see Figure 6). Strongyloides stercoralis disseminated hyperinfection is a particularly
Figure 10 Non-reactive tuberculosis in advanced HIV disease e this concentrated mass of mycobacteria appeared as necrosis on H&E stains. Ziehl-Neelsen stain.
Figure 13 Viral inclusion bodies e JC virus encephalitis. The bizarre astrocytes and the purple intranuclear inclusion bodies are characteristic. H&E stain.
Figure 11 Mass necrosis of lung due to herpes simplex 1 infection e anti-HSV immunohistochemistry.
DIAGNOSTIC HISTOPATHOLOGY --:-
7
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003
MINI-SYMPOSIUM: AUTOPSY PATHOLOGY
REFERENCES 1 HSE. The approved list of biological agents - health and safety executive. www.hse.gov.uk/pubns/misc208.pdf. 2 Royal College of Pathologists. Guidelines on autopsy practice: precautions for high-risk infectious autopsies. In preparation, for publication in 2019. 3 Lucas SB. The maternal death autopsy. In: Pignatelli M, Gallagher P, eds. Recent advances in histopathology, vol. 23. London: JP Medical Pulishers, 2014; 17e30. 4 Lucas SB. The role of the autopsy in the diagnosis of sepsis and related fatal syndromes. In: Flanagan AM, ed. Recent advances in histopathology, vol. 24. London: JP Medical Publishers, 2016; 115e29. 5 Ramos-Casals M, Brita-Zeron P, Lopez-Gjuillermo A, Khamashtra MA, Bosch X. Adult haemophagocytic syndrome. Lancet 2014; 383: 1503e16. 6 Pathak S, McDermott MF, Savic S. Autoinflammatory diseases: update on classification diagnosis and management. J Clin Pathol 2017; 70: 1e8. 7 Lucas SB. Histopathology of fungal diseases. In: Kibbler C, Barton R, Gow NAR, Howell S, MacCallum DM, Manuel RJ, eds. Oxford textbook of medical mycology. Oxford: OUP, 2018; 289e97 [chapter 40].
Figure 14 Cerebral malaria e abundant parasites in the capillary red cells. H&E stain.
lethal infection in people with T-cell lymphoma, HTLV-1 infection, or on steroid therapy; they may have been infected recently or decades ago. The characteristic larvae are seen in the gut mucosa, lung and elsewhere. An uncommon problem is finding a large eosinophilic necrotic lesion, e.g. in liver, with a possible degenerate worm within. The causes of these lesions do include worms (Fasciola, Toxocara canis); more frequently they are allergic reactions to drugs, or a lymphoma, or part of a hypereosinophilia syndrome. Serology on autopsy blood to look for worm antibodies is helpful.A
Acknowledgements Most of the information presented in this review comes from personal experience and reflection since 1976, bolstered by discussion with my departmental colleagues at St Thomas’ who have also taken up infectious disease morbid anatomy and histopathology. I gratefully acknowledge: the essential role of the many coroners who asked me and my colleagues at St Thomas’ to investigate infection-related deaths in their jurisdictions - crucially, they never placed obstacles in the path of fully investigating the deaths with histopathology and microbiology; my UK clinical infectious disease colleagues who organised consented autopsies following deaths under their care; the large-scale autopsy-based collaborative research projects undertaken in several countries in Africa; and very importantly, all the pathology colleagues in UK and elsewhere who send us case consultations on their infection autopsy cases. I have no funding sources to aknowledge.
Practice points C C
C
C
C
Sample everything for cellular pathology microscopy Take blood cultures in all cases of possible systemic sepsis; and seek out all pre-mortem microbiology test results Apply the standard trio of stains: Gram, Ziehl-Neelsen, Grocott silver to lesions In systemic ? sepsis syndromes, consider the autoinflammatory syndromes when investigations for an infectious agent are negative When you are baffled, seek expert pathology consultation help; also cultivate the local infectious disease networks for clinical and laboratory advice.
DIAGNOSTIC HISTOPATHOLOGY --:-
8
Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Lucas S, Investigating infectious diseases at autopsy, Diagnostic Histopathology (2018), https://doi.org/ 10.1016/j.mpdhp.2018.07.003