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Fatal disseminated histoplasmosis presenting as FUO in an immunocompetent Italian host
Legal Medicine 25 (2017) 66–70
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Case Report
Fatal disseminated histoplasmosis presenting as FUO in an immunocompetent Italian host Alessandro Bonsignore a, Giulio Fraternali Orcioni b, Rosario Barranco a, Francesco De Stefano a, Jean Louis Ravetti b, Francesco Ventura a,⇑ a b
University of Genova, Department of Legal and Forensic Medicine, Via De Toni 12, Genova 16132, Italy San Martino Hospital, Department of Clinical Pathology, Largo Rosanna Benzi 10, Genova 16132, Italy
a r t i c l e
i n f o
Article history: Received 11 October 2016 Received in revised form 18 January 2017 Accepted 21 January 2017 Available online 24 January 2017 Keywords: Disseminated histoplasmosis Immunocompetent host Fever of unknown origin
a b s t r a c t Histoplasmosis is a relatively rare infectious disease endemic to certain geographic areas such as East Africa, eastern and central United States, western Mexico, Central and South America. Disseminated histoplasmosis has been reported mainly in immunocompromised hosts and in AIDS patients. In this paper we report on a fatal case of undiagnosed disseminated histoplasmosis presenting as fever of unknown origin (FUO) in a 43-year-old Italian woman who, although splenectomized 5 years earlier due to a motor vehicle accident, was otherwise immunocompetent. This case report highlights the fact that, even in Europe, histoplasmosis is an emerging sporadic infection which needs be considered in the differential diagnosis of given clinical scenarios. The proposed case is of blatant forensic concern as it addresses the hypothesis of professional responsibility due to a missed diagnosis of histoplasmosis. A timely diagnosis, with appropriate therapies, could have prevented death. The role of the forensic pathologist is also crucial because the post-mortem diagnosis of histoplasmosis (never considered in the differential diagnosis during prior hospitalization) highlights the importance of a meticulous and thorough autopsy to elucidate the cause of death. Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction Histoplasmosis, or Darling’s disease, is a relatively rare fungal disease caused by Histoplasma capsulatum, an ubiquitous saprophytic fungus in soil, endemic in certain parts of the world such as East Africa, eastern and central United States, western Mexico, Central and South America [1,2]. Although discovered in 1905 by the world renown pathologist, Samuel T. Darling, it continued to be commonly misdiagnosed as miliary tuberculosis until the 1930s [3]. Human histoplasmosis is attributable to the inhalation of the spores of either of two varieties of the pathogen, Histoplasma capsulatum var. capsulatum and Histoplasma capsulatum var. duboisii. [4,5]. In addition, infection can also occur upon contact with mucosal surfaces or non-intact epidermis. Symptoms of the mycosis may vary greatly, but the disease primarily affects the upper respiratory tract. Occasionally other organs are involved, leading to disseminated histoplasmosis, which can prove fatal when undiagnosed or untreated [6]. Histoplasmosis
⇑ Corresponding author. E-mail address: [email protected] (F. Ventura). http://dx.doi.org/10.1016/j.legalmed.2017.01.008 1344-6223/Ó 2017 Elsevier B.V. All rights reserved.
is common in patients with AIDS, due to their impaired immune response. Most affected individuals have clinically silent forms of infection with no apparent manifestations of disease. The acute phase of histoplasmosis is characterized by non-specific upper respiratory symptoms, often with cough, or a flu-like syndrome with normal Chest X-ray findings in 40–70% of cases [7]. Histoplasmosis may be further divided into the following 4 clinical types [8]: primary pulmonary histoplasmosis, progressive disseminated histoplasmosis, primary cutaneous histoplasmosis and so-called African histoplasmosis. Although the disease can be diagnosed by a number of methods, each presents inherent limitations. A definitive diagnosis relies on either isolating H. capsulatum on special culture media or visualizing the yeast form, i.e. microconidia ranging in size from 2 lm to 6 lm in diameter, upon direct examination of clinical specimens by way of specific fungal staining techniques. The former procedure, however, is lengthy, usually lasting 2–4 weeks, and lacks sensitivity in the case of self-limiting disease. Visualization of the yeast form using fungal staining of tissues and blood is less timeconsuming, but the sensitivity is lower than with culture [9–11]. Among the non-culture techniques, antibody detection methods are the major diagnostic tool worldwide given their general
A. Bonsignore et al. / Legal Medicine 25 (2017) 66–70
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availability, rapid turnaround time and accuracy. The two routine methodologies are complement fixation and immunodiffusion. There are false negatives, however, in immunocompromised patients and throughout the first six weeks of illness, when antibodies may still be lacking [11]. All the more, false positive results may eventuate due to known cross-reactivity with other fungi. ELISA and PCR are rapid techniques to detect fungal antigens, characterized by high sensitivity as well [12]. In contrast, Histoplasmin skin test is rarely useful as a diagnostic method [13].
2. Case report A 43-year-old Italian woman was admitted to a Regional hospital presenting with fever (37.8 °C) of unknown origin (FUO) of a few days’ duration. Albeit splenectomized 5 years earlier due to a motor vehicle accident, she was immunocompetent, having been in good health with a good medical history and serology. Specifically, HIV, HCV and HBV infections had been excluded. Moreover, white blood cells counts were within the normal range at a periodic check-up for work-related purposes, two months prior to the onset of any symptoms. Biochemical lab tests showed no obvious abnormalities of the immune response. The woman, a floriculturist employed at a florist shop, was 170 cm tall and weighed 60 kg. During the clinical investigation, standard hematological tests ruled out even mild or transient leukopenia. HIV antibody titers were repeatedly negative. A week later, the suspicion of a possible lymphoproliferative disease was investigated via mediastinoscopy with incisional biopsy in order to evaluate three small paratracheal lymphnodes. The biopsy specimens revealed the presence of small areas of necrosis with nuclear debris, granulocytes, plasma cells and several histiocytes, in the absence of any detectable neoplastic foci or specific granulomatous formations effacing node architecture. In the meantime a number of imaging tests were performed: contrast-enhanced PET (cePET/CT) (contrast enhancement at enlarged lymph nodes), cerebral CT (hypodense corticosubcortical areas in paramedian left occipital-parietal regions) and abdominal and thoracic CT (pleural thickening and bilateral apical infiltrates with interstitial thickening and bilateral micronodulations; enlarged mediastinal and retrocrural lymph nodes). Besides the persistent fever, the clinical picture further evolved with the onset of hepatic manifestations, with hepatomegaly accompanied by altered liver function tests, i.e. biochemical indices of cholestasis (without jaundice). A liver biopsy was performed revealing the presence of multiple foci of fibrinoid necrosis, with a predominantly periportal distribution, characterized by nuclear debris, lymphocytes, neutrophils, eosinophils and histiocytes. Histochemical investigation via Acid Fast staining technique produced Ziehl–Neelsen negative results, leaving necrotizing granulomatous inflammation as the sole histologic finding. A sudden deterioration of the patient’s general conditions ensued, due to the rapid onset of septic shock culminating in her death 3 days later (35 days from the onset of symptoms). A full post-mortem examination was performed 24 h later so as to ascertain the cause of death. The external examination showed multiple haemorrhagic petechiae distributed over thoracic and abdominal regions as well on the upper extremities. Lividity was evident on dependent regions of her back and was fixed. The internal examination revealed bilateral hydrothorax (200 cc in each of the pleural cavities) and increased weight of the lungs (left: 820 g, right: 810 g) which were both edematous. A round nodular 1.5 cm encapsulated, blackish-brown lesion was observed at the upper lobe of the right lung (Fig. 1). Multiple other spots of
Fig. 1. Macroscopic findings. Nodular pulmonary lesion observed at autopsy.
increased density were also observed throughout the pulmonary parenchyma. The heart weighed 330 g, revealing no gross alterations. The liver weighted 1850 g and was rather yellow in color. There was evidence of prior abdominal surgery with splenectomy. Lastly, as for the remaining visceral organs, only the kidneys were noteworthy for the presence of haemorrhagic petechiae. Histopathological studies were conducted on formalin-fixed, paraffin-embedded, 4-lm thick tissue sections, examining haematoxylin-eosin (H&E) and Grocott methenamine silver (GMS)-stained specimens. The results of the latter were indicative of disseminated histoplasmosis with multivisceral involvement. Post-mortem blood culture findings confirmed (3–4 weeks later) positivity for the pathogen. Specifically, by Grocott staining a multivisceral infiltrate of histiocyte-macrophage cells was evident. The cytoplasm of these phagocytes was laden with black granules, whose morphology was consistent with Histoplasma capsulatum (mainly at the occipital-parietal subcortical area of the brain) (Fig. 2). Lung specimens showed microfoci of confluent, cheese-like necrosis with sero-fibrinous exudate and a weak surrounding inflammatory response; Grocott staining identified fungal hyphae and spores indicative of complicating Candida albicans infection (i.e. terminal superinfection) (Fig. 3A and B). Bone marrow specimens revealed
Fig. 2. Histological findings. Brain: histiocyte-macrophage cells in brain tissue, with granule-laden cytoplasm attributable to H. capsulatum. 100 Grocott.
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Fig. 3. Histological findings. A) Lung: microfoci of confluent cheese-like necrosis with sero-fibrinous exudate and weak inflammatory response. 20 H&E. B) Lung: fungal spores and hyphae, morphologically indicative of C. albicans. 40 Grocott. C) Bone marrow: necrotic focus with sero-fibrinous and granulocytic exudate. 40 H&E. D) Bone marrow: histiocyte-macrophage cells in bone marrow, with granule-laden cytoplasm attributable to H. capsulatum. 40 Grocott.
necrotic focus with sero-fibrinous and granulocytic exudate (Fig. 3C and D). There were evident and widespread foci of necrosis with sero-fibrinous exudate in lymph nodes and liver (Fig. 4).
3. Discussion Transmission of H. capsulatum generally relies on aerosolized spores that germinate at body temperature and are transformed into the pathogenic yeast form. At the alveolar level, the H. capsulatum yeasts are engulfed by macrophages, after opsonization with the antibody, then multiply within phagolysosomes and ultimately lyse host cells [9,14]. The pathogens, once in hilar and mediastinal lymph nodes, can access the bloodstream and then be disseminated to various organs. Macrophages of the reticuloendothelial system engulf the pathogens and sequester them [9,15].
Fig. 4. Histological Findings. Liver: necrotic focus with sero-fibrinous and granulocytic exudate. 40 H&E.
The infection is controlled by CD4+ T cells that recognize antigens such as fungal cell-wall and heat-shock proteins. Consequently these T cells secrete cytokines that stimulate macrophages to destroy intracellular yeasts. Patients may harbor silent foci of infection even after developing the cell-mediated response, similarly to tuberculosis. The pathogens are not killed completely and thus, even after years, reactivation may occur [4,16]. Microscopically, histoplasmosis appears as budding ovalshaped cells of 1–5 lm in diameter. Methenamine silver staining techniques, such as Grocott’s, identify the 3- to 5-lm smoothwalled cyst of the fungus, which can persist for years. The lesions resemble tubercular granulomas which are particularly prone to calcification, often with a concentric, laminar pattern. In a few cases the pulmonary lesions progress or become reactivated, leading to progressive fibrotic and necrotic lesions that closely mimic reactivation tuberculosis [7,17]. Severe infections can cause hepatosplenomegaly, lymphadenopathy, and adrenal enlargement. Infection by H. capsulatum produces granuloma with coagulative necrosis that subsequently undergo fibrosis and concentric calcification. Sometimes, especially in the initial stages of the disease, histoplasmosis eludes histological detection by routine techniques and, absent any microbiological characterization via cultural methods, the infection remains undiagnosed. Whenever there are grounds to suspect histoplasmosis, methenamine silver staining or periodic acid–Schiff stain are likely to facilitate the histopathologic diagnosis [4]. Histoplasma infection in healthy and immunocompetent individuals is self-limiting, or asymptomatic in 95% of cases. Disseminated histoplasmosis arises due to reactivation of a previous infection [18]. This form is extremely rare and most patients are immunosuppressed, especially in individuals with CD4 lymphocyte counts below 150–200 per mm3. Other risk factors for disseminated disease are corticosteroid, cytotoxic and immunosuppressive therapies [9,19,20]. Also, a deficit in the inter ferone-c/interleuchina-12 pathway also represents a rare cause [21,22]. The clinical course varies depending on the patient’s
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immune status. In immunocompetent individuals, disseminated disease usually has a chronic or intermittent course with focal organ distribution. An acute and rapidly fatal course is instead observed in young children and with severely immunosuppressed patients. Symptoms of the progressive disseminated form are nonetheless nonspecific. The patient may experience fever, weight loss, headaches, and respiratory disorders, whereas intestinal symptoms are rare. Physical examination often elicits findings of hepatomegaly, splenomegaly and lymphadenopathy. More severe cases may lead to shock and multiorgan failure. The brain and the meninges are involved in about 5–10% of cases [23,24]. An infrequent manifestation is endocarditis, generally occurring with systemic emboli [25]. Rarely involved anatomical sites include the kidneys, ureters, bones and joints, and the gall bladder. Case reports of disseminated histoplasmosis described in the literature are relatively few and almost entirely confined to a limited number of geographical regions. Harnalikar et al. [26] describe a case of disseminated cutaneous histoplasmosis in a 60-year-old immunocompetent Indian host who died 2 days after the onset of symptoms. There are, however, several reports in the literature regarding cases of disseminated histoplasmosis in immunocompetent patients of Indian origin [27–29]. Salfelder et al. [30] report on 90 cases of fatal disseminated histoplasmosis. According to the authors the cause of death varied, but most cases were due to organ failure associated with the mycosis. In Europe there have been few reports of disseminated histoplasmosis generally concerning immunodepressed patients. Antinori et al. [31] report on one case of progressive disseminated histoplasmosis in a 35-year-old Italian HIV-positive individual. To the best of our knowledge the case herein reported is unprecedented inasmuch as cases of fatal disseminated histoplasmosis in immunocompetent hosts have never been previously described in Western countries. In all likelihood the woman contracted the fungal infection while handling contaminated soil due to her occupation as a floriculturist. Asplenic patients may have decreased production of properdin and tuftin and, consequently, a decreased opsonization capacity. This could entail an altered clearance of bacterial pathogens (pneumococcus, meningococcus). Moreover, her prior splenectomy (5 years earlier), with the ensuing implications for peripheral immune surveillance, likely favored the onset of disseminated histoplasmosis, an otherwise rare occurrence in immunocompetent patients, even though there had been no overt signs of impaired immunity (two months prior to the onset of the symptoms related to the infection, all routinely performed hematological and serological tests resulted normal). With this case report we aim to highlight the diagnostic challenges posed by a suspected case of histoplasmosis, particularly in the initial stages of disease. All the more, given the lack of sufficiently specific clinical-radiological features, the diagnosis often proves quite elusive, requiring a high index of suspicion. Indeed, a host of other conditions enter in the differential diagnosis with histoplasmosis. These include diseases that are epidemiologically even more frequent (at least in Europe) such as interstitial pneumonia of unknown origin, toxoplasmosis, leishmaniasis, tuberculosis, brucellosis and - particularly in the immunocompromised and/ or in transplant recipients - cryptococcosis, candidiasis and coccidioidomycosis. Furthermore, besides the clinical pitfalls listed above, the already ample differential diagnosis must also take into account the possibility of a subclinical histoplasmosis superinfection with other pathogens and/or neoplasms.
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Our case report therefore highlights the possibility of including histoplasmosis as an emerging sporadic disease in Europe, thus eligible to enter in differential diagnosis with FUO in immunocompetent hosts, even in the absence of known risk factors (i.e. transplant recipients, contact with soil having suitable moisture and contaminated by bird or bat droppings). The circumstance herein presented emphasizes the need to maintain a high index of suspicion when analyzing all granulomatous lesions of unknown etiology. The proposed case is of blatant forensic concern as it addresses the hypothesis of professional responsibility due to a missed diagnosis of histoplasmosis. A timely diagnosis, with appropriate therapies, could have prevented death. The role of the forensic pathologist is also crucial because the post-mortem diagnosis of histoplasmosis (never considered in the differential diagnosis during prior hospitalization) highlights the importance of a meticulous and thorough autopsy to elucidate the cause of death.
References [1] R.P. Rapini, J.L. Bolognia, J.L. Jorizzo, Dermatology, Vol. II, Mosby, St. Louis, 2007. [2] K.J. Ryan, C.G. Ray, Sherris Medical Microbiology, fourth ed., McGraw Hill, New York, 2004. [3] P. Tong, W.C. Tan, M. Pang, Sporadic disseminated histoplasmosis simulating miliary tuberculosis, Br. Med. J. (Clin. Res. Ed.) 287 (6395) (1983) 822–823. [4] C.A. Kauffman, Histoplasmosis: a clinical and laboratory update, Clin. Microbiol. Rev. 20 (1) (2007) 115–132. [5] C.A. Kauffman, Histoplasmosis, in: W.E. Dismukes, P.G. Pappas, J.D. Sobel (Eds.), Clinical Mycology, Oxford University Press, New York, 2003, pp. 285– 298. [6] H.C. Gugnani, F. Muotoe-Okafor, African histoplasmosis: a review, Rev. Iberoam. Micol. 14 (4) (1997) 155–159. [7] R.S. Cotran, V. Kumar, N. Fausto, S.L. Robbins, A.K. Abbas, Robbins and Cotran Pathologic Basis of Disease, ninth ed., Elsevier/Saunders, St. Louis, 2005. [8] C.A. Kauffman, Histoplasmosis: a clinical and laboratory update, Clin. Microbiol. Rev. 20 (1) (2007) 115–132. [9] R. Kurowski, M. Ostapchuk, Overview of Histoplasmosis, Am. Fam. Physician 66 (12) (2002) 2247–2253. [10] B.L. Gomez, J.I. Figueroa, A.J. Hamilton, B.L. Ortiz, M.A. Robledo, A. Restrepo, R.J. Hay, Development of a novel antigen detection test for histoplasmosis, J. Clin. Microbiol. 35 (1997) 2618–2622. [11] B.L. Gomez, J.I. Figueroa, A.J. Hamilton, S. Diez, M. Rojas, A. Tobon, A. Restrepo, R.J. Hay, Detection of the 70-kilodalton histoplasma capsulatum antigen in serum of histoplasmosis patients: correlation between antigenemia and therapy during follow-up, J. Clin. Microbiol. 37 (1999) 675–680. [12] L.J. Wheat, C.A. Kauffman, Histoplasmosis, Infect. Dis. Clin. North Am. 17 (2003) 1–19. [13] R.W. Bradsher, Histoplasmosis and blastomycosis, Clin. Infect. Dis. 22 (Suppl. 2) (1996) S102–S111. [14] J. Wheat, G. Sarosi, D. McKinsey, R. Hamill, R. Bradsher, P. Johnson, J. Loyd, C. Kauffman, Practice guidelines for the management of patients with histoplasmosis, Infect. Dis. Soc. Am. Clin. Infect. Dis. 30 (2000) 688–695. [15] D.J. Conces Jr., Histoplasmosis, Semin. Roentgenol. 31 (1996) 14–27. [16] M. Saidinejad, M.M. Burns, M.B. Harper, Disseminated histoplasmosis in a nonendemic area, Pediatr. Infect. Dis. J. 23 (2004) 781–782. [17] J. Wheat, Histoplasmosis. Experience during outbreaks in Indianapolis and review of the literature, Medicine 76 (1997) 339–354. [18] P. Zhou, G. Miller, R.A. Seder, Factors involved in regulating primary and secondary immunity to infection with histoplasma capsulatum: TNF-alpha plays a critical role in maintaining secondary immunity in the absence of IFNgamma, J. Immunol. 160 (1998) 1359–1368. [19] J.W. Gurney, D.J. Conces, Pulmonary histoplasmosis, Radiology 199 (1996) 297–306. [20] A.N. Manepalli, L. Rush, Disseminated histoplasmosis and Wegener’s granulomatosis, South. Med. J. 91 (1998) 1156–1158. [21] C.C. Kennedy, A.H. Limper, Redefining the clinical spectrum of chronic pulmonary histoplasmosis: a retrospective case series of 46 patients, Medicine (Baltimore) 86 (2007) 252–258. [22] J.W. Wynne, G.N. Olsen, Acute histoplasmosis presenting as the adult respiratory distress syndrome, Chest 66 (1974) 158–161. [23] L.J. Wheat, P.A. Connolly-Stringfield, R.L. Baker, M.F. Curfman, M.E. Eads, K.S. Israel, S.A. Norris, D.H. Webb, M.L. Zeckel, Disseminated histoplasmosis in the acquired immune deficiency syndrome: clinical findings, diagnosis and treatment, and review of the literature, Medicine (Baltimore) 69 (1990) 361–374. [24] L.J. Wheat, B.E. Batteiger, B. Sathapatayavongs, Histoplasma capsulatum infections of the central nervous system: a clinical review, Medicine 69 (1990) 244–260.
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[25] C.A. Hage, S. Bowyer, S.E. Tarvin, D. Helper, M.B. Kleiman, W.L. Joseph, Recognition, diagnosis, and treatment of histoplasmosis complicating tumor necrosis factor blocker therapy, Clin. Infect. Dis. 50 (2010) 85–92. [26] M. Harnalikar, V. Kharkar, U. Khopkar, Disseminated cutaneous histoplasmosis in an immunocompetent adult, Indian J. Dermatol. 57 (3) (2012) 206–209. [27] S. Kathuria, M.R. Capoor, S. Yadav, A. Singh, V. Ramesh, Disseminated histoplasmosis in an apparently immunocompetent individual from north India: a case report and review, Med. Mycol. 51 (7) (2013) 774–778. [28] D. De, U.K. Nath, Disseminated histoplasmosis in immunocompetent individuals- not a so rare entity, in India, Mediterr. J. Hematol. Infect. Dis. 7 (1) (2015) e2015028.
[29] M.V.S. Subbalaxmi, P. Umabala, P. Roshni, N. Chandra, Y.S. Raju, S.M. Rudramurthyd, A rare presentation of progressive disseminated histoplasmosis in an immunocompetent patient from a non-endemic region, Med. Mycol. Case Rep. 2 (2013) 103–107. [30] K. Salfelder, K. Brass, G. Doehnert, R. Doehnert, E. Sauerteig, Fatal disseminated histoplasmosis. Anatomic study of autopsy cases, Virchows Arch. A Pathol. Pathol. Anat. 350 (4) (1970) 303–335. [31] S. Antinori, L. Galimberti, C. Bonaccorso, L. Vago, M. Nebuloni, R. Esposito, A case of fatal disseminated histoplasmosis of autochthonous origin in an Italian AIDS patient, Eur. J. Clin. Microbiol. Infect. Dis. 16 (7) (1997) 545–546.
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Case Report
Fatal disseminated histoplasmosis presenting as FUO in an immunocompetent Italian host Alessandro Bonsignore a, Giulio Fraternali Orcioni b, Rosario Barranco a, Francesco De Stefano a, Jean Louis Ravetti b, Francesco Ventura a,⇑ a b
University of Genova, Department of Legal and Forensic Medicine, Via De Toni 12, Genova 16132, Italy San Martino Hospital, Department of Clinical Pathology, Largo Rosanna Benzi 10, Genova 16132, Italy
a r t i c l e
i n f o
Article history: Received 11 October 2016 Received in revised form 18 January 2017 Accepted 21 January 2017 Available online 24 January 2017 Keywords: Disseminated histoplasmosis Immunocompetent host Fever of unknown origin
a b s t r a c t Histoplasmosis is a relatively rare infectious disease endemic to certain geographic areas such as East Africa, eastern and central United States, western Mexico, Central and South America. Disseminated histoplasmosis has been reported mainly in immunocompromised hosts and in AIDS patients. In this paper we report on a fatal case of undiagnosed disseminated histoplasmosis presenting as fever of unknown origin (FUO) in a 43-year-old Italian woman who, although splenectomized 5 years earlier due to a motor vehicle accident, was otherwise immunocompetent. This case report highlights the fact that, even in Europe, histoplasmosis is an emerging sporadic infection which needs be considered in the differential diagnosis of given clinical scenarios. The proposed case is of blatant forensic concern as it addresses the hypothesis of professional responsibility due to a missed diagnosis of histoplasmosis. A timely diagnosis, with appropriate therapies, could have prevented death. The role of the forensic pathologist is also crucial because the post-mortem diagnosis of histoplasmosis (never considered in the differential diagnosis during prior hospitalization) highlights the importance of a meticulous and thorough autopsy to elucidate the cause of death. Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction Histoplasmosis, or Darling’s disease, is a relatively rare fungal disease caused by Histoplasma capsulatum, an ubiquitous saprophytic fungus in soil, endemic in certain parts of the world such as East Africa, eastern and central United States, western Mexico, Central and South America [1,2]. Although discovered in 1905 by the world renown pathologist, Samuel T. Darling, it continued to be commonly misdiagnosed as miliary tuberculosis until the 1930s [3]. Human histoplasmosis is attributable to the inhalation of the spores of either of two varieties of the pathogen, Histoplasma capsulatum var. capsulatum and Histoplasma capsulatum var. duboisii. [4,5]. In addition, infection can also occur upon contact with mucosal surfaces or non-intact epidermis. Symptoms of the mycosis may vary greatly, but the disease primarily affects the upper respiratory tract. Occasionally other organs are involved, leading to disseminated histoplasmosis, which can prove fatal when undiagnosed or untreated [6]. Histoplasmosis
⇑ Corresponding author. E-mail address: [email protected] (F. Ventura). http://dx.doi.org/10.1016/j.legalmed.2017.01.008 1344-6223/Ó 2017 Elsevier B.V. All rights reserved.
is common in patients with AIDS, due to their impaired immune response. Most affected individuals have clinically silent forms of infection with no apparent manifestations of disease. The acute phase of histoplasmosis is characterized by non-specific upper respiratory symptoms, often with cough, or a flu-like syndrome with normal Chest X-ray findings in 40–70% of cases [7]. Histoplasmosis may be further divided into the following 4 clinical types [8]: primary pulmonary histoplasmosis, progressive disseminated histoplasmosis, primary cutaneous histoplasmosis and so-called African histoplasmosis. Although the disease can be diagnosed by a number of methods, each presents inherent limitations. A definitive diagnosis relies on either isolating H. capsulatum on special culture media or visualizing the yeast form, i.e. microconidia ranging in size from 2 lm to 6 lm in diameter, upon direct examination of clinical specimens by way of specific fungal staining techniques. The former procedure, however, is lengthy, usually lasting 2–4 weeks, and lacks sensitivity in the case of self-limiting disease. Visualization of the yeast form using fungal staining of tissues and blood is less timeconsuming, but the sensitivity is lower than with culture [9–11]. Among the non-culture techniques, antibody detection methods are the major diagnostic tool worldwide given their general
A. Bonsignore et al. / Legal Medicine 25 (2017) 66–70
67
availability, rapid turnaround time and accuracy. The two routine methodologies are complement fixation and immunodiffusion. There are false negatives, however, in immunocompromised patients and throughout the first six weeks of illness, when antibodies may still be lacking [11]. All the more, false positive results may eventuate due to known cross-reactivity with other fungi. ELISA and PCR are rapid techniques to detect fungal antigens, characterized by high sensitivity as well [12]. In contrast, Histoplasmin skin test is rarely useful as a diagnostic method [13].
2. Case report A 43-year-old Italian woman was admitted to a Regional hospital presenting with fever (37.8 °C) of unknown origin (FUO) of a few days’ duration. Albeit splenectomized 5 years earlier due to a motor vehicle accident, she was immunocompetent, having been in good health with a good medical history and serology. Specifically, HIV, HCV and HBV infections had been excluded. Moreover, white blood cells counts were within the normal range at a periodic check-up for work-related purposes, two months prior to the onset of any symptoms. Biochemical lab tests showed no obvious abnormalities of the immune response. The woman, a floriculturist employed at a florist shop, was 170 cm tall and weighed 60 kg. During the clinical investigation, standard hematological tests ruled out even mild or transient leukopenia. HIV antibody titers were repeatedly negative. A week later, the suspicion of a possible lymphoproliferative disease was investigated via mediastinoscopy with incisional biopsy in order to evaluate three small paratracheal lymphnodes. The biopsy specimens revealed the presence of small areas of necrosis with nuclear debris, granulocytes, plasma cells and several histiocytes, in the absence of any detectable neoplastic foci or specific granulomatous formations effacing node architecture. In the meantime a number of imaging tests were performed: contrast-enhanced PET (cePET/CT) (contrast enhancement at enlarged lymph nodes), cerebral CT (hypodense corticosubcortical areas in paramedian left occipital-parietal regions) and abdominal and thoracic CT (pleural thickening and bilateral apical infiltrates with interstitial thickening and bilateral micronodulations; enlarged mediastinal and retrocrural lymph nodes). Besides the persistent fever, the clinical picture further evolved with the onset of hepatic manifestations, with hepatomegaly accompanied by altered liver function tests, i.e. biochemical indices of cholestasis (without jaundice). A liver biopsy was performed revealing the presence of multiple foci of fibrinoid necrosis, with a predominantly periportal distribution, characterized by nuclear debris, lymphocytes, neutrophils, eosinophils and histiocytes. Histochemical investigation via Acid Fast staining technique produced Ziehl–Neelsen negative results, leaving necrotizing granulomatous inflammation as the sole histologic finding. A sudden deterioration of the patient’s general conditions ensued, due to the rapid onset of septic shock culminating in her death 3 days later (35 days from the onset of symptoms). A full post-mortem examination was performed 24 h later so as to ascertain the cause of death. The external examination showed multiple haemorrhagic petechiae distributed over thoracic and abdominal regions as well on the upper extremities. Lividity was evident on dependent regions of her back and was fixed. The internal examination revealed bilateral hydrothorax (200 cc in each of the pleural cavities) and increased weight of the lungs (left: 820 g, right: 810 g) which were both edematous. A round nodular 1.5 cm encapsulated, blackish-brown lesion was observed at the upper lobe of the right lung (Fig. 1). Multiple other spots of
Fig. 1. Macroscopic findings. Nodular pulmonary lesion observed at autopsy.
increased density were also observed throughout the pulmonary parenchyma. The heart weighed 330 g, revealing no gross alterations. The liver weighted 1850 g and was rather yellow in color. There was evidence of prior abdominal surgery with splenectomy. Lastly, as for the remaining visceral organs, only the kidneys were noteworthy for the presence of haemorrhagic petechiae. Histopathological studies were conducted on formalin-fixed, paraffin-embedded, 4-lm thick tissue sections, examining haematoxylin-eosin (H&E) and Grocott methenamine silver (GMS)-stained specimens. The results of the latter were indicative of disseminated histoplasmosis with multivisceral involvement. Post-mortem blood culture findings confirmed (3–4 weeks later) positivity for the pathogen. Specifically, by Grocott staining a multivisceral infiltrate of histiocyte-macrophage cells was evident. The cytoplasm of these phagocytes was laden with black granules, whose morphology was consistent with Histoplasma capsulatum (mainly at the occipital-parietal subcortical area of the brain) (Fig. 2). Lung specimens showed microfoci of confluent, cheese-like necrosis with sero-fibrinous exudate and a weak surrounding inflammatory response; Grocott staining identified fungal hyphae and spores indicative of complicating Candida albicans infection (i.e. terminal superinfection) (Fig. 3A and B). Bone marrow specimens revealed
Fig. 2. Histological findings. Brain: histiocyte-macrophage cells in brain tissue, with granule-laden cytoplasm attributable to H. capsulatum. 100 Grocott.
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Fig. 3. Histological findings. A) Lung: microfoci of confluent cheese-like necrosis with sero-fibrinous exudate and weak inflammatory response. 20 H&E. B) Lung: fungal spores and hyphae, morphologically indicative of C. albicans. 40 Grocott. C) Bone marrow: necrotic focus with sero-fibrinous and granulocytic exudate. 40 H&E. D) Bone marrow: histiocyte-macrophage cells in bone marrow, with granule-laden cytoplasm attributable to H. capsulatum. 40 Grocott.
necrotic focus with sero-fibrinous and granulocytic exudate (Fig. 3C and D). There were evident and widespread foci of necrosis with sero-fibrinous exudate in lymph nodes and liver (Fig. 4).
3. Discussion Transmission of H. capsulatum generally relies on aerosolized spores that germinate at body temperature and are transformed into the pathogenic yeast form. At the alveolar level, the H. capsulatum yeasts are engulfed by macrophages, after opsonization with the antibody, then multiply within phagolysosomes and ultimately lyse host cells [9,14]. The pathogens, once in hilar and mediastinal lymph nodes, can access the bloodstream and then be disseminated to various organs. Macrophages of the reticuloendothelial system engulf the pathogens and sequester them [9,15].
Fig. 4. Histological Findings. Liver: necrotic focus with sero-fibrinous and granulocytic exudate. 40 H&E.
The infection is controlled by CD4+ T cells that recognize antigens such as fungal cell-wall and heat-shock proteins. Consequently these T cells secrete cytokines that stimulate macrophages to destroy intracellular yeasts. Patients may harbor silent foci of infection even after developing the cell-mediated response, similarly to tuberculosis. The pathogens are not killed completely and thus, even after years, reactivation may occur [4,16]. Microscopically, histoplasmosis appears as budding ovalshaped cells of 1–5 lm in diameter. Methenamine silver staining techniques, such as Grocott’s, identify the 3- to 5-lm smoothwalled cyst of the fungus, which can persist for years. The lesions resemble tubercular granulomas which are particularly prone to calcification, often with a concentric, laminar pattern. In a few cases the pulmonary lesions progress or become reactivated, leading to progressive fibrotic and necrotic lesions that closely mimic reactivation tuberculosis [7,17]. Severe infections can cause hepatosplenomegaly, lymphadenopathy, and adrenal enlargement. Infection by H. capsulatum produces granuloma with coagulative necrosis that subsequently undergo fibrosis and concentric calcification. Sometimes, especially in the initial stages of the disease, histoplasmosis eludes histological detection by routine techniques and, absent any microbiological characterization via cultural methods, the infection remains undiagnosed. Whenever there are grounds to suspect histoplasmosis, methenamine silver staining or periodic acid–Schiff stain are likely to facilitate the histopathologic diagnosis [4]. Histoplasma infection in healthy and immunocompetent individuals is self-limiting, or asymptomatic in 95% of cases. Disseminated histoplasmosis arises due to reactivation of a previous infection [18]. This form is extremely rare and most patients are immunosuppressed, especially in individuals with CD4 lymphocyte counts below 150–200 per mm3. Other risk factors for disseminated disease are corticosteroid, cytotoxic and immunosuppressive therapies [9,19,20]. Also, a deficit in the inter ferone-c/interleuchina-12 pathway also represents a rare cause [21,22]. The clinical course varies depending on the patient’s
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immune status. In immunocompetent individuals, disseminated disease usually has a chronic or intermittent course with focal organ distribution. An acute and rapidly fatal course is instead observed in young children and with severely immunosuppressed patients. Symptoms of the progressive disseminated form are nonetheless nonspecific. The patient may experience fever, weight loss, headaches, and respiratory disorders, whereas intestinal symptoms are rare. Physical examination often elicits findings of hepatomegaly, splenomegaly and lymphadenopathy. More severe cases may lead to shock and multiorgan failure. The brain and the meninges are involved in about 5–10% of cases [23,24]. An infrequent manifestation is endocarditis, generally occurring with systemic emboli [25]. Rarely involved anatomical sites include the kidneys, ureters, bones and joints, and the gall bladder. Case reports of disseminated histoplasmosis described in the literature are relatively few and almost entirely confined to a limited number of geographical regions. Harnalikar et al. [26] describe a case of disseminated cutaneous histoplasmosis in a 60-year-old immunocompetent Indian host who died 2 days after the onset of symptoms. There are, however, several reports in the literature regarding cases of disseminated histoplasmosis in immunocompetent patients of Indian origin [27–29]. Salfelder et al. [30] report on 90 cases of fatal disseminated histoplasmosis. According to the authors the cause of death varied, but most cases were due to organ failure associated with the mycosis. In Europe there have been few reports of disseminated histoplasmosis generally concerning immunodepressed patients. Antinori et al. [31] report on one case of progressive disseminated histoplasmosis in a 35-year-old Italian HIV-positive individual. To the best of our knowledge the case herein reported is unprecedented inasmuch as cases of fatal disseminated histoplasmosis in immunocompetent hosts have never been previously described in Western countries. In all likelihood the woman contracted the fungal infection while handling contaminated soil due to her occupation as a floriculturist. Asplenic patients may have decreased production of properdin and tuftin and, consequently, a decreased opsonization capacity. This could entail an altered clearance of bacterial pathogens (pneumococcus, meningococcus). Moreover, her prior splenectomy (5 years earlier), with the ensuing implications for peripheral immune surveillance, likely favored the onset of disseminated histoplasmosis, an otherwise rare occurrence in immunocompetent patients, even though there had been no overt signs of impaired immunity (two months prior to the onset of the symptoms related to the infection, all routinely performed hematological and serological tests resulted normal). With this case report we aim to highlight the diagnostic challenges posed by a suspected case of histoplasmosis, particularly in the initial stages of disease. All the more, given the lack of sufficiently specific clinical-radiological features, the diagnosis often proves quite elusive, requiring a high index of suspicion. Indeed, a host of other conditions enter in the differential diagnosis with histoplasmosis. These include diseases that are epidemiologically even more frequent (at least in Europe) such as interstitial pneumonia of unknown origin, toxoplasmosis, leishmaniasis, tuberculosis, brucellosis and - particularly in the immunocompromised and/ or in transplant recipients - cryptococcosis, candidiasis and coccidioidomycosis. Furthermore, besides the clinical pitfalls listed above, the already ample differential diagnosis must also take into account the possibility of a subclinical histoplasmosis superinfection with other pathogens and/or neoplasms.
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Our case report therefore highlights the possibility of including histoplasmosis as an emerging sporadic disease in Europe, thus eligible to enter in differential diagnosis with FUO in immunocompetent hosts, even in the absence of known risk factors (i.e. transplant recipients, contact with soil having suitable moisture and contaminated by bird or bat droppings). The circumstance herein presented emphasizes the need to maintain a high index of suspicion when analyzing all granulomatous lesions of unknown etiology. The proposed case is of blatant forensic concern as it addresses the hypothesis of professional responsibility due to a missed diagnosis of histoplasmosis. A timely diagnosis, with appropriate therapies, could have prevented death. The role of the forensic pathologist is also crucial because the post-mortem diagnosis of histoplasmosis (never considered in the differential diagnosis during prior hospitalization) highlights the importance of a meticulous and thorough autopsy to elucidate the cause of death.
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