Medical Mycology Case Reports 2 (2013) 103–107
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A rare presentation of progressive disseminated histoplasmosis in an immunocompetent patient from a non-endemic region M.V.S. Subbalaxmi a,n, P. Umabala b, Roshni Paul c, Naval Chandra a, Y.S. Raju a, Shivaprakash M. Rudramurthy d a
Department of General Medicine, Nizam's Institute of Medical Sciences, Hyderabad 500082, Andhra Pradesh, India Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad 500082, Andhra Pradesh, India c Department of Pathology, Nizam's Institute of Medical Sciences, Hyderabad 500082, Andhra Pradesh, India d Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 January 2013 Received in revised form 12 April 2013 Accepted 15 April 2013
Histoplasmosis is an important systemic fungal infection in endemic areas. In India, the disease has been reported from several parts of the country, most cases being from eastern India considered to be endemic for the disease. There have been very few cases reported from the state of Andhra Pradesh, in the southern part of India. We report a case of progressive disseminated histoplasmosis presenting with bleeding manifestations in an immune competent patient from the state of Andhra Pradesh. & 2013 International Society for Human and Animal Mycology. Published by Elsevier B.V All rights reserved.
Keywords: Histoplasmosis PDH Immunocompetent host Thrombocytopenia India
1. Introduction Histoplasmosis caused by the dimorphic fungus Histoplasma capsulatum presents most commonly as pulmonary and progressive disseminated (PDH) forms [1]. Approximately 10% of individuals infected with histoplasmosis may develop progressive disseminated histoplasmosis which usually presents with fever, malaise, hepatosplenomegaly and lymphadenopathy. Other manifestations of PDH include pancytopenia, renal failure, disseminated intra vascular coagulation (DIC), skin lesions, gastrointestinal manifestations like diarrhea, vomiting, neurologic manifestations like encephalopathy, focal parenchymal lesions and sometimes adrenal insufficiency [1]. Progressive disseminated histoplasmosis is usually seen in immunocompromised patients such as persons with HIV infection [1]. In India histoplasmosis has been reported from different pockets in the eastern, north eastern, northern, western and southern parts of the country [2–10].We report a case of progressive disseminated histoplasmosis in an immunocompetent patient, presenting with bleeding manifestations from the state of Andhra Pradesh in the southern part of India. This case merits discussion due to the rarity of the presentation mimicking immune thrombocytopenic purpura,
n
Corresponding author. Tel.: +91 409490457909. E-mail address: [email protected] (M.V.S. Subbalaxmi).
in a patient with no underlying risk factor for progressive disseminated histoplasmosis. 2. Case A male farmer aged 56 years from Adilabad district, in Andhra Pradesh, was admitted on day 0 to a tertiary care hospital in Andhra Pradesh, south India with complaints of hematuria and epigastric pain for last 15 days. Epigastric pain was more after taking food but there was no history of radiation. He complained of nausea though there was no history of vomiting and fever. Patient was admitted to another hospital 5 days prior to the present admission for evaluation of his epigastric pain. Upper gastrointestinal endoscopy was done there, which revealed gastritis. In view of very low platelet counts he was referred to our hospital. There was no history of smoking or substance abuse or travel outside the state of Andhra Pradesh to any regions known to be endemic for histoplasmosis. At admission on day 0, general physical examination revealed petechiae on his right shoulder and tenderness in the epigastric area. Rest of the general examination and systemic examination were normal. Laboratory work up done on day +1 revealed hemoglobin 12.1 g/dl; mean corpuscular volume 86 fl; total leukocyte count 4000/cmm with normal differential count; platelet count 10,000/cmm.
2211-7539/$ - see front matter & 2013 International Society for Human and Animal Mycology. Published by Elsevier B.V All rights reserved. http://dx.doi.org/10.1016/j.mmcr.2013.04.003
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ESR was within normal limits. Peripheral blood smear showed thrombocytopenia (Fig. 1A) with giant platelets (Fig. 1B). Complete urine examination showed plenty of red blood cells. Random blood sugar, glycosylated hemoglobin, coagulation profile, liver and renal function tests serum lipase were all within normal limits; serum amylase was 150 U/ml and LDH 2260 U/L. ELISAs for anti-nuclear antibodies, HIV antibody and HIV antigen were all negative. In view of the age and persisting thrombocytopenia, a bone marrow aspiration and biopsy were done on day +3 to rule out lymphoma. Bone marrow aspirate was particulate with normal cellularity, M:E ratio was 2:1, with normal erythropoeisis and myelopoeisis. Megakaryocytes were slightly increased in number
(Fig. 2A). Large number of histiocytes, some foamy, filled with tiny intracytoplasmic yeasts were seen. There were a few yeasts lying extracellularly. These were oval, 2–3 m in size with a slightly eccentric nucleus and a clear zone around the nucleus (Fig. 2B). As these organisms had to be differentiated from others with similar morphology, trephine sections were subjected to special staining with periodic acid schiff (PAS) (Fig. 3A) and silver methanamine (SM) (Fig. 3B),which showed yeast cells morphologically suggestive of H. capsulatum. As the bone marrow aspirate was not sent for culture at the first instance, repeated bone marrow aspiration was done on day +7 for confirmation of histoplasmosis. The aspirate was inoculated on to brain heart infusion agar, Sabouraud's dextrose agar with
Fig. 1. Leishman stain: peripheral blood smear showing (A) thrombocytopenia ( 400) with (B) giant platelets ( 1000).
Fig. 2. (A) Giemsa 400: BMA particulate with normal cellularity, M:E ratio 2:1, normal erythropoiesis, myelopoiesis, megakarycytes slightly increased in number with normal morphology. (B) Giemsa 400 (inset Giemsa 1000): histiocytes showing intracytoplasmic yeasts.
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Fig. 3. Trephine section showing yeast cells stained with PAS (A) and SM (B) ( 400).
2.1. Imaging Chest radiograph and ultrasound scanning of abdomen which were done on day +3 of hospital stay were normal. 2.2. Treatment
Fig. 4. Lactophenol cotton blue mount ( 400): large, rounded, single-celled, tuberculate macroconidia and small, pyriform shaped microconidia of Histoplasma capsulatum. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this article.)
chloramphenicol, and Sabouraud's dextrose agar with cycloheximide and chloramphenicol. As H. capsulatum is a CDC/NIH risk group 3 agent, handling of clinical material and isolates were done in the Class II Bio Safety Cabinet with BSL2 practices, containment equipment and facilities. On Sabouraud's dextrose agar at 25 1C, colonies appeared after 1 week of incubation i.e. on day +14. The colonies were highly folded, adherent, initially white, turning buffbrown. Microscopic morphology showed the presence of characteristic large, rounded, single-celled, tuberculate macroconidia and small, pyriform shaped microconidia (Fig. 4), based on which the fungus provisionally was identified as H. capsulatum. The isolate was sent to the National Culture Collection of Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research, Chandigarh, for confirmation. At NCCPF, the identity of the isolate was confirmed by morphology, conversion to yeast phase and sequencing of rDNA. For extraction of DNA, the isolate was inoculated in Sabouraud dextrose broth and incubated in a shaker incubator (130 rpm) at 35 1C for 5 days. The cells were killed by adding thiomersal at a final concentration of 0.02% (wt/v) and incubating it at room temperature for 1 h. The hyphal mass was separated by filtration and then transferred to a sterile mortar and pestle. Whole cell DNA from the fungi was extracted by crushing the mycelial mat with liquid nitrogen followed by extraction using phenol: chloroform extraction method as described previously by Baghela et al. [11]. Sequencing of 28s region of rDNA was performed using primer pairs NL1 (5′-GCATATCAATAAGCGGAGGAAAAG) and NL-4 (5′-GGTCCGTGTTT CAAGACGG). Consensus sequences were prepared by using sequence obtained by forward and reverse primers with the help of Bionumerics software Version 6.6 (Applied Maths, Ghent, Belgium). Pair wise sequence alignment with the database showed 499% similarity with the CBS 136.72 strain of H. capsulatum. The isolate is deposited at NCCPF as NCCPF 230016.
The patient’s platelet counts at admission were 10,000/cmm and were continuing to fall further for which he received platelet transfusions on alternate days. On day +8, patient was started on treatment with injection amphotericin B deoxycholate at a daily dose of 1 mg/kg b wt. Pain abdomen gradually subsided and the patient’s appetite improved. Platelet count increased to 30,000/ cmm on day +13 i.e. after 5 days of starting of amphotericin. Patient was given amphotericin B deoxycholate for 14 days followed by itraconazole 200 mg twice a day with a plan to continue for 1 year. Platelet count was 50,000/cmm at discharge on day +22. 2.3. Follow up and outcome Patient reported to the general medicine outpatient department for follow up on day +52. Patient showed marked clinical improvement and platelet counts on that day were 150,000/cmm. Platelet count was 160,000/cmm at the next folloup on day+112. The patient's renal function and liver function were monitored throughout the course of treatment with antifungals till his last visit (day 112) to the outpatient department for follow up and were found to be within normal limits.
3. Discussion H. capsulatum causing histoplasmosis is presently classified into six distinct clades based on combined data analysis of partial sequences of four protein encoding genes of 46H. capsulatum isolates. These include five clades of H. capsulatum var capsulatum: (i) class 1 North American;(ii) class 2 North American; (iii) Central American; (iv) South American group A (which includes the previous H. capsulatum var farciminosum; (v) South American group B; and one clade for H. capsulatum var duboisii [12]. The first case of histoplasmosis in India was reported in 1954 [2]. Subsequently several cases have been reported from different regions of India [3–10]. In India, the fungus was isolated from the environment of an old building along the Ganges River close to Calcutta [13]. There have been sporadic reports of histoplasmosis in patients from Andhra Pradesh [9,14]. In our hospital, prior to the present case, progressive disseminated histoplasmosis was diagnosed in two patients with HIV infection, in whom H. capsulatum var capsulatum was isolated from lymph node biopsy (unpublished data). Both patients hailed from the eastern part of the country known to be endemic for
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histoplasmosis. In the present case, the patient hails from the Adilabad district in the state of Andhra Pradesh in southern India and has never traveled outside the district before his illness started. The climate of the district is characterized by hot summer and in general is dry except during the south-west monsoon season from June to September when 80% of the yearly rainfall is received. This patient became symptomatic in July when the district has a precipitation of 10 in., temp.—max 310 1C and min 240 1C, humidity 84%. The present patient is a farmer by occupation and could have been exposed to contaminated soil during the course of his work. In their review of endemic fungal infections including histoplasmosis in the Asia-Pacific region, Chakrabarti and slavin [15] conclude that an accurate epidemiological picture of endemic fungal infections remains unclear due to inadequate laboratory facilities, limited experience with fungal infections and few epidemiological surveys performed in these regions. PDH develops mostly in patients who are immunocompromised and are unable to mount an effective CMI response to the organism. This includes patients with AIDS [1,9,16,17] transplant recipients [1,9,14], those with hematologic malignancies [1,9], and those on corticosteroids [1,9]. In the present case, the patient had no major identifiable underlying immunocompromising condition. Thrombocytopenia is reported in histoplasmosis along with other cytopenias whenever there is bone marrow involvement [1,9] but reports of patients presenting with isolated thrombocytopenia as in the present case are rare [18,19]. Hood et al. [18] reported two patients with disseminated histoplasmosis and predominant thrombocytopenia. One patient presented with severe thrombocytopenic purpura and splenomegaly. Investigations did not reveal any evidence of histoplasmosis. She died on the sixth hospital day from a massive intra-cerebral hemorrhage and one kidney was removed for homotransplantation. Histoplasmosis was diagnosed at autopsy. The second patient, with chronic glomerulonephritis and uremia, who received the renal homograft from the first patient, developed initial signs of homograft rejection, 5 days postoperatively. Diffuse thrombocytopenic purpura occurred shortly thereafter. Spores of Histoplasma capsulatum were observed in blood smears, in leukocyte concentrates, and in 5-day leukocyte cultures from the blood obtained prior to death. Disseminated histoplasmosis was found also at autopsy. Both patients received corticosteroids, in the first case as treatment for thrombocytopenia and in the second patient as immunosuppressive therapy following renal transplantation. Armitage et al. [19] reported two cases of disseminated histoplasmosis with thrombocytopenia dominating the clinical picture. In one case, the patient was diagnosed with histoplasmosis and recovered after being treated with amphotericin B. In the other case the patient was treated with steroids for a mistaken diagnosis of immune thrombocytopenic purpura (ITP) and succumbed, the diagnosis of histoplasmosis being made at autopsy. These studies conclude that disseminated histoplasmosis should always be considered in the differential diagnosis of thrombocytopenia, as the use of corticosteroids for treatment of the thrombocytopenia with failure to diagnose the underlying disease can prove fatal. In our case, though ITP was considered as an admitting diagnosis we did not administer corticosteroids. The reasons being the age of the patient, complaint of epigastric pain and loss of appetite. An elevated LDH (2260 U/L) which was done as part of work up for thrombocytopenia substantiated our decision not to administer steroids empirically and a decision was made to defer specific therapy till the bone marrow examination reports were available. In the present case PDH was confirmed by direct demonstration of the organism in the bone marrow and growth of the organism in culture.
Des Prez et al. [20] in their studies on the pathogenesis of thrombocytopenia in histoplasmosis conclude that the yeast from H. capsulatum activates the platelets resulting in serotonin release reaction and aggregation without the involvement of the complement pathway. The plasma cofactors which are involved are Ig G which is necessary for induction of release reaction. Another feature observed in this patient was that despite involvement of bone marrow our patient was afebrile throughout hospital course. The gastrointestinal tract is involved in 70–90% of cases of disseminated histoplasmosis. The present patient complained of persistent epigastric pain which subsided after receiving 2 weeks of amphotericin B. Biopsy of the upper gastrointestinal endoscopy could not be done due to persistent thrombocytopenia. Though ultrasound of abdomen did not reveal evidence of pancreatitis, serum lipase and amylase were elevated. Pancreatitis is reported to be a rare manifestation of disseminated histoplasmosis. A patient with histoplasmosis and pancreatitis [21] diagnosed at autopsy is reported where the patient had very high lipase and moderately elevated amylase levels. For the management of moderately severe to severe histoplasmosis, liposomal amphotericin B (3.0 mg/kg daily) is recommended for 1–2 weeks, followed by oral itraconazole (200 mg three times daily for 3 days and then 200 mg twice daily for a total of at least 12 months [22]. The deoxycholate formulation of amphotericin B (0.7–1.0 mg/kg daily) is recommended as an alternative to a lipid formulation in patients who are at a low risk for nephrotoxicity. In the present case as the patient could not afford liposomal amphotericin B, he was treated with amphotericin B deoxycholate for 2 weeks followed by oral itraconazole. PDH should always be considered in the differential diagnosis of thrombocytopenia, irrespective of the patient's immune status and endemicity of the disease in the region. Recognition of the varied and confounding clinical presentations of histoplasmosis, good diagnostic laboratory facilities and extensive population based studies to know the endemicity of the disease in different regions are all necessary for improved understanding of the epidemiology of histoplasmosis.
Conflict of interest statement There are none.
Acknowledgments We acknowledge Prof. Arunaloke Chakrabarti, In Charge, National Culture Collection of Pathogenic Fungi (supported by Indian Council of Medical Research, New Delhi) and Head Department of Medical Microbiology, PGIMER, Chandigarh for confirming the identity of the isolate. References [1] Kauffman CA. Histoplasmosis: a clinical and laboratory update. Clinical Microbiology Reviews 2007;20(1):115–132. [2] Panja G, Sen S. A unique case of histoplasmosis. Journal of the Indian Medical Association 1954;6:257–258. [3] Randhawa HS. Occurrence of histoplasmosis in Asia. Mycopathologia et Mycologia Applicata 1970;41:75–89. [4] Padhye AA, Pathak AA, Katkar VJ, Hazare VK, Kauffman L. Oral histoplasmosis in India: a case report and an overview of cases reported during 1968–92. Journal of Medical and Veterinary Mycology 1994;32:93–103. [5] Randhawa HS, Khan ZU. Histoplasmosis in India: current status. The Indian Journal of Chest Diseases and Allied Sciences 1994;36:193–213. [6] Randhawa HS, Chaturvedi S, Khan ZU, et al. Epididymal histoplasmosis diagnosed by isolation of Histoplasma capsulatum from semen. Mycopathologia 1995;131:173–177.
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[7] Sen S, Bajaj MS, Vijayaraghavan M. Histoplasmosis of the eyelids—a case report. Indian Journal of Pathology and Microbiology 1999;42:495–497. [8] Goswami RP, Pramanik N, Banerjee D, et al. Histoplasmosis in eastern India: the tip of the iceberg? Transactions of the Royal Society of Tropical Medicine and Hygiene 1999;93:540–542. [9] Subramanyan S, Abraham OC, Rupali P, Zacharariah A, Mathews MS, Mathai D. Disseminated histoplasmosis. Journal of the Association of Physicians of India 2005;53:185–189. [10] Joshi SA, Kagal AS, Bharadwaj RS, Kulkarni SS, Jadhav MV. Disseminated histoplasmosis. Indian Journal of Medical Microbiology 2006;24:297–298. [11] Baghela A, Thungapathra M, Shivaprakash MR, Chakrabarti A. Multilocus microsatellite typing for Rhizopus oryzae. Journal of Medical Microbiology 2010;59:1449–1455. [12] Kasuga T, Taylor JW, White TJ. Phylogenetic relationships of varieties and geographical groups of the human pathogenic fungus Histoplasma capsulatum Darling. Journal of Clinical Microbiology 1999;37:653–663. [13] Sanyal M, Thammayya A. Histoplasma capsulatum in the soil of Gangetic Plain in India. Indian Journal of Medical Research 1975;63:1020–1028. [14] Rao R. Recurrent primary cutaneous histoplasmosis in a post renal transplant patient. Journal of Nephrology and Renal Transplantation 2008;1(1):53–58. [15] Chakrabarti A, Slavin MA. Endemic fungal infections in the Asia pacific region. Medical Mycology 2011;49:337–344.
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[16] Johnson PC, Kardori N, Najjar A, et al. Progressive disseminated histoplasmosis in patients with acquired immunodeficiency syndrome. American Journal of Medicine 1988;88:152–158. [17] Kahi CJ, Wheat JR, Allen SD, et al. Gastrointestinal histoplasmosis. American Journal of Gastroenterology 2005;100:220–231. [18] Hood AB, Inglis FG, Lowenstein L. Histoplasmosis and thrombocytopenic purpura: transmission by renal homotransplantation. Canadian Medical Association Journal 1965;93:587–592. [19] Armitage JO, Sheets RF. Idiopathic thrombocytopenic purpura. Journal of the American Medical Association 1977;237(21):2323–2324. [20] Des Prez RM, Steckley S, Stroud RM, Hawiger J. Interaction of Histoplasma capsultaum with human platelets. The Journal of Infectious Diseases 1980;142:32. [21] Mc Kinsey DS, Gupta MR, Riddler SA, Driks MR, Smith DL, et al. Long term amphotericin B therapy for disseminated histoplasmosis in patients with acquired immunodeficiency syndrome. Annals of Medicine 1989;111:655–659. [22] L. Wheat J, Freifeld AG, Kleiman MB, Baddley JW, McKinsey DS, Loyd JE, et al. Clinical practice guidelines for the management of patients with histoplasmosis: update by the infectious. Diseases Society of America. Clinical Infectious Diseases 2007;45:807–825.
Contents lists available at ScienceDirect
Medical Mycology Case Reports journal homepage: www.elsevier.com/locate/mmcr
A rare presentation of progressive disseminated histoplasmosis in an immunocompetent patient from a non-endemic region M.V.S. Subbalaxmi a,n, P. Umabala b, Roshni Paul c, Naval Chandra a, Y.S. Raju a, Shivaprakash M. Rudramurthy d a
Department of General Medicine, Nizam's Institute of Medical Sciences, Hyderabad 500082, Andhra Pradesh, India Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad 500082, Andhra Pradesh, India c Department of Pathology, Nizam's Institute of Medical Sciences, Hyderabad 500082, Andhra Pradesh, India d Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 January 2013 Received in revised form 12 April 2013 Accepted 15 April 2013
Histoplasmosis is an important systemic fungal infection in endemic areas. In India, the disease has been reported from several parts of the country, most cases being from eastern India considered to be endemic for the disease. There have been very few cases reported from the state of Andhra Pradesh, in the southern part of India. We report a case of progressive disseminated histoplasmosis presenting with bleeding manifestations in an immune competent patient from the state of Andhra Pradesh. & 2013 International Society for Human and Animal Mycology. Published by Elsevier B.V All rights reserved.
Keywords: Histoplasmosis PDH Immunocompetent host Thrombocytopenia India
1. Introduction Histoplasmosis caused by the dimorphic fungus Histoplasma capsulatum presents most commonly as pulmonary and progressive disseminated (PDH) forms [1]. Approximately 10% of individuals infected with histoplasmosis may develop progressive disseminated histoplasmosis which usually presents with fever, malaise, hepatosplenomegaly and lymphadenopathy. Other manifestations of PDH include pancytopenia, renal failure, disseminated intra vascular coagulation (DIC), skin lesions, gastrointestinal manifestations like diarrhea, vomiting, neurologic manifestations like encephalopathy, focal parenchymal lesions and sometimes adrenal insufficiency [1]. Progressive disseminated histoplasmosis is usually seen in immunocompromised patients such as persons with HIV infection [1]. In India histoplasmosis has been reported from different pockets in the eastern, north eastern, northern, western and southern parts of the country [2–10].We report a case of progressive disseminated histoplasmosis in an immunocompetent patient, presenting with bleeding manifestations from the state of Andhra Pradesh in the southern part of India. This case merits discussion due to the rarity of the presentation mimicking immune thrombocytopenic purpura,
n
Corresponding author. Tel.: +91 409490457909. E-mail address: [email protected] (M.V.S. Subbalaxmi).
in a patient with no underlying risk factor for progressive disseminated histoplasmosis. 2. Case A male farmer aged 56 years from Adilabad district, in Andhra Pradesh, was admitted on day 0 to a tertiary care hospital in Andhra Pradesh, south India with complaints of hematuria and epigastric pain for last 15 days. Epigastric pain was more after taking food but there was no history of radiation. He complained of nausea though there was no history of vomiting and fever. Patient was admitted to another hospital 5 days prior to the present admission for evaluation of his epigastric pain. Upper gastrointestinal endoscopy was done there, which revealed gastritis. In view of very low platelet counts he was referred to our hospital. There was no history of smoking or substance abuse or travel outside the state of Andhra Pradesh to any regions known to be endemic for histoplasmosis. At admission on day 0, general physical examination revealed petechiae on his right shoulder and tenderness in the epigastric area. Rest of the general examination and systemic examination were normal. Laboratory work up done on day +1 revealed hemoglobin 12.1 g/dl; mean corpuscular volume 86 fl; total leukocyte count 4000/cmm with normal differential count; platelet count 10,000/cmm.
2211-7539/$ - see front matter & 2013 International Society for Human and Animal Mycology. Published by Elsevier B.V All rights reserved. http://dx.doi.org/10.1016/j.mmcr.2013.04.003
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ESR was within normal limits. Peripheral blood smear showed thrombocytopenia (Fig. 1A) with giant platelets (Fig. 1B). Complete urine examination showed plenty of red blood cells. Random blood sugar, glycosylated hemoglobin, coagulation profile, liver and renal function tests serum lipase were all within normal limits; serum amylase was 150 U/ml and LDH 2260 U/L. ELISAs for anti-nuclear antibodies, HIV antibody and HIV antigen were all negative. In view of the age and persisting thrombocytopenia, a bone marrow aspiration and biopsy were done on day +3 to rule out lymphoma. Bone marrow aspirate was particulate with normal cellularity, M:E ratio was 2:1, with normal erythropoeisis and myelopoeisis. Megakaryocytes were slightly increased in number
(Fig. 2A). Large number of histiocytes, some foamy, filled with tiny intracytoplasmic yeasts were seen. There were a few yeasts lying extracellularly. These were oval, 2–3 m in size with a slightly eccentric nucleus and a clear zone around the nucleus (Fig. 2B). As these organisms had to be differentiated from others with similar morphology, trephine sections were subjected to special staining with periodic acid schiff (PAS) (Fig. 3A) and silver methanamine (SM) (Fig. 3B),which showed yeast cells morphologically suggestive of H. capsulatum. As the bone marrow aspirate was not sent for culture at the first instance, repeated bone marrow aspiration was done on day +7 for confirmation of histoplasmosis. The aspirate was inoculated on to brain heart infusion agar, Sabouraud's dextrose agar with
Fig. 1. Leishman stain: peripheral blood smear showing (A) thrombocytopenia ( 400) with (B) giant platelets ( 1000).
Fig. 2. (A) Giemsa 400: BMA particulate with normal cellularity, M:E ratio 2:1, normal erythropoiesis, myelopoiesis, megakarycytes slightly increased in number with normal morphology. (B) Giemsa 400 (inset Giemsa 1000): histiocytes showing intracytoplasmic yeasts.
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Fig. 3. Trephine section showing yeast cells stained with PAS (A) and SM (B) ( 400).
2.1. Imaging Chest radiograph and ultrasound scanning of abdomen which were done on day +3 of hospital stay were normal. 2.2. Treatment
Fig. 4. Lactophenol cotton blue mount ( 400): large, rounded, single-celled, tuberculate macroconidia and small, pyriform shaped microconidia of Histoplasma capsulatum. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this article.)
chloramphenicol, and Sabouraud's dextrose agar with cycloheximide and chloramphenicol. As H. capsulatum is a CDC/NIH risk group 3 agent, handling of clinical material and isolates were done in the Class II Bio Safety Cabinet with BSL2 practices, containment equipment and facilities. On Sabouraud's dextrose agar at 25 1C, colonies appeared after 1 week of incubation i.e. on day +14. The colonies were highly folded, adherent, initially white, turning buffbrown. Microscopic morphology showed the presence of characteristic large, rounded, single-celled, tuberculate macroconidia and small, pyriform shaped microconidia (Fig. 4), based on which the fungus provisionally was identified as H. capsulatum. The isolate was sent to the National Culture Collection of Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research, Chandigarh, for confirmation. At NCCPF, the identity of the isolate was confirmed by morphology, conversion to yeast phase and sequencing of rDNA. For extraction of DNA, the isolate was inoculated in Sabouraud dextrose broth and incubated in a shaker incubator (130 rpm) at 35 1C for 5 days. The cells were killed by adding thiomersal at a final concentration of 0.02% (wt/v) and incubating it at room temperature for 1 h. The hyphal mass was separated by filtration and then transferred to a sterile mortar and pestle. Whole cell DNA from the fungi was extracted by crushing the mycelial mat with liquid nitrogen followed by extraction using phenol: chloroform extraction method as described previously by Baghela et al. [11]. Sequencing of 28s region of rDNA was performed using primer pairs NL1 (5′-GCATATCAATAAGCGGAGGAAAAG) and NL-4 (5′-GGTCCGTGTTT CAAGACGG). Consensus sequences were prepared by using sequence obtained by forward and reverse primers with the help of Bionumerics software Version 6.6 (Applied Maths, Ghent, Belgium). Pair wise sequence alignment with the database showed 499% similarity with the CBS 136.72 strain of H. capsulatum. The isolate is deposited at NCCPF as NCCPF 230016.
The patient’s platelet counts at admission were 10,000/cmm and were continuing to fall further for which he received platelet transfusions on alternate days. On day +8, patient was started on treatment with injection amphotericin B deoxycholate at a daily dose of 1 mg/kg b wt. Pain abdomen gradually subsided and the patient’s appetite improved. Platelet count increased to 30,000/ cmm on day +13 i.e. after 5 days of starting of amphotericin. Patient was given amphotericin B deoxycholate for 14 days followed by itraconazole 200 mg twice a day with a plan to continue for 1 year. Platelet count was 50,000/cmm at discharge on day +22. 2.3. Follow up and outcome Patient reported to the general medicine outpatient department for follow up on day +52. Patient showed marked clinical improvement and platelet counts on that day were 150,000/cmm. Platelet count was 160,000/cmm at the next folloup on day+112. The patient's renal function and liver function were monitored throughout the course of treatment with antifungals till his last visit (day 112) to the outpatient department for follow up and were found to be within normal limits.
3. Discussion H. capsulatum causing histoplasmosis is presently classified into six distinct clades based on combined data analysis of partial sequences of four protein encoding genes of 46H. capsulatum isolates. These include five clades of H. capsulatum var capsulatum: (i) class 1 North American;(ii) class 2 North American; (iii) Central American; (iv) South American group A (which includes the previous H. capsulatum var farciminosum; (v) South American group B; and one clade for H. capsulatum var duboisii [12]. The first case of histoplasmosis in India was reported in 1954 [2]. Subsequently several cases have been reported from different regions of India [3–10]. In India, the fungus was isolated from the environment of an old building along the Ganges River close to Calcutta [13]. There have been sporadic reports of histoplasmosis in patients from Andhra Pradesh [9,14]. In our hospital, prior to the present case, progressive disseminated histoplasmosis was diagnosed in two patients with HIV infection, in whom H. capsulatum var capsulatum was isolated from lymph node biopsy (unpublished data). Both patients hailed from the eastern part of the country known to be endemic for
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histoplasmosis. In the present case, the patient hails from the Adilabad district in the state of Andhra Pradesh in southern India and has never traveled outside the district before his illness started. The climate of the district is characterized by hot summer and in general is dry except during the south-west monsoon season from June to September when 80% of the yearly rainfall is received. This patient became symptomatic in July when the district has a precipitation of 10 in., temp.—max 310 1C and min 240 1C, humidity 84%. The present patient is a farmer by occupation and could have been exposed to contaminated soil during the course of his work. In their review of endemic fungal infections including histoplasmosis in the Asia-Pacific region, Chakrabarti and slavin [15] conclude that an accurate epidemiological picture of endemic fungal infections remains unclear due to inadequate laboratory facilities, limited experience with fungal infections and few epidemiological surveys performed in these regions. PDH develops mostly in patients who are immunocompromised and are unable to mount an effective CMI response to the organism. This includes patients with AIDS [1,9,16,17] transplant recipients [1,9,14], those with hematologic malignancies [1,9], and those on corticosteroids [1,9]. In the present case, the patient had no major identifiable underlying immunocompromising condition. Thrombocytopenia is reported in histoplasmosis along with other cytopenias whenever there is bone marrow involvement [1,9] but reports of patients presenting with isolated thrombocytopenia as in the present case are rare [18,19]. Hood et al. [18] reported two patients with disseminated histoplasmosis and predominant thrombocytopenia. One patient presented with severe thrombocytopenic purpura and splenomegaly. Investigations did not reveal any evidence of histoplasmosis. She died on the sixth hospital day from a massive intra-cerebral hemorrhage and one kidney was removed for homotransplantation. Histoplasmosis was diagnosed at autopsy. The second patient, with chronic glomerulonephritis and uremia, who received the renal homograft from the first patient, developed initial signs of homograft rejection, 5 days postoperatively. Diffuse thrombocytopenic purpura occurred shortly thereafter. Spores of Histoplasma capsulatum were observed in blood smears, in leukocyte concentrates, and in 5-day leukocyte cultures from the blood obtained prior to death. Disseminated histoplasmosis was found also at autopsy. Both patients received corticosteroids, in the first case as treatment for thrombocytopenia and in the second patient as immunosuppressive therapy following renal transplantation. Armitage et al. [19] reported two cases of disseminated histoplasmosis with thrombocytopenia dominating the clinical picture. In one case, the patient was diagnosed with histoplasmosis and recovered after being treated with amphotericin B. In the other case the patient was treated with steroids for a mistaken diagnosis of immune thrombocytopenic purpura (ITP) and succumbed, the diagnosis of histoplasmosis being made at autopsy. These studies conclude that disseminated histoplasmosis should always be considered in the differential diagnosis of thrombocytopenia, as the use of corticosteroids for treatment of the thrombocytopenia with failure to diagnose the underlying disease can prove fatal. In our case, though ITP was considered as an admitting diagnosis we did not administer corticosteroids. The reasons being the age of the patient, complaint of epigastric pain and loss of appetite. An elevated LDH (2260 U/L) which was done as part of work up for thrombocytopenia substantiated our decision not to administer steroids empirically and a decision was made to defer specific therapy till the bone marrow examination reports were available. In the present case PDH was confirmed by direct demonstration of the organism in the bone marrow and growth of the organism in culture.
Des Prez et al. [20] in their studies on the pathogenesis of thrombocytopenia in histoplasmosis conclude that the yeast from H. capsulatum activates the platelets resulting in serotonin release reaction and aggregation without the involvement of the complement pathway. The plasma cofactors which are involved are Ig G which is necessary for induction of release reaction. Another feature observed in this patient was that despite involvement of bone marrow our patient was afebrile throughout hospital course. The gastrointestinal tract is involved in 70–90% of cases of disseminated histoplasmosis. The present patient complained of persistent epigastric pain which subsided after receiving 2 weeks of amphotericin B. Biopsy of the upper gastrointestinal endoscopy could not be done due to persistent thrombocytopenia. Though ultrasound of abdomen did not reveal evidence of pancreatitis, serum lipase and amylase were elevated. Pancreatitis is reported to be a rare manifestation of disseminated histoplasmosis. A patient with histoplasmosis and pancreatitis [21] diagnosed at autopsy is reported where the patient had very high lipase and moderately elevated amylase levels. For the management of moderately severe to severe histoplasmosis, liposomal amphotericin B (3.0 mg/kg daily) is recommended for 1–2 weeks, followed by oral itraconazole (200 mg three times daily for 3 days and then 200 mg twice daily for a total of at least 12 months [22]. The deoxycholate formulation of amphotericin B (0.7–1.0 mg/kg daily) is recommended as an alternative to a lipid formulation in patients who are at a low risk for nephrotoxicity. In the present case as the patient could not afford liposomal amphotericin B, he was treated with amphotericin B deoxycholate for 2 weeks followed by oral itraconazole. PDH should always be considered in the differential diagnosis of thrombocytopenia, irrespective of the patient's immune status and endemicity of the disease in the region. Recognition of the varied and confounding clinical presentations of histoplasmosis, good diagnostic laboratory facilities and extensive population based studies to know the endemicity of the disease in different regions are all necessary for improved understanding of the epidemiology of histoplasmosis.
Conflict of interest statement There are none.
Acknowledgments We acknowledge Prof. Arunaloke Chakrabarti, In Charge, National Culture Collection of Pathogenic Fungi (supported by Indian Council of Medical Research, New Delhi) and Head Department of Medical Microbiology, PGIMER, Chandigarh for confirming the identity of the isolate. References [1] Kauffman CA. Histoplasmosis: a clinical and laboratory update. Clinical Microbiology Reviews 2007;20(1):115–132. [2] Panja G, Sen S. A unique case of histoplasmosis. Journal of the Indian Medical Association 1954;6:257–258. [3] Randhawa HS. Occurrence of histoplasmosis in Asia. Mycopathologia et Mycologia Applicata 1970;41:75–89. [4] Padhye AA, Pathak AA, Katkar VJ, Hazare VK, Kauffman L. Oral histoplasmosis in India: a case report and an overview of cases reported during 1968–92. Journal of Medical and Veterinary Mycology 1994;32:93–103. [5] Randhawa HS, Khan ZU. Histoplasmosis in India: current status. The Indian Journal of Chest Diseases and Allied Sciences 1994;36:193–213. [6] Randhawa HS, Chaturvedi S, Khan ZU, et al. Epididymal histoplasmosis diagnosed by isolation of Histoplasma capsulatum from semen. Mycopathologia 1995;131:173–177.
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