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Infections in Hemopoietic Stem Cell Transplant Recipients, Part I
Clinical Microbiology Newsletter Vol. 32, No. 18
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September 15, 2010
Infections in Hemopoietic Stem Cell Transplant Recipients, Part I* Rafik Samuel, M.D.,1 Allan L. Truant, Ph.D.,2 and Byungse Suh, M.D., Ph.D.,1 Section of Infectious Diseases, Department of Medicine, Temple University School of Medicine,1 and Clinical Microbiology, Immunology, and Virology Laboratories, Department of Pathology and Laboratory Medicine, Temple University Hospital and School of Medicine,2 Philadelphia, Pennsylvania
Abstract Hemopoietic stem cell transplantation, often called bone marrow transplantation, is a relatively new medical procedure that is now used to treat patients who were diagnosed with diseases once thought incurable, such as hematologic malignancies, immune deficiencies, solid tumors such as breast and ovarian cancers, sickle cell disease, and genetic-defect disorders. Patients who receive bone marrow transplantation also receive severe immunosuppression therapy as part of their care to minimize the risk of donor stem cell rejection. As a consequence of the patient’s profound immunosuppression, opportunistic infections emerge as common causes of disease. Part I of this article will review some of the more common opportunistic fungal infections that can occur in these immunocompromised patients, with an emphasis on methods for their laboratory diagnosis.
Introduction Hemopoietic stem cell transplantation (HSCT), more commonly known as bone marrow transplantation (BMT), is a relatively new medical procedure. The procedure is used to treat patients diagnosed with diseases once thought incurable, such as hematologic malignancies, immune deficiencies, solid tumors such as breast and ovarian cancers, sickle cell disease, and genetic-defect disorders. There are several different types of HSCT. In autologous transplantation, patients function as their own donors; allogeneic transplantation uses HLAmatched donors of related or unrelated origins; and syngeneic transplantation *Editor’s Note: Part II of this article will be published in the October 1, 2010 issue of CMN (Vol. 32, No. 19).
Corresponding Author: Allan L. Truant, Ph.D., Clinical Microbiology Laboratories, Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3401 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-3210. Fax: 215-7072053. E-mail: [email protected]
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involves identical twins as donor and recipient. According to a recent report, the total number of patients undergoing HSCT worldwide was approximately 45,000, of which those with autologous and allogeneic transplants represent 30,000 and 15,000 cases, respectively (1). Major inherent problems in HSCT recipients that contribute to mortality and morbidity, include relapse of the original condition, graft-versus-host disease (GVHD), infections, and multiorgan failure. Overall, in patients with HSCT, relapse of underlying diseases contributes to 21 to 78% of the mortality, while infections and GVHD are responsible for 5 to 21% and 15% of deaths (1). This article will focus on infectious complications commonly encountered in HSCT recipients. Infections in this patient population are seen as a consequence of profound immunosuppressive treatment necessary to remove as many pathologic cells as possible and to suppress the immunologic ability to reject the transfused cells. This profound immunosuppression in the HSCT recipients results in severe neutropenia, © 2010 Elsevier
defects in immunity, and disruption of the integrity of mucous membranes, leading to mucositis. The types and timing of the infections encountered in HSCT recipients in relation to the transplantation procedure are depicted in Fig. 1 (2) and provide the basis for this article. Available identification methods for pathogens described in the review can be found in the Manual of Clinical Microbiology (3), the Manual of Molecular and Clinical Laboratory Immunology (4), the Manual of Commercial Methods in Clinical Microbiology (5), and the Clinical Microbiology Procedures Handbook (6).
Immunizations Immunizations, one of the most significant milestones in preventive medicine, are the most effective means
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Pneumocystis jirovecii
Figure 1. Phases of opportunistic infections among allogeneic HSCT recipients (n = 8), (modified from reference 2).
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of reducing a variety of infections in humans. Immunization can be divided into two major categories, active and passive. Active immunization is defined as the administration of a vaccine, which causes the host to develop immunity to the agent contained in the vaccine. Vaccines are available in many different forms: attenuated live, inactivated whole organism, cellular subunit or component, or toxoid. Passive immunization is defined as the administration of preformed antibodies, which are protective against the disease under consideration. Active immunization usually results in long-term protection lasting several decades to a lifetime. Passive immunization produces immediate protection, but the immunity is relatively brief and is commonly used to prevent the onset of clinical disease. It may either be administered following exposure or prior to an anticipated exposure. HSCT recipients have a diminished level of immune function and therefore respond to antigenic challenge poorly and rather unpredictably. Also, antibody titers to vaccine-preventable diseases are known to decline during the first 1 to 4 years after transplantation and may require re-vaccination. A 1994 national survey of transplantation centers participating in the National Marrow Donor Program (7) led to national guidelines for vaccinations for HSCT recipients. These guidelines were published in 2000 under the co-sponsorship of the CDC, the Infectious Disease Society of America, and the American Society of Blood and Marrow Transplantation (2). The modified vaccination schedule published in the guidelines also can be found in reference 8.
Mycotic Infections Fungal infections that are frequently encountered in HSCT recipients are divided into three categories: yeast, mold, and endemic fungal infections. Recommendations on the therapeutic management of these infections are summarized in Table 1, and additional information on therapeutic options for all infections discussed in this review can be obtained from reference 8. However, current therapeutic recommendations should always be obtained prior to final decisions for any specific patient.
Yeast Infections Candida species are commonly Clinical Microbiology Newsletter 32:18,2010
encountered during the early post-transplantation stage. Conditioning regimens cause severe neutropenia and mucositis, and HSCT recipients are susceptible to Candida infections. Subsequently, the host defense mechanisms of the skin, intestine, and the liver are further compromised due to GVHD. Candidemia can cause a high mortality rate ranging from 40 to 60%. Among Candida species, Candida albicans is most frequently encountered, representing approximately 50%, and the other species, including Candida glabrata Candida tropicalis, Candida parapsilosis, Candida krusei, and Candida lusitaniae, account for the rest. Of these species, C. parapsilosis has been associated more often with vascular devices. Another Candida species, Candida dubliniensis, can be misidentified as C. albicans because both species are capable of germ tube formation (9). Chromogenic isolation media are available to correctly detect and identify C. albicans, C. tropicalis, and C. krusei. Candida bloodstream infections may lead to either acute or chronic disseminated diseases. Acute disseminated candidiasis may involve multiple organs, including the kidney, heart, skin, and musculoskeletal system. C. tropicalis is often isolated from neutropenic patients with this syndrome (10). Chronic disseminated candidiasis, which may be a complication of severe Candida mucositis and often affects the liver and spleen in addition to other organs, is often referred to as “hepatosplenic candidiasis.” Most infections caused by Cryptococcus neoformans are acquired through the respiratory system with colonization in the lungs. In the immunocompetent host, C. neoformans is eliminated by the normal host defense mechanisms. In the immunocompromised host, however, the organism spreads hematogenously to extrapulmonary organs, where it has a predilection for the central nervous system (CNS). Prolonged hypercortisolism, either endogenous or exogenous in nature, is known to predispose patients to cryptococcal infections, which may be the case in allogeneic HSCT recipients with GVHD. The most common presentation of CNS cryptococcosis is meningitis, usually subacute, although acute at times in onset. Complications include © 2010 Elsevier
hydrocephalus, elevated intracranial pressure, encephalitis, hearing or visual loss, facial weakness, and seizure. Occasionally, mass lesions in the brain or spinal cord can be observed. The clinical course may vary greatly depending on the immunocompetence of the host. A definitive diagnosis of cryptococcosis is made by culturing the organism from specimens obtained from the involved site. The detection of cryptococcal capsular polysaccharide antigen by latex agglutination or enzyme immunoassay (EIA) in serum or cerebrospinal fluid (CSF), is used to diagnose cryptococcosis rapidly, with specificity and sensitivity approaching 100%. The India ink preparation may be positive in 25 to 50% of patients with cryptococcal meningitis. Trichosporon beigelii is occasionally isolated as part of the normal flora of human skin and is known to colonize the gastrointestinal and respiratory tracts in immunosuppressed hosts. T. beigelii grows on routine media used in the mycology laboratory. In profoundly immunosuppressed hosts with neutropenia, it may cause disseminated disease with a high mortality rate of 74% (11). Disseminated trichosporonosis may cause fungemia, funguria, endocarditis, cutaneous lesions, renal failure, and pneumonia. The genus Malassezia, formerly known as Pityrosporum, consists of lipophilic yeasts, and the carriage rate of Malassezia spp. on the normal skin approaches 100% in adults. They live on the skin surface, as well as in the ducts of hair follicles and sebaceous glands. Malassezia is associated with a folliculitis that is usually maculopapular but occasionally is pustular. The lesion is commonly seen in immunocompromised patients, such as solid organ transplant recipients, but the incidence of Malassezia infections in HSCT recipients is rare (12). Folliculitis, caused by Malassezia, is usually diagnosed by the microscopic examination of skin scrapings from the lesion in a KOH preparation or by using Calcifluor white stain. To isolate Malassezia furfur, the growth medium must be supplemented with a lipid component by overlaying Sabouraud’s glucose agar with a few drops of sterile olive oil. Mold infections remain the most dreaded infectious complications of 0196-4399/00 (see frontmatter)
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Table 1. Recommended therapies for fungal and parasitic infections post-HSCT Clinical presentation 1. Non-neutropenic patients with candidiasis or disseminated candidiasis
2. Neonatal candidiasis 3. Cryptococcal meningitis (based on HIV-related infections) 4. Trichosporon infections 5. Malassezia infections 6. Invasive aspergillosis
7. Fusarium infections
8. Zygomycosis 9. Histoplasmosis: moderate to severe pulmonary
Histoplasmosis: disseminated Histoplasmosis: CNS 10. Coccidioides: extensive infection
11. Blastomycosis: moderate to severe pulmonary or disseminated extrapulmonary Blastomycosis: mild to moderate disease or mild to moderate disseminated or osteoarticular disease Blastomycosis: CNS 12. Pneumocystis jirovecii infections
13. Toxoplasmosis
14. Malaria
Malaria prophylaxis for HSCT recipients 15. Strongyloidiasis 16. Chagas’ disease 17. Cryptosporidiosis
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Recommended therapy a. Fluconazole or b. Caspofungin, micafungin, or anidulafungin c. Deoxycholate amphotericin B (DAmB), liposomal amphotericin B, or amphotericin B lipid complex (LFAmB) d. Voriconazole a. Amphotericin B for disseminated candidiasis b. Liposomal amphotericin B if urinary tract involvement. a. DAmB or LFAmB with or without flucytosine (2 wk), followed by fluconazole (8 wk), followed by maintenance daily fluconazole a. Amphotericin B plus fluconazole or voriconazole a. Little data available; possibly fluconazole a. Voriconazole b. LFAmB, DAmB, or posaconazole, or itraconazole, or caspofungin, or micafungin as salvage therapy a. DAmB alone or with flucytosine or rifampin or b. LFAmB or c. Voriconazole or posaconazole a. Usually surgery plus antifungal therapy of DAmB or LFAmB or b. Possibly posaconazole from in vitro and animal studies a. LFAmB or DAmB (1-2 wk), possibly with methylprednisolone followed by itraconazole (12 wk) b. For chronic cavity pulmonary histoplasmosis; itraconazole (1-2 yr) a. LFAmB or DAmB (1-2 wk), followed by oral itraconazole (1 yr or longer for immunosuppressed patients) a. LFAmB (4-6 wk), followed by oral itraconazole (min. 1 yr or until resolution of CSF abnormalities) a. Possible surgical debridement plus antifungal therapy with fluconazole (especially for CNS infections), itraconazole, or DAmB (as second alternate); AmB usually reserved for patients with respiratory failure, pregnant individuals, or rapidly progressing infection a. LFAmB or DAmB (1-2 wk), followed by oral itraconazole (6-12 mo) a. Oral itraconazole (6-12 mo) a. LFAmB or DAmB (4-6 weeks) followed by oral azole (minimum 12 mo). In pregnancy, LFAmB is recommended (avoid azoles) a. Trimethoprim-sulfamethoxazole (TMP/SMZ) b. Alternative therapy includes pentamidine, atovaquone, trimetrexate, trimethoprim plus dapsone, or high-dose clindamycin. a. Pyrimethamine with folinic acid and sulfadiazine b. Prophylaxis in seropositive HSCT recipients: TMP/SMZ or pyrimethamine/sulfadoxine a. Chloroquine for susceptible strains b. For chloroquine-resistant Plasmodium vivax, mefloquine or halofantrine c. For mefloquine-resistant Plasmodium falciparum, quinine sulfate or quinidine and doxycycline d. Alternate therapy, atovaquone/proguanil with or without artesunate or artesunate alone a. Chloroquine or b. Mefloquin, atovaquone/proguanil, or doxycycline a. Ivermectin or b. Thiabendazole a. Nifurtimox (available as an investigational drug from CDC) b. Benznidazole a. Nitazoxanide (documented use in immunocompetent but controversial in immunocompromised patients) b. Spiramycin or paromomycin with azithromycin, TMP/SMZ, or somatostatin
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HSCT recipients. These infections carry very high mortality rates, approaching 100% unless the immune status recovers. The majority of HSCT recipients remain severely immunocompromised, immediately before and after the HSCT procedure for up to approximately 180 days, rendering them at high risk for mold infections. Aspergillus and other molds, including Fusarium, Scedosporium, and Zygomycetes also play an important role as pathogens in these patients. Aspergillus species are ubiquitous in the environment worldwide and are responsible for the second most common systemic fungal infections requiring hospitalization in the United States, next to candidiasis. The common species infecting humans are Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans. Of these, A. fumigatus causes approximately 90% of invasive aspergillosis. Aspergillus species are usually susceptible to amphotericin B; however, A. terreus may be resistant. Risk factors for invasive aspergillosis (IA) include neutropenia, high-dose corticosteroid therapy, and cytotoxic therapy. The respiratory tract is the usual portal of entry by inhalation of airborne conidia. Damaged skin, surgical wounds, or the external ear may also serve as entry sites. In HSCT recipients, the firstline defense is compromised due to the reduced number of macrophages, monocytes, and neutrophils. Hence, the incidences of IA in allogeneic and autolgous HSCT patients are reported to be 4 to 9% and 0.5 to 6%, respectively, and pulmonary infections account for 80 to 90% of invasive aspergillosis (13). The most characteristic early lesions, which may be demonstrated by CT scans, are small, pleural-based nodules with surrounding low attenuation (the “halo” sign). As patients recover from neutropenia, the nodules may cavitate and reveal an “air-crescent” sign. The halo and crescent signs are typical of aspergillosis but are not definitive. Aspergillus may disseminate hematogenously to extapulmonary sites, including the brain, myocardium, liver, spleen, skin, and kidneys. The brain is the most commonly involved site in 25 to 40% of invasive pulmonary aspergillosis cases, and MRI images may reveal lesions that were not detected by CT scans. The demonstration of Aspergillus Clinical Microbiology Newsletter 32:18,2010
hyphae in tissue, usually best visualized by silver staining, is the best means of diagnosis. Aspergillus hyphae are 2 to 4 μm wide, frequently septate, and branch acutely at 45°. Blood cultures of patients with IA are invariably negative. The monitoring of galactomannan, a cell wall component of Aspergillus, by EIA is a potentially useful method for diagnosis. Fusarium is the second-most-common filamentous fungal pathogen in HSCT recipients after Aspergillus. The portal of entry and risk factors for fusariosis are similar to those of aspergillosis. Fusariosis shows a bimodal distribution in its onset of clinical manifestations in HSCT recipients. The most common species to cause human infections is Fusarium solani, accounting for approximately 50% of the cases, while other species, such as Fusarium moniliformis, Fusarium oxysporum, Fusarium dimerum, and Fusarium proliferatum account for the rest. The most common presentation of fusariosis in immunocompromised hosts is pulmonary infections, with dissemination to extrapulmonary sites occuring in as many as 70% of cases. The most common extrapulmonary site is the skin. Diagnosis is usually made by biopsy demonstrating the presence of acutely branched and septate hyphae when prepared with methenamine silver or periodic acid-Schiff (PAS) stain. Fungal cultures are mandatory to confirm and to differentiate it from Aspergillus species. Blood cultures are positive in approximately 50% of disseminated cases of fusariosis. Zygomycosis, previously known as phycomycosis or mucormycosis, refers to uncommon fungal infections caused by a group of fungi belonging to the class Zygomycetes. The majority of organisms causing the infections belong to three genera: Rhizopus, Mucor, and Rhizomucor. These fungi are ubiquitous in the environment. When histologically examined, the Zygomycetes are broad, nonseptate or pauciseptate, thick-walled, irregularly shaped hyphae with rightangle branching. Culture is positive in only 25% of histologically confirmed cases of pulmonary zygomycosis. Final identification to the species level requires culture of the fungus, although species identification is not necessary for treatment. © 2010 Elsevier
There is a bimodal distribution of onset, early and late, but zygomycosis tends to occur later after allogeneic HSCT transplantation, corresponding to the severity of GVHD and the intensity of corticosteroid treatment (14). The major mode of transmission is believed to be the respiratory route, although the traumatized-skin and gastrointestinal routes play minor roles. Host factors predisposing to zygomycosis include immunosuppression, uncontrolled diabetes mellitus, deferoxamine therapy, skin breakdown, intravenous drug abuse, and malnourishment. The best-known condition associated with zygomycosis is uncontrolled diabetes mellitus with ketoacidosis. There is evidence that the incidence of zygomycosis is increasing in HSCT recipients. Clinical manifestations of zycomycosis are usually classified by the site involved: rhinocerebral, sinus, pulmonary, gastrointestinal, cutaneous, abdominopelvic, and disseminated. Rhinocerebral zygomycosis typically originates in the nares, palate, or paranasal sinuses with gradual or rapid involvement of the eye and brain. Symptoms may be nonspecific and may progress to facial and periorbital swelling and numbness with blurred vision, corneal anesthesia, orphthalmoplegia, and facial anhidrosis. Once the brain is involved, there may be a deterioration of mental status, onset of seizures, or cerebrovasular accidents. Radiographic procedures, such as CT scan and MRI, are required to determine the extent of disease process. Pulmonary zygomycosis resembles aspergillosis. Zygomycosis also tends to cause vascular invasion as with aspergillosis, resulting in thrombosis, hemorrhage, and infarction. If a lung biopsy specimen reveals the presence of definite fungal elements but culture fails to grow, the presumptive diagnosis favors zygomycosis over aspergillosis. Cutaneous zygomycosis can be caused by direct invasion of preexisting wounds. The lesions may involve not only superficial layers, but also deeper tissue, such as muscle, fascia, and bone. The diagnosis is made primarily by direct histopathologic examination and by fungal culture. Endemic mycoses conventionally refer to histoplasmosis, blastomycosis, and coccidioidomycosis because they occur in a geographically specific area. 0196-4399/00 (see frontmatter)
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These infections are caused by Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis, respectively. These organisms exhibit thermal dimorphism: at a temperature of 25 to 30°C in the laboratory and in the natural habitat, they grow as molds, but when grown at 37°C, they are yeasts, except C. immitis, which in tissue phase is a spherule. The initial infections can be dormant for many years, and they may reactivate to reveal clinical manifestations as the host undergoes immunosuppression. In healthy hosts with low-level exposure to H. capsulatum, fewer than 5% develop symptomatic disease, whereas most patients develop symptomatic infection following heavy exposure. Flu-like pulmonary illnesses, pericarditis, arthritis, or arthralgias with erythema nodosum are the most common manifestations of histoplasmosis. In immunosuppressed patients, such as transplant recipients, however, widely disseminated infection occurs in the majority of patients (88%), with predominant involvement of the lungs and other organs throughout the reticuloendothelial system. Clinical presentation of disseminated histoplasmosis is nonspecific, and usually the symptom complex consists of fever, chills, fatigue, anorexia, and weight loss. On examination, one should look for hepatosplenomegaly, oropharyngeal ulcers (25 to 75%), and cutaneous lesions, including papules, pustules, plaques, ulcers, abscesses, and cellulitis. When the CNS is involved, patients may present with headache, confusion, and seizures. Supportive laboratory findings may involve pancytopenia, elevated alkaline phosphatase, elevated erythrocyte sedimentation rate, and electrolyte abnormalities suggestive of adrenal insufficiency. The definitive diagnosis of histoplasmosis can be made by culture confirmation of the etiologic agent, but routine blood cultures in broth do not yield positive results. The Isolator system (Wampole Laboratories, Princeton, NJ.), also known as the lysis-centrifugation blood culture method, appears to be superior for Candida, Cryptococcus, and Histoplasma compared to automated systems. Effective Histoplasma antibody levels often do not appear in immunosup140
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pressed patients; therefore, classical methods, such as complement fixation and immunodiffusion tests measuring the patient’s antibody levels, are not as useful as in healthy hosts. The detection of H. capsulatum antigen by EIA in body fluids, such as serum, is extremely useful in the initial diagnosis and subsequent monitoring of disease progression during therapy in patients with disseminated histoplasmosis. C. immitis is a dimorphic fungus. The mold form represents a saprophytic phase and grows in soil, whereas inhalation of the conidia into the lungs results in the morphologic change into large, thick-walled spherules that become filled with hundreds of endospores. Each endospore has the potential to form a new spherule, thus continuing the infection cycle. Patients with compromised cellular immunity, including transplant recipients and HIV/AIDS patients, are at higher risk for developing extrapulmonary disseminated disease. Also, pregnant individuals and persons of African and Filipino descent have a higher risk for disseminated disease. Two disseminated and one localized pulmonary cases of coccidioidomycosis have been described in HSCT recipients; both disseminated cases died, while the localized case survived. Acute pulmonary infections are asymptomatic in 60% of healthy individuals, but patients with symptoms present with a flu-like illness. Arthralgias and myalgias may be prominent, and erythema multiforme or erythema nodosum may also be present. Chest radiograph is abnormal in 50% of the symptomatic patients, and eosinophilia in peripheral blood is frequently observed. Symptoms usually clear in a few weeks. In immunocompromised patients, coccidioidomycosis tends to disseminate, with the skin, subcutaneous tissues, bones, and meninges being the most common sites involved. Blood cultures are usually positive in this patient population. Coccidioidomycosis in solid organ transplant recipients carries a dismal prognosis. The definitive diagnosis of coccidioidomycosis is made by isolating the organism from appropriate clinical specimens, such as tissue aspirate or biopsy specimens, sputum, and body © 2010 Elsevier
fluids. The fungus grows well on routine mycology culture media, and it takes only 2 to 7 days. A DNA-specific probe is now available, and correct identification is possible as soon as growth is obtained. Demonstration of typical spherules, measuring 20 to 80 μm in diameter on wet mounts with KOH preparation, may lead to more rapid diagnosis. Serology plays an important role in the diagnosis and follow-up of immunocompromised patients with coccidioidomycosis, whereas the response in immunocompromised hosts may be poor. Tube precipitin-reacting antibodies, which represent IgM antibodies, are present in primary infection and disappear as the infection enters the chronic phase. IgG antibodies detected by complement fixation or other methods appear instead during the chronic phase of infection and persist. B. dermatitidis, which is the asexual stage of Ajellomyces dermatitidis, is a dimorphic fungus. It grows at room temperature as a whitish/tan mold and grows within the host or at 37°C as budding, yeast-like cells with a thick, refractile cell wall that reproduce by single broad-based buds. The organism proliferates in warm and moist soil rich with decaying organic matter, and persons with occupational exposure to soil appear to have the highest risk of developing this infection. Blastomycosis usually affects more non-compromised hosts; however, increasing numbers of cases are being described among transplant recipients (15). Human blastomycosis usually occurs through inhalation of conidia into the lungs, which usually leads to asymptomatic or self-limited infections but occasionally leads to chronic pneumonia. Although some patients with pulmonary blastomycosis recover spontaneously, most patients require treatment. Blastomycosis may involve the lungs, skin, bone, and genitourinary tract, in decreasing order of frequency. Pulmonary blastomycosis in transplant recipients may occasionally disseminate to involve the skin, skeletal system, and male genitourinary tract. Mortality rates approach 30 to 40%, and most deaths occur during the first few weeks of therapy. Demonstration of the yeast from clinical specimens or growth of the fungus Clinical Microbiology Newsletter 32:18,2010
in culture usually makes a diagnosis, but culture may take 2 to 4 weeks of incubation. The examination of KOH preparations and/or tissue biopsy specimens stained with PAS or methenamine silver stain may be used for direct specimen diagnosis. The organism can also be identified by employing highly specific and sensitive DNA probes. Neither serology nor skin testing for blastomycosis is specific or sensitive, and therefore, they have low clinical utility. Another technique, known as calcofluor white stain, that enhances the detection of fungi by binding to β-1,3- and β-1,4polysaccharide of the cell wall in fungi is available. This method, however, needs a fluorescence microscope equipped with a barrier filter to protect the eye. Fungi exhibit green fluorescence (16). The gene sequence studies on Pneumocystis carinii indicate that it should be categorized as a fungus. These organisms show 60 and 20% homology with fungi and protozoa, respectively. They are currently placed in the phylum Ascomycota within a newly described class, Aschiascomycetes. The new nomenclature for the Pneumocystis species that causes human disease is Pneumocystis jirovecii, since P. carinii can cause disease only in rats (17). Very few Pneumocystis organisms (<10 organisms) are required to initiate a fulminant infection in immunocompromised rats. Most humans become seropositive by 2 to 4 years of age, and the organism can survive indefinitely without causing any ill effect until the host immune system is severely compromised. In AIDS patients, 95% of Pneumocystis cases occur in patients with CD4 counts of less than 200/mm3, with an annual incidence of 8%. Clinical signs and symptoms of Pneumocystis pneumonia are those of atypical pneumonia, which has a rather slow onset over a 2- to 4-week period of fever, nonproductive cough, and increasing dyspnea. The clinical picture, however, may be very variable. The chest radiograph typically shows
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bilateral diffuse infiltrates, perihilar in distribution. Although there is no large prospective study comparing signs and symptoms in HSCT recipients, it is prudent to think that the clinical picture would be similar to that seen in HIVpositive patients. Histopathologic staining of a sputum or preferably a bronchoalveular lavage specimen makes a definitive diagnosis. Methenamine silver, toluidine blue, and cresyl echt violet are most commonly used to selectively stain the cell wall. The Wright-Giemsa stain is used to stain the nuclei of all developmental stages of the organism. In addition, specific immunofluorescence has been used with success. In allogeneic HSCT recipients, Pneumocystis infection develops in 10 to 20% of patients, typically during the period immediately after engraftment until 6 months following HSCT. During this period and beyond for those requiring immunosuppressive agents, Pneumocystis prophylaxis should be maintained. Editor’s Note: Part II of this article will be published in the October 1, 2010 issue of CMN (Vol. 32, No. 19). References 1. Loberiza, F., Jr. 2003. Report on state of the art in blood and marrow transplantation — part 1 of the IBMTR/ABMTR summary slides with guide. IBMTR/ ABMTR Newsl. 10:7-10. 2. Center for Disease Control and Prevention. 2000. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Disease Society of America, and the American Society of Blood and Marrow Transplantation. MMWR Morb. Mortal. Wkly. Rep. 49(RR-10):1-128. 3. Murray, P.R. et al. (ed.) 2007. Manual of clinical microbiology, 9th ed. ASM Press, Washington, DC. 4. Detrick, B., R.G. Hamilton, and J.D. Folds (ed.) 2006. Manual of molecular and clinical laboratory immunology, 7th ed. ASM Press, Washington, D.C.
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5. Truant, A.L. (ed.) 2002. Manual of commercial methods in clinical microbiology. ASM Press, Washington, DC. 6. Isenberg, H.D. (ed.) 2004. Clinical microbiology procedures handbook. ASM Press, Washington, DC. 7. Henning, K.J. et al. 1997. A national survey of immunization practices following allogeneic bone marrow transplantation. JAMA 277:1148-1151. 8. Samuel, R.A., A.L. Truant, and B. Suh. 2005. Cumitech 42, Infections in hemopoietic stem cell transplant recipients. Coordinating ed., A.L. Truant. ASM Press, Washington, DC. 9. Gutierrez, J. et al. 2002. Candida dubliniensis, a new fungal pathogen. J. Basic Microbiol. 42:207-227. 10. Wingard, J.R., W.G. Merz, and R. Saral. 1979. Candida tropicalis: a major pathogen in immunocompromised patients. Ann. Intern. Med. 91:539-543. 11. Hoy, J. et al. 1986. Trichosporon beigelii infection: a review. Rev. Infect. Dis. 8:959-967. 12. Morrison, V.A. and D. J. Weisdorf. 2000. The spectrum of Malassezia infections in the bone marrow transplant population. Bone Marrow Transplant. 26:645648. 13. Denning, D. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781-805. 14. Maertens, J. et al. 1999. Mucormycosis in allogeneic bone marrow transplant recipients: report of five cases and review of the role of iron overload in the pathogenesis. Bone Marrow Transplant. 24:307-312. 15. Kauffman, C.A. 2003. Endemic mycosis after hemopoietic stem cell or solid organ transplantation, p. 524-532. In R.A. Brown, P. Ljungman, and C.V. Paya (ed.), Transplant infections, 2nd ed. Lippincott Williams & Wilkins, Philadelphia, PA. 16. LaRocco, M.T. 2003. Reagents, stains, and media: mycology, p. 1686-1692. In P.R. Murray et al. (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, DC. 17. Cushion, M.D. 2003. Pneumocystis, p. 1712-1725. In P.R. Murray et al. (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, DC.
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Infections in Hemopoietic Stem Cell Transplant Recipients, Part I* Rafik Samuel, M.D.,1 Allan L. Truant, Ph.D.,2 and Byungse Suh, M.D., Ph.D.,1 Section of Infectious Diseases, Department of Medicine, Temple University School of Medicine,1 and Clinical Microbiology, Immunology, and Virology Laboratories, Department of Pathology and Laboratory Medicine, Temple University Hospital and School of Medicine,2 Philadelphia, Pennsylvania
Abstract Hemopoietic stem cell transplantation, often called bone marrow transplantation, is a relatively new medical procedure that is now used to treat patients who were diagnosed with diseases once thought incurable, such as hematologic malignancies, immune deficiencies, solid tumors such as breast and ovarian cancers, sickle cell disease, and genetic-defect disorders. Patients who receive bone marrow transplantation also receive severe immunosuppression therapy as part of their care to minimize the risk of donor stem cell rejection. As a consequence of the patient’s profound immunosuppression, opportunistic infections emerge as common causes of disease. Part I of this article will review some of the more common opportunistic fungal infections that can occur in these immunocompromised patients, with an emphasis on methods for their laboratory diagnosis.
Introduction Hemopoietic stem cell transplantation (HSCT), more commonly known as bone marrow transplantation (BMT), is a relatively new medical procedure. The procedure is used to treat patients diagnosed with diseases once thought incurable, such as hematologic malignancies, immune deficiencies, solid tumors such as breast and ovarian cancers, sickle cell disease, and genetic-defect disorders. There are several different types of HSCT. In autologous transplantation, patients function as their own donors; allogeneic transplantation uses HLAmatched donors of related or unrelated origins; and syngeneic transplantation *Editor’s Note: Part II of this article will be published in the October 1, 2010 issue of CMN (Vol. 32, No. 19).
Corresponding Author: Allan L. Truant, Ph.D., Clinical Microbiology Laboratories, Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3401 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-3210. Fax: 215-7072053. E-mail: [email protected]
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involves identical twins as donor and recipient. According to a recent report, the total number of patients undergoing HSCT worldwide was approximately 45,000, of which those with autologous and allogeneic transplants represent 30,000 and 15,000 cases, respectively (1). Major inherent problems in HSCT recipients that contribute to mortality and morbidity, include relapse of the original condition, graft-versus-host disease (GVHD), infections, and multiorgan failure. Overall, in patients with HSCT, relapse of underlying diseases contributes to 21 to 78% of the mortality, while infections and GVHD are responsible for 5 to 21% and 15% of deaths (1). This article will focus on infectious complications commonly encountered in HSCT recipients. Infections in this patient population are seen as a consequence of profound immunosuppressive treatment necessary to remove as many pathologic cells as possible and to suppress the immunologic ability to reject the transfused cells. This profound immunosuppression in the HSCT recipients results in severe neutropenia, © 2010 Elsevier
defects in immunity, and disruption of the integrity of mucous membranes, leading to mucositis. The types and timing of the infections encountered in HSCT recipients in relation to the transplantation procedure are depicted in Fig. 1 (2) and provide the basis for this article. Available identification methods for pathogens described in the review can be found in the Manual of Clinical Microbiology (3), the Manual of Molecular and Clinical Laboratory Immunology (4), the Manual of Commercial Methods in Clinical Microbiology (5), and the Clinical Microbiology Procedures Handbook (6).
Immunizations Immunizations, one of the most significant milestones in preventive medicine, are the most effective means
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Pneumocystis jirovecii
Figure 1. Phases of opportunistic infections among allogeneic HSCT recipients (n = 8), (modified from reference 2).
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of reducing a variety of infections in humans. Immunization can be divided into two major categories, active and passive. Active immunization is defined as the administration of a vaccine, which causes the host to develop immunity to the agent contained in the vaccine. Vaccines are available in many different forms: attenuated live, inactivated whole organism, cellular subunit or component, or toxoid. Passive immunization is defined as the administration of preformed antibodies, which are protective against the disease under consideration. Active immunization usually results in long-term protection lasting several decades to a lifetime. Passive immunization produces immediate protection, but the immunity is relatively brief and is commonly used to prevent the onset of clinical disease. It may either be administered following exposure or prior to an anticipated exposure. HSCT recipients have a diminished level of immune function and therefore respond to antigenic challenge poorly and rather unpredictably. Also, antibody titers to vaccine-preventable diseases are known to decline during the first 1 to 4 years after transplantation and may require re-vaccination. A 1994 national survey of transplantation centers participating in the National Marrow Donor Program (7) led to national guidelines for vaccinations for HSCT recipients. These guidelines were published in 2000 under the co-sponsorship of the CDC, the Infectious Disease Society of America, and the American Society of Blood and Marrow Transplantation (2). The modified vaccination schedule published in the guidelines also can be found in reference 8.
Mycotic Infections Fungal infections that are frequently encountered in HSCT recipients are divided into three categories: yeast, mold, and endemic fungal infections. Recommendations on the therapeutic management of these infections are summarized in Table 1, and additional information on therapeutic options for all infections discussed in this review can be obtained from reference 8. However, current therapeutic recommendations should always be obtained prior to final decisions for any specific patient.
Yeast Infections Candida species are commonly Clinical Microbiology Newsletter 32:18,2010
encountered during the early post-transplantation stage. Conditioning regimens cause severe neutropenia and mucositis, and HSCT recipients are susceptible to Candida infections. Subsequently, the host defense mechanisms of the skin, intestine, and the liver are further compromised due to GVHD. Candidemia can cause a high mortality rate ranging from 40 to 60%. Among Candida species, Candida albicans is most frequently encountered, representing approximately 50%, and the other species, including Candida glabrata Candida tropicalis, Candida parapsilosis, Candida krusei, and Candida lusitaniae, account for the rest. Of these species, C. parapsilosis has been associated more often with vascular devices. Another Candida species, Candida dubliniensis, can be misidentified as C. albicans because both species are capable of germ tube formation (9). Chromogenic isolation media are available to correctly detect and identify C. albicans, C. tropicalis, and C. krusei. Candida bloodstream infections may lead to either acute or chronic disseminated diseases. Acute disseminated candidiasis may involve multiple organs, including the kidney, heart, skin, and musculoskeletal system. C. tropicalis is often isolated from neutropenic patients with this syndrome (10). Chronic disseminated candidiasis, which may be a complication of severe Candida mucositis and often affects the liver and spleen in addition to other organs, is often referred to as “hepatosplenic candidiasis.” Most infections caused by Cryptococcus neoformans are acquired through the respiratory system with colonization in the lungs. In the immunocompetent host, C. neoformans is eliminated by the normal host defense mechanisms. In the immunocompromised host, however, the organism spreads hematogenously to extrapulmonary organs, where it has a predilection for the central nervous system (CNS). Prolonged hypercortisolism, either endogenous or exogenous in nature, is known to predispose patients to cryptococcal infections, which may be the case in allogeneic HSCT recipients with GVHD. The most common presentation of CNS cryptococcosis is meningitis, usually subacute, although acute at times in onset. Complications include © 2010 Elsevier
hydrocephalus, elevated intracranial pressure, encephalitis, hearing or visual loss, facial weakness, and seizure. Occasionally, mass lesions in the brain or spinal cord can be observed. The clinical course may vary greatly depending on the immunocompetence of the host. A definitive diagnosis of cryptococcosis is made by culturing the organism from specimens obtained from the involved site. The detection of cryptococcal capsular polysaccharide antigen by latex agglutination or enzyme immunoassay (EIA) in serum or cerebrospinal fluid (CSF), is used to diagnose cryptococcosis rapidly, with specificity and sensitivity approaching 100%. The India ink preparation may be positive in 25 to 50% of patients with cryptococcal meningitis. Trichosporon beigelii is occasionally isolated as part of the normal flora of human skin and is known to colonize the gastrointestinal and respiratory tracts in immunosuppressed hosts. T. beigelii grows on routine media used in the mycology laboratory. In profoundly immunosuppressed hosts with neutropenia, it may cause disseminated disease with a high mortality rate of 74% (11). Disseminated trichosporonosis may cause fungemia, funguria, endocarditis, cutaneous lesions, renal failure, and pneumonia. The genus Malassezia, formerly known as Pityrosporum, consists of lipophilic yeasts, and the carriage rate of Malassezia spp. on the normal skin approaches 100% in adults. They live on the skin surface, as well as in the ducts of hair follicles and sebaceous glands. Malassezia is associated with a folliculitis that is usually maculopapular but occasionally is pustular. The lesion is commonly seen in immunocompromised patients, such as solid organ transplant recipients, but the incidence of Malassezia infections in HSCT recipients is rare (12). Folliculitis, caused by Malassezia, is usually diagnosed by the microscopic examination of skin scrapings from the lesion in a KOH preparation or by using Calcifluor white stain. To isolate Malassezia furfur, the growth medium must be supplemented with a lipid component by overlaying Sabouraud’s glucose agar with a few drops of sterile olive oil. Mold infections remain the most dreaded infectious complications of 0196-4399/00 (see frontmatter)
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Table 1. Recommended therapies for fungal and parasitic infections post-HSCT Clinical presentation 1. Non-neutropenic patients with candidiasis or disseminated candidiasis
2. Neonatal candidiasis 3. Cryptococcal meningitis (based on HIV-related infections) 4. Trichosporon infections 5. Malassezia infections 6. Invasive aspergillosis
7. Fusarium infections
8. Zygomycosis 9. Histoplasmosis: moderate to severe pulmonary
Histoplasmosis: disseminated Histoplasmosis: CNS 10. Coccidioides: extensive infection
11. Blastomycosis: moderate to severe pulmonary or disseminated extrapulmonary Blastomycosis: mild to moderate disease or mild to moderate disseminated or osteoarticular disease Blastomycosis: CNS 12. Pneumocystis jirovecii infections
13. Toxoplasmosis
14. Malaria
Malaria prophylaxis for HSCT recipients 15. Strongyloidiasis 16. Chagas’ disease 17. Cryptosporidiosis
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Recommended therapy a. Fluconazole or b. Caspofungin, micafungin, or anidulafungin c. Deoxycholate amphotericin B (DAmB), liposomal amphotericin B, or amphotericin B lipid complex (LFAmB) d. Voriconazole a. Amphotericin B for disseminated candidiasis b. Liposomal amphotericin B if urinary tract involvement. a. DAmB or LFAmB with or without flucytosine (2 wk), followed by fluconazole (8 wk), followed by maintenance daily fluconazole a. Amphotericin B plus fluconazole or voriconazole a. Little data available; possibly fluconazole a. Voriconazole b. LFAmB, DAmB, or posaconazole, or itraconazole, or caspofungin, or micafungin as salvage therapy a. DAmB alone or with flucytosine or rifampin or b. LFAmB or c. Voriconazole or posaconazole a. Usually surgery plus antifungal therapy of DAmB or LFAmB or b. Possibly posaconazole from in vitro and animal studies a. LFAmB or DAmB (1-2 wk), possibly with methylprednisolone followed by itraconazole (12 wk) b. For chronic cavity pulmonary histoplasmosis; itraconazole (1-2 yr) a. LFAmB or DAmB (1-2 wk), followed by oral itraconazole (1 yr or longer for immunosuppressed patients) a. LFAmB (4-6 wk), followed by oral itraconazole (min. 1 yr or until resolution of CSF abnormalities) a. Possible surgical debridement plus antifungal therapy with fluconazole (especially for CNS infections), itraconazole, or DAmB (as second alternate); AmB usually reserved for patients with respiratory failure, pregnant individuals, or rapidly progressing infection a. LFAmB or DAmB (1-2 wk), followed by oral itraconazole (6-12 mo) a. Oral itraconazole (6-12 mo) a. LFAmB or DAmB (4-6 weeks) followed by oral azole (minimum 12 mo). In pregnancy, LFAmB is recommended (avoid azoles) a. Trimethoprim-sulfamethoxazole (TMP/SMZ) b. Alternative therapy includes pentamidine, atovaquone, trimetrexate, trimethoprim plus dapsone, or high-dose clindamycin. a. Pyrimethamine with folinic acid and sulfadiazine b. Prophylaxis in seropositive HSCT recipients: TMP/SMZ or pyrimethamine/sulfadoxine a. Chloroquine for susceptible strains b. For chloroquine-resistant Plasmodium vivax, mefloquine or halofantrine c. For mefloquine-resistant Plasmodium falciparum, quinine sulfate or quinidine and doxycycline d. Alternate therapy, atovaquone/proguanil with or without artesunate or artesunate alone a. Chloroquine or b. Mefloquin, atovaquone/proguanil, or doxycycline a. Ivermectin or b. Thiabendazole a. Nifurtimox (available as an investigational drug from CDC) b. Benznidazole a. Nitazoxanide (documented use in immunocompetent but controversial in immunocompromised patients) b. Spiramycin or paromomycin with azithromycin, TMP/SMZ, or somatostatin
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HSCT recipients. These infections carry very high mortality rates, approaching 100% unless the immune status recovers. The majority of HSCT recipients remain severely immunocompromised, immediately before and after the HSCT procedure for up to approximately 180 days, rendering them at high risk for mold infections. Aspergillus and other molds, including Fusarium, Scedosporium, and Zygomycetes also play an important role as pathogens in these patients. Aspergillus species are ubiquitous in the environment worldwide and are responsible for the second most common systemic fungal infections requiring hospitalization in the United States, next to candidiasis. The common species infecting humans are Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans. Of these, A. fumigatus causes approximately 90% of invasive aspergillosis. Aspergillus species are usually susceptible to amphotericin B; however, A. terreus may be resistant. Risk factors for invasive aspergillosis (IA) include neutropenia, high-dose corticosteroid therapy, and cytotoxic therapy. The respiratory tract is the usual portal of entry by inhalation of airborne conidia. Damaged skin, surgical wounds, or the external ear may also serve as entry sites. In HSCT recipients, the firstline defense is compromised due to the reduced number of macrophages, monocytes, and neutrophils. Hence, the incidences of IA in allogeneic and autolgous HSCT patients are reported to be 4 to 9% and 0.5 to 6%, respectively, and pulmonary infections account for 80 to 90% of invasive aspergillosis (13). The most characteristic early lesions, which may be demonstrated by CT scans, are small, pleural-based nodules with surrounding low attenuation (the “halo” sign). As patients recover from neutropenia, the nodules may cavitate and reveal an “air-crescent” sign. The halo and crescent signs are typical of aspergillosis but are not definitive. Aspergillus may disseminate hematogenously to extapulmonary sites, including the brain, myocardium, liver, spleen, skin, and kidneys. The brain is the most commonly involved site in 25 to 40% of invasive pulmonary aspergillosis cases, and MRI images may reveal lesions that were not detected by CT scans. The demonstration of Aspergillus Clinical Microbiology Newsletter 32:18,2010
hyphae in tissue, usually best visualized by silver staining, is the best means of diagnosis. Aspergillus hyphae are 2 to 4 μm wide, frequently septate, and branch acutely at 45°. Blood cultures of patients with IA are invariably negative. The monitoring of galactomannan, a cell wall component of Aspergillus, by EIA is a potentially useful method for diagnosis. Fusarium is the second-most-common filamentous fungal pathogen in HSCT recipients after Aspergillus. The portal of entry and risk factors for fusariosis are similar to those of aspergillosis. Fusariosis shows a bimodal distribution in its onset of clinical manifestations in HSCT recipients. The most common species to cause human infections is Fusarium solani, accounting for approximately 50% of the cases, while other species, such as Fusarium moniliformis, Fusarium oxysporum, Fusarium dimerum, and Fusarium proliferatum account for the rest. The most common presentation of fusariosis in immunocompromised hosts is pulmonary infections, with dissemination to extrapulmonary sites occuring in as many as 70% of cases. The most common extrapulmonary site is the skin. Diagnosis is usually made by biopsy demonstrating the presence of acutely branched and septate hyphae when prepared with methenamine silver or periodic acid-Schiff (PAS) stain. Fungal cultures are mandatory to confirm and to differentiate it from Aspergillus species. Blood cultures are positive in approximately 50% of disseminated cases of fusariosis. Zygomycosis, previously known as phycomycosis or mucormycosis, refers to uncommon fungal infections caused by a group of fungi belonging to the class Zygomycetes. The majority of organisms causing the infections belong to three genera: Rhizopus, Mucor, and Rhizomucor. These fungi are ubiquitous in the environment. When histologically examined, the Zygomycetes are broad, nonseptate or pauciseptate, thick-walled, irregularly shaped hyphae with rightangle branching. Culture is positive in only 25% of histologically confirmed cases of pulmonary zygomycosis. Final identification to the species level requires culture of the fungus, although species identification is not necessary for treatment. © 2010 Elsevier
There is a bimodal distribution of onset, early and late, but zygomycosis tends to occur later after allogeneic HSCT transplantation, corresponding to the severity of GVHD and the intensity of corticosteroid treatment (14). The major mode of transmission is believed to be the respiratory route, although the traumatized-skin and gastrointestinal routes play minor roles. Host factors predisposing to zygomycosis include immunosuppression, uncontrolled diabetes mellitus, deferoxamine therapy, skin breakdown, intravenous drug abuse, and malnourishment. The best-known condition associated with zygomycosis is uncontrolled diabetes mellitus with ketoacidosis. There is evidence that the incidence of zygomycosis is increasing in HSCT recipients. Clinical manifestations of zycomycosis are usually classified by the site involved: rhinocerebral, sinus, pulmonary, gastrointestinal, cutaneous, abdominopelvic, and disseminated. Rhinocerebral zygomycosis typically originates in the nares, palate, or paranasal sinuses with gradual or rapid involvement of the eye and brain. Symptoms may be nonspecific and may progress to facial and periorbital swelling and numbness with blurred vision, corneal anesthesia, orphthalmoplegia, and facial anhidrosis. Once the brain is involved, there may be a deterioration of mental status, onset of seizures, or cerebrovasular accidents. Radiographic procedures, such as CT scan and MRI, are required to determine the extent of disease process. Pulmonary zygomycosis resembles aspergillosis. Zygomycosis also tends to cause vascular invasion as with aspergillosis, resulting in thrombosis, hemorrhage, and infarction. If a lung biopsy specimen reveals the presence of definite fungal elements but culture fails to grow, the presumptive diagnosis favors zygomycosis over aspergillosis. Cutaneous zygomycosis can be caused by direct invasion of preexisting wounds. The lesions may involve not only superficial layers, but also deeper tissue, such as muscle, fascia, and bone. The diagnosis is made primarily by direct histopathologic examination and by fungal culture. Endemic mycoses conventionally refer to histoplasmosis, blastomycosis, and coccidioidomycosis because they occur in a geographically specific area. 0196-4399/00 (see frontmatter)
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These infections are caused by Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis, respectively. These organisms exhibit thermal dimorphism: at a temperature of 25 to 30°C in the laboratory and in the natural habitat, they grow as molds, but when grown at 37°C, they are yeasts, except C. immitis, which in tissue phase is a spherule. The initial infections can be dormant for many years, and they may reactivate to reveal clinical manifestations as the host undergoes immunosuppression. In healthy hosts with low-level exposure to H. capsulatum, fewer than 5% develop symptomatic disease, whereas most patients develop symptomatic infection following heavy exposure. Flu-like pulmonary illnesses, pericarditis, arthritis, or arthralgias with erythema nodosum are the most common manifestations of histoplasmosis. In immunosuppressed patients, such as transplant recipients, however, widely disseminated infection occurs in the majority of patients (88%), with predominant involvement of the lungs and other organs throughout the reticuloendothelial system. Clinical presentation of disseminated histoplasmosis is nonspecific, and usually the symptom complex consists of fever, chills, fatigue, anorexia, and weight loss. On examination, one should look for hepatosplenomegaly, oropharyngeal ulcers (25 to 75%), and cutaneous lesions, including papules, pustules, plaques, ulcers, abscesses, and cellulitis. When the CNS is involved, patients may present with headache, confusion, and seizures. Supportive laboratory findings may involve pancytopenia, elevated alkaline phosphatase, elevated erythrocyte sedimentation rate, and electrolyte abnormalities suggestive of adrenal insufficiency. The definitive diagnosis of histoplasmosis can be made by culture confirmation of the etiologic agent, but routine blood cultures in broth do not yield positive results. The Isolator system (Wampole Laboratories, Princeton, NJ.), also known as the lysis-centrifugation blood culture method, appears to be superior for Candida, Cryptococcus, and Histoplasma compared to automated systems. Effective Histoplasma antibody levels often do not appear in immunosup140
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pressed patients; therefore, classical methods, such as complement fixation and immunodiffusion tests measuring the patient’s antibody levels, are not as useful as in healthy hosts. The detection of H. capsulatum antigen by EIA in body fluids, such as serum, is extremely useful in the initial diagnosis and subsequent monitoring of disease progression during therapy in patients with disseminated histoplasmosis. C. immitis is a dimorphic fungus. The mold form represents a saprophytic phase and grows in soil, whereas inhalation of the conidia into the lungs results in the morphologic change into large, thick-walled spherules that become filled with hundreds of endospores. Each endospore has the potential to form a new spherule, thus continuing the infection cycle. Patients with compromised cellular immunity, including transplant recipients and HIV/AIDS patients, are at higher risk for developing extrapulmonary disseminated disease. Also, pregnant individuals and persons of African and Filipino descent have a higher risk for disseminated disease. Two disseminated and one localized pulmonary cases of coccidioidomycosis have been described in HSCT recipients; both disseminated cases died, while the localized case survived. Acute pulmonary infections are asymptomatic in 60% of healthy individuals, but patients with symptoms present with a flu-like illness. Arthralgias and myalgias may be prominent, and erythema multiforme or erythema nodosum may also be present. Chest radiograph is abnormal in 50% of the symptomatic patients, and eosinophilia in peripheral blood is frequently observed. Symptoms usually clear in a few weeks. In immunocompromised patients, coccidioidomycosis tends to disseminate, with the skin, subcutaneous tissues, bones, and meninges being the most common sites involved. Blood cultures are usually positive in this patient population. Coccidioidomycosis in solid organ transplant recipients carries a dismal prognosis. The definitive diagnosis of coccidioidomycosis is made by isolating the organism from appropriate clinical specimens, such as tissue aspirate or biopsy specimens, sputum, and body © 2010 Elsevier
fluids. The fungus grows well on routine mycology culture media, and it takes only 2 to 7 days. A DNA-specific probe is now available, and correct identification is possible as soon as growth is obtained. Demonstration of typical spherules, measuring 20 to 80 μm in diameter on wet mounts with KOH preparation, may lead to more rapid diagnosis. Serology plays an important role in the diagnosis and follow-up of immunocompromised patients with coccidioidomycosis, whereas the response in immunocompromised hosts may be poor. Tube precipitin-reacting antibodies, which represent IgM antibodies, are present in primary infection and disappear as the infection enters the chronic phase. IgG antibodies detected by complement fixation or other methods appear instead during the chronic phase of infection and persist. B. dermatitidis, which is the asexual stage of Ajellomyces dermatitidis, is a dimorphic fungus. It grows at room temperature as a whitish/tan mold and grows within the host or at 37°C as budding, yeast-like cells with a thick, refractile cell wall that reproduce by single broad-based buds. The organism proliferates in warm and moist soil rich with decaying organic matter, and persons with occupational exposure to soil appear to have the highest risk of developing this infection. Blastomycosis usually affects more non-compromised hosts; however, increasing numbers of cases are being described among transplant recipients (15). Human blastomycosis usually occurs through inhalation of conidia into the lungs, which usually leads to asymptomatic or self-limited infections but occasionally leads to chronic pneumonia. Although some patients with pulmonary blastomycosis recover spontaneously, most patients require treatment. Blastomycosis may involve the lungs, skin, bone, and genitourinary tract, in decreasing order of frequency. Pulmonary blastomycosis in transplant recipients may occasionally disseminate to involve the skin, skeletal system, and male genitourinary tract. Mortality rates approach 30 to 40%, and most deaths occur during the first few weeks of therapy. Demonstration of the yeast from clinical specimens or growth of the fungus Clinical Microbiology Newsletter 32:18,2010
in culture usually makes a diagnosis, but culture may take 2 to 4 weeks of incubation. The examination of KOH preparations and/or tissue biopsy specimens stained with PAS or methenamine silver stain may be used for direct specimen diagnosis. The organism can also be identified by employing highly specific and sensitive DNA probes. Neither serology nor skin testing for blastomycosis is specific or sensitive, and therefore, they have low clinical utility. Another technique, known as calcofluor white stain, that enhances the detection of fungi by binding to β-1,3- and β-1,4polysaccharide of the cell wall in fungi is available. This method, however, needs a fluorescence microscope equipped with a barrier filter to protect the eye. Fungi exhibit green fluorescence (16). The gene sequence studies on Pneumocystis carinii indicate that it should be categorized as a fungus. These organisms show 60 and 20% homology with fungi and protozoa, respectively. They are currently placed in the phylum Ascomycota within a newly described class, Aschiascomycetes. The new nomenclature for the Pneumocystis species that causes human disease is Pneumocystis jirovecii, since P. carinii can cause disease only in rats (17). Very few Pneumocystis organisms (<10 organisms) are required to initiate a fulminant infection in immunocompromised rats. Most humans become seropositive by 2 to 4 years of age, and the organism can survive indefinitely without causing any ill effect until the host immune system is severely compromised. In AIDS patients, 95% of Pneumocystis cases occur in patients with CD4 counts of less than 200/mm3, with an annual incidence of 8%. Clinical signs and symptoms of Pneumocystis pneumonia are those of atypical pneumonia, which has a rather slow onset over a 2- to 4-week period of fever, nonproductive cough, and increasing dyspnea. The clinical picture, however, may be very variable. The chest radiograph typically shows
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bilateral diffuse infiltrates, perihilar in distribution. Although there is no large prospective study comparing signs and symptoms in HSCT recipients, it is prudent to think that the clinical picture would be similar to that seen in HIVpositive patients. Histopathologic staining of a sputum or preferably a bronchoalveular lavage specimen makes a definitive diagnosis. Methenamine silver, toluidine blue, and cresyl echt violet are most commonly used to selectively stain the cell wall. The Wright-Giemsa stain is used to stain the nuclei of all developmental stages of the organism. In addition, specific immunofluorescence has been used with success. In allogeneic HSCT recipients, Pneumocystis infection develops in 10 to 20% of patients, typically during the period immediately after engraftment until 6 months following HSCT. During this period and beyond for those requiring immunosuppressive agents, Pneumocystis prophylaxis should be maintained. Editor’s Note: Part II of this article will be published in the October 1, 2010 issue of CMN (Vol. 32, No. 19). References 1. Loberiza, F., Jr. 2003. Report on state of the art in blood and marrow transplantation — part 1 of the IBMTR/ABMTR summary slides with guide. IBMTR/ ABMTR Newsl. 10:7-10. 2. Center for Disease Control and Prevention. 2000. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Disease Society of America, and the American Society of Blood and Marrow Transplantation. MMWR Morb. Mortal. Wkly. Rep. 49(RR-10):1-128. 3. Murray, P.R. et al. (ed.) 2007. Manual of clinical microbiology, 9th ed. ASM Press, Washington, DC. 4. Detrick, B., R.G. Hamilton, and J.D. Folds (ed.) 2006. Manual of molecular and clinical laboratory immunology, 7th ed. ASM Press, Washington, D.C.
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5. Truant, A.L. (ed.) 2002. Manual of commercial methods in clinical microbiology. ASM Press, Washington, DC. 6. Isenberg, H.D. (ed.) 2004. Clinical microbiology procedures handbook. ASM Press, Washington, DC. 7. Henning, K.J. et al. 1997. A national survey of immunization practices following allogeneic bone marrow transplantation. JAMA 277:1148-1151. 8. Samuel, R.A., A.L. Truant, and B. Suh. 2005. Cumitech 42, Infections in hemopoietic stem cell transplant recipients. Coordinating ed., A.L. Truant. ASM Press, Washington, DC. 9. Gutierrez, J. et al. 2002. Candida dubliniensis, a new fungal pathogen. J. Basic Microbiol. 42:207-227. 10. Wingard, J.R., W.G. Merz, and R. Saral. 1979. Candida tropicalis: a major pathogen in immunocompromised patients. Ann. Intern. Med. 91:539-543. 11. Hoy, J. et al. 1986. Trichosporon beigelii infection: a review. Rev. Infect. Dis. 8:959-967. 12. Morrison, V.A. and D. J. Weisdorf. 2000. The spectrum of Malassezia infections in the bone marrow transplant population. Bone Marrow Transplant. 26:645648. 13. Denning, D. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781-805. 14. Maertens, J. et al. 1999. Mucormycosis in allogeneic bone marrow transplant recipients: report of five cases and review of the role of iron overload in the pathogenesis. Bone Marrow Transplant. 24:307-312. 15. Kauffman, C.A. 2003. Endemic mycosis after hemopoietic stem cell or solid organ transplantation, p. 524-532. In R.A. Brown, P. Ljungman, and C.V. Paya (ed.), Transplant infections, 2nd ed. Lippincott Williams & Wilkins, Philadelphia, PA. 16. LaRocco, M.T. 2003. Reagents, stains, and media: mycology, p. 1686-1692. In P.R. Murray et al. (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, DC. 17. Cushion, M.D. 2003. Pneumocystis, p. 1712-1725. In P.R. Murray et al. (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, DC.
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