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Penicillium marneffei (Penicilliosis)
Updated July, 2009
History Penicillium marneffei was described as a new species by Gabriel Segretain in 1959 (13). G. Segretain was head of the Mycology Unit at the Pasteur Institute (Paris, France) he received a Penicillium strain isolated from bamboo rats (Rhizomys sinensis) for species level identification. This strain had been isolated by Capponi et al. from rats native to the highlands of Central Vietnam, and maintained in captivity for experimental infections at the Pasteur Institute of Indochina (a former French colony) at Dalat, South Vietnam (3). Three rats died from a spontaneous disseminated mycosis involving the reticuloendothelial system. The Penicillium strain was isolated and an experimentally infected mouse was sent by airplane to the Pasteur Institute in Paris for further studies. The fungus was identified by G. Segretain of a new species called marneffei, in honor of Hubert Marneffe, Director of the Pasteur Institute in Indochina (13). Segretain accidentally pricked his finger with a needle used to inoculate hamsters with the yeast form. Nine days later a small nodule appeared at the site of inoculation followed by lymphangitis and axillary lymph nodes hypertrophy. The excised nodule gave a positive culture, the puncture aspiration of the lymph node did not grow (personal communication from G. Segretain) (14). This was the first indication of the possible pathogenicity to human. At that time E. Drouhet was doing clinical studies with nystatin. He demonstrated the high in vitro susceptibility of the strain to this polyene which was given orally to G. Segretain: 20 millions units a day for 30 days. The infection resolved, the role of nystatin is doubtful, however very high dose of another polyene: amphotericin B, given orally can be absorbed and able to cure some systemic mycoses (14). The first natural infection was described in 1973 by Di Salvo et al. in a patient with Hodgkin’s disease who underwent a splenectomy (5). In 1984-88 several autochtonus cases were published in Southern China including Hong Kong -- some of them initially misdiagnosed as histoplasmosis -- and in Thailand, (4,9) and imported cases in non AIDS patient were recognized. P. marneffei was isolated from other rat species: Rhizomys pruinosus, R. sumatrensis and Cannomys badius and from soil in burrows of bamboo rats (6). In 1988, the first cases were reported in AIDS patients who had travelled in South East Asia and were diagnosed in Europe, USA, Australia (6). Penicilliosis became a major problem for HIV positive patients living in endemic area (20). Cooper, C. R., Jr. 1998. From bamboo rats to humans: the odyssey of Penicillium marneffei. ASM News 64:390-397. Microbiology Guided Medline Search P. marneffei is a thermal dimorphic fungus. In vitro at 37°C the yeast form which seems to be an arthroconidia is unicellular, oval or elongated, it does not reproduce by budding as most yeasts but by fission. This typical form is easily seen in histopathological section of biopsies or in smear of bone marrow aspirate: a septum divides the cells in two parts -- Their size is 2-3 x 2 µm. The mycelial phase obtained at 27°C has a bluish gray-green center. In 24 to 48 H a red pigment is produced, it diffuses into all the agar medium in a Petri dish. This Penicillium is biverticillate either symmetric or asymmetric, the conidia are smooth-walled in short chains (6). Epidemiology Guided Medline Search The epidemiology of penicilliosis is still mysterious. The bamboo rats are the only animals known to be naturally infected by this fungus. It is thought that P. marneffei is a natural part of the flora inhabiting vegetation and soil. The fungus is present in internal organs of rats with an infection rate varying with the species. How rats are colonized or infected is still unclear. How human beings get infected is still unresolved however a case control study could not establish exposure to bamboo rats as a risk factor for acquiring penicilliosis, exposure to soil, especially during the rainy season, could be the critical factor (2). The portal of entry of infection in man is probably the lung. Autochtonus cases were reported from Northern Thailand, Vietnam, Laos, Taiwan, Indonesia, South China (Guangxi Province), Hong Kong (6) and more recently from Eastern India (state of Manipur) (12). One case was also reported from Ghana (Africa) (9). Imported cases in Europe, the United Sates, Canada, Australia and Japan have been described in immunocompromised patients, mainly with AIDS, with a past history of traveling in Southeast Asia, weeks, months or years earlier (6). Clinical Manifestations Guided Medline Search The disease affects mostly patients with impaired cellular immunity. Apparently healthy persons can contract the disease. In AIDS it occurs at an advanced stage of HIV disease with a CD4 cell count below 100/mm3. In Northern Thailand, in patients infected with HIV, penicilliosis is the third most common opportunistic infection, accounting for 15 to 20% of all AIDS-related illness, after tuberculosis and cryptococcosis (18). The disease is characterized by fever, weight loss, lung infiltrates, papulonecrotic skin lesions, hepatosplenomegaly, lymphadenopathy, diarrhea and anemia or pancytopenia (4, 8, 20). The disease is lethal if left untreated, particularly in disseminated infections and in immunocompromised hosts; localized infections can resolve spontaneously. Laboratory Diagnosis Guided Medline Search The fungus can be detected by direct examination and/or culture of bone marrow aspirate or skin or lymph node biopsy, bronchoalveolar lavage, or blood culture (20). On smear or tissue section, the small non budding septate yeast are characteristic; in culture, the filamentous phase produces a diffusing red pigment and must be differentiated from other species of "red Penicillium". A specific fluorescent-antibody test and an antigen detection test have been developed. Review Article: Gauthier G, et al. Insights into Fungal Morphogenesis and Immune Evasion. Microbe 2008;3(9):416-423.
SUSCEPTIBILITY TESTING IN VITRO AND IN VIVO Guided Medline Search In Vitro and In Vivo In Vitro Studies Penicillium marneffei is classified as a hazardous microorganism to handle and should be manipulated under a safety cabinet or in a biosafety level 3 laboratory according to national or international regulations. In vitro testing for susceptibility to antifungal agents is not standardized for dimorphic fungi. Although major progress has been made for yeasts, the result of in vitro testing of filamentous or dimorphic fungi is still controversial with respect to reproducibility and relevance to clinical outcome. When tested in broth, rapid production of a red pigment confounds reading results either by the naked eye or by a photometer. A limited number of studies have tested in vitro susceptibility of isolates of P. marneffei. Thirty isolates of P. marneffei from clinical specimens were tested for amphotericin B and 5-fluorocytosine (5-FC) in a modified macrobroth dilution bioassay using a microtiter plate with a semisolid growth medium for azoles (19). All isolates were highly susceptible to 5-FC, miconazole, ketoconazole and itraconazole. Fluconazole was the least active, 20 strains had an MIC of 20 mg/L. Geometric mean of MICs were: amphotericin B: 0.976 mg/L (range 0.25-4), 5-FC: 0.25 mg/L, miconazole 0.001 mg/L, itraconazole 0.009 mg/L, and fluconazole 7.9 mg/L. Initial therapy failed in 8 of 35 (22.6%) patients treated with amphotericin B, 3 of 12 (25%) patients treated with itraconazole and 7 of 11 (63.6%) patients treated with fluconazole. Although patients were not randomly assigned to receive therapy with the various antifungal agents, a good correlation was found between in vitro and in vivo data for azoles; results with amphotericin B were more diffi cult to assess (19). We tested 6 isolates from AIDS patients and 4 isolates from non-AIDS patients with amphotericin B, 5-FC, ketoconazole, itraconazole and fluconazole (5), using Steer’s technique on agar plates, with an inoculum of 104 spores/ml. Incubation time was 24 hours at 35°C. Casitone agar medium was used for amphotericin B and azole derivatives and yeast morphology agar (YMA), a synthetic medium was used for 5-FC. There was no difference with respect to the immune status (HIV) of the patients. MICs to amphotericin B were 0.04 mg/L (3 isolates), 0.78 mg/L (3 isolates) and 1.56 mg/L (4 isolates). All isolates had an MIC of 0.04 mg/L or below to 5-FC, ketoconazole and itraconazole, and all isolates had MICs of 50 mg/L or above for fluconazole. The high MICs to fluconazole may be due to use of an inappropriate medium (7). Sekhon et al. used different methods to test the susceptibility of mycelial and yeast forms (15). The MIC and minimum fungicidal concentration (MFC) for mycelial forms to amphotericin B were 0.78 to 1.56 and 0.78 to 3.125 mg/L, respectively. MICs of yeast forms were 3.125 to 25 mg/L and MFCs were one dilution higher. MICs to fluconazole were generally lower for the yeast forms (6.25-25 mg/L) than the mycelial form (25-50 mg/L) while MFCs for mycelial cultures were above 100 mg/L compared with 6.25 to 100 mg/L for the yeast form. MICs for the mycelial form to 5-FC ranged from less than 0.195 to 0.39 mg/L , and a higher MIC (6.25 mg/L) were recorded for the yeast forms. MFCs to 5-FC for the yeast forms were 25 to 100 mg/L. MICs for the mycelial forms to itraconazole ranged from less than 0.195 to 3.125 mg/L and higher values (< 0.195 to 50 mg/L) were recorded for the yeast-like forms. MFCs to itraconazole for mycelial and yeast forms ranged from less than 0.195 to 0.39 and 25 to 100 mg/L, respectively (15). MICs to fluconazole tended to be higher for mycelial forms than for yeast forms: 50 versus 25 mg/L (22). In one report, one strain had an MIC to nystatin of 0.65 mg/L in a semisynthetic medium with a reading after 24 h of incubation; the MIC increased to 3.2 mg/L at the 4th day of incubation (6). In other anecdotal reports one isolate was reported susceptible to amphotericin B and 3 isolates susceptible to amphotericin B and 5-FC (9). Boon-Long et al. tested 8 strains to amphotericin B, 5-FC, fluconazole, itraconazole, miconazole and D 0870-a new triazole antifungal agent-by a microbroth dilution method in RPMI, brain heart infusion and yeast nitrogen base medium. Although there were differences depending on the medium used, itraconazole had the lowest MICs (range 0.003-0.016 mg/L), and miconazole and D 0870 had similar values (range 0.013-0.5 mg/L and 0.003-0.1 mg/L, respectively). MICs were amphotericin B 0.06 to 0.5 mg/L; fluconazole 2 to 16 mg/L and flucytosine 0.25 to 32 mg/L (1). In the first reported case of natural human infection, the MIC of amphotericin B was 0.78 mg/L (5). For the isolate of the first imported case in Canada, MICs to fluconazole, 5-FC, itraconazole and miconazole were 12.5, 0.39, less than 0.195 and less than 0.195 mg/L, respectively (16). These results indicate that polyenes, 5-FC , itraconazole and ketoconazole are effective in vitro. Limited data are available for fluconazole which seemed less active than itraconazole. In Vivo Studies Animal studies have shown that in experimentally infected hamsters, nystatin can prolong survival time. Oral itraconazole in immunosuppressed guinea pigs achieved a cure, even at a low dosage (22). In this animal model of infection a good correlation was established between in vitro and in vivo data.
ANTIMICROBIAL THERAPY Guided Medline Search Smart search At the current time, the therapy of choice for invasive infection appears to be intravenous amphotericin B followed by oral itraconazole. Three patients were cured by intravenous amphotericin B alone or in combination with oral 5-FC or oral ketoconazole (4, 9). One patient with delayed treatment because of misdiagnosis died despite receiving a combination of amphotericin B and flucytosine for 9 days (9).
In a review of 44 AIDS patients and 44 non HIV-infected patients all untreated
patients and those with disseminated infections died (Table 1) (6).
Amphotericin B, with or without flucytosine and itraconazole, was effective as
well as ketoconazole, but only two patients were treated with ketoconazole.
Amphotericin B was given at a total cumulative dose of approximately 40 mg/kg.
Itraconazole (400 mg/day) was successful. Maintenance therapy was given
successfully to a small number of patients (6). In one AIDS patient, itraconazole appeared to be superior to Combination Therapy There are no studies comparing treatment with a single agent and with a combination. Some patients obtained good results with amphotericin B and flucytosine. There is no published experience with itraconazole plus flucytosine. HIV Patients In an open nonrandomized study in Thailand, 74 HIV-infected patients with disseminated P. marneffei infection were treated with IV amphotericin B 0.6 mg/kg/d for 2 weeks followed by oral itraconazole 200 mg x 2/d for 10 weeks: 72 (97.3%) patients responded to this treatment. Fever and other signs and symptoms resolved within the first 2 weeks. Delayed treatment can result in death (17). In a double-blind trial 71 of these successfully treated patients received a maintenance therapy with itraconazole 200 mg/d or placebo. None of the 36 patients assigned to itraconazole relapsed, 20 of 35 patients (57%) assigned to placebo had relapsed within one year (21). Localized Infection In Immunocompetent Hosts No data are available on the treatment of the localized form of the disease in which spontaneous cure can occur, particularly in nonimmunocompromised hosts. In this setting, itraconazole is a logical first-line treatment. If this drug is not available, ketoconazole seems a good alternative, although few patients have been treated with ketoconazole.
PREVENTION Guided Medline Search Smart search A primary prophylaxis trial with itraconazole is scheduled in the Chang Mai (Thailand) area. Due to the severity of the disease and its high incidence, such an approach seems reasonable. Itraconazole prophylaxis could also decrease the incidence of cryptococcosis and histoplasmosis. However, due to the high incidence of tuberculosis, the concomitant use of rifampin is likely to adversely affect the efficacy of itraconazole by accelerating its metabolism. Traveling in an endemic area is not recommended for severely immunocompromised individuals, particularly AIDS patients. No data are available showing the efficacy of HAART in reducing the incidence of penicilliosis or continuing maintenance therapy in HIV positive patients, as it is the case for other opportunistic infection when the immunity is partly restored.
Table 1. Outcome for
Penicillium marnefeii Infections (6).
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