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We report the first two proven cases of cavitary pulmonary zygomycosis caused by Rhizopus homothallicus. The diagnosis in each case was based on histology, culture of the causal agent, and the nucleotide sequence of the D1/D2 region of the 28S ribosomal DNA.
Case 1 involved a 47-year-old male with a history of type II diabetes mellitus for 13 years who developed diabetic nephropathy leading to end-stage renal disease. He had undergone renal transplantation and was on triple-drug immunosuppression (cyclosporine, azathioprine, and prednisolone). One month posttransplantation, he developed an intermittent fever with chills and pleuritic chest pain. A chest X-ray revealed a cavitary lesion in the right upper lobe with bilateral nodular infiltrates, for which he was treated with a primary drug regimen of antituberculosis therapy (ATT), although multiple sputum examinations were negative for acid-fast bacilli. The patient did not respond to the ATT and was referred to our institute (Postgraduate Institute of Medical Education and Research [PGIMER], Chandigarh, India). At our institute, contrast-enhanced computed tomography (CT) of the thorax revealed a thick, smooth-walled cavitary lesion of the posterior segment of the right upper lobe and multiple nodules in both lungs (Fig. (Fig.1).1). No pleural effusion was noticed. Echocardiography ruled out endocarditis. His blood sugar levels ranged from 153 to 226 mg/dl. While he was at the hospital, he was treated with human insulin therapy. ATT was continued along with vancomycin and tazobactam. Ultrasound-guided fine-needle aspiration of the cavitary lesion of the lung was performed. Direct microscopic examination of calcofluor white-stained mounts of the aspirated fluid was done. Part of the fluid was cultured on Sabouraud dextrose agar (SDA) and brain heart infusion agar (HiMedia, Mumbai, India). Direct microscopy of the aspirated fluid did not reveal any fungal elements, nor did the cultures yield any fungal colonies during 4 weeks of incubation. As the patient did not improve until the 20th day of his hospital stay, an open-lung biopsy was performed. It revealed pus in the pleural cavity with a large cavitary lesion in the right upper lobe. No apparent mass was noticed. A wedge-shaped biopsy sample was taken from the site of the lesion. Tissue slides stained by the hematoxylin-and-eosin and periodic acid-Schiff procedures showed dense acute inflammatory infiltrates across the interstitial septa, a dense fibrosis surrounding the alveoli, extensive necrosis with nuclear debris and broad, aseptate, ribbon-like hyphae (Fig. (Fig.2).2). The causal fungus was observed invading the vessel wall. Culture of the biopsy tissue on SDA (HiMedia, Mumbai, India) grew a fast-growing, floccose white colony turning grayish. Microscopic examination of the colony showed broad, aseptate hyphae with lateral not-well-developed sporangiophores bearing globose sporangia containing a small number of sporangiospores. In addition, a large number of golden brown zygospores with stellate walls and with unequal suspensor cells were observed. The isolate was presumptively identified as Rhizopus homothallicus Hesseltine and Ellis (8). Liposomal amphotericin B (Fungisome; Lifecare Innovations, India) was given at a dose of 1.5 mg/kg of body weight/day. Follow-up after a cumulative dose of 5.0 g of liposomal amphotericin B therapy showed a striking improvement of the patient both clinically and on radiological investigation. No relapse or recurrence of the infection was noticed during 5 months of follow-up after recovery.
Case 2 involved a 70-year-old male who presented at the King George Medical University Hospital, Lucknow, India, with a history of fever, cough, chest pain, mucopurulent expectoration, and recurrent hemoptysis for 25 days. On general examination, he had signs of glossitis and stomatitis. Examination of his respiratory system revealed bronchial breath sound over the left mammary area, and the rest of the physical examination was unremarkable. Chest radiography revealed the presence of air space consolidation with eccentric cavitation in the midzone of the left lung. CT of the thorax revealed a large, thick-walled cavity in the left upper lobe abutting the chest wall and encroaching upon the arch of the aorta. On routine investigation, uncontrolled blood sugar levels (range, 232 to 360 mg/dl) were noted. Sputum examination did not reveal acid-fast bacilli. The patient was treated with oral antibiotics (625 mg of amoxicillin-clavulanic acid twice daily and 150 mg of clindamycin four times a day for 2 weeks). He did not show clinical or radiological improvement. The patient refused to undergo bronchoscopy. Transthoracic needle aspiration of the left cavitary lesion revealed ribbon-like, broad, coenocytic hyphae on direct KOH examination, as well as smears subjected to periodic acid-Schiff staining and Gomori's methenamine silver staining procedure. Fungal culture of the aspirate on SDA (HiMedia, Mumbai, India) grew white, cottony colonies. Direct examination of a teased mount of the colony stained with lactophenol cotton blue showed numerous golden brown, globose, spiny zygospores with suspensor cells. The isolate was tentatively identified as R. homothallicus. The patient was treated with injectable insulin for glycemic control and intravenous conventional amphotericin B (50 mg/day) for 15 days. After a cumulative dose of 750 mg of amphotericin B, the patient developed acute renal failure. After the withdrawal of amphotericin B, his renal function improved rapidly. However, he refused subsequent treatment with amphotericin B and left the hospital against medical advice. He returned after 3 months with massive hemoptysis and succumbed to his illness rapidly before any treatment with antifungal agents could be initiated.
Colonies of both isolates on SDA were fast growing, white, floccose, and devoid of pigmentation on the reverse side. Within 10 to 14 days of incubation at 28 to 30°C, colonies turned grayish. Slide culture mounts of the isolate from case 1 stained with lactophenol cotton blue (PGIMER MCCL [Mycology Culture Collection Laboratory] 710076) on SDA incubated at 30°C for 10 days showed broad, hyaline, aseptate, branching hyphae producing very few sporangiophores opposite poorly developed rhizoids. The sporangiophores measured 5 to 27 μm in diameter and 50 to 150 μm in length bearing globose to subglobose sporangia measuring 50 to 150 μm in diameter. The sporangiospores were angular-globose, grayish, 3.5 to 5.0 μm in length, and 4.0 to 6.5 μm in width. The striking feature was abundant homothallic, thick-walled zygospores that were reddish brown in color and measured 40 to 100 μm in diameter, including stellate spines. Suspensor cells were uneven in size, the larger ones being globose (Fig. (Fig.33).
The isolate from case 2 (MCCL 710099) was studied at the CDC. Slide cultures on potato dextrose agar and malt extract agar after 2 weeks at 25°C did not show any fertile sporangia containing sporangiospores. Sporangiophores were poorly developed opposite poorly developed rhizoids in tufts of two to five at several locations. Due to the lack of any sporulation, agar blocks (2 by 2 cm) with mycelial growth from the culture plates were transferred aseptically to a plate containing 20 ml of sterilized distilled water to which 3 to 5 drops of 15% filter-sterilized yeast extract solution was added. After 5 days of incubation at 37°C, examination of growth over the surface of water showed abundant globose, golden brown, spiny zygospores supported by uneven-size suspensor cells. Both isolates were thermotolerant and grew at 46 to 48°C. Both isolates were identified as R. homothallicus Hesseltine and Ellis (8).
The identities of both isolates were further confirmed by nucleotide sequencing of the 28S ribosomal DNA (rDNA) region. Whole-cell DNA was extracted from the isolates by a slightly modified version of the small-scale fungal DNA extraction method described by Lee and Taylor (10). Briefly, pure cultures of the isolates were grown in Sabouraud dextrose broth (HiMedia, Mumbai, India) and incubated at 37°C on a rotary shaker (HT Infors, Germany) at 120 rpm for 3 to 5 days. The mycelial mat (0.4 to 0.5 g) was prepared and crushed to a fine powder with a mortar and pestle in the presence of liquid nitrogen and lysis buffer. The DNA was extracted by the standard phenol-chloroform (25:24) extraction method. DNA precipitation was carried out using 2 volumes of chilled absolute alcohol and 1/5 volume of 10 M-ammonium acetate, followed by washing with 70% alcohol. The DNA pellet was dissolved in 100 μl of TE buffer. DNA preparations were stored at −20°C until use. PCR was performed in a reaction mixture of 10 μl containing 2 mM MgCl2, 200 μM each deoxynucleoside triphosphate (Bangalore Genie, Bangalore, India), 0.25 μM each primer NL1 (5′-GCATATCAATAAGCGGAGGAAAAG) and primer NL4 (5′-GGTCCGTGTTTCAAGACGG) (Integrated DNA Technologies, Inc., Coralville, IA), 0.25 U of Taq polymerase (Bangalore Genie), and 10 ng of fungal genomic DNA. The amplification reactions were performed in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany). PCR amplification was performed by 36 cycles of annealing at 52°C, extension at 72°C for 2 min, and denaturation at 94°C for 1 min. PCR products were purified with a gel extraction kit (Qiagen Hilden, Germany), and both strands were sequenced by the BigDye terminator cycle sequencing ready reaction kit, version 3.1 (Applied Biosystems, Foster City, CA) with primers NL1 and NL4. The reaction products were analyzed on Genetic Analyzer 3130 (Applied Biosystems). The basic local alignment search tool (BLAST) was used to compare the sequences obtained with those in the GenBank database and to see the similarity of the two isolates. The sequences of both isolates gave 99% identity with each other and 98% identity with the ex-type strain of R. homothallicus (AB 250198, NRRL 2538 = CBS 336.62). The two Indian isolates have been deposited in the CBS Fungal Biodiversity Centre, Utrecht, Netherlands, with the following accession numbers: MCCL 710076 = CBS 125071; MCCL 710099 = CBS 125072.
Antifungal susceptibility testing of both isolates was performed by the microdilution broth technique following the protocol of Clinical and Laboratory Standards Institute document M-38A (5). The MICs for both isolates were similar: amphotericin B, 0.5 μg/ml; flucytosine, >64.0 μg/ml; fluconazole, 64.0 μg/ml; itraconazole, >16.0 μg/ml; voriconazole, 4.0 μg/ml; caspofungin, 16 μg/ml.
Among the different agents of zygomycosis, Rhizopus spp. are the most commonly implicated agents causing human infections, and R. oryzae is the predominant species, being implicated in 90% of the reported cases of invasive zygomycosis (3, 4, 17). The other Rhizopus spp. less commonly reported as causal agents are R. microsporus (16), R. azygosporus (6), R. schipperae (2), and R. stolonifer (7). To our knowledge, the present report describes the first two cases of invasive zygomycosis caused by R. homothallicus. Hesseltine and Ellis described R. homothallicus in 1961 based on the ex-type strain (NRRL 2538) isolated from a soil sample from Guatemala in 1956 (8). Subsequently, the species was isolated in India from soil samples from several areas, from dung, and from stored grains of Triticum sp. (http://www.cabri.org/CABRI/srs-doc/index.html). When isolated from soil or other environmental sources, R. homothallicus closely resembles R. microsporus in general morphology, especially asexual sporangiophores, sporangia, sporangiospores, and maximum growth temperatures (19). However, strains maintained under laboratory conditions often lose their sporulation ability, including the ability to form zygospores. According to Schipper and Stalpers, the ex-type strain of R. homothallicus (NRRL 2538 = CBS 336.62) no longer produces zygospores (19). In 1970, Scholer attempted to produce experimental infection in mice using R. homothallicus but was not successful. The reasons for this failure were considered to be an insufficient number of sporangiospores in the inoculum and a failure to inject large-size zygospores intravenously (20).
Schipper (18) classified Rhizopus spp. into three groups, namely, the R. stolonifer group, the R. oryzae group, and the R. microsporus group, based on phenotypic characters and maximum growth temperatures (18, 19). Recent studies by Abe et al. (1) based on the molecular phylogeny of Rhizopus spp. have concurred with Schipper's treatment of Rhizopus sp. groups.
Earlier observations by Scholer (20) and recent observations by Jennessen (9) have stressed that in R. homothallicus, rhizoids, sporangiophores, sporangia, and sporangiospores are often poorly developed. We also observed similar findings in our two isolates. Production of abundant zygospores was the main phenotypic character that was helpful in the identification of the isolate from case 1. The isolate from case 2 failed to produce zygospores when grown on routinely used potato dextrose agar and malt extract agar. A low-nutrient medium had to be used to induce zygospore production (13). R. homothallicus can also be confused with another homothallic species, namely, R. sexualis, which also produces abundant zygospores. However, R. homothallicus grows at temperatures as high as 46 to 48°C, while R. sexualis does not grow at 37°C.
Given the limitation of phenotypic identification methods, rDNA-based gene sequences have been used extensively for the molecular identification of fungi, including zygomycetes (1, 11, 23). The rDNA comprises a small-subunit gene (18S), a large-subunit gene (28S), and internal transcribed spacer (ITS) regions (ITS1 and ITS2). The ITS region is generally used for the species identification of fungi, as the sequences of closely related taxa can be aligned with confidence. To obtain similar resolution with the 18S and 28S genes, a large portion of the DNA must be sequenced. In the present study, many attempts to sequence the ITS region of the rDNA failed (results not shown). Hence, sequencing of the D1/D2 region of the 28S rDNA was performed. The failure to obtain pure sequence of the ITS region may be attributed to the homothallic nature of the fungal species under study, which led to multiple distinct ITS regions in a single strain. The presence of multiple bands did not allow proper analysis of the sequences. The presence of multiple distinct ITS regions in another homothallic Rhizopus species, R. sexualis, has been described earlier. Therefore, sequencing of the D1/D2 region of the 28S rDNA may be a more useful and easy method for the identification of homothallic R. homothallicus (1, 11, 23).
Pulmonary zygomycosis is considered second in frequency after the rhino-orbital-cerebral type among different categories of zygomycosis and has rarely been reported without any predisposing factor. Coughing, fever, and pleuritic chest pain are the common presenting symptoms of patients with pulmonary zygomycosis (22). Pulmonary zygomycosis may have a wide variety of lesions, including an isolated solitary nodule, lobar involvement, and cavitary or disseminated lesions (14, 15, 22). Pulmonary consolidation, cavitation, or an effusion is less frequently seen (15, 22). Both patients in the present report had cavitary lesions. Tedder et al. (22), in a review of 156 cases of pulmonary zygomycosis, observed that only 6.0% had radiographic findings of cavitation of the lung. In our earlier two series of reports on zygomycosis from PGIMER, 25 patients had pulmonary zygomycosis and none had cavitary lesions (3, 4), although a contrasting claim of approximately 40% cavitary lesions among patients with pulmonary zygomycosis has been made (12). However, it is not clear whether the type of lesion depends on the virulence of the causal agent, the host's immune status, or both. It was suggested that such cavities represent liquefaction of pulmonary infarcts (15). A correlation between the type of fungi of Mucorales isolated from the pulmonary lesion and the type of lesion has never been established. Interestingly, a few species such as R. stolonifer (7) and Cunninghamella bertholletiae (24) have been isolated from patients with cavitary lesions.
Hemoptysis in patients with zygomycosis may be fatal and may occur due to erosion of the cavitary lesion into the bronchus (23). The patient in case 2 had a similar fate. Sputum or bronchoalveolar lavage analysis, though frequently employed, rarely leads to confirmation of the diagnosis. Procedures such as open-lung biopsy, surgical extirpation, and transthoracic needle aspiration provide better samples for diagnosis (22). In the present two cases, invasive procedures helped in the definitive diagnosis.
Without prompt therapeutic management, invasive zygomycosis invariably proves fatal. Aggressive surgical treatment, appropriate medical therapy, and control of predisposing factors are of vital importance in the treatment of such cases (3). Amphotericin B is the first-line drug of choice for most of the cases of zygomycosis. In both of the cases presented here, the patients were treated with amphotericin B using either a conventional or a liposomal formulation. The patient in case 1 responded well to the therapy. The second patient succumbed to the infection, possibly due to inadequate treatment. Both isolates of R. homothallicus had amphotericin B MICs of 0.5 μg/ml. The MIC patterns observed with the two isolates of R. homothallicus were consistent with those reported for other Rhizopus species (21).
Nucleotide sequence data for the 28S rDNA regions of both isolates were submitted to GenBank (accession no. EU128745 for MCCL 710076 and EU491016 for MCCL 710099).
We thank the Indian Council of Medical Research, New Delhi, India, for their support in purchasing the sequencer for the Centre of Advance Research in Medical Mycology.
None of us has an association that might pose a conflict of interest relevant to this report.
Published ahead of print on 3 March 2010.