Outbreak of intestinal infection due to Rhizopus microsporus

Abstract

Sinopulmonary and rhinocerebral zygomycosis has been increasingly found in patients with hematological malignancies and bone marrow transplantation, but intestinal zygomycosis remains very rare in the literature. We investigated an outbreak of intestinal infection due to Rhizopus microsporus in 12 patients on treatment for hematological malignancies over a period of 6 months in a teaching hospital. The intake of allopurinol during hospitalization (P < 0.001) and that of commercially packaged ready-to-eat food items in the preceding 2 weeks (P < 0.001) were found to be independently significant risk factors for the development of intestinal zygomycosis. A total of 709 specimens, including 378 environmental and air samples, 181 food samples, and 150 drug samples, were taken for fungal culture. Among them, 16 samples of allopurinol tablets, 3 prepackaged ready-to-eat food items, and 1 pair of wooden chopsticks were positive for Rhizopus microsporus, which was confirmed by ITS1-5.8S-ITS2 rRNA gene cluster (internal transcribed spacer [ITS]) sequencing. The mean viable fungal counts of allopurinol obtained from wards and pharmacy were 4.22 x 10(3) CFU/g of tablet (range, 3.07 x 10(3) to 5.48 x 10(3)) and 3.24 x 10(3) CFU/g of tablet (range, 2.68 x 10(3) to 3.72 x 10(3)), respectively, which were much higher than the mean count of 2 x 10(2) CFU/g of food. Phylogenetic analysis by ITS sequencing showed multiple clones from isolates of contaminated allopurinol tablets and ready-to-eat food, of which some were identical to patients' isolates, and with one isolate in the cornstarch used as an excipient for manufacture of this drug. We attempted to type the isolates by random amplification of polymorphic DNA analysis, with limited evidence of clonal distribution. The primary source of the contaminating fungus was likely to be the cornstarch used in the manufacturing of allopurinol tablets or ready-to-eat food. Rhizopus microsporus is thermotolerant and can multiply even at 50 degrees C. The long holding time of the intermediates during the manufacturing process of allopurinol amplified the fungal load. Microbiological monitoring of drugs manufactured for highly immunosuppressed patients should be considered.

FIG. 1.

Microbiological and histopathological examination of the resected intestinal specimen of case 5. (a) Potassium hydroxide (KOH)-digested wet mount of tissue stained by calcofluor white, showing bluish-white fluorescence of branching fungal elements without septum. (b) Hematoxylin-and-eosin-stained section of the mesoappendix with a vessel showing extensive thrombosis, with the lumen filled by blood cells and fungal elements. (c) Pale-staining hyphae appear as holes or bubbles in the section. Only red cells are seen in the thrombosed vessel due to severe neutropenia. Invasion into the vessel wall by fungal hyphae is seen. (d) Grocott silver-stained section of a vessel, showing wide wavy ribbon-like aseptate hyphae with broad-angle branching.

FIG. 2.

(a) Clinical specimen was cultured for 48 h at 37°C; a fluffy colony with a pale gray color filled up the whole Sabouraud dextrose agar plate (80 mm). (b) With lactophenol cotton blue preparation, globose sporangia (up to 80 μm in diameter) were found with simple rhizoids directly under unbranched, short (up to 500 μm in length) sporangiophores. (c) In three-week-old culture, azygospores (up to 30 μm in diameter) were not found in all patient, food, and drug isolates except in the control strain. (d) For scanning electron microscopy examination of spore wall ornamentation, R. microsporus var. rhizopodiformis was observed as small, regularly spaced, and cylindrical spines formed on the wall of globose spores. R. microsporus var. rhizopodiformis was identified in patients' isolates (P3, P7, and P9) and in allopurinol (D2). (e) R. microsporus var. chinensis; rows of blunt warts were arranged mainly in line from pole to pole on the wall of ellipsoidal spores. R. microsporus var. chinensis was identified in patients' isolates (P1, P2, P4 to P6, P8, P10, and P11), food isolates (F3 and F4), and raw material (cornstarch) of allopurinol (R1). The presence of R. microsporus var. chinensis and that of R. microsporus var. rhizopodiformis were found simultaneously in isolates from patient (P12) and allopurinol (D1, D3, and D4). (f) Morphological transition of spores between R. microsporus var. chinensis and R. microsporus var. rhizopodiformis was observed in the purified colonies by electron microscopy.

FIG. 3.

Phylogenetic tree showing the relationship of 24 strains isolated from patients, foods, drugs and cornstarch, inferred from ITS (648 nucleotide positions) sequence data by the neighbor-joining method and rooted using sequence of Pirella circinans (AM778602). The scale bar indicates the estimated number of substitutions per 50 bases. Numbers at nodes indicated levels of bootstrap support calculated from 1,000 trees. All names and accession numbers are given as cited in the GenBank database.