outbreak of intestinal infection due to rhizopus...

1

Outbreak of Intestinal Infection due to Rhizopus microsporus 1

Vincent CC Cheng1,2

, Jasper FW Chan1, Antonio HY Ngan

1, Kelvin KW To

1, SY 2

Leung1, HW Tsoi

1, WC Yam

1, Josepha WM Tai

2, Samson SY Wong

1, Herman Tse

1, 3

Iris WS Li1, Susanna KP Lau

1, Patrick CY Woo

1, Anskar YH Leung

3, Albert KW Lie

3, 4

Raymond HS Liang3, TL Que

4, PL Ho

1, KY Yuen

1* 5

6

From Department of Microbiology1, Infection Control Unit

2, Department of 7

Medicine3, Queen Mary Hospital, and Department of Pathology, Tuen Mun Hosptial

4, 8

Hong Kong Special Administrative Region, China. 9

Correspondence to: 10

KY Yuen 11

Carol Yu’s Centre for Infection and Division of Infectious Diseases, Department of 12

Microbiology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, 13

Pokfulam, Hong Kong Special Administrative Region, China. 14

(Tel: +852-28553206, Fax: +852-28724555, E-mail: [email protected]) 15

16

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00908-09 JCM Accepts, published online ahead of print on 29 July 2009

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ABSTRACT 17

Sinopulmonary and rhinocerebral zygomycosis has been increasingly reported 18

in patients with hematological malignancies and bone marrow transplantation, but 19

intestinal zygomycosis remains very rare in the literature. We investigated an outbreak 20

of intestinal infection due to Rhizopus microsporus in 12 patients on treatment for 21

hematological malignancies over a period of 6 months in a teaching hospital. The 22

intake of allopurinol during hospitalization (P<0.001) and commercially packaged 23

ready-to-eat food items in the preceding 2 weeks (P<0.001) were found to be 24

independently significant risk factors for the development of intestinal zygomycosis. 25

A total of 709 specimens, including 378 environmental and air samples, 181 food 26

samples and 150 drug samples, were taken for fungal culture. Among them, 16 27

samples of allopurinol tablets, 3 pre-packaged ready-to-eat food items and 1 pair of 28

wooden chopsticks, were positive for Rhizopus microsporus, which were confirmed 29

by ITS1-5.8S-ITS2 rRNA gene cluster (ITS) sequencing. The mean viable fungal 30

counts of allopurinol obtained from wards and pharmacy were 4.22 × 103 cfu/g of 31

tablet (range 3.07–5.48 × 103) and 3.24 × 10

3 cfu/g of tablet (range 2.68–3.72 × 10

3) 32

respectively which were much higher than the mean count of 2 × 102 cfu/g of food. 33

Phylogenetic analysis by ITS showed multiple clones from isolates of contaminated 34

allopurinol tablets and ready-to-eat food of which some were identical to patients’ 35


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isolates, and with one isolate in the corn starch used as excipient for manufacturing 36

this drug. We attempted to type the isolates by random amplification of polymorphic 37

DNA (RAPD) analysis with limited evidence of clonal distribution. The primary 38

source of the contaminating fungus was likely to be the corn starch used in the 39

manufacturing of allopurinol tablets or ready-to-eat food. Rhizopus microsporus was 40

thermo-tolerant and could multiply even at 50ºC. The long holding time of the 41

intermediates during the manufacturing process of allopurinol has amplified the 42

fungal load. Microbiological monitoring of drugs manufactured for highly 43

immunosuppressed patients should be considered. 44

45

Key words: Rhizopus microsporus, intestinal zygomycosis, drug contamination, 46

allopurinol, RAPD, immunosuppressed host47


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INTRODUCTION 48

Zygomycosis has become an important emerging infection in patients with 49

hematological malignancy undergoing chemotherapy or bone marrow transplantation 50

(24), especially after the availability of voriconazole as antifungal prophylaxis (25, 28, 51

44). Patients with breakthrough zygomycosis while on voriconazole prophylaxis 52

usually manifested as sinopulmonary or rhinocerebral disease since the fungal spores 53

were ubiquitously found in the environment and could therefore be acquired through 54

inhalation or traumatic inoculation through the skin or mucosa. Gastrointestinal 55

zygomycosis was a rare clinical entity (50, 54), but cases or outbreak of tongue, 56

gastric or cutaneous zygomycosis due to Rhizopus microsporus after exposure to 57

contaminated wooden tongue depressors had been reported (23, 26, 33). 58

The species Rhizopus microsporus group which belongs to the Class 59

Zygomycetes and Order Mucorales can cause life-threatening infections in 60

immunocompromised patients (9, 39, 46). The laboratory diagnosis of R. microsporus 61

relies mainly on the characteristic microscopic and phenotypic features of the fungal 62

mycelium culture on agar medium. Molecular diagnostic test for this fungus is not 63

widely available. PCR amplification and sequencing of the 18S and 28S rRNA genes 64

(58, 64), internal transcribed spacer (ITS) region of rRNA (27), and recently 65

PCR-restriction fragment length polymorphism (22) and high-affinity iron permease 66


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gene sequence have been used to identify members of clinically important 67

Zygomycetes (30). 68

Here, we report an unprecedented outbreak of intestinal zygomycosis due to R. 69

microsporus as a result of the oral intake of contaminated allopurinol tablets and 70

ready-to-eat food items in patients with hematological malignancies undergoing 71

chemotherapy and bone marrow transplantation. The clinical spectrum of this 72

outbreak of intestinal infection by R, microsporus ranges from asymptomatic 73

colonization, mucosal involvement to invasive disease. 74

75

MATERIALS and METHODS 76

Setting. The hematological oncology unit at Queen Mary Hospital, a university 77

affiliated teaching hospital in Hong Kong, serves as a tertiary referral center for 78

treatment of refractory hematological malignancies and bone marrow transplantation. 79

During the outbreak period, there were two pediatric (ward C6 and K8N) and two 80

adult (ward K20N and J8N) medical wards providing care for these patients or for 81

bone marrow transplantation. Except for C6, all these wards were equipped with 82

positive pressure high efficiency particulate filtered air in protective isolation rooms 83

(K8N with 7 rooms, K20N with 14 rooms, and J8N with 10 rooms) for patients with 84

neutropenia. 85


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Outbreak investigation. An outbreak of intestinal infection due to R. microsporus 86

occurred among these in-patients over a period of 6 months. Invasive intestinal 87

zygomycosis was defined as a symptomatic case having intestinal tissue specimen 88

showing characteristic histological tissue invasion by aseptate hyphae with wide but 89

varying diameter and a positive culture for R. microsporus with or without 90

extra-intestinal dissemination. Mucosal zygomycosis was defined as a case with 91

abdominal symptom, positive stool culture for R. microsporus and radiological 92

evidence of bowel mucosal thickening but without histological proof of tissue 93

invasion. A case of colonization was defined as an asymptomatic case with 94

surveillance stool culture positive for R. microsporus. Microbiological laboratory data 95

was also retrieved to identify any unrecognized case and the past incidence of this rare 96

infection. The medical records of the case patients were reviewed by clinical 97

microbiologists and the infection control team as described in our previous outbreak 98

investigations (5, 6, 7, 66). The hematologists were interviewed for any change in 99

chemotherapeutic regimen which might have altered the degree of 100

immunosuppression in this group of patients, and the nursing staffs were inspected for 101

any changes in patient care practice. Patients and their relatives, if available, were 102

interviewed in detail for food, drinks, eating utensils and drug history. A case control 103

study was performed to identify the risk factors for the outbreak. Since their 104


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immunocompromised state is a known risk factor for zygomycosis, patients staying in 105

the hematology and bone marrow transplant units with stool culture negative for the 106

fungus were chosen as control. 107

Screening of stool samples. During the period of outbreak investigation, stool 108

samples from all hospitalized patients requesting for bacterial culture in Queen Mary 109

Hospital were also screened for Mucorales. To avoid potential contamination by 110

wooden spatula used during specimen collections (15, 57), stool samples were 111

collected with sterile swabs and transported in sterile bottles. Pea-sized formed stool 112

of about one gram or 0.5 mL of watery stool was streaked out onto Sabouraud 113

dextrose agar plus chloramphenicol 50 mg/L (Becton Dickinson and Company) and 114

incubated at 37ºC for 7 days. 115

Screening of environmental, food and drug samples. Environmental samples 116

from clinical areas, kitchen and pharmacy, and air samples from clinical areas, kitchen 117

and the corridors were collected for fungal culture. Food items including fresh fruits, 118

pre-packaged ready-to-eat food and taste additives were tested as well. Food items 119

were cultured as previously described with modification (51, 52). Tested items and 120

swabs were inoculated into enrichment broth (containing peptone 10 g/L, yeast extract 121

1 g/L, dextrose 25g/L, chloramphenicol 50 mg/L) and Sabouraud agar for incubation 122

at 37°C. All broths were checked for mycelium formation every 24 hours and 123


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subcultured onto Sabouraud dextrose agar at day 5 for 48 hours of incubation at 37°C. 124

Quantitative culture was performed for the unopened refrigerated items of same batch 125

number if there are positives on culture for Rhizopus spp. 126

For screening of drugs, after swabs for fungal culture were taken from surface of 127

an opened vial, 3 tablets from each bottle were immersed into enrichment broth 128

(containing peptone 10 g/L, yeast extract 1 g/L, dextrose 25 g/L, chloramphenicol 50 129

mg/L) incubated at 37°C for up to 7 days. Aliquots were taken for subculture onto 130

Sabouraud agar either if the broth became turbid or after 7 days, whichever is earlier. 131

Quantitation of mold was performed by dissolving 1 tablet into 1 mL of enrichment 132

broth, aliquoting 10 µL in triplicate and spreading onto Sabouraud dextrose agar with 133

modification as described (53). The mean colony forming unit (cfu) was the average 134

of those from 3 different tablets in the same batch. The result was expressed as cfu/g 135

of tablet. 136

Laboratory identification of Rhizopus isolates. Fungal colonies were examined 137

under direct microscopy for morphological characteristic of Rhizopus spp. Isolates of 138

R. microsporus group were identified according to standard morphological criteria (17) 139

and stored in 1 mL of sterile water at room temperature for genotypic analysis. 140

Scanning electron microscopy was performed to identify the variety of R. 141

microsporus group isolates. 142


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Antifungal susceptibility and treatment outcome. Susceptibility of R. microsporus 143

to amphotericin B and poscaonazole were determined by E-test (AB Biodisk, Sweden) 144

according to the manufacturer’s instructions. Aspergillus flavus ATCC 204304 was 145

used as control. The treatment outcome of our cases was recorded. 146

DNA extraction. Fungal DNA extraction was performed as described in our 147

previous publications (60, 61). Briefly, DNA was extracted from 1 g of fungal cells in 148

10 mL of distilled water using the DNeasy plant mini kit according to the 149

manufacturer’s instructions (QIAGEN, Hilden, Germany). The extracted DNA was 150

eluted in 50 µL of buffer AE, the resultant mixture was diluted 10 times, and 1 µL of 151

the diluted extract was used for PCR. 152

ITS1-5.8S-ITS2 rRNA gene cluster (ITS) sequencing. PCR amplification and 153

DNA sequencing of the ITS were performed according to published protocols (59, 62). 154

Briefly, DNase I-treated distilled water and PCR master mix, which contains 155

deoxynucleoside triphosphates (dNTPs), PCR buffer, and Taq polymerase, were used 156

in all PCR reactions by adding 1 U of DNase I (Roche, USA) to 40 µl of distilled 157

water or PCR master mix. The mixture were incubated at 25°C for 15 min, and 158

subsequently at 95°C for 10 min to inactivate the DNase I. The fungal DNA extract 159

and controls were amplified with 0.5 µM primers (ITS1 160

5’-TCCGTAGGTGAACCTGCGG-3’ and ITS4 5’-TCCTCCGCTTATTGATATGC-3’) 161


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(Gibco BRL, Rockville, MD, USA). The PCR mixture (25 µL) contained fungal DNA, 162

PCR buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 2 mM MgCl2 and 0.01% gelatin), 163

200 µM of each dNTPs and 1.0 U Taq polymerase (Applied Biosystem, Foster City, 164

CA, USA). The mixtures were amplified in 40 cycles of 94°C for 1 min, 55°C for 1 165

min and 72°C for 2 mins, and a final extension at 72°C for 10 mins in an automated 166

thermal cycler (Applied Biosystem, Foster City, CA, USA). DNase I-treated distilled 167

water was used as the negative control. Ten microliters of each amplified product was 168

electrophoresed in 1.5% (w/v) agarose gel, with a molecular size marker (φX174 169

HaeIII digest, Boehringer Mannheim, Germany) in parallel. Electrophoresis in 170

Tris-borate-EDTA buffer was performed at 100 V for 1.5 h. The gel was stained with 171

ethidium bromide (0.5 µg/mL) for 15 min, rinsed and photographed under ultraviolet 172

light illumination. 173

The PCR products were purified using the QIAquick Gel Extraction kit 174

(QIAgen, Hilden, Germany). Both strands of the PCR products were sequenced with 175

an ABI Prism 3700 DNA Analyzer (Applied Biosystems), using the PCR primers. The 176

sequences of the PCR products were compared with sequences of closely related 177

species in the GenBank by multiple sequence alignment using Clustal_X 1.83 (49). 178

Phylogenetic characterization. Phylogenetic tree construction was performed 179

using the neighbor-joining method with Clustal_X 1.83. A segment of 648 bp 180


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nucleotide of ITS were included in the analysis. 181

Typing by Random amplification of polymorphic DNA (RAPD). The DNA of the 182

Rhizopus isolates was extracted using the PrepMan® Ultra Sample Preparation kit 183

(Applied Biosystems) according to the manufacturer’s instructions. PCR was 184

performed as described previously with modifications (55). Reaction mixtures 185

contained 1× PCR buffer, 3mM MgCl2, 200µM each of the dNTPs, 0.2µM primer, 186

0.625U of AmpliTaq Gold DNA polymerase (Applied Biosystems) and 50ng of 187

genomic DNA in a 25 µl volume. Amplification was carried out in a GeneAmp 9700 188

thermal cycler (Applied Biosystems) with cycle conditions as follows: 95°C hot start 189

for 10 mins, 45 cycles of 92°C for 1 min, 35°C for 1 min, 72°C for 2 mins, with a 190

final extension at 72°C for 7 mins. 10µl of PCR products from each reaction were 191

analyzed by electrophoresis in 1.2% agarose gel in 1× TBE buffer and stained with 1× 192

SYBR Gold nucleic acid stain (Invitrogen). 193

RAPD banding patterns were compared based on the presence or absence of 194

observed amplification products on gel electrophoresis. Numerical analysis was 195

performed with GelCompar II version 3.0 (Applied Maths). Cluster analysis was 196

performed with calculation of Jaccard coefficients, and a dendrogram was constructed 197

using the UPGMA (unweighted pair-group method using arithmetic averages) method. 198

However, no interpretative standards for typing of filamentous fungus by RAPD 199


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currently exist. 200

Statistical analysis. The statistical analysis is performed to show the absence of 201

differences in the clinical characteristics between the patient and the control groups, 202

apart from the risk factors proposed. Non-parametric Mann-Whitney U test was 203

chosen for the continuous variables because there is no assumption of normal 204

distribution of values in the sample variable as distinct from parametric tests. The 205

Fisher’s exact test was used to compare independent categorical variables between 206

groups, and is considered superior to the chi-square goodness of fit test because it 207

does not utilize a normal approximation to the binomial distribution which could lead 208

to errors when the sample numbers are small. All reported P values were two-sided. A 209

P value of <0.05 was considered statistically significant. Computation was performed 210

using the Statistical Package for the Social Sciences (SPSS) Version 15.0 for 211

Windows. 212

213

RESULTS 214

Outbreak investigation. Since the opening of bone marrow transplant (BMT) 215

center at Queen Mary Hospital in 1990, a total of 44,940 fungal surveillance cultures 216

including 5319 stool samples were performed. Up to 31 December 2005, only 1 217

Mucorales (an Absidia sp.) was recovered from a sputum sample in the year 2004. 218


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During the same period, a total of 100,312 fungal cultures were performed in 219

non-BMT patients, Mucorales were positive in 25 specimens from 11 patients but 220

none of them was isolated from stool or intestinal tissue. During the outbreak period, 221

14 surveillance stool samples from 7 patients out of a total of 557 stool samples were 222

found to have R. microsporu. For septic work up of symptomatic cases, 1 wound swab 223

from 1 patient, 1 abdominal fluid from 1 patient, 20 operative tissues from 3 patients 224

were obrtained. After the retrospective review of clinical and laboratory data, a total 225

of 12 cases (5 invasive, 2 mucosal, and 5 colonization) due to R. microsporus were 226

identified during the study period (figure 1a-d). They were aged between 6 to 73 years 227

and the male to female ratio was 9:3 (table 1). All patients had sole intestinal 228

involvement without clinical or radiological evidence of sinopulmonary involvement. 229

The cases were scattered in the four hematology-oncology wards (C6, K8N, K20N 230

and J8N) and in one other general adult medical ward (E3) where these patients may 231

stay temporarily before moving into hematology-oncology wards. Case-control study 232

showed that the use of allopurinol during hospitalization and the intake of 233

commercially packaged ready-to-eat food in the preceding 2 weeks were found to be 234

independent statistically significant risk factors for developing intestinal infection by 235

R. microsporus (table 2). 236


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A total of 709 non-patient specimens were obtained for fungal culture. These 237

included 378 environmental and air samples taken from the wards (246 samples), 238

pharmacy (17 samples), kitchen and catering service (63 samples), store room (40 239

samples), corridor (5 samples), and microbiology laboratory (7 samples). Another 181 240

food samples and 150 drug samples were also tested (table 3). Cultures revealed 241

Paecilomyces species (8 samples), Penicillium species (8 samples), Exophiala species 242

(3 samples), Aspergillus niger (5 samples), Aspergillus fumigatus (1 sample), Absidia 243

species (1 sample), and Rhizopus microsporus (1 sample) from environmental 244

specimens, 5 samples positive for Mucorales (one isolate of R. oryzae and Mucor 245

species each, and three isolates of R. microsporus) from food specimens, and 16 246

samples positive for Rhizopus species from drug specimens. Further analysis 247

demonstrated that all 16 samples of allopurinol tablets, 3 pre-packaged ready-to-eat 248

food items, and 1 pair of wooden chopsticks were positive for R. microsporus. None 249

of the cases had ever used wooden chopsticks and therefore this factor is no longer 250

included in further analysis. The mean viable fungal counts of allopurinol tablet 251

obtained from ward and pharmacy were 4.22 × 103

cfu/g of tablet (ranged 3.07–5.48 × 252

103) and 3.24 × 10

3 cfu/g of tablet (ranged 2.68–3.72 × 10

3) respectively, whereas the 253

mean viable counts in the food items were about 2 × 102 cfu/g. 254


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The Hong Kong government and the manufacturer which is a local 255

pharmaceutical company were notified of this important public health incident 256

involving Rhizopus-contaminated allopurinol. All production lines of this 257

pharmaceutical company were immediately suspended for further investigation. The 258

raw materials and finished tablets of allopurinol were retrieved for microbiological 259

culture by our laboratory as well as the government laboratory. A territory wide recall 260

and suspension of the use of this brand of allopurinol tablets were urgently 261

implemented. Subsequent laboratory test also showed that the corn starch used as part 262

of the excipients for allopurinol was found to contain 2 cfu/g of R. microsporus. 263

During the site visit, it was found that the active and inactive ingredients of the drug 264

granules were mixed and heated up to 50oC in oven for 4 hours, and subsequently 265

held at 20oC with 3% water content for 5 to 14 days. This prolonged holding time was 266

due to inevitable delays as a result of recent overbooking of the tabletting machine. 267

Such long holding time has provided an excellent opportunity for this thermotolerant 268

fungus to multiply to a high level. 269

In view of this unusual happening of fatal cases due to intestinal zygomycosis in 270

our hospital, the Hospital Authority of Hong Kong initiated a retrospective review and 271

2-week prospective enhanced surveillance for all hospitalized hematology patients at 272

all regional hospitals in Hong Kong. Ten other patients with intestinal infection by R. 273


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microsporus were identified in 4 regional hospitals. Two patients succumbed in 2 274

other regional hospitals were confirmed to have invasive intestinal zygomycosis due 275

to R. microsporus by post-mortem histopathological and microbiological examination. 276

Laboratory identification of Rhizopus isolates. All the patient strains were 277

identified as R. microsporus by their morphological appearance of fluffy, rapidly 278

growing mold with aseptate, broad, readily twisted, ribbon-like hyphae, the presence 279

of rhizoids directly under sporangiophores with unbranched sporangiophore, 280

sporangium’s size of less than 100 µm, sporangiophore’s length of less than 1000 µm 281

(figure 2a–c), the absence of azygospore, and optimal growth at 42ºC. R. microsporus 282

var. chinensis and R. microsporus var. rhizopodiformis were differentiated by the 283

spore appearance on electron microscopy (figure 2d–e). However morphological 284

transitions of spores between R. microsporus var. chinensis and R. microsporus var. 285

rhizopodiformis were observed in the purified colonies under electron microscopy 286

which rendered the differentiation useless for typing purpose (figure 2f). 287

Antifungal susceptibility and treatment outcome. Susceptibility of R. microsporus 288

to amphotericin B and posaconazole of 12 patients isolates were determined by 289

E-tests. The MIC of amphotericin B and poscaonazole ranged from 0.06 µg/mL to 1.5 290

µg/mL and 0.5 µg/mL to 2 µg/mL respectively. The MIC of the control strain of A. 291

flavus ATCC 204304 against amphotericin B and poscaonazole was 0.5 µg/mL and 292


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0.25 µg/mL respectively. 293

Based on these MIC results and the previous reports of in vitro synergism 294

between liposomal amphotericin B (AmBisome) and posaconazole (34) and between 295

caspofungin and posaconazole (11), our patients were treated with these 3 antifungal 296

agents, either in combination or with posaconazole alone, in addition to surgical 297

intervention where possible, depending on the extent of involvement (table 1). For the 298

5 patients with invasive intestinal disease, 3 (cases 1, 2 and 5) were identified 299

prospectively and were given the combination therapy. Cases 1 and 5 succumbed 300

despite surgery and antifungal agents due to the extensive involvement and failure of 301

neutrophil recovery, whereas case 2 survived with appendicectomy, antifungal therapy 302

and recovery of neutrophils. The other 2 patients with invasive intestinal disease 303

(cases 3 and 8) were identified retrospectively and were treated with fluconazole and 304

voriconazole respectively as empirical antifungal therapy. The 2 patients (cases 4 and 305

12) with mucosal zygomycosis were treated with combination therapy (case 4 306

received anidulafungin instead of caspofungin) and both responded clinically with 307

microbiological clearance of Rhizopus microsporus in subsequent stool samples. One 308

of them (case 4) succumbed 44 days after the onset of symptom due to severe 309

veno-occlusive disease with liver failure and non-engraftment. All except one (case 9) 310

of the 5 patients (cases 6, 7, 10 and 11) with asymptomatic colonization were treated 311


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with posaconazole because of upcoming chemotherapy or conditioning for bone 312

marrow transplant. All, including the patient who did not receive treatment, remained 313

asymptomatic with documented clearance of R. microsporus in subsequent stool 314

cultures. After stopping the use of this contaminated brand of allopurinol by switching 315

to that produced by another manufacturer and the strict enforcement of taking only 316

boiled food items by this group of patients, there were no reports of any further cases 317

in the subsequent 4 months of surveillance. 318

ITS sequencing. PCR of the ITS region of the fungal isolates produced amplicons 319

of about 600 bp. Sequencing of the amplicons showed unambiguous sequence in 24 320

isolates. Phylogenetic analysis showed that there were >98% nucleotide identity 321

between the sequence of any of the 24 isolates and those of Rhizopus microsporus var. 322

rhizopodiformis (DQ641306), Rhizopus azygosporus (DQ119008), Rhizopus 323

homothallicus (AF115728) and Rhizopus oryzae (AB097378). Among the 26 isolates, 324

there were 8 sequence types (figure 3). The first one consisted of 4 patient isolates (P2, 325

P3, P6, and P7), 5 drug isolates (D1-1, D1-2, D1-3, D2 and D3-2), 2 food isolates (F3 326

and F5) and 1 corn starch isolate (R1); the second one consisted of 2 patient isolates 327

(P8 and P9), 1 drug isolate (D4-2) and 2 food isolates (F4-1, F4-2); the third one 328

consisted of 2 food isolates (F2-1 and F2-2); and the other 5 consisted of 1 isolate 329

each (P1, P4, P5, P10-1 and D5). 330


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RAPD: PCR was performed with 4 random primers (OPC-01: 331

TTCGAGCCAG; OPC-02: GTGAGGCGTC; OPC-11: AAAGCTGCGG; OPC-19: 332

GTTGCCAGCC). Each set of reaction was performed in duplicate. Weak and 333

indeterminate bands were excluded from the numerical analysis. For the R. 334

microsporus isolates P9 and F1, we failed to produce consistent band patterns using 335

the above primers and hence they were excluded from RAPD analysis. A total of 61 336

RAPD markers were established from the present study (unpublished data). We 337

attempted to type the isolates by RAPD analysis with limited evidence of clonal 338

distribution. 339

340

DISCUSSION 341

We have reported the first major outbreak of intestinal infection by R. 342

microsporus in patients with hematological malignancies and bone marrow 343

transplantation. Rhizopus spp. has a worldwide distribution and is found in soils from 344

cultivated grassland and forest locations. This organism has been isolated from a 345

variety of food items including corn, barley, sorghum, wheat, oat, rice, onions, 346

groundnuts, sweet potatoes, pecans and tomatoes (1, 36). However, reports on R. 347

microsporus are relatively uncommon compared with other Rhizopus species. It has 348

been associated with the moldy lumber as evidenced by the presence of serological 349


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response to Rhizopus species among sawmill workers (37, 38). It has also been found 350

in the wooden material used for patient care. The use of contaminated wooden tongue 351

depressors as splinters for intravenous catheter fixation in premature neonates (26) 352

and wooden applicators for the preparation of drugs administered via the nasogastric 353

tubes in critically ill patients (23) have been incriminated in nosocomial outbreaks of 354

cutaneous and gastric zygomycosis respectively. A pseudoepidemic has also been 355

reported among patients with hematological conditions due to the use of contaminated 356

wooden sticks during stool collection (57). To the best of our knowledge, outbreak of 357

R. microsporus due to contaminated oral medication and less likely food items, as 358

shown in our case control and molecular analysis, has not been reported in the 359

literature. As the phylogenetic tree of ITS showed that the patient isolates closely 360

cluster with both drug and food isolates, it is not possible to blame either as the sole 361

common source for this outbreak. However, the mean viable fungal counts in 362

allopurinol tablets were much higher than the European Pharmacopoeia standard of 363

less than 100 fungal counts per 1 gram or 1 mL of non-sterile pharmaceuticals 364

products (4, 10), whereas the mean viable fungal count in the positive food items were 365

much lower. Upon further investigation of the source of drug contamination (31), the 366

allopurinol tablet was found to contain corn starch as part of its excipients which 367

contained 2 cfu/g of R. microsporus. During the time of dehydration of the 368


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granulations containing both active and inactive ingredients, the mixture was 369

processed at 50ºC in oven for 4 hours with the subsequent holding at 20ºC with 3% 370

water content for 5 to 14 days. This long holding time has provided an excellent 371

opportunity for the fungus to multiply to a high level within this starch containing 372

mixture which is nutritionally favorable for R. microsporus. Moreover, the 373

thermotolerance of the fungus allows its survival at 50ºC. Allopurinol and colchicine 374

are commonly used in patients with hyperuricemia and gout. Their side effects are 375

mainly related to hypersensitivity and drug interactions (16). Infective complications 376

due to contamination have never been reported. After these cases were reported from 377

our hospital, 10 similar cases were then found in other hospitals in Hong Kong. 378

At the moment, there is no standard typing method for zygomycetes which poses 379

severe limitations on the study of their molecular epidemiology (45). In a recent study 380

reporting 27 cases of zygomycosis in a tertiary-care cancer centre in the USA (21), 381

amplification of repetitive genomic sequences adopted from Aspergullus typing was 382

used (13). However, the strains of zygomycetes can only be differentiated at genus 383

level. Although random amplified polymorphic DNA (RAPD) analysis was applied in 384

Rhizopus stolonifer (55), the molecular typing of R. microsporus had never been 385

reported in the literature. In this study, we actually tested more primers than reported 386

but some were excluded because of the lack of variability or reproducibility in pattern. 387


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This is consistent with the practice of other research using RAPD as a tool to 388

differentiate rhizopus and rhizomucor species (55, 56). Our presently reported typing 389

method by ITS sequencing or RAPD typing can differentiate our isolates into many 390

types but their usefulness still await further validation. In future outbreaks, more data 391

can be accumulated to determine the cut-off criteria for clonality. At the moment, 392

primer sets used in this study appear to be most discriminative and reproducible. At 393

this stage, it is not possible to prioritize between ITS sequence typing from RAPD 394

typing. PCR sequence analysis for one loci such as ITS is easily reproducible, 395

comparable between laboratories and illustrative of evolutionary relationships but less 396

discriminative for clonality. RAPD involves PCR of multiple loci which does not 397

require sequencing but is less reproducible and results are not easy to compare 398

between laboratories. RAPD usually has higher discriminatory power but it shows 399

clustering rather than evolutionary relationships. For ITS sequencing, even one 400

nucleotide difference can be considered unique because of fidelity of DNA 401

polymerase in eukaryotes. As for RAPD, there are no interpretive standards for 402

zygomycetes. Unlike the typing method for bacteria (19), the interpretative standards 403

are based on large-scale comparison of many different strains with other typing 404

methods, such scale is not available for most molds at this stage. 405

Gastrointestinal manifestation was rare in patients with zygomycosis. It mostly 406


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occurred in premature neonates and was often associated with disseminated diseases 407

(8, 18, 35, 41). Necrotizing enterocolitis was described in premature neonates (29, 43, 408

63) and less frequently in neutropenic adults (47, 48). All our five cases of invasive 409

intestinal zygomycosis had severe necrotizing ileocecitis leading to intestinal 410

perforation with or without retroperitoneal necrosis, which was in contrast to the 411

classical reports of the stomach being the most frequently involved site in adults with 412

gastrointestinal zygomycosis (32). A recent study implicated that the suppression of 413

the gastric acidity by proton pump inhibitors in the critically ill patients might serve as 414

a predisposing factor for the gastric predilection (23). Since most of our patients did 415

not have concomitant pulmonary, rhinocerebral or disseminated involvement, this 416

suggested that the route of acquisition is oral ingestion rather than inhalation of fungal 417

spores. Furthermore, the infecting dose was likely to be rather substantial because in 418

at least two of our cases (cases 1 and 2), the onset of invasive diseases occurred 419

immediately after the first cycle of induction chemotherapy for acute leukemia. This 420

phenomenon was also shown in a recent case report (40). Since the duration of 421

neutropenic phase was less than 2 weeks in our cases (cases 1 and 2), the risk of 422

invasive fungal infection should not be high unless there was a significant load of 423

microbial exposure (14). 424

Zygomycosis has become an emerging infectious disease in the 425


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immunocompromised patients. Prior to this outbreak, zygomycosis was uncommon in 426

our locality (65). According to our laboratory information system, over 140,000 427

fungal cultures were performed in 15 years, only 11 patients had culture proven 428

zygomycosis with respiratory or cutaneous involvement and Rhizopus spp. had only 429

been encountered in 2 patients before this outbreak. In a recent epidemiologic study 430

involving 16 bone marrow transplant centers in North America, zygomycosis was 431

found in 18 (7.2%) out of 250 episodes of invasive fungal infection among adult bone 432

marrow transplant recipients between 2004 and 2007. Rhizopus species was found in 433

10 of these episodes (28). There was also a trend of increasing proportion of mold 434

infection by zygomycetes (24). This phenomenon may be related to the introduction 435

of voriconzaole, a broad spectrum antifungal agent, as prophylactic regimen during 436

bone marrow transplantation (25, 28, 44). In our bone marrow transplant center, 437

voriconazole had not been used as routine antifungal prophylaxis. Thus the emergence 438

of zygomycosis in our locality at this stage is not related to selection by voriconazole. 439

This phenomenon is therefore dissimilar to the emergence of Candida krusei or C. 440

glabrata after routine fluconazole prophylaxis in this group of patients (12). 441

The clinical spectrum of intestinal infection by R. microsporus ranges from 442

asymptomatic colonization, mucosal involvement to invasive disease as illustrated in 443

this outbreak. Our enhanced surveillance of stool culture picked up an 6 additional 444


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patients (cases 6, 7, 9-12) with asymptomatic colonization of R. microsporus and one 445

other (case 4) with mucosal involvement as evidenced by segmental thickening of 446

proximal jejunal wall in computerized tomography study. In contrast to the previous 447

reports of invasive disease (2, 3, 20, 42) the organism could act as a colonizer or 448

produce mucosal invasion in the gastrointestinal tract in the immunocompromised 449

host, similar to that of Candida species. However, further investigation should be 450

conducted to understand the natural evolution of clinical progression from 451

colonization to invasion, and whether there is any intracellular latency of Mucorales 452

in the gastrointestinal tract, in order to determine the optimal treatment and preventive 453

strategy for this new clinical entity. 454

455

ACKNOWLEDGMENT 456

We are grateful to Mr Hon-kit Chui and Miss Sally SM Leung for their assistance in 457

performing microbiological testing of the environmental and patient samples. We also 458

thank the hard working and conscientious frontline health care workers for their active 459

participation in the patient management and infection control. We thank Dr. York Y. N. 460

Chow, Secretary for Food and Health and Dr. Ping-Yan Lam, director of health, Hong 461

Kong SAR, The People’s Republic of China for his kind support. This work was 462

partly funded by the HKSAR Research Fund commissioned block grant for the 463


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Control of Infectious Diseases and the Consultancy Service for Enhancing Laboratory 464

Surveillance of Emerging Infectious Disease for Department of Health, the Health, 465

Welfare, and Food Bureau of the Hong Kong Special Administrative Region of China. 466

467


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66. Yuen, K. Y., W. H. Seto, T. Y. Ching, W. C. Cheung, Y. Kwok, and Y. B. Chu. 759

1992. An outbreak of Candida tropicalis peritonitis in patients on intermittent 760

peritoneal dialysis. J Hosp Infect. 22:65-72. 761

762

763


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Figure legend 764

765

Figure 1. Microbiological and histopathological examination of the resected intestinal 766

specimen of case 5. 1a, Potassium hydroxide (KOH) digested wet mount of tissue 767

stained by calcofluor white, showing bluish white fluorescence of branching fungal 768

elements without septum; 1b, H&E stained section of the mesoappendix with a vessel 769

showing extensive thrombosis with the lumen filled by blood cells and fungal 770

elements; 1c, Pale-staining hyphae appear as holes or bubbles in the section. Only red 771

cells are seen in the thrombosed vessel due to severe neutropenia. Invasion into the 772

vessel wall by fungal hyphae is seen; 1d, Grocott silver stained section of a vessel 773

showing wide wavy ribbon-like aseptated hyphae with broad-angle branching. 774

775

Figure 2. 2a, Clinical specimen was cultured for 48 hours culture at 37°C (optimal 776

temperature), fluffy colony with pale grey color filled up the whole Sabouraud 777

Dextrose Agar plate (80 mm); 2b, With lactophenol cotton blue preparation, globose 778

sporangia (up to 80 µm diameter) were found with simple rhizoids directly under 779

unbranched, short (up to 500 µm in length) sporangiophores; 2c, In three weeks old 780

culture, azygospores (up to 30 µm diameter) were not found in all patient, food, and 781

drug isolates except in the control strain; 2d, For scanning electron microscopy 782

examination of spore wall ornamentation, R. microsporus var. rhizopodiformis was 783

observed as small, regularly spaced and cylindrical spines formed on the wall of 784

globose spores. R. microsporus var. rhizopodiformis was identified in patients’ 785

isolates (P3, P7, and P9), and allopurinol (D2).; 2e, R. microsporus var. chinensis, 786

rows of blunt warts were arranged mainly in line from pole to pole on the wall of 787

ellipsoidal spores. R. microsporus var. chinensis was identified in patients’ isolates 788

(P1, P2, P4-6, P8, P10, and P11), food isolates (F3, F4), and raw material (corn starch) 789

of allopurinol (R1). Presence of R. microsporus var. chinensis and R. microsporus var. 790

rhizopodiformis were found simultaneously in isolates from patient (P12) and 791

allopurinol (D1, D3, D4); 2f, Morphological transition of spores between R. 792

microsporus var. chinensis and R. microsporus var. rhizopodiformis was observed in 793

the purified colonies under electron microscopy. 794

795

Figure 3. Phylogenetic tree showing the relationship of 24 strains isolated from 796

patients, foods, drugs and corn starch, inferred from ITS (648 nucleotide positions) 797

sequence data by the neighbor-joining method and rooted using Pirella circinans 798

(AM778602). The scale bar indicates the estimated number of substitutions per 50 799

bases. Numbers at nodes indicated levels of bootstrap support calculated from 1000 800

trees. All names and accession numbers are given as cited in the GenBank database. 801


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Table 1. Clinical characteristics of patients with intestinal zygomycosis due to Rhizopus microsporus isolated in operative tissue or stool

Case

(type)

Sex /

Age

(ward)

Hemic

malignancy &

(chemotherapy)

Symptoms & key

blood tests

Abdominal CT and laparotomy findings Histopathological findings Antifungal regimen &

clinical outcome

1(I) M/6

(C6)

ALL

(V, D, Met, Leu

& Dexa)

Abdominal pain;

ANC: 0.34; ALT:

13; Cr: 44

CT: distended bowel loops at central

abdominal region, swollen R psoas

muscle with abscess formation;

moderate R hydronephrosis

Laparotomy: necrotic small, large bowel,

R posas & paraspinal muscles.

Extensive involvement of all the tissues by

fungal elements with pronounced

angiotrophism with thrombosis, invasion of

the blood vessel wall by fungal hyphae, and

extensive full-thickness infarction of the

bowel walls.

Posa, AmB, Caspo 3 days &

Des 27 days after symptom

onset; died 36 days after

symptom onset

2(I) M/11

(K8N)

AML

(Met, Ida, Cyt &

E)

RLQ pain; ANC:

0.05; ALT: 9; Cr:

48

CT: swollen appendix measuring up to

2.5cm with thickened wall of 7mm.

Laparotomy: infarction & perforation of

paracaecal appendix and adjacent

omentum.

Transmural infarction of the appendix with an

abundant amount of fungal organisms

suggestive of mucorales by their branching at

right angles and invasion of the blood vessel

walls.

Posa, AmB, Caspo, Des 1

day after symptom onset;

fever & abdominal pain

resolved 16 days after

therapy

3(I)! M/57

(E3)

DLBCL

(V, D, M, A , &

Dexa)

Abdominal pain;

ANC: 0.26; ALT:

152

Cr: 126

CT: intraperitoneal gas suggestive of

perforated bowel.

Laparotomy: two perforations at cecum

with indurated edge.

Mucosal infiltration and necrosis due to fungal

element.

Fluconazole; died 8 days

after symptom onset


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4(M) M/35

(J8N)

Fanconi’s

anemia / MDS

(TBI, ATG, F, &

Cyc)

Diarrhea; ANC:

0.48; ALT: 57; Cr:

67

CT: segmental thickening of proximal

jejunal wall.

NA Posa, AmB, Anidulafungin 1

day after symptom onset;

died 44 days after symptom

onset due to complications

of BMT

5(I) M/38

(K20N)

ALL

(Clo)

RLQ pain; ANC:

0.03; ALT: 833;

Cr: 94

CT: bowel wall thickening of the

terminal ileum, cecum and proximal

ascending colon.

Laparotomy: swollen cecum & terminal

ileum; acute gangrenous appendicitis.

Extensive invasion of the terminal ileum,

cecum, appendix and omentum by fungal

elements composed of aseptate hyphae. The

fungi stain with Grocott silver stain and

Periodic acid-Schiff digested stain are

morphologically consistent with Mucorales.

Posa, AmB, Caspo 3 days

after symptom onset; died 28

days after symptom onset

6(C) M/38

(J8N)

AML (B & Cyc) Asymptomatic;

ANC: 6.54; ALT:

37; Cr: 68

CT: no abnormal bowel dilatation or

bowel wall thickening.

NA Posa 5 days after isolation of

Rhizopus sp. in stool; stable

7(C)

M/50

(J8N)

Precursor T-ALL

(TBI + Cyc)

Asymptomatic;

ANC: 2.73; ALT:

60 Cr: 67



NA

Posa 2 days after isolation of


8(I)! F/42

(K20N)

NK cell

lymphoma (A)

Abdominal

distention; ANC:

3.72; ALT 12; Cr

79

CT: moderate amount of ascites,

thickened peritoneal surface with

omental cake suggestive of

lymphomatous infiltration.

Laparotomy: thinning of cecal wall

Extensive involvement of the small intestine,

stomach, urinary bladder, omentum,

mesentery, large intestine, kidney, lung, liver,

spleen, and pancreas by branching aseptate

fungal elements

Voriconazole; died 23 days

after symptom onset;

Rhizopus sp. isolated in

peritoneal fluid 1 day after

patient succumbed


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without perforation; inflamed omentum

with adhesion to pelvic cavity.

9(C)

M/73

(K20N)

DLBCL (Rit,

Cyc, Epi, V, P)

Asymptomatic



NA No antifungal agent given;

stable

10(C)

M/66

(J8N)

Mantle cell

lymphoma (Cyc,

Car, E)

Asymptomatic

Not done



11(C) F/55

(J8N)

B-cell

lymphoma

(Cyc, Car, E)

Asymptomatic

Not done



12(M) F/59

(K20N)

AML (Cyt, D) Diarrhea

CT: mural thickening at cecum &

terminal ileum.

NA Posa, AmB, Caspo 1 day

after symptom onset; stable

Note. A, asparaginase; AmB, amBisome; ALL, acute lymphoblastic leukemia; ALT, alanine aminotransferase (U/L); ANC, absolute neutrophil count (109/L); ATG,

anti-thymocyte globulin; B, busulphan; BMT, bone marrow transplant; C, colonization case; Car, Carmustine; Caspo, caspofungin; Clo, clofarabine; Cr, creatinine (µmol/L);

CT, computer tomography; Cyc, cyclophosphamide; Cyt, cytarabine; D, daunorubicin; Des, Deferasirox; Dexa, dexamethasone; DLBCL, diffuse large B cell lymphoma; E,

etoposide; Epi, epirubicin; F, fludarabine; I, invasive case; Ida, idarubicin; M, mucosal case; Met, methotrexate; MDS, myelodysplastic syndrome; NA, not applicable; NK,

natural killer; Leu, leunase; L, left; P, prednisolone; Posa, posaconazole, R, right; Rit, rituximab; RLQ, right lower quadrant; T, thioguanine; TBI, total body irradiation; V,

vincristine; !Retrospectively identified.


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Table 2. Characteristics of patients with or without intestinal zygomycosis

Case (n=9) Control (n=45) P value

Age (median, range) 50 (11–73) 46 (19–70) 0.552

Sex (male) 7 25 0.283

Underlying disease

Acute leukemia

Lymphoma

Myeloma

Myelodysplastic syndrome

Severe aplastic anemia

5

3

0

1

0

23

13

6

1

2

0.512

Bone marrow transplantation

4 9 0.195

Hematological parameters on admission

Absolute neutrophil count (median, range) x109/L 2.3 (0.3–29.0) 2.1 (0.0–8.7) 0.710

Hemoglobin (median, range) g/dL 11.1 (4.6–13.5) 10.7 (6.2–14.6) 0.642

Platelet count (median, range) x109/L

145 (7–267) 157 (7–443) 0.410

Biochemical parameters on admission

Urea (median, range) mmol/L 4.8 (2.9–11.5) 4.9 (2.0–13.5) 0.816

Creatinine (median, range) µmol/L 67 (50–106) 67 (43–140) 0.710

Random glucose (median, range) mmol/L 5.6 (4.2–11.6) 5.9 (4.4–8.9) 0.794

Albumin (median, range) g/L 41 (26–45) 40 (20–48) 0.584

Globulin (median, range) g/L 31 (20–41) 31 (23–63) 0.592

Total bilirubin (median, range) µmol/L 9.0 (3.0–13.0) 7.5 (2.0–42.0) 0.934

Alanine transaminase (median, range) U/L 32 (8–103) 26 (7–66) 0.508

Aspartate transaminase (median, range) U/L

Lactate dehydrogenase (median, range) U/L

39 (15–90)

228.5 (139.0–4300.0)

25 (16–58)

195.0 (97.0–1199.0)

0.068

0.563

Epidemiological exposure

Intake of allopurinol in hospital (yes/no)

Intake of specific ready-to-eat food (yes/no)

Use of wooden tongue depressor (yes/no)

Use of wooden chopstick (yes/no)

9 / 0

7 / 2

0

0

9 / 36

9 / 36

0

0

<0.001

<0.001

NA

NA

NA, not applicable


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Table 3. Screening of food and drug samples for Rhizopus microsporus group

Item

(total no. of

sample)

Description of sample (total no. and site of collection)

Fresh fruits (2) Apple (1 from kitchen)

Orange (1 from kitchen)

Ready-to-eat

food (179)

Additives (14 packs from kitchen),

Biscuits (70 packs from kitchen and 12 packs from convenience store)*

Cakes (5 from kitchen and 12 from convenience store)

Cereals (3 packs from kitchen)

Macaroni (1 pack from kitchen)

Raisins (1 pack from kitchen)

Sandwiches (3 from kitchen and 38 from convenience store)*

Juice (2 bottles from kitchen and 1bottle from convenience store)

Milk (15 bottles from kitchen and 2 bottles from convenience store )

Drugs (150) Acyclovir tablets (3 vials from wards, 14 vials from pharmacy)

Allopurinol tablets (5 vials from wards, 11 vials from pharmacy)*

Bactidol mouthwash (11 bottles from wards)

Chinese herbal medicine (6 bottles from patients)

Ciprofloxacin tablets (5 vials from pharmacy)

Cotrimoxazole tablets (4 vials from wards, 6 vials from pharmacy)

Dexamethasone tablets (6 vials from pharmacy)

Esomeprazole (3 vials from wards, 21 vials from pharmacy)

Famotidine tablets (1 vial from ward, 8 vials from pharmacy)

Gastrocaine (1 vial from ward)

Itraconazole tablets (1 vial from ward, 5 vials from pharmacy)

Itraconazole syrup (1 vial from ward, 1 vial from pharmacy)

Lactulose (1 vial from ward)

Paracetamol (3 vials from wards, 11 vials from pharmacy)

Phenytoin (2 vials from wards, 11 vials from pharmacy)

Potassium syrup (2 vials from wards)

Prednisolone (2 vials from wards, 5 vials from pharmacy)

* Growth of Rhizopus species


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Rhizopus stolonifer [AB097373]

Rhizopus japonicus [AB097347]

Rhizopus caespitosus [AF115730]

Rhizopus caespitosus [DQ641325]

P5

D5

P1

P4

Rhizopus homothallicus [AF115728]

Rhizopus microsporus var. rhizopodiformis [DQ641306]

Rhizopus oryzae [EF136360]

Rhizopus azygosporus [DQ119008]

R1

P6

P2

P3

D1-3

F5

F3

D3-2

D2

P7

D1-2

D1-1

971

787

1000

1000

1000

1000

P9

F4-1

F4-2

P8

D4-2

F2-2

F2-1

P10-1

0.02


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outbreak of intestinal infection due to rhizopus...

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