Download A Case of Cutaneous Penicilliosis in a Child with Acute Myeloid

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Title Page
Title: A Case of Cutaneous Penicilliosis in a Child with
Acute Myeloid Leukaemia
Running Title: Paediatric Case of Penicilliosis
Sandhya G. Krishnan1, Nancy Wen Sim Tee2, Ai Ling Tan3, Ah Moy Tan4, Mark Jean Aan Koh5, Chia Yin
Chong6, 7,8, Koh Cheng Thoon6, 7,8, Natalie Woon Hui Tan6, 7,8
Department of Paediatrics, KK Women’s and Children’s Hospital, Singapore, 100 Bukit Timah Road,
Singapore 229889, email: [email protected], Phone number: +65 91390574
2
Department of Pathology and Laboratory Medicine, KK Women’s and Children’s Hospital, Singapore,
100 Bukit Timah Road, Singapore 229889
3
Department of Pathology, Singapore General Hospital, Singapore, Outram Road, Singapore 169608
4
Haematology and Oncology Service, Department of Paediatric Subspecialties, KK Women’s and
Children’s Hospital, Singapore, 100 Bukit Timah Road, Singapore 229889
5
Dermatology Service, KK Women’s and Children’s Hospital, Singapore, 100 Bukit Timah Road,
Singapore 229889
6
Infectious Disease Service, Department of Paediatrics, KK Women’s and Children’s Hospital, Singapore,
100 Bukit Timah Road, Singapore 229889
7
Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857
8
Yong Loo Lin School of Medicine, National University of Singapore, 1E Kent Ridge Road, National
University Health System Building (NUH), Singapore 119228
1
Corresponding author:
Sandhya G Krishnan
Department of Emergency Medicine, K K Women’s and Children’s Hospital
100 Bukit Timah Road, Singapore 229899
Telephone Number: +65 91390574
Fax Number: +65 6394 1172
Email address: [email protected]
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Disease/Indication: Penicilliosis
Pathology/Symptoms: Penicillium citrinum, cutaneous fungal infection, galactomannan
Treatment: caspofungin, itraconazole
ABSTRACT
Introduction: We present a case of cutaneous Penicilliosis in a paediatric patient with Acute myeloid
leukaemia (AML).
Case report: A 2-year-old boy with AML first developed probable pulmonary Aspergillosis during induction
chemotherapy in an overseas center in May 2013, and he was treated with ambisome and voriconazole. When
he was admitted to our centre with relapsed AML in October 2013, he was given a 5th course of
chemotherapy, and treated with ambisome for probable pulmonary Aspergillosis in view of pulmonary
nodular opacities on CT. He thereafter developed an erythematous skin lesion with central eschar on his right
hand and left calf. Serum and bronchoalveolar lavage galactomannan antigen (GM Ag) indices increased to
>10 Ag Index. Ambisome was changed to voriconazole, and caspofungin was added for 10 days. The left calf
skin biopsy showed abundant fungal hyphae with septations. Skin culture grew Penicillium citrinum –with
Minimum Inhibitory Concentration in µg/mL as follows: caspofungin 0.016, itraconazole 0.5, amphotericin
1.5, voriconazole > 256. Caspofungin and itraconazole were commenced, and voriconazole discontinued. The
skin lesions and serial GM Ag indices improved. The patient later developed increasing GM Ag indices to >10
Ag Index which was attributed to Aspergillus flavus left pulmonary mycetoma, which was surgically resected.
He eventually succumbed to relapsed AML after bone marrow transplant.
Conclusion: To the best of our knowledge, we report probably the first paediatric case of Penicillium citrinum
infection. Rising GM Ag indices were attributed to cross-reactivity of Penicillium spp. with GM Ag enzyme
immunoassays.
(246 words)
INTRODUCTION
With the increasing prevalence of primary and secondary immunodeficiency, e.g. AIDS, and the increasing
use of immunosuppressive therapy, the incidence of opportunistic infections has been on the rise. Infections,
particularly that of Penicillium marneffei, have been reported in multiple centres in South East Asia. However,
Penicillium citrinum, which is mainly isolated from the environment, has only rarely been reported to cause
infection in humans. To the best of our knowledge, we report here probably the first paediatric case of
Penicillium citrinum infection and we looked into the diagnosis and treatment of the infection in this patient.
(96 words)
CASE REPORT
A 2-year-old boy was diagnosed with acute myeloid leukaemia (AML – M5 subtype) at another overseas
centre in May 2013. He received 4 chemotherapy cycles as per the Children Oncology Group Australia Acute
Myeloid Leukaemia Protocol (COGAAML 531) consisting of cytarabine, daunorubicin, etoposide and
mitoxantrone. During his induction chemotherapy, his CT thorax showed a nodule containing an air fluid
level in the anterior right upper lobe and peripheral peribronchial changes, highly suggestive of a fungal
infection. A broncho-alveolar lavage (BAL) was processed and was positive for Galactomannan antigen (the
exact levels were not available to us) although the fungal cultures of the BAL fluid were negative. He was
thus diagnosed with probable pulmonary Aspergillosis and was initially treated with amphotericin B
liposome (AmBisome) and voriconazole followed by voriconazole monotherapy (uncertain dosage) for 3
months. At the end of the chemotherapy, he was in complete remission. CT Thorax also showed
improvement. He was discharged on prophylactic oral co-trimoxazole.
He was subsequently readmitted to our centre in October 2013 for relapsed AML and was given a fifth
chemotherapy course. CT Thorax revealed two pulmonary nodular opacities in the right middle and lower
lobes. He was then commenced on AmBisome (3mg/kg/day).
Subsequently, he developed two tender, erythematous skin nodules with central eschar on his left calf and
right hand in November 2013, with no concomitant fever. Within 2 days, the nodules developed central
necrosis and his serum and BAL Galactomannan antigen simultaneously increased to >10 Antigen index.
AmBisome was then changed to voriconazole (18 mg/kg/ day) and caspofungin (50 mg m2/day) was added
for 10 days. An excisional biopsy of the skin lesion on the left calf was performed, and histology showed
abundant fungal hyphae with septations which were highlighted on Periodic Acid Schiff (PAS) and GomoriMethenamine silver (GMS) stains. There was only mild surrounding chronic inflammation (Fig. 1a). The
tissue was cultured on Sabouraud Dextrose agar (SDA) and in Brain Heart Infusion Broth (BHIB) and
incubated at 30 degrees Celsius. Both the SDA and BHIB grew a mould identified to be Penicillium species
on microscopy. Polymerase-Chain Reaction (PCR) and Internal Transcribed Spacer (ITS) sequencing were
done to identify the species with a result of Penicillium citrinum with a score of 99% similarity using Basic
Local Alignment Search Tool (BLAST) analysis. Antifungal susceptibility was done using the E test on
RPMI media [RPMI –1640 medium with MOPS solution and 2% glucose (Sigma-Aldrich, USA)]. The
Minimum Inhibitory Concentrations (MIC) in g/mL were 0.016 for caspofungin, 0.5 for itraconazole, 1.5
for amphotericin, >256 for voriconazole. At this juncture, CT Thorax also showed a left upper lobe nodule
with peripheral cavitation suggestive of mycetoma and right pleural thickening with a small pleural effusion.
Voriconazole was promptly discontinued; caspofungin (50 mg m2/day) and itraconazole (5 mg/kg/day) were
commenced. With the new antifungal regime, the skin lesion on the right hand improved. Serum
Galactomannan antigen indices, which were regularly trended, also decreased to 6.96 Antigen index.
(487 words)
OUTCOME AND FOLLOW-UP
In February 2014, the patient’s serum Galactomannan antigen index was noted to again rise to >10 Antigen
index. Subsequent CT Thorax showed a left upper lobe nodule with internal air fluid level and a stable rightsided effusion. He then underwent resection of the left lung mycetoma, which grew Aspergillus flavus, with
MIC in g/mL of 0.006 for caspofungin, 1 for itraconazole, 2 for voriconazole and 4 for amphotericin. After
surgical resection of the mycetoma, the serum Galactomannan antigen index decreased to 0.91 Antigen
index.
The patient was subsequently referred to another tertiary centre for bone marrow transplant, which he
underwent at the end of 2014. Unfortunately, his AML relapsed and he passed away in early 2015.
(116 words)
DISCUSSION
Penicillium species are endemic in the environment, particularly in the South East Asian region. Of these,
species, Penicillium marneffei has been the most commonly reported pathogen, especially amongst
immunocompromised individuals. On the other hand, Penicillium citrinum has only been reported in a
handful of cases – causing urinary tract infection (Guze et al., 1958), mycotic keratitis (Gugnani et al., 1978),
bronchopulmonary infection (Mori et al., 1987), pneumonia with pericarditis in a patient with acute
leukaemia (Mok et al., 1997) and chronic sinusitis (Twaruzek et al., 2014). It was noteworthy that all the
studies thus far had been reported only on adults and the cases of Penicillium citrinum causing
bronchopulmonary infection, pneumonia with pericarditis and urinary tract infection were identified mainly
on autopsy. There have been no previous reports of Penicillium citrinum infections in children. We believe
this is probably the first reported paediatric case of Penicillium citrinium infection to the best of our
knowledge.
In this study, we found that the temporal trend of Galactomannan Antigen index (GM Ag) levels can be used
to monitor disease activity of both Penicillium and Aspergillus infections. Studies have shown that the
monoclonal antibody present in the commercial sandwich ELISA cross-reacts with the heteropolysaccharide
galactomannan complex antigen, which is present in the cell walls of Aspergillus, Penicillium and
Cryptococcus species (Huang et al., 2007). One study reported the GM Ag indices (Optical Density) to be
significantly higher in Penicilliosis (with or without fungaemia) than in Cryptococcosis (Huang et al., 2007).
However there are no studies that compared GM Ag indices of Penicillium infections to Aspergillus
infections. In our case study, the rising galactomannan level indicated increasing fungal activity and the
patient likely had both invasive Penicillium and Aspergillus infections. The GM Ag index rose when he
developed the cutaneous Penicillium infection and it decreased appropriately after the excision of the lesion
and after starting appropriate anti-fungal drugs. The Penicillium was obtained from an excisional biopsy and
corresponding histological findings make it less likely a contaminant. Subsequently, the GM Ag index level
rose again and correlated with radiological development of a left lung mycetoma due to Aspergillus flavus. It
was therefore difficult to attribute the rising GM Ag levels preferentially to either the Penicillium activity or
the concomitant Aspergillus activity.
Our study was a case report of paediatric Penicilliosis. More studies with a substantial sample size are needed
to evaluate further the role of GM Antigen assay in monitoring the disease activity of Penicilliosis. Patients
suffering from Penicilliosis often present with non-specific symptoms and definitive diagnosis is often by
identification or isolation of Penicillium sp. in tissue biopsies. However, the invasive nature and cultural
methods of these investigations often delay diagnosis, which may be some of the reasons why Penicillium
citrinum was detected only on autopsy in the cases of urinary tract infection (Guze et al., 1958) and
bronchopulmonary Penicilliosis (Mori et al., 1987). Early serodiagnosis of Penicilliosis would guide rapid
treatment and reduce mortality associated with disseminated penicilliosis. There have so far been no specific
serodiagnostic tests available for Penicillium citrinum but several tests have been investigated thoroughly for
Penicillium marneffei. For instance, specific monoclonal antibody-based ELISA tests have been developed
with Penicillium marneffei cell wall antigens in serum and urine (Cao et al., 1999; Panichakul et al., 2002) to
aid the serodiagnosis of Penicilliosis. However these tests are not available commercially. Clinical trials
should be conducted to study further the applicability of these investigations.
Thus far, there have been no reports on effective antifungals against Penicillium citrinum, possibly because
the microbe is often identified only on post mortem. There have however been studies demonstrating that
other Penicillium sp, especially Penicillium marneffei, can be treated with parenteral amphotericin B and oral
itraconazole (Sirisanthana et al., 1998). In addition, voriconazole, which is an extended spectrum triazole,
has also been reported to be an effective therapeutic option for systemic Penicillium marneffei infections in
AIDS patients (Supparatpinyo et al., 2007). In our case, the empirical use of AmBisome and voriconazole
were ineffective against Penicillium citrinum. Instead, the use of caspofungin and itraconazole guided by the
MIC were effective in treating the infection. Therefore, early serodiagnosis, pathological diagnosis and
appropriate antifungal susceptibility testing are essential for appropriate treatment in order to improve
clinical outcomes.
In summary, to the best of our knowledge, we report probably the first paediatric case of Penicillium citrinum
infection in a child with acute myeloid leukaemia. A high GM Ag index, though highly suggestive of
Aspergillus infection, can also be contributed by Penicillium infection, which could be a concomitant
pathogen especially in immunocompromised individuals. More studies are needed to elucidate the role of
GM Ag in Penicilliosis. Specific tests for serodiagnosis of Penicillium infections may help early diagnosis,
and thereby improve clinical outcomes in the future.
(791 words)
REFERENCES
Cao, L., Chan, K.M., Chen, D.L., Vanittanakom, N., Lee, C., Chan, C.M., Sirisanthana, T., Tsang,
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marneffei and in sera of penicilliosis patients. J.Clin. Microbiol, 37: 981-986.
Gugnani, H.C., Gupta, S., Talwar, R.S. (1978). Role of opportunistic fungi in ocular infections in
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Sirisanthana, T., Supparatpinyo, K., Perriens, J., Nelson, K.E. (1998). Amphotericin B and
itraconazole for treatment of disseminated Penicillium marneffei infection in human immunodeficiency
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Supparatpinyo, K., Schlamm, H.T. (2007). Voriconazole as therapy for systemic Penicillium marneffei
infections in AIDS patients. Am. J. Trop. Med. Hyg. 77: 350-353
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Figure/Table Captions
Fig. 1(a). GMS stain of patient’s skin biopsy showing fungal hyphae with septations.
Abbreviations ( non standard)
1. Acute Myeloid Leukaemia (AML)
2. Broncho-Alveolar Lavage (BAL)
3. Galactomannan Antigen index (GM Ag)
Ethical Statement
This is a retrospective review of a case of Penicilliosis and no experimental procedure was performed on the
patient involved.
Acknowledgements/Declaration of Interest
None of the authors have any conflict of interest to report.
Signature: Sandhya Krishnan (on behalf of all authors)
Date: 05/07/2015
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