Journal of Applied Microbiology ISSN 1364-5072
REVIEW ARTICLE
A review of melanized (black) fungal contamination in pharmaceutical products—incidence, drug recall and control measures* R. Vijayakumar1, M. Saleh Al-Aboody1 and T. Sandle2 1 Department of Medical Laboratory, College of Science AlZulfi, Majmaah University, AlZulfi, Saudi Arabia 2 Microbiology, Bio Products Laboratory, Elstree, Hertfordshire, UK
Keywords biocides, fungi, moulds, mycology, pharmaceuticals. Correspondence Tim Sandle, Microbiology, Bio Products Laboratory, Dagger Lane, Elstree, Hertfordshire, WD6 3BX, UK. E-mail:
[email protected] * In the Virulence factors of black fungi section there have been some changes since first publication. 2015/0758: received 17 April 2015, revised 22 June 2015 and accepted 22 June 2015 doi:10.1111/jam.12888
Summary The aim of this study was to describe the incidence of contamination of pharmaceutical products by melanized fungi and to consider control measures in relation to bioburden and cleanrooms. This study reviews and analyses pharmaceutical product recalls and offers incidence rates of fungal detection from a typical cleanrooms. The recalls include some serious cases which resulted in the loss of life. Of different types of fungal contamination incidences some of the most damaging have been due to melanized fungi (‘black mould’), such as Exserohilum rostratum. The focus of the article is with melanized fungi. The study concludes that, from the review of recent pharmaceutical product recalls, fungal contamination is either increasingly common within cleanroom environments or the accuracy of sampling and the level of reporting has risen. The prevalence of melanized fungi in pharmaceutical facilities rests on specific virulence factors particular to these types of fungi, which are outlined. The article identifies a gap in the way that such fungi are screened for using available cultural methods. The article provides some control strategies, including assessing the suitability of disinfectants and biocides, for reducing the risk of melanized fungal incidences within the pharmaceutical facility. Understanding the fungal risk to pharmaceutical products remains a poorly understood and often overlooked aspect of pharmaceutical microbiology. This article helps to identify this risk and offer some guidance to those involved with pharmaceutical products manufacture in relation to bio-contamination control strategies.
Introduction Fungi (moulds and yeasts) are an important group of micro-organisms. Fungi are responsible for various infections especially with the immunocompromised host. In addition to their medical importance, these organisms are associated with contamination of surfaces and spoilage of pharmaceutical, cosmetic and food products. Fungal contamination of pharmaceutical products can cause not only serious economic losses to the manufacturer but can also lead to serious health problems to customers (patients) (Pitt and Hocking 1997; Dupont 2002). Over the years, several mould issues associated with pharmaceutical cleanrooms, cold rooms and controlled Journal of Applied Microbiology © 2015 The Society for Applied Microbiology
areas have been reported. For example, several vaccine and pharmaceutical companies in Europe have experienced an increase in mould contamination due to an increase in ambient temperatures and issues with items brought into cleanrooms (Lopolito et al. 2007; Sandle 2011; Vijayakumar et al. 2012). A review of fungal contamination of pharmaceutical products reported by various authors, together with recall data relating to more than 100 pharmaceutical products collated by the Federal Drug Administration (FDA) for the years between 2000 and 2010, shows that contamination by mould and yeast was found in 21% of samples (Jimenez 2007; Vijayakumar et al. 2012; Smith et al. 2013). More recently in the year of 2012, the most serious event ever— 1
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due to fungal contamination of sterile pharmaceuticals— occurred. This was with methylprednisolone acetate steroid injections manufactured by the New England Compounding Center (NECC, Boston, MA, USA) and the incident led to 64 patients dying from fungal meningitis. This was through receiving contaminated product by being administered the drug as a treatment for arthritis via the spinal cord. On December 2014, 14 former NECC executives and technicians, including co-founder and president, were indicted on a host of federal charges related to this outbreak. This outbreak was the result of contamination of the product by Exserohilum rostratum. This organism is a member of the melanized fungi (dematiaceous fungi) group, which constitutively produce melanin pigment during infection. This group of fungi are extremely difficult to treat with antifungal drugs and they are far harder infections to eliminate compared with hyaline fungi (Brandt and Warnock 2003; Smith et al. 2013). The majority of the fungal contamination product recalls are due to contamination by hyaline fungi such as Aspergillus and Fusarium (Chang et al. 2006; Jimenez 2007; Vijayakumar et al. 2012; Ahearn and Stulting 2014). When compared to the hyaline fungi, melanized fungi are very difficult to treat and they are resistant to common antifungal drugs. For this reason, it is important for cleanroom personnel to know about black fungi incidence and appropriate control measures. This task is made more difficult because there are no major studies available about different melanized fungi and their incidence within pharmaceutical cleanrooms and controlled environments. Hence, in this review, we analyse recent drug recalls due to melanized (black) fungi and consider their incidence in cleanroom environments. To add to this, we also assess virulence factors and appropriate control measures. What are ‘Melanized fungi’? The terms used to describe these types of fungi have evolved over the past decades. As Sporothrix schenckii was one of the earliest melanized fungi described, ‘sporotrichoid’ was often used to describe similar fungi. ‘Phaeoid’, ‘phaeo-sporotrichose’ and ‘dematiaceous’ have also been mentioned in the literature (Pappagianis and Ajello 1994). ‘Phaeo’ comes from the Greek meaning ‘dark’ and has been commonly used, particularly when describing infections due to these fungi as ‘phaeohyphomycosis’, i.e. infection caused by dark-walled fungi, as suggested by Ajello et al. (1974), Rinaldi (1996). It has been suggested that the term ‘dematiaceous’ is not appropriate given its etymologic derivation from the Greek ‘deme’, meaning bundle, although it has become fairly entrenched in medical mycological literature and the term will likely continue to persist in nomenclature (Pappagianis and Ajello 1994). The 2
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term ‘melanized’ has become utilized more recently, given its specific meaning. Hence, in this review, the terms ‘dematiaceous’, ‘melanized’ and ‘dark’ are used interchangeably to denote the fungal elements containing melanin. Melanized or dematiaceous fungi, as defined above, are frequently considered ubiquitous saprobes inhabiting living and dead plant material and, for the most part, residing in the soil (De Hoog et al. 2000). The ability of some species in this genus, such as Exophiala xenobiotica, to grow in high concentrations of xenobiotics (a foreign chemical substance found within an organism) such as xylene, toluene or creosote-treated utility poles, as well as to cause human disease, is noteworthy (Prenafeta-Boldu et al. 2006). More than a hundred species and sixty genera of these melanized fungi are connected to a broad spectrum of human infections (Revankar 2007). The main clinical forms of infections as a result of these dematiaceous fungi include phaeohyphomycoses (cutaneous, subcutaneous and systemic), that affects both immunocompetent and immunocompromised individuals; chromoblastomycosis, mycetoma, sinusitis and bloodstream infections. Among these, chromoblastomycosis and mycetoma are considered occupational diseases due to the tendency for some rural labourers working in tropical and subtropical climates to contract associated fungal infections. Melanized fungi in cleanroom environments Moulds are ubiquitous in nature and therefore pose a risk to pharmaceutical manufacturing operations. Wet or damp areas are a further factor which can encourage fungal growth in cleanrooms (Lopolito et al. 2007). Such moulds can be detected by effective environmental monitoring regimes, deploying a range of air and surface monitoring techniques using appropriate microbiological culture media and appropriate incubation conditions (Sandle 2014). There are very few articles available assessing mould contamination incidences from pharmaceutical cleanroom environments (Lopolito et al. 2007; Sandle 2011; Vijayakumar et al. 2012). Sandle (2011), in a cleanroom survey, observed 1% of fungi, from all detected microbial contamination events, in EU GMP Grade C/ ISO 14644 class 7 and 8% in EU GMP Grade D environments in the United Kingdom. In addition, various researchers have reported that Aspergillus spp., Penicillium spp., Trichophyton spp. and other moulds have caused significant microbial contamination issues in production environments and to manufactured products. For example, Lopolito et al. (2007) reported that the most common fungi in cleanrooms are species of Penicillium, Cladosporium and Aspergillus. They also noted that these fungi come from many sources within the cleanroom, Journal of Applied Microbiology © 2015 The Society for Applied Microbiology
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including door kick plates, bags, boxes, markers, intervention equipment, cart wheel, ceiling tiles, poorly maintained flooring and in some cases, high-pressure impingement application devices for applying germicides. From these data, it can be presumed that a wide variety of fungal isolates are present in the cleanroom environment. The most commonly occurring fungal isolates and their sources are listed in Table 1 (Sandle and Vijayakumar 2014). In the absence of reports that discuss the incidence of melanized fungi in cleanroom environment, we sampled and analysed air samples collected within pharmaceutical cleanroom environments and obtained a list of melanized fungal isolates (Table 2). The cleanroom examined was located in India. The cleanrooms were EU GMP Grade C and D/ISO 14644 class 7 and 8. Samples were taken using a MAS-100 NT volumetric impaction air sampler. A total of 36 samples were taken, with 1000 l of air drawn for each sample, using agar plates filled with tryptone soya agar (TSA). Plates were incubated at 20–25°C for 5–7 days. From this, 185 different fungal isolates were recovered. It is recognized that the culture medium, incubation times, and incubation temperatures can affect the recovery and growth of fungi (Sandle 2014). The results are designed to be illustrative of an industry standard cleanroom in terms of the types of fungi that can be recovered from such environments; in relation to other facilities the results will differ due to geographical and industrial factors. The overall incidence of melanized fungi in pharmaceutical environment observed, in this data set, was 49%. While it is recognized that pharmaceutical cleanrooms will differ, it is of interest that among the isolates, Cladosporium and Curvularia were observed predominantly. It can be concluded from our literature review and direct observations that melanized fungi will be found within many cleanrooms. Table 1 Predominant fungal contaminants and possible the sources within the pharmaceutical environment Air/surface
Personnel
Walls/floors
Water
Cladosporium spp. Penicillium spp. Aspergillus spp. Alternaria spp. Curvularia spp. Fusarium spp.
Pityrosporum ovale P. orbiculare Trichophyton* Epidermophyton*, Microsporon*
Cladosporium spp. Penicillium spp. Aspergillus spp.
Cladosporium spp. Penicillium spp. Aspergillus spp.
*Dermatophytes present if people infected with skin diseases.
Journal of Applied Microbiology © 2015 The Society for Applied Microbiology
Table 2 Incidence of hyaline and dematiaceous fungi isolated from the air samples in pharmaceutical cleanroom environment Fungal isolates (n = 185)
% of occurrence
Dematiaceous fungi Cladosporium spp. Curvularia spp. Alternaria spp. Exserohilum spp. Bipolaris spp. Hyaline fungi (Penicillium, Aspergillus, Rhizopus etc.)
2757 1027 595 324 162 5140
However, it stands that some organizations are not identifying the fungal isolates due to a lack of experience in mycology techniques together with inadequate facilities for conducting identifications. A further reason may also relate to the type of culture media used to collect environmental samples or due to the incubation regime selected for environmental monitoring plates, both of which may not be optimal (Gebala and Sandle 2013). More scientific studies are required about exact incidence of fungal species in cleanroom facilities and understanding those that are detected can be enhanced by the use of molecular based identification techniques. There are two major sequencing targets for fungal identification. These are the D1/D2 region of the large ribosome subunit (LSU) and the internal transcribed spacer regions (ITS1/ITS2) (Nelsson et al. 2011). These methods are very expensive and need technically skilled personnel and thus they cannot be performed in basic level laboratories. These issues are beyond the scope of this review and are not discussed further. Melanized fungi associated with contamination and drug recall As per available literature, hyaline fungi are the predominant type of contamination reported from pharmaceutical products recalled due to fungal contamination. Melanized fungi are involved very rarely in recalls of formulated pharmaceutical products. However, recently melanized fungi associated with drug recalls and rare disease outbreaks have been observed in various countries where pharmaceutical products are repacked by compounding pharmacies. Compounding pharmacies Pharmacy compounding is the process of combining drug ingredients to prepare medications that are not commercially available or to alter commercially available medications to meet specific patient needs, such as with dye-free or liquid formulations (Anon. 2015). The practice of compounding has been reported to be increasing with an estimated 43 000 compounded medications prepared daily 3
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in the United States (Smith 2002). Pharmacists traditionally have prepared medications to fulfil individual prescription requests or manipulated reasonable quantities of human drugs on receipt of a valid prescription for an individually identified patient from a licensed practitioner. This practice has latterly become more commercial. The past decade has seen a trajectory, with the USA, away from small-scale hospital compounding to compounding on a larger, commercial and independent basis. Some compounding is legal under state laws, and when appropriate, FDA can exercise its enforcement discretion regarding new drugs and certain other requirements of the Federal Food, Drug, and Cosmetic Act (http://www.fda.gov/ora/compliance_ ref/cpg/cpgdrg/cpg460-200.html). Contamination concerns with compounding pharmacies The Institute of Safe Medication Practices (ISMP) has published a list of selected pharmacy compounding concerns related to sterile products that have occurred since the 1990s. This list relates to both compounding and hospital pharmacies and it describes 200 adverse events including vision loss, blindness, infection and death from 71 different compounded products (Anon. 2012a). As per the available literatures, melanized fungal contamination events have been extracted and listed in Table 3. Referring back to the NECC case, remarkably, the same drug, methylprednisolone, was involved in meningitis
outbreaks due to a fungal contamination by black fungi Exophiala dermatitidis and Exserohilum in the years of 2002 and 2012. Evidently the compounding of preservative-free corticosteroids requires meticulous sterility assurance to prevent fungal contamination. If sterility control is lacking, the concentrated steroids are a suitable media to support the aggressive growth of pathogenic fungi. Various dematiaceous fungi and issues in pharmaceutical contamination are discussed below. Review of fungal contamination incidences Exophiala in 2002 The black yeast E. dermatitidis has been isolated from plant debris and soil and is a recognized causative agent of mycetoma and phaeohyphomycosis in humans. Clinical manifestations include subcutaneous cystic lesions, endocarditis and brain abscesses. E. dermatitidis is neurotropic and cerebral infections are frequently seen. It is an uncommon aetiological agent, involved with fatal infections of the central nervous system in otherwise healthy, mainly adolescent patients in East Asia (Hiruma et al. 1993; Matsumoto et al. 1993; Chang et al. 2000). In the USA., cases have been reported where inoculation of patients with medical fluids containing contaminated water led to nosocomial common-source outbreaks leading to sometimes fatal neurological implications (Woollons et al. 1996; Engemann et al. 2002).
Table 3 Selected Pharmacy Sterile Compounding outbreaks and pharmaceuticals recall due to dematiaceous fungal contamination Severity of contamination
Year
State
Drug contaminated
Contaminant
2002
North Carolina, South Carolina
Methyl prednisolone (Compounded drug)
Exophiala
5 patients infected – one died
2010
India, USA Nationwide
Cladosporium
No information’s available
2012
New York, USA
Biopolaris hawaiiensis
2012
Nationwide
2013
Nationwide
2014
Nationwide
Intravenous bags of metronidazole, ondansetron and ciprofloxacin Triamcinolone acetonide – Ophthalmic injections (Compounded drug) Methylprednisolone acetate injections (compounded drug) Risperdal Consta (Risperidone) Gel-E Donut pillows and Squishon 2 cushions— devices are used in Neonatal and Paediatric ICU settings
40 patients developed fungal endophthalmitis – 39 patients lost vision 750 patients developed fungal meningitis – 64 died No complications reported No complications reported
4
Exserohilum rostratum Alternaria alternata Cladosporium
Manufacturer details
Reference
Prepared from compounding pharmacy A Claris Life sciences, Ahmedabad, India
Engemann et al. (2002)
Franck’s compounding lab, FL
Anon. (2012c), Mikosz et al. (2014)
New England Compounding Center Johnson & Johnson company Children’s Medical Ventures, Monroeville, PA
Anon. (2012b)
Anon. (2010) Anon. (2013)
Rockoff (2013) http://www.fda.gov/ MedicalDevices/ Safety/ucm410964.htm
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In a pharmaceutical products contamination issue, on September 2002 the North Carolina Division of Public Health (NCDPH) was notified of two cases of meningitis caused by a rare fungus in patients who had received epidural injections at outpatient pain management clinics. The subsequent report describes five cases of fungal infection associated with contaminated drugs prepared at a compounding pharmacy. Cases occurred up to 152 days following an injection. With further investigation, compounding pharmacy A was identified as the source of the methylprednisolone acetate administered to all five patients with Exophiala infections. On-site investigation of compounding pharmacy A by the South Carolina Board of Pharmacy (SCBP) and federal regulators found that improper sterilization processes had occurred. This included no written procedures for sterilizer operation, no testing for sterility or appropriate checking of quality indicators and inadequate cleanroom practices as outlined in the American Society of Health-System Pharmacists (ASHP) guidance for pharmacy-prepared sterile products (Anon. 2000). Microbiological culture at CDC and the Food and Drug Administration (FDA) of unopened vials from three separate lots of injectable methylprednisolone obtained from compounding pharmacy A yielded E. dermatitidis (Matsumoto et al. 1993). The severity of this fungal contamination caused the death from meningitis in one patient, sacroiliitis in another, and meningitis in three other patients. Each patient had received either epidural or intra-articular injections of methylprednisolone compounded at pharmacy A. Exserohilum rostratum outbreak in 2012 One of the most tragic melanized fungal contamination incidents relating to sterile products was the incident stemming from the New England Compounding Center (NECC) in 2012. A series of sterility assurance failures triggered a multistate outbreak of fungal infections among patients who received contaminated preservative-free methylprednisolone acetate steroid injections manufactured by the NECC (Anon. 2012b). The infections identified included fungal meningitis, a form of meningitis that is not contagious; localized spinal or paraspinal infections; and infections associated with injections in a peripheral joint space, such as a knee, shoulder or ankle (Smith et al. 2013). On October 2012, following distribution of the medicine, near 14 000 individuals were potentially exposed to three lots of contaminated steroids resulting in more than 750 infections and 64 deaths (Kainer et al. 2012; Bell et al. 2013; Chiller et al. 2013; Gade et al. 2013; Kerkering et al. 2013; Lockhart et al. 2013). These infections were due to contamination of the product by dematiaceous fungi—E. rostratum. Tests carried out at CDC and FDA Journal of Applied Microbiology © 2015 The Society for Applied Microbiology
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laboratories on the preservative-free MPA vials confirmed the presence of E. rostratum in unopened vials from two of the three recalled lots (Anon. 2012b). These laboratory test results strengthen the link between preservative-free MPA vials and the outbreak. While investigating, CDC laboratories recovered another melanized fungus—Cladosporium cladosporioides—in one unopened vial which was sent from the state of Tennessee. This fungus was also recovered from five patient isolates and from one Cerebrospinal fluid (CSF) sample, each of which was sent to CDC for testing. The significant reason for this incident was due to the sterility assurance failures, subsequently identified by the FDA, together with reported concerns with cleanroom management (including disinfection practices), aseptic filling practices, and the improper use of autoclaves for equipment sterilization. This contaminant genus Exserohilum consists of phaeoid or dematiaceous filamentous fungi characterized by long, multidistoseptate conidia with protruding hilum and the formation of dark colonies (McGinnis et al. 1986). Approximately 35 species belong to the genus Exserohilum. Some are host specific; however, only three, E. rostratum, Exserohilum longirostratum, and Exserohilum mcginnisii, are pathogenic for humans. Recent taxonomic and molecular studies have shown a high homology between these three species, indicating that they could, in fact, be the same species (Lau et al. 2007; da Cunha et al. 2012). Exserohilum species are environmental fungi that are common in grass and soil and less common in marine environments. They are considered to be cosmopolitan pathogens, although they occur mainly in warm tropical and subtropical area (Kainer et al. 2012). Exserohilum infections have been identified in both immunosuppressed and immunocompetent patients and cause a wide range of topical and systemic diseases (Revankar et al. 2002; Adler et al. 2006; Revankar and Sutton 2010). From our study within the pharmaceutical environment, Exserohilum was the fourth commonest fungus present. The incidence of these fungi was observed with 3% of the samples taken (Table 2). However, the incidence rate in practice will vary depending upon the climatic conditions, geographical locations, the nature of the product processed and maintenance of cleanrooms. Bipolaris in 2012 In March 2012, two concurrent outbreaks of fungal endophthalmitis were associated with two environmental moulds contaminating two compounded medications: intraocular dye Brilliant Blue G (BBG) and triamcinolone acetonide (triamcinolone) each labelled as sterile from the same compounding pharmacy (Franck’s Compounding Lab, Ocala, FL, USA). Together, these two outbreaks 5
Melanized fungal contamination in pharmaceuticals
represent the largest reported outbreak of infectious endophthalmitis and one of the largest outbreaks in the United States attributed to contamination of a compounded medication. Fungal endophthalmitis is inflammation of the intraocular cavities. On March 5, 2012, the Healthcare Associated Infections Program of the California Department of Public Health was alerted to a cluster of nine cases of fungal endophthalmitis. During the investigation, cases were defined as laboratory-confirmed or suspected fungal endophthalmitis among patients who had undergone vitrectomy at the Los Angeles County ambulatory surgical centre during October 2011–January 2012. Here, the implicated BBG lot was used for the eye treatment. On further investigation, microbiological testing conducted by CDC of an unopened BBG vial from the same lot yielded the environmental mould Fusarium incarnatum-equiseti species complex. These data collectively turned the investigation focus on Franck’s products. Franck’s records indicated that BBG from the contaminated lot was shipped to 22 facilities in 15 states. On March 9, 2012, Franck’s recalled all lots of BBG (Anon. 2012c, Mikosz et al. 2014). Fusarium species are hyaline fungi; hence this organism is excluded from further discussion in this review. In the meantime, on March 26, 2012, CDC was notified of a patient in whom fungal endophthalmitis had developed during February 2012. This followed an intravitreal injection of triamcinolone at a New York ophthalmology practice. Triamcinolone is a corticosteroid used to treat a variety of ophthalmic conditions. Three additional patients with suspected fungal endophthalmitis were identified at the practice; all four had received intravitreal injections of triamcinolone from a single lot manufactured at Franck’s on November 4, 2011. Preliminary CDC laboratory testing identified Bipolaris hawaiiensis, a rare environmental mould infrequently described as a human pathogen, in ocular specimens from these casepatients. Because Franck’s invoices indicated that triamcinolone from the same lot had been shipped to five ophthalmology practices in four states, the investigation was expanded. On March 31, 2012, Franck’s recalled this lot of triamcinolone. Bipolaris hawaiiensis mould was identified in specimens from triamcinolone-exposed case-patients. Exposure to either product was the only factor associated with the case. Of the 40 case-patients for whom data were available, 39 (98%) lost their vision. Overall, 47 cases were identified and reported in these two outbreaks in nine states of USA, among these 21 cases of fungal endophthalmitis were associated with BBG exposure contaminated with Fusarium mould and 26 cases were associated with Franck’s triamcinolone exposure contaminated with dematiaceous Bipolaris mould (Mikosz et al. 2014). 6
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However, 1 year later in 2013, the same episode reoccurred in California, as reported by Small and his co-workers. Fungal endophthalmitis developed in the eyes of 82% (14 out of 17) patients, following the administration of intravitreal triamcinolone via injection. The triamcinolone was obtained from the same suspect lot. The causative fungus was B. hawaiiensis (Small et al. 2014). Bipolaris is a dematiaceous, filamentous fungus. It is cosmopolitan in nature and is isolated from plant debris and soil. Clinically significant species inciting human disease include B. spicifera, B. hawaiiensis and B. australiensis. It is the most common mycosis attributed to allergic fungal sinusitis (Castelnuovo et al. 2004; Kobayashi et al. 2008; Revankar and Sutton 2010). From our review, in the cleanroom pharmaceutical environment the incidence rate of these fungi are less than 2%. Alternaria in 2013 On September 2013, a Johnson & Johnson company (NJ, USA) recalled 5000 vials of its Risperdal Consta, a longacting drug for patients with schizophrenia, after laboratory testing found the medicine was no longer sterile. Routine analysis showed one lot of the injected drug was contaminated with Alternaria alternata, a common melanized mould found in the cleanroom environment. Fortunately, there were not any reported complications among patients who received the medicine (Rockoff 2013). A. alternata, a cosmopolitan saprophyte commonly found in soil and plants, is usually considered an outdoor allergen (Bush and Prochnau 2004). It is, from our review, the third most predominant dematiaceous fungi in pharmaceutical cleanroom environment. The overall incidence was 6% of cleanroom air samples. The production of melanin-like pigment is one of its major characteristics. Cladosporium in 2010 & 2014 Cladosporium is a dematiaceous mould that is common in the environment. Outdoors, it can be found on plants and other organic matter. Indoors, it is common in the air and on surfaces such as wallpaper or carpet, particularly where moisture is present. In our study of pharmaceutical cleanroom environment, it was the predominant fungal contaminant, found from 28% of air samples. Clinically, Cladosporium is a very rare cause of human illness. Nonetheless, it has been known to cause several different types of infections including skin, eye, sinus and brain infections especially in vulnerable populations such as neonates, critically ill patients and immunocompromised patients. It has also been associated with allergies and asthma. Moreover, the fungus was identified Journal of Applied Microbiology © 2015 The Society for Applied Microbiology
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by CDC and FDA, in clinical specimens, as one of the pathogens associated with the NECC multistate outbreak of fungal meningitis and other fungal infections associated with contaminated steroid injections. Furthermore, Cladosporium has been reported in a few cases of contamination in sterile pharmaceutical products and associated recalls by FDA. On May 29, 2010, the US FDA alerted healthcare professionals not to use certain intravenous (IV) bags of metronidazole, ondansetron and ciprofloxacin because of potential contamination due to Cladosporium species. FDA has received reports of floating matter in IV bags manufactured by Claris Lifesciences Limited (Ahmedabad, India). These potentially affected products were sold under the Claris, Sagent Pharmaceuticals, Pfizer and West-Ward Pharmaceuticals labels (Anon. 2010; Anon 2013). Laboratory analysis of the foreign matter showed that the products were contaminated with dematiaceous mould (Cladosporium). On the basis of the findings, Pfizer halted distribution for all sterile-injectables in Claris Manufactured IV bags while the FDA monitored the recall. Of the 17 million Claris IV bags affected, only about 20% were distributed before the foreign matter was discovered. No information has been released to reveal if all of the contaminated bags were recovered or if any were administered to patients. Fortunately, to date, no complications have been reported because of this contaminated medication (Anon. 2013). Later, on May 2014, FDA and Children’s Medical Ventures, Monroeville, PA notified health professionals and their health delivery organizations of a class 1 recall of this product due to complaints about visible mould on the outer surface of Gel-E Donut and Squishon 2 gelfilled products. These products are pillows and cushion supportive medical devices used for neonatal and paediatric intensive care unit settings. The contaminants isolated from the product were Cladosporium and Penicillium. These are commonly found moulds and can cause difficulty in breathing or allergic reactions. The use of such contaminated product may cause serious adverse health consequences, including death. Further analysed reports not yet available (http://www.fda.gov/MedicalDe vices/Safety/ucm410964.htm). Virulence factors of black fungi Based on the above reviews, melanized fungi are ubiquitous and present in cleanroom environment and such fungi can cause serious contamination should they be transferred into products. In terms of survival in cleanrooms, melanized fungi are typically more resistant than hyaline fungi. Cleanroom quality personnel and microbiologists should understand the incidence rate and have Journal of Applied Microbiology © 2015 The Society for Applied Microbiology
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knowledge of appropriate control measures in relation to these types of fungi. The virulence factors of melanized fungi are discussed below. One of the likely virulence factors is the presence of melanin in the dematiaceous fungal cell wall (Paolo et al. 2006; Santos et al. 2007). Melanin is believed to contribute to microbial virulence by reducing a pathogen’s susceptibility to being killed by host antimicrobial mechanisms and by influencing the host immune response to infection. These fungi constitutively produce melanin pigment during infection and are extremely difficult to treat with antifungal drugs (Brandt and Warnock 2003). Melanin is very resistant to a variety of physicochemical agents, including free radical compounds, toxic metals, desiccation, and even ionizing radiation (Haselwandter and Ebner 1994; Fogarty and Tobin 1996; Dadachova and Casadevall 2008). Hence, it has been called an ‘an antifungal resistance factor’, given its ability to reduce the susceptibilities of melanized cells to antifungal drugs (Ikeda et al. 2003). Notably, there is no evidence for the involvement of melanin in drug efflux pumps or in alterations in the synthesis of ergosterols or b-glucans in fungal cell wall/cell membrane structures. In agreement with and paraphrasing Ahearn and Stulting (Ahearn and Stulting, 2014), it is noted that various reports have indicate that in compost and hot springs, melanized fungi can persist in metabolically active states at temperatures in excess of the 62–65°C (based on studies recorded in vitro for maximal growth of thermophilic fungi (Morsy et al. 2010; Loro et al. 2012) For example, E. rostratum may produce chlamydoconidia and ascospores and it is recognized as thermotolerant; nevertheless, the role of these morphotypes (potential precursors for recurrent contaminations) in the epidemiology of a meningitis outbreak (although possible) is unknown. With the NECC case, the three contaminated lots of methylprednisolone yielded a variety of bacteria and fungi usually susceptible to routine disinfection and sterility procedures. This reaffirms that the failure or lack of sterility and quality control procedures rather than any especial morphological characteristic of the contaminating fungus. Exophiala is a thermotolerant, constitutive producer of melanin, is consistently able to grow at temperatures above 37°C and it produces extracellular polysaccharide capsules, which all are regarded to be virulence factors (Yurlova and de Hoog 2002; Langfelder et al. 2003). Exophiala dermatitidis is sporadically isolated as an aerial contaminant or from general environmental sampling; moreover, it is a profuse colonizer of steam baths and dishwashers and is capable of surviving repeated exposures to 60–80°C (Zalar et al. 2011; Woo et al. 2013). In addition, dried fungal morphotypes, including conidia and mycelia of various genera, may demonstrate pro7
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longed temperature/survival tolerances up to and exceeding 100°C (Suryanarayanan et al. 2011; Carlsson et al. 2012; Ahearn and Stulting, 2014). Various authors have reported that resistant and dormant morphotypes, albeit in low numbers, may be present in many laminated cardboard materials, cellulosic packaging fillers, hospital linens, and paperboard for aseptic packaging (Houbraken et al. 2010; Delgado et al. 2012; Duffy et al. 2014). In the pharmaceutical facility, antiseptics and disinfectant are used to control the growth of bacteria and fungi. In comparison with bacteria, very little is known about the ways in which fungi can circumvent the action of antiseptics and disinfectants. Here, very few rigorous studies have been performed with yeasts and moulds and antiseptics and disinfectants (Miller 1969; Dekker 1987). Moulds (filamentous fungi) are generally more resistant than yeasts and they are considerably more resistant than most nonsporulating bacteria. Mould spores, however, are less resistant than bacterial spores to biocides (Russell and Furr 1996). It can be speculated that the cell wall composition in moulds confers a high level of intrinsic resistance on these organisms. Due to such incomplete understanding, the activity of biocides against fungal micro-organisms is not as well documented as the activity of biocides against bacteria. Only a few studies are available for lethal concentrations of antiseptics and disinfectants towards yeasts and moulds (Wallhausser 1984; Day et al. 2009). Sandle et al. (2014) studied the in vitro fungicidal activity of biocides against a large series of pharmaceutical environmental fungal isolates. They reported that minimum inhibitory concentrations of chlorhexidine, benzalkonium chloride and cetrimide against hyaline fungi were not more than 16 lg/ml, while Alternaria showed >32 lg/ml and other dematiaceous fungi fell within the range 8–16 lg/ml. Based on these available literatures, the melanized fungi are more resistant than other fungi. Thus, cleanroom personnel should implement the effective disinfection programme to minimize occurrences of these types of fungi, paying close attention to efficacious concentrations and contact times. Control measures Chemical disinfection is a vital part of the contamination control programme in pharmaceutical and compounding pharmacy areas. To decide which method or combinations of methods are to be employed for disinfecting processing areas, it is important to understand the types of micro-organisms that are the prime sources of contamination (Nagarkar et al. 2001). Moreover, understanding the types of micro-organisms provides clues about their points of origin; therefore regular reviews of cleanroom 8
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microflora are important. Since there are numerous ways that cleanrooms can become contaminated with fungi, effective disinfection methods, by use of an agent with fungicidal activity, is extremely important. The disinfection programme requires the selection of the appropriate disinfectants, their proper application and validation of the disinfectant’s capability to inactivate or destroy vegetative cells. Importantly, not all disinfectants typically used in the cleanroom are effective against fungal spores and it may be necessary to use an additional sporicidal agent to control fungi. USP chapter Disinfectants and Antiseptics provide some guidance as to how to structure disinfectant validation studies (Anon. 2011). A series of tests to demonstrate the efficacy of a disinfectant/sporicidal agent against fungal spores within the pharmaceutical manufacturing cleanroom environment is necessary since many agents that are effective against vegetative fungi are not effective against fungal spores. Cleanroom personnel should aware about selection of disinfectants and the need to assess disinfectant efficacy against environmental fungi. Conclusion This article has presented an overview of recent fungal contamination issues affecting pharmaceutical products together with a review of the most common types of fungi found within pharmaceutical cleanrooms. This review has shown that many of the fungal incidences have occurred in relation to products prepared in compounding pharmacies. The article has focused upon some recent contamination incidences where melanized fungi have been a particular concern. Our reason for doing so is because understanding of fungal contamination within pharmaceutical facilities remains poorly understood and due to the particular challenges that melanized fungi pose. Strategies for control, we contend, are based on better understanding of what is present within the cleanroom (by undertaking microflora reviews); stronger regulatory oversight; and care in the selection and qualification of cleanroom disinfectants. With microflora reviews, it is important that the data collected are meaningful. It is our contention that routine environmental monitoring regimes may not be sufficient for detection of resistant morphotypes. Consideration should be given to the types of culture media used and the incubation conditions. From the concurrent serious outbreaks of compounded pharmacy products during last few years, enhanced regulatory overview of compounding pharmacies with multiple infractions should be considered as part of the efforts to improve compounded medication safety. Compounding pharmacies should ensure that pharmacy staff is Journal of Applied Microbiology © 2015 The Society for Applied Microbiology
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trained appropriately and that proper aseptic techniques are followed. FDA has outlined specific activities that help distinguish the role of compounding pharmacies from pharmaceutical manufacturing. These concerns should not be ignored by pharmaceutical organizations either, for although controls may be stricter fungi can occur within any cleanroom. To choose the most effective disinfectant, microbiologists must take a multitude of factors into consideration relating to disinfectant suitability, in-use concentrations and contact times. Furthermore, it is important to include melanized fungi as test strains when qualifying disinfectants: as part of suspension tests, coupon surface studies and examinations into minimum inhibitory concentration determinations. With these efficacy tests the two fungi commonly cited in many disinfectant test protocols: Aspergillus brasiliensis (as a representative filamentous fungus) and Candida albicans (despite its dimorphic physiology, a representative unicellular yeast) may not be sufficient. Thus, consideration should be given to the inclusion of a melanized fungus. Here, most species of Cladosporium can be handled with the same ease as Aspergillus, and most have a biohazard rating of class I or II. Moreover, given its ubiquity, Cladosporium is representative of the types of outdoor moulds that could be transferred into the pharmaceutical facility. When good aseptic techniques and effective disinfection programme are followed in purposely designed cleanrooms, supported by regular isolate trending, in the compound pharmacies and pharmaceutical cleanroom facilities, a more robust contamination control programme can be developed to prevent melanized mould occurrences. This strategy will reduce risk of excursions in a facility, and ultimately safeguard product quality and protect patients. Conflict of Interest None declared. References Adler, A., Yaniv, I., Samra, Z., Yacobovich, J., Fisher, S., Avrahami, G. and Levy, I. (2006) Exserohilum: an emerging human pathogen. Eur J Clin Microbiol Infect Dis 25, 247–253. Ahearn, D.G. and Stulting, R.D. (2014) Fungi associated with drug recalls and rare disease outbreaks. J Ind Microbiol Biotechnol 41, 1591–1597. Ajello, L., Georg, L.K., Steigbigel, R.T. and Wang, C.J. (1974) A case of phaeohyphomycosis caused by a new species of Phialophora. Mycologia 66, 490–498. Anon. (2000) American Society of Health-System Pharmacists. ASHP guidelines on quality assurance for pharmacy-
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prepared sterile products. Am J Health Syst Pharm 57, 1150–1169. Anon. (2010) IV bags from Claris Lifesciences contaminated. Reactions wkly 1305, 4. Anon. (2011) United States Pharmacopeia (USP) 34, Chapter Disinfection and Antiseptics – General Information pp. 579–580. May 1, 2011. Bethesda, MD, USA. Anon. (2012a) Institute for Safe Medication Practices. Medication safety alert/acute care. “Sterile compounding tragedy is a symptom of a broken system on many levels”. 2012;17(21): Available at: http://www.ismp.org/Newsletters/ acutecare/showarticle.asp?id=34 Accessed April 1, 2013. Anon. (2012b) Centers for Disease Control and Prevention. “Multistate fungal meningitis outbreak investigation”. Available at: http://www.cdc.gov/hai/outbreaks/ currentsituation Accessed December 20, 2012. Anon. (2012c) Centers for Disease Control and Prevention (CDC). Notes from the field: multistate outbreak of postprocedural fungal endophthalmitis associated with a single compounded pharmacy—United States, March– April 2012. MMWR Morb Mortal Wkly Rep 61, 310–311. Anon. (2013) FDA Approved Risk Evaluation and Mitigation Strategies (REMS). Bethesda, MD: FDA. Available at: http://www.fda.gov/Drugs/DrugSafety/ PostmarketDrugSafetyInformationforPatientsandProviders/ ucm214035.htm Accessed April 6, 2015. Anon. (2015) International Academy of Compounding Pharmacists. “About compounding”. Available at: http:// www.iacprx.org/about_compounding/index.html Accessed April 6, 2015. Bell, W.R., Dalton, J.B., McCall, C.M., Karram, S., Pearce, D.T., Memon, W., Lee, R. and Carroll, K.C. et al. (2013) Iatrogenic Exserohilum infection of the central nervous system mycological identification and histopathological findings. Mod Pathol 26, 166–170. Brandt, M.E. and Warnock, D.W. (2003) Epidemiology, clinical manifestations, and therapy of infections caused by dematiaceous fungi. J Chemother 15, 36–47. Bush, R.K. and Prochnau, J.J. (2004) Alternaria-induced asthma. J Allergy Clin Immunol 113, 227–234. Carlsson, F., Edman, M., Holm, S., Eriksson, A.M. and Jonsson, B.G. (2012) Increased heat resistance in mycelia from wood fungi prevalent in forests characterized by fire: a possible adaptation to forest fire. Fungal Biol 116, 1025–1031. Castelnuovo, P., De Bernardi, F., Cavanna, C., Pagella, F., Bossolesi, P., Marone, P. and Farina, C. (2004) Invasive fungal sinusitis due to Bipolaris hawaiiensis. Mycoses 47, 76–81. Chang, H.L., Kim, D.S., Park, D.J., Kim, H.J., Lee, C.H. and Shin, H.J. (2000) Acute cerebral phaeohyphomycosis due to Wangiella dermatitidis accompanied by cerebrospinal eosinophilia. J Clin Microbiol 38, 1965–1966. Chang, D.C., Grant, G.B., O’Donnell, K., Wannemuehler, K.A., Noble-Wang, J., Rao, C.Y., Jacobson, L.M., Crowell, C.S.
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