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EVERY STEP OF THE WAY

MICROBIAL SOLUTIONS

The Utility of ITS2 Sequencing for Identifying Fungal Contaminants In the past decade, the pharmaceutical manufacturing

industry has witnessed pronounced growth, accompanied

by an increase in oversight and new manufacturing

regulations. Industrial settings today are also faced with

pronounced prevalence of molds, a situation that can lead

to environmental monitoring excursions and suspension of

operations. Interestingly, recalls due to fungal contamination

are also on the rise.

Of the 45 recalls by the US Food and Drug Administration

(FDA) in 2012, 16% were due to mold or yeast (Table 1).

The multistate outbreak of fungal meningitis in 2012 that

claimed 48 lives was caused by product contamination

by a common mold, Exserohilum rostratum, in

methylprednisolone acetate injections. Alarmingly, this is

not the first instance; a similar outbreak of fungal infections,

including meningitis, was previously reported from a

contaminated lot of injectable methylprednisolone from a

compounding pharmacy in North Carolina. In 2006, Bausch

& Lomb made headlines when an investigation by the

United States Centers for Disease Control and Prevention

(CDC) reported an increased incidence of fungal keratitis,

which resulted in a major recall for Bausch & Lomb and

upwards of $250 million in settlements from lawsuits.

SummaryPhenotypic methods are widely

used for fungal identification.

However, identification of

these fungal isolates from

pharmaceutical environments

using standard identification

procedures is often subjective.

We have found that the use

of ITS2 gene sequencing is

substantially more accurate and

reproducible.

The Utility of ITS2 Sequencing for Identifying Fungal Contaminants

While any type of fungal contamination is undoubtedly a

cause for concern, a species-level identification is crucial to

providing a definitive root cause as part of an investigation.

Indoor air, water, personnel, and materials are the primary

sources for pharmaceutical fungal contamination. Given

the potential risk, the current good manufacturing practices

(cGMP) require that the quality of a finished pharmaceutical

product has to be assured through controlled processes.

Establishing an environmental monitoring program is

one of the most important components of an effective

manufacturing production and process control system for

aseptic production of pharmaceuticals, as well as non-

sterile products.

To aid in demonstrating compliance, many manufacturers

undertake species characterization of every environmental

isolate to provide accurate information to measure the

state of control of the manufacturing facility through the

specific detection, identification, and quantification of

microorganisms. Bacterial microorganisms found in the

manufacturing environment are frequently identified to

species level. Historically, fungal identification, especially

mold identification, has not been held to the same

standards as bacterial identification. This is primarily due

to the limitations in the existing conventional methods

in identifying fungi to the species level. Additionally, the

recognition of the pharmaceutical properties of mushrooms

established a unique place for fungal species as dietary

supplements or nutraceuticals and the identification of

fungal species has become more significant in the light of

new FDA regulations for the dietary supplement industry

(21 CFR Part 111).

Recall Date Company Product Description Reason/ProblemCompany

Status

3/20/2103 Med Prep Consulting, Inc. All compounded products Potential fungal contamination

3/17/2013 Med Prep Consulting, Inc. All compounded products Potential fungal contamination

3/16/2013 Med Prep Consulting, Inc.Magnesium sulfate 2 g in dextrose 5% in water, 50 mL for injection

Confirmed fungal contamination

10/6/2012New England Compounding Center (NECC)

Methylprednisolone acetate, betamethasone, bupivicaine, more

Sterility; linkage to meningitis outbreak

Closed

10/5/2012 Hospira, Inc. Lactated Ringer’s and 5% dextrose Container leak and mold contamination

7/13/2012 Westone Laboratories, Inc. Ear lubricant Potential contamination with pathogenic bacteria and mold

5/25/2012 Franck’s Compounding LabSterile human and veterinary prescription drugs

Presence of microorganisms and fungal growth

Closed

5/2/2012 Franck’s Compounding LabTriamcinolone acetonide P.F. 80 mg/mL

Potential fungal contamination Closed

3/31/2012 Franck’s Compounding LabTriamcinolone acetonide P.F. 80 mg/mL

Potential fungal contamination Closed

3/9/2012 Franck’s Compounding Lab Brilliant Blue G Potential fungal contamination Closed

Table 1: Recent FDA drug recalls due to fungal contamination

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Conventional Fungal Identification and Its LimitationsFungi are a diverse group of eukaryotic microorganisms

with approximately 100,000 species. The two groups of

fungi that have practical importance in the cleanroom are

molds and yeasts. Phenotypic methods are widely used

by many laboratories for fungal identification due to their

relatively lower costs. However, identification of these fungal

isolates from pharmaceutical environments using standard

identification procedures requires experienced, skilled

technologists, as the expression of the fungal phenotype

frequently depends on the media and growth conditions

that have been used. A further limitation with phenotypic

methods is the size and type of reference database used.

Many of these databases are orientated towards clinical

diagnostics and not necessarily well represented for

industrial application. Therefore, extreme care is required

while interpreting microbiological identification test results

and the trending of data. For instance, Black aspergilla,

a member of the genus Aspergillus section Nigri comprises

one of the most confusing and difficult groups to identify

due to subtle differences between the species. Aspergillus

niger, a member of the Aspergillus section Nigri, has always

been problematic in cold rooms where pharmaceutical

and biotech companies store raw materials for their

cleanroom operations. Differentiation of the members of

the A. niger aggregate complex (Table 2) has been difficult

to accomplish using morphological criteria (Samson et al.

2007).

Accurate identification is very important when out-of-

specification results are obtained, and if the contamination

source has to be determined and tracked. Remediation

efforts are not effective if inaccurate information is used

to solve a given problem. However, with the advent

of molecular identification methods, a more accurate

identification of the fungal contaminant can be obtained.

Species Conidial size (mm) Vesicle size (µm) Color and size of sclerotia (mm)

Uniseriate species

A. aculeatinus 2.5-4.5 45-80 White to cream, 0.4-0.6

A. aculeatus 3.5-5 60-80 Cream, up to 0.5

A. japonicus 3.5-5 20-35 White to cream, up to 0.5

A. uvarum 3-4 20-30 Dark brown to black, 0.5-0.8

Biseriate species

A. brasiliensis 3.5-4.5 30-45 White, 1-1.5

A. carbonarius 7-9 40-80 Pink to yellow, 1.2-1.8

A. costaricaensis 3.1-4.5 40-90 Pink to grayish yellow, 1.2-1.8

A. ellipticus 3.3-5.5 75-100 Dull yellow to brown, 0.5-1.5

A. foetidus 3.5-4.5 50-80 White, 1.2-1.8

A. heteromorphus 3.5-5 15-30 White, 0.3-0.6

A. homomorphus 5-7 50-65 -

A. ibericus 5-7 50-60 -

A. lacticoffeatus 3.4-4.1 40-65 -

A. niger 3.5-5 45-80 -

A. piperis 2.8-3.6 40-55 Yellow to pink-brown, 0.5-0.8

A. sclerotiicarbonarius 4.8-9.5 45-90 Yellow to orange to red-brown

A. sclerotioniger 4.5-6.4 30-50 Yellow to orange to red-brown

A. tubingensis 3-5 40-80 White to pink, 0.5-0.8

A. vadensis 3-4 25-35 - 1. Studies in Mycology (2007) 59:129-145.

Table 2: Morphological characteristics of different species belonging to Aspergillus section Nigri.1

Genotypic IdentificationThe use of ribosomal DNA sequences for the purposes

of organismal taxonomic classification has been in use

for many decades. More recently, ribosomal genes have

been used to study the phylogenetic relationships of fungi.

Although fungal taxonomists have been using phylogenetic

analysis to characterize, classify, and re-classify fungi

for many years, it is only recently that this approach has

gained popularity for the routine identification of fungi in

the pharmaceutical manufacturing environment. This is

primarily due to the fact that while the technology existed

for performing these analyses, applications that were

developed in a compliant manner and were able to be

validated in a cGMP laboratory have only recently been

made available through products and contract service

laboratories. The introduction of MicroSEQ®, a commercial

product available from Applied Biosystems, as well as a

number of laboratories using DNA sequencing for fungal

identification, initially provided a solution for increasing the

quality of a species-level identification for fungal organisms.

DNA sequencing provides data for identification that are

substantially more accurate and reproducible than relying

solely on visual phenotypic characteristics. This is generally

well understood and accepted. The FDA recommended the

use of genetic methods in their 2004 update to the guidance

document, “FDA Guidance for Industry. Sterile Drug

Products Produced by Aseptic Processing – Current Good

Manufacturing Practice.” In this document, the FDA states

in the section on environmental monitoring, “Genotypic

methods have been shown to be more accurate and precise

than traditional biochemical and phenotypic techniques.

These methods are especially valuable for investigations

into failures (e.g., sterility test; media fill contamination).”

Limitations with the “Out of the Box” Identification SystemDespite the acceptance and support from the scientific

community and regulatory agencies, there are still

opportunities for improvement with the current

commercially available sequence-based identification

system. The first is the coverage of the database for known

fungal species. This should not be confused with the size of

the database, as a database filled with hundreds, or even

thousands, of clinical species not encountered in the

pharmaceutical manufacturing environment adds no value

to the identification system (Rozynek et al. 2004 and Hall

et al. 2003).

A second limitation of the commercially available system is

the gene target chosen to build the DNA sequence library.

This is not a limitation of the technology, but rather a

limitation of the application. When choosing an appropriate

gene target for phylogenetic analysis, one needs to find a

target that undergoes enough genetic mutation for there to

be measurable differences in the DNA sequences of similar

but different species. However, the nucleotide differences

should not be so big that truly related species appear to

be more dissimilar than they really are. This is the great

challenge in choosing the appropriate target, and in many

cases it is simply a case of trial and error.

Limits of D2 Gene ResolutionThe D2 expansion segment of the Large Subunit (LSU) of

the ribosomal gene, as utilized in the MicroSEQ system,

typically does a good job of placing an unknown fungal

isolate into the appropriate higher level taxa (Genus, Family,

Order,) and can differentiate many species reasonably, but

not in all cases. Due to these limitations in the resolution

of the D2 segment, closely related organisms may have

identical or very similar DNA sequences. For example,

several Aspergillus niger strains were reclassified as

Aspergillus brasiliensis (Varga et al. 2007). Most significant

among the isolates was Aspergillus niger ATCC 16404 that

was reclassified as Aspergillus brasiliensis. This organism

is cited in several USP chapters as a QC organism,

including USP <61> “Microbial Limits Test – Enumeration”

and USP <71> “Sterility Test.” Because of the number of

pharmaceutical companies performing these USP tests,

it is very important to be able to correctly identify the QC

organism.


Unfortunately, this is another example of where virtually

all phenotypic tests, as well as D2 DNA sequencing, are

unable to differentiate these two species from one another.

Phenotypically, it has always been difficult to differentiate

between A. niger strains due to a lack of diversity in

morphological features, unstable phenotypic characters,

and the significant influence of culture conditions on the

phenotype (Rinyu et al. 1995). Furthermore, D2 sequences

provide no additional information, as the DNA sequences for

all observed A. niger and A. brasiliensis strains are identical

(Figure 1).

Merits of Using ITS2 Gene Sequencing The Internal Transcribed Spacer (ITS) regions of the

ribosomal operon have been used for fungal systematics

and classification. There are two ITS regions in the fungal

rRNA operon. The first, ITS1, is found between the 18S and

5.8S rRNA genes. The second, ITS2, is located between

the 5.8S and the 28S rRNA genes. The entire rRNA operon

is transcribed. However, after transcription, the two ITS

sequences are excised and are therefore not used for any

functional purpose. Since the ITS sequences are important

enough as spacer regions to be maintained by the cell,

but not used for any functional purpose, they accumulate

mutations at a faster rate than the 5.8S, 18S, and 28S

rRNA genes. This slightly increased rate of accumulated

mutations allows the ITS sequences to provide an improved

level of resolution as compared with the D2 sequence.

It is therefore customary among the fungal phylogeneticists

to sequence the entire stretch of ITS1-5.8S-ITS2 to use in

fungal classification. However, for the routine identification

purposes, our laboratory has found that the use of ITS2

alone is usually sufficient for species-level identification.

In support for ITS2 sequencing, Houseknecht et al.

concluded that although A. niger and A. brasiliensis are

very similar, sequencing of the entire ITS1-5.8S-ITS2 DNA

sequence shows there are five differences between the

strains of A. niger and A. brasiliensis. When comparing the

ITS2 sequence alone, there is one nucleotide difference

between the species (Figure 2); however, this difference has

been shown to be extremely reproducible and is therefore

considered to be a diagnostic indicator of the species.

Figure 1. The D2 expansion region lacks species-level resolution for the two Aspergillus spp.

N Join: 4.250 %

Aspergillus niger ATCC 16888 Type

Aspergillus brasiliensis ATCC 16404

Aspergillus brasiliensis ATCC MYA-4553 Type

Aspergillus brasiliensis NRRL 35542

Aspergillus niger NRLL 1956

Aspergillus heteromorphus

Aspergillus phoenicis

Aspergillus flavus

Aspergillus fumigatus

Figure 2. The ITS2 region clearly distinguishes the two Aspergillus spp.

N Join: 3.420 %




Aspergillus brasiliensis ATCC MYA-4553 Type

Aspergillus brasiliensis NRRL 35542

Aspergillus niger NRLL 1956

Aspergillus heteromorphus

Aspergillus phoenicis

Aspergillus flavus

Aspergillus fumigatus



The Impact of the Expansion of the ITS2 Reference Database and the Frequency of Fungal ID for Customer SamplesThe accuracy of an identification is not only dependent

on the genetic target used for the identification, but

also equally dependent on the library against which the

data is compared. Having access to the most relevant,

accurate, and compliant microbial libraries is crucial to

obtaining correct species-level identifications for regulated

manufacturing operations. As a company, we recognized

early on that the libraries associated with commercial

systems were not sufficient. Since that time, we have

established the most comprehensive bacterial and fungal

sequence databases for the organisms encountered in

manufacturing environments, allowing us to provide the

highest quality identification results for unknown isolates.

The Accugenix® library generation and maintenance

activities are designed to ensure that the bacterial and

fungal reference libraries contain all possible organisms

that are relevant to the industries we serve. They are also

intended to verify that existing library entries are classified

in accordance with current literature so that tracking and

trending of environmental and objectionable organisms can

be performed efficiently. Incorrect library entries can lead to

inaccurate and inconsistent identifications and misdirected

remediation efforts.

We can demonstrate a key advantage to the continued

development of our proprietary fungal library by examining

the rates of identification of unknown organisms isolated

from pharmaceutical manufacturing facilities. We have

recently seen a dramatic increase in the number of fungal

samples submitted for genotypic identification, up to

almost 30,000 in 2012 (Figure 3). During that time, we

have quadrupled the size of the Accugenix® proprietary

ITS2 fungal identification database. The addition of novel

organisms, as well as updates in taxonomic descriptions

to the proprietary library, increases the probability of a

species-level identification. As a result of this dramatic

increase in our library database, we are now able to report

93.5% of our customer samples to a species-level ID and

the percentage of “No Match” reports has dropped from

greater than 10% to 2.6% (Figure 4).


Figure 3. Total number of fungal samples analyzed per year

Figure 4. Fungal library entries and the impact on customer sample identification

30000

25000

20000

15000

10000

5000

2009 2010 2011 2012

60%

50%

40%

30%

20%

10%

0%

500

600

400

300

200

100

0

90%

100%

80%

70%

2009 2010 2011 2012

New Library Entries Species

Genus No Match

ITS

2 se

quen

ce id

entif

icat

ion

(%)

Num

ber

of n

ew e

ntrie

s to

ITS

2 lib

rary

per

yea

r(2

012

tota

l ent

ries:

146

1)

Continuous maintenance requires timely evaluation of

sequences that are not identified to the species level.

Investigating groups or clusters of sequences that do not

provide a species-level identification is essential to building

a library that contains relevant organisms with a broad

range of coverage. The importance of an updated library is

further demonstrated by considering the species Penicillium

chrysogenum, a commonly occurring mold in indoor

environments. This species has gained much attention in

the pharmaceutical industry for production of the antibiotic

penicillin. However, recent phylogenetic studies showed

that the penicillin-producing strains originally described by

Alexander Fleming are not P. chrysogenum, but Penicillium

rubens (Houbraken et al. 2011). The P. rubens organism is

not present in the MicroSEQ D2 library and the phylogenetic

neighbor joining tree constructed after searching the D2

database with the P. rubens sequence is disjointed and

provides inaccurate information (Figure 5).

It is critical that the identification libraries against which

data are compared contain the most current taxonomic

classifications, have a breadth of coverage and contain

relevant organisms — those that have been identified

as a result of EM programs. If the library lacks depth of

coverage, the interpretation of the data may not always

be reliable. Over the last 20 years, the Charles River

Accugenix® group has identified more than one million

microorganisms primarily isolated from manufacturing

environments. As such, we understand the biodiversity

present in these environments. We know the frequency

with which organisms are recovered and the variety of

organisms that are isolated from manufacturing facilities.

We continually strive to improve our genotypic reference

databases to improve the frequency of species-level

identification by encompassing the organisms that are

relevant to the industries we serve. Our goal is to always

provide the highest level of accuracy and reliability.

N Join: 0.375 %

Penicillum rubens CBS 205.57

Specimen Penicillum rubens CBS 205.57

MicroSeqID Phylogenetic TreeITS2 Phylogenetic Tree

Penicillum chrysogenum

Penicillum melanoconidum

Penicillum glandicola

Penicillum rubens

Penicillum verrucosum

Penicillum commune

Penicillum venetum

Penicillum turbatum

Penicillum griseofulvum

Penicillum cyclopium

Penicillum camembertii CBS 190.67

Penicillum griseofulvum DSM 896

Penicillum aurantiogriseum aurantiogriseum CBS 324.89

Penicillum commune CBS 474.92

Penicillum histrum DSM 62833

Penicillum hordei CBS 701.68

Penicillum nalgiovense CBS 352.48

Penicillum verrucosum verrucosum CBS 603.74

Penicillum rubens CBS 205.57

Penicillum chrysogenum CBS 306.48

Penicillum viridicatum DSM 2447

N Join: 1.0 %

Figure 5. Impact of updated libraries and species identification


[email protected] • www.criver.com © 2017, Charles River Laboratories International, Inc.

ReferencesExophiala infection from contaminated injectable steroids

prepared by a compounding pharmacy -- United States.

MMWR Morb Mortal Wkly Rep 2002; 51:1109-1112.

Hall L, Wohlfiel S, Roberts GD. Experience with the

MicroSEQ D2 Large-Subunit Ribosomal DNA Sequencing

Kit for Identification of Filamentous Fungi Encountered

in the Clinical Laboratory. J Clinical Microbiology 2003;

42(2):622-626.

Houbraken J, Frisvad JC, Samson RA. Fleming’s penicillin

producing strain is not Penicillium chrysogenum but

P. rubens. IMA Fungus 2011; 2(1):87-95.

Houseknecht J, Stamenova E, Suh S-O, Beck B, McKee M,

Zhou J. Reclassification of ATCC® 16404™ and ATCC®

9642™ as Aspergillus brasiliensis. Pharm Micro Forum

Newsletter 2008; 14(10):2-8.

Rinyu E, Varga J, Ferenczy L. Phenotypic and Genotypic

Analysis of Variability in Aspergillus fumigatus. J Clinical

Microbiology 1995; 33(10):2567-2575.

Rozynek P, Gilges S, Brüning T, Wilhelm M. Quality Test

of the MicroSEQ D2 LSU Fungal Sequencing Kit for the

Identification of Fungi. Int J Hyg Environ Health 2004;

207(3):297-299.

Samson RA, Noonim P, Meijer M, Houbraken J, Frisvad JC,

Varga J. Diagnostic tools to identify black Aspergilli. Studies

in Mycology 2007; 59:129–145.

Varga J, Kocsube S, Toth B, Frisvad JC, Perrone G, Susca

A, Meiijjer M, Samson R. Aspergillus brasiliensis sp. Nov.,

a biseriate black Aspergillus species with world-wide

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