Treatment of Infections by Cryptic Aspergillus Species

Wagner L. Nedel • Alessandro C. Pasqualotto

Received: 23 May 2014 / Accepted: 28 August 2014 / Published online: 13 September 2014

� Springer Science+Business Media Dordrecht 2014

Abstract The best treatment for patients with inva-

sive aspergillosis caused by cryptic Aspergillus spe-

cies remains uncertain, mainly due to the limited

clinical data that have been published so far. In face of

this limitation, patients should be treated with standard

first-line therapy for invasive aspergillosis, with

therapy being modified according to in vitro suscep-

tibility testing. In this review, we summarize the

importance of cryptic Aspergillus species in modern

medicine, including their prevalence, methods for

detection and response to antifungal drugs.

Keywords Invasive aspergillosis � Cryptic species �Treatment

Introduction

Aspergillus spp are opportunistic molds that can be

involved in a wide variety of clinical syndromes, from

allergic to invasive syndromes. Most cases of asper-

gillosis are caused by a few Aspergillus species, in

particular Aspergillus fumigatus, Aspergillus flavus,

Aspergillus terreus and Aspergillus niger [1]. Asper-

gillus species have emerged as important causes of

life-threatening infections in immunocompromised

hosts, including patients with prolonged neutropenia,

HIV infection, stem cell and organ transplantation [2].

More recently, investigators have revealed the pre-

sence of uncovered ‘‘cryptic’’ Aspergillus species in

clinical samples. Even though these fungi are mor-

phologically indistinguishable within main Aspergil-

lus sections, the advantage of a proper speciation in

order to find them remains a matter of debate.

Voriconazole is considered as first-line therapy for

invasive aspergillosis, but the performance of this

azole antifungal drug is uncertain against the ‘‘cryp-

tic’’ Aspergillus species, since some of these have

revealed higher MICs to the azoles. This short review

will deal with the evidence on how to best treat

patients infected with ‘‘cryptic’’ Aspergillus species.

What are We Dealing With?

Fungal diseases have increased in importance in recent

years. Accordingly, a greater knowledge now exists on

the epidemiology of such infections, and physicians

W. L. Nedel � A. C. Pasqualotto

Universidade Federal de Ciencias da Saude de Porto

Alegre (UFCSPA), Porto Alegre, Brazil

W. L. Nedel

Hospital Nossa Senhora da Conceicao (HNSC),

Porto Alegre, Brazil

A. C. Pasqualotto (&)

Molecular Biology Laboratory, Irmandade Santa Casa de

Misericordia de Porto Alegre, Av Independencia 155,

Porto Alegre 90430-020, Brazil

e-mail: pasqualotto@santacasa.tche.br

123

Mycopathologia (2014) 178:441–445

DOI 10.1007/s11046-014-9811-z

seem to better understand the importance of a proper

speciation for medically relevant fungi [3]. Species

characterization in medical mycology has traditionally

been based on morphological fungal characteristics.

However, this largely depends on growth conditions

and requires considerable expertise to achieve a good

level of discrimination. The story of the cryptic

Aspergillus species started with Aspergillus lentulus.

Balajee and colleagues investigated seven atypical

clinical isolates of A. fumigatus that showed poor

sporulation and had low in vitro susceptibility to

several antifungal drugs. By using multilocus

sequence typing (MLST), four of these isolates were

found to be part of a new fungal species, named A.

lentulus [4]. Additional reports soon followed from

different geographical areas, evaluating both clinical

and environmental samples [5]. In a recent publication

[6], it was reported that a 36-year-old renal transplant

patient had a pneumonia 4 month after transplanta-

tion. A. lentulus was recovered on sputum, and the

patient was said to have ‘‘recovered on voriconazole’’

(MIC of 0.5 lg/ml). In their manuscript, the authors

present a table showing the literature data from five

previous patients with A. lentulus pneumonia. As

expected, most of these studies were case reports only,

with limited microbiological data and anecdotal

clinical information regarding antifungal treatment.

Soon after A. lentulus became notorious, a new

species emerged within the Fumigati section: Neosar-

torya pseudofischeri [7]. Balajee et al. were actually

searching for A. lentulus by studying three poorly

sporulating A. fumigatus isolates. Very limited data

were provided for these patients. One patient was a

stem cell transplant recipient who was treated with

amphotericin B lipid complex, followed by vorico-

nazole plus caspofungin. The patient died due to

progressive leukemia. The other two patients in the

study had cystic fibrosis, and N. pseudofischeri was

isolated from sputum. The authors performed a

literature review, and since the year 1929, N. pseudo-

fischeri had been associated with invasive diseases in

only seven patients—and for three of these, no data on

antifungal treatment were available. What can we

learn from that, from the therapeutic point of view?

Not much, to be honest.

With the emergence of A. lentulus and N. pseudofisc-

heri, the list of cryptic Aspergilus species increased

exponentially. In the Fumigati section, this now include

A. udagawae, A. viridinutans, A. fumigatiaffini, and

A. novofumigatus. Other cryptic species include A. alli-

aceus (Flavi section); A. carneu and A. alabamensis

(Terrei section); A. tubingensis, A. awamori, and

A. acidus (Nigri section); A. sydowii (Versicolores

section); A. westerdijkia and A. persii (Circumdati

section); and A. calidoustus, A. insuetus, and A. keveii

(Usti section). There is certainly more than that. Again,

only a limited number of isolates of each of these cryptic

Aspergillus species has described clinically, and the data

regarding antifungal treatment are even more scarce.

How Can We Identify the Cryptic Species?

This is usually performed by fungal DNA sequencing

[7, 8, 9, 10, 3]. Most studies have used ITS sequenc-

ing—the standard locus for fungal species identifica-

tion—which allows for Aspergillus identification at

the complex level. Sequencing additional genes is

usually required for proper speciation, including Beta-

tubulin, calmodulin, and Rodlet A genes [8]. This

process, however, has some remarkable limitations,

including the limited data available on fungal sequenc-

ing (less than 1%), annotation errors in the databanks

and a lack of standardized, universally adopted

process of translating the comparison into species

names [11]. The main point appears to be: Should we

sequence every clinical isolate? Are the mycology

laboratories serving tertiary hospitals around the globe

prepared to perform large scale fungal DNA sequenc-

ing? Or should this be restricted to reference labora-

tories? The answers to these questions will depend

directly on the expected frequency of cryptic species

in the clinical practice.

So, are These Cryptic Aspergillus Species

Frequent? Are they Clinically Relevant?

Let’s first take a look at the results of some large

randomized trials in which patients with invasive

aspergillosis were enrolled. In the Herbrecht study

(n = 277) [12], culture was positive for 149 patients

(53.8 %), but speciation was made by classical meth-

ods only. Therefore, the frequency of cryptic species

was not reported (indeed, the trial was conducted far

before the concept of ‘‘cryptic’’ species was in place).

In the AmBiLoad trial (n = 201) [13], only 14.9 % of

patients had positive culture and/or histologic findings.

The vast majority of patients entered the trial based on

imaging alone (58.7 %), while galactomannan was

442 Mycopathologia (2014) 178:441–445

123

positive for 22.4 %. In the most recent randomized trial

(n = 277) [14], designed to evaluate the use of

combination therapy for patients with invasive asper-

gillosis, most patients were enrolled based on positive

galactomannan results and culture was positive for

only 50 individuals (18.0 %). These data reveal what

seems obvious nowadays: Most patients in clinical

studies are enrolled based on antigen testing (e.g.,

galactomannan); so, culture is usually not obtained and

the frequency of ‘‘cryptic’’ species cannot be deter-

mined. Hopefully real-life cohort studies could provide

us with a better evidence on the subject.

TRANSNET (Transplant-Associated Infection

Surveillance Network) was a multicenter prospective

surveillance study of invasive fungal infections

involving transplant patients. Badley et al. [15]

collected data on antifungal therapy and length of

hospitalization in a cohort of 361 transplant patients

with invasive aspergillosis, including stem cell trans-

plant recipients (n = 228) and solid organ transplant

recipients (n = 133). Despite mentioning that ‘‘etio-

logic Aspergillus species data were collected,’’ no

detailed information on Aspergillus species distribu-

tion was provided on the manuscript. In two additional

TRANSNET studies (both published in the year 2010)

[16, 17], again Aspergillus isolates were identified at

the complex level only. There was not a single

mention in the manuscripts to ‘‘Aspergillus section’’,

‘‘cryptic’’ or ‘‘siblings’’ species.

The complete identification of the Aspergilli at the

species level in the TRANSNET studies was presented

in a different publication [18], in partnership with the

American Center for Disease Control and Prevention

(CDC, Atlanta). The author’s main conclusion in that

publication was that ‘‘over 10 % of the isolates

associated with invasive aspergillosis in transplant

recipients were found to be cryptic species.’’ That is

quite an alarming proportion. However, a closer look

at the data shows that within the Fumigati section (in

which 67.4 % of isolates were included), A. fumigatus

represented no less than 93.9 % of the isolates.

A. fumigatus were followed in frequency by A. lentulus

(2.7 % of the isolates within the Fumigati section),

and all A. lentulus isolates were recovered from a

single medical center. Other (less frequent) species

included A. udagawae (2.0 % within section) and

N. pseudofischeri (0.8 %). In the Flavi and Terrei

sections, isolates showed 100 % homology with the

control strains (which means no cryptic species were

found). Nevertheless in the Nigri section (that collec-

tively represented only 8.7 % of isolates), 31.6 % of

isolates were found to be A. tubingensis. These isolates

all that had low MICs to antifungal drugs, putting a

question on the importance of these findings. All

isolates of A. ustus (n = 6) were re-identified as

A. calidoustus, 40 % of A. versicolor isolates (n = 5)

were found to be A. sydowii, and the only isolate of

A. nidulans was renamed Emericella quadrilineata.

Overall, the data suggest that in most cases the

occurrence of cryptic species is of limited importance

in transplant patients with invasive aspergillosis.

More recently, results of the FILPOP study (Pop-

ulation-Based Survey of Filamentous Fungi and

Antifungal Resistance in Spain) [3] were published.

This was the first study to systematically evaluate the

frequency of cryptic Aspergillus species in Europe.

The overall frequency of cryptic species in the study

was 14.4 %, which was reduced to 7.7 % if isolates

belonging to the Nigri section were excluded.

Are They More Resistant?

They certainly are. The FILPOP study [3] demonstrated

quite clearly that resistant to multiple antifungal drugs

was frequent among cryptic species, particularly in

A. lentulus, A. alliaceus, A. sydowii, A. calidoustus,

A. keveii, A. insuetus and A. fumigatiaffinis. However,

together these species represented as low as 5 % of all

Aspergillus isolates (n = 277) recovered in the study.

One may argue that performing antifungal susceptibility

testing would be more important than identifying all

clinical isolates of Aspergillus at the species level, by the

means of molecular methods. Others would say for sure

that both would generate important data, particularly

from the epidemiological point of view. Both opinions

seem to be rigth in some aspects.

Do Patients Infected with Cryptic Aspergillus

Species Respond Less to Antifungal Drugs?

There is no clear answer to this question. Even though

some of the cryptic Aspergillus species demonstrate

higher MICs to a variety of antifungal drugs, clinical

data are still very limited. Some patients died of

invasive aspergillosis due to a cryptic species while

being very sick, so the importance of the underlying

disease cannot be underestimated. In several publica-

tions patients were only colonized by Aspergillus

Mycopathologia (2014) 178:441–445 443

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species, creating even more uncertainty. Also, it is

possible (although not properly investigated) that

some of the cryptic species may show reduced

virulence, in comparison to the ‘‘classical’’ Aspergil-

lus species. There is a whole world to be discovered

here.

Should We Systematically Search for Them?

Based on the high MIC to several antifungal agents, it

would be logical to assume that patients with aspergil-

losis will directly benefit from the identification of these

isolates at the species level. But is this a cost-effective

intervention? A recent expert panel [10] concluded that

‘‘Taken together, data regarding differences in patho-

genicity and in vivo drug susceptibilities of the various

species within Aspergillus and Fusarium complex do

not categorically suggest that identification within these

taxa will impact clinical and therapeutic decision

making, at least at the present time … However,

identification to the species/strain level could inform the

epidemiology of fungal infections and can be critical in

outbreak investigations.’’ We do believe this message is

still valid nowadays.

So How Should We Treat the Cryptic Aspergillus

Species?

Unfortunately there is no solid clinical data to guide us

on this regard. Due to the apparent low frequency of

cryptic Aspergillus species in the clinical practice,

they are not likely to influence the choice of empirical

or primary antifungal use. Combination antifungal

therapy seems attractive, but still no data are available.

At this point, we must rely mostly on in vitro

information. As mentioned earlier, MIC to azoles is

usually high for the cryptic Aspergillus species, while

amphotericin B commonly retains activity. However,

that varies largely among fungal isolates and species,

reinforcing the importance of in vitro susceptibility

studies for every isolate.

Conclusion

With the increasing incidence of mycoses and reports

of invasive fungal infections in unusual populations,

like critically ill patients, physicians are currently

more aware of the need of a rapid and precise fungal

identification at the species level. Widespread avail-

ability of modern molecular techniques has allowed

for the characterization of cryptic Aspergillus species,

some of which revealed reduced susceptibility to

several important antifungal drugs. How to best treat

these isolates remain a challenge, since clinical data

are still very limited. In general, it seems reasonable to

conclude that cryptic Aspergillus species occur in low

frequency. The final message of this review could be:

‘‘don’t panic. Keep studying your isolates.’’ The future

will certainly tell us more about the importance of

these ‘‘bizarre’’ Aspergillus species. In the meanwhile,

it is reasonable to say that we should keep treating our

patients as usual, pretending we have never been

introduced to the ‘‘cryptic’’ concept. The need to

sequence all molds in clinical practice also remains

uncertain. Despite providing us with additional infor-

mation, this increments the overall costs associated to

patient care and adds more complexity to the diagno-

sis. As shown in this review, there is a limited

knowledge on the meaning of the DNA sequencing

results, so it would be not a complete mistake–at least

for the moment being–to label these tests as ‘‘for

research use only.’’

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