Introduction

According to the World Health Organization definition: “a disease outbreak is the occurrence of cases of disease in excess of what would normally be expected in a defined community, geographical area or season. An outbreak may occur in a restricted geographical area, or may extend over several countries. It may last for a few days or weeks, or for several years. A single case of a communicable disease long absent from a population, or caused by an agent not previously recognized in that community or area, or the emergence of a previously unknown disease, may also constitute an outbreak and should be reported and investigated” [http://www.who.int/topics/disease_outbreaks/en/; accessed 22/07/2015].

Fungi are eukaryotic microorganisms, of which certain species are involved in human diseases, primarily opportunistic infections, including health care-associated infections, in a growing number of immunocompromised patients. These highly susceptible patients often develop severe, rapidly progressive, and difficult to diagnose or treat fungal invasive disease. The increase in susceptible or at-risk patient populations and enhanced diagnostics led to a global increase in the incidence of health care-associated fungal invasive diseases. Additionally, numerous reports of health care-associated fungal outbreaks were published [1•, 2–5]. In the clinical practice, yeast are distinguished from filamentous fungi, including dermatophytes and molds, This categorization has no taxonomical relevance, but yeast and molds have distinct environmental reservoirs, transmission and contamination routes, clinical presentations, and susceptibility profiles to antifungal treatments.

The clinical presentation of invasive fungal disease is usually aspecific, and in contrast to other diseases, such as cholera or Ebola virus for instance, the case definition of fungal infections cannot usually rely only on patient clinical presentation. Therefore, precise identification of the fungus involved is critical to detect and investigate a fungal infection outbreak. In practice, the identification of a fungal outbreak usually relies on the detection of an unusually high number of infections due to a fungus species diagnosed in the laboratory. In many cases, species identification is not precise enough to understand the spreading mechanism of a fungus involved in an outbreak. Appropriate investigation relies on the identification of specific strains or types within the species involved in the outbreak. For instance, El Tor, a particular biotype of the Vibrio cholerae species, is the dominant strain responsible for the ongoing seventh cholera pandemic [6]. The Mycobacterium tuberculosis Beijing strain, which is multidrug-resistant, is responsible for tuberculosis epidemics in Europe and Asia [7].

Most typing techniques were developed to assess the link between closely related isolates for population genetic studies, epidemiological investigation of outbreaks, or surveillance of human diseases. Typing is used to examine the genetic relationships between environmental and clinical isolates involved in colonization or infection. In epidemiological investigations, understanding pathogen distribution and measuring the genetic relatedness between strains is critical to trace outbreak sources, identify disease transmission routes, and monitor health care-associated infections. The number of scientific publications addressing fungal typing, as retrieved in the PubMed database [http://www.ncbi.nlm.nih.gov/pubmed] by using (Mycological Typing Techniques [Mesh]) search terms, has steadily increased since 1991 (Fig. 1). This paper reviews the current understanding of molecular typing approaches used to investigate clinical fungal outbreaks.

Fig. 1
figure 1

Trends in the 2480 publications addressing mycological typing techniques in PubMed [http://www.ncbi.nlm.nih.gov/pubmed with the query (“Mycological Typing Techniques” [Mesh]), July 22th 2015]

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Yeast Infections

Many yeast species are commensal or colonize human mucous membranes; however, invasive yeast disease is a growing issue among immunocompromised and intensive care unit patients. Strain-typing studies are required to avoid implicating an environmental source based only on the presence of a single yeast species. Indeed, differentiation between exogenous and endogenous exposure to yeast is important to trace the source of infection and implement appropriate control measures.

Candidiasis

Among fungal outbreak reports, the majority investigated health care-associated transmission of Candida spp., the most frequent genus involved in human colonization and infections. The main epidemiological findings of yeast outbreak investigations using various typing schemes, based on a non-exhaustive inventory of the literature, are described below.

Resistogram Typing

Analysis of strain resistance profiles to various chemical products, referred to as resistogram typing, was used to investigate a Candida albicans outbreak involving 12 neonates. The study concluded that transmission likely occurred via the hands of staff [8].

Restriction Fragment Length Polymorphism Typing

Many studies have used restriction endonuclease fragment length polymorphism (RFLP) analysis of genomic C. albicans DNA. RFLP analysis with EcoRI and BstNI enzymes highlighted the genetic relatedness between C. albicans strains isolated from the bloodstream and the retrograde medication syringe fluids in neonates [9]. Application of BssHII RFLP enabled the identification of a C. albicans genetic cluster involved in 50 % of the studied candidiasis cases [10]. EcoRI RFLP was used to rule out a common origin of the C. albicans strains involved in an endophthalmitis outbreak among heroin addicts [11].

Pulsed Field Gel Electrophoresis Typing

Pulsed Field Gel Electrophoresis (PFGE) karyotyping with SfiI restriction endonuclease analysis proved effective in discriminating between temporally related C. albicans isolates and investigating suspected health care-associated invasive candidiasis outbreaks in neonatal intensive care units [4]. PFGE typing was also useful to investigate outbreaks caused by the major human pathogenic yeast Candida glabrata. A C. glabrata hospital-acquired fungemia outbreak among children was traced to a unique genotype that had contaminated milk bottles during a disinfection unit failure episode [12]. In contrast, Xba RFLP analysis demonstrated the heterogeneous genetic background of C. glabrata isolates involved in an outbreak that affected 23 patients [13]. Using BssHII and EagI RFLP followed by PFGE analysis, Candida parapsilosis prosthetic valve endocarditis was traced to the contaminated hands of health-care workers. These findings indicated that glove tears were likely the cause of C. parapsilosis transmission during valve replacement surgery [14].

Random Amplified Polymorphic DNA Typing

Outbreaks of health care-associated fungemia involving the skin commensal yeast species C. parapsilosis were investigated using random amplified polymorphic DNA (RAPD) typing. Although RAPD typing lacks discriminatory power and reproducibility, the technique was instrumental in identifying contamination sources and the findings reinforced targeted infection control measures [15–18].

Although less frequently involved in human infections and colonization than C. albicans, C. glabrata, and C. parapsilosis, Candida krusei infections are nevertheless particularly dreaded, primarily due to the unfavorable antifungal drug resistance profile of this species. Using RFLP, a study highlighted the endogenous origin of C. krusei infectious episodes. In this study, HinfI RFLP typing revealed indistinguishable restriction patterns between C. krusei strains isolated from each patient and their respective environment, while the isolate patterns compared between patients were dissimilar [19]. C. krusei was also involved in health care-associated outbreaks. In particular, a C. krusei fungemia outbreak in a neonatal intensive care unit was traced to a contaminated infusate batch, as the strains cultured from the patients and the infusate displayed an identical M-13 primer-RAPD profile [20]. Similarly, in pre-term infants, RAPD analysis traced a Candida pelliculosa fungemia outbreak to a single strain [21].

MALDI-TOF Mass Spectrometry Typing

MALDI-TOF mass spectrometry protein fingerprinting was recently assessed on C. parapsilosis [22], C. glabrata [23], and C. albicans [24]. As the technique proved highly reliable for rapid and high throughput species-level identification, this identification tool is very useful for fungal outbreak investigations. However, further studies are required before it can be used for fungal subspecies typing.

Multilocus Microsatellite Typing

Multilocus microsatellite markers genotyping, also referred as short tandem repeats (STRs) or multiple-locus variable number tandem repeat analysis (MLVA), is a very powerful and reproducible technique that was used to investigate the spread of C. albicans within a surgical intensive care unit. Analysis of three polymorphic microsatellite markers ruled out an outbreak caused by a unique C. albicans genotype, thereby demonstrating the absence of cross-transmission among patients [25]. Microsatellite typing analysis proved effective in detecting C. parapsilosis outbreaks [3, 26–28].

Multilocus Sequence Typing

In 2006, Viviani et al. have reported the first application of multilocus sequence typing (MLST) to investigate a suspected outbreak of C. albicans bloodstream infections within one hospital ward. MLST analysis demonstrated that eight bloodstream infections were caused by a unique genotype, which was endemic in the ward over the course of a 4-year period [29].

Whole-Genome Sequence Typing

As high throughput sequencing facilities are now widely available, whole-genome sequence analysis is feasible, and whole-genome single nucleotide polymorphisms typing (WGST) can be used, instead of MLST, to infer the genetic relatedness of fungal isolates. Whole-genome sequencing provides maximal discriminatory power and has the advantage of revealing genomic polymorphisms that may be associated with strain pathogenicity. WGST was used to investigate a recent nationwide Saprochaete clavata outbreak in France. Whole-genome sequence data enabled the design of a clade-specific genotyping method, which detected a particular clone involved in the outbreak, and allowed for a case-case study analyzing the risk factors associated with infection linked with this specific clone [30•].

rRNA Intergenic Spacer Region Typing

Outbreaks of Pichia anomala fungemia among pediatric patients were assessed using IGS-PCR, a PCR-based method targeting the intergenic spacer region 1 (IGS1) of the ribosomal RNA (rRNA) gene. The genome comprises approximately 100 rRNA gene copies that are repeated in tandem head-to-tail with non-transcribed IGS regions in between. IGS-PCR discriminates between rRNA gene IGS region length polymorphisms. This technique confirmed the point source of a P. anomala outbreak [31].

Repetitive Sequence-Based PCR

Trichosporon asahii is an emerging opportunistic agent involved in invasive infections, primarily affecting immunocompromised patients. An outbreak of urinary tract infections following urinary catheter manipulations was investigated via repetitive sequence-based PCR (rep-PCR) using the DiversiLab system, which amplifies many different-sized DNA amplicons within the non-coding, repetitive sequences of the genome. The rep-PCR fingerprinting profiles identified four genotypic clusters, although no specific epidemiological relationship could be demonstrated [32].

Other Rare Yeast Outbreaks

Blastoschizomyces capitatus isolates from four neutropenic patients and their respective environment were analyzed using four molecular typing methods: PFGE, RFLP, RAPD, and polymerase chain reaction fingerprinting using a single primer specific for one minisatellite or two microsatellite DNAs. This health care-associated outbreak was traced to clonal dissemination of a B. capitatus strain from vacuum flasks used for milk distribution [33].

Cryptococcus gattii is an emerging human and animal pathogen in Canada and the US Pacific Northwest region. MLST was used to identify four major C. gattii lineages (VGI to VGIV) by analyzing the genetic loci URA5, IGS1, LAC1, CAP59, GPD1, PLB1, and SOD1. A C. gattii type VGIIa and VGIIb outbreak started in 1999 in Vancouver Island (Canada) and subsequently spread to mainland British Columbia and the states of Washington and Oregon (USA) [34]. Currently, species harboring the VGIIc genotype have only been isolated in Oregon [35].

Dermatophytosis

Dermatophytes may be transmitted via shared use of contaminated fomites or direct physical contact with an infected person. Tinea capitis outbreaks occur among communities of children, especially in schools. Molecular typing of Microsporum canis isolates collected from a group of children and their environment, using RAPD, PCR amplification of sub-repeat elements in the rRNA non-transcribed spacer (NTS), and the rRNA intergenic transcribed spacer (ITS) sequence analysis, highlighted the genetic similarity of all M. canis isolates. The study showed that fomites, such as carpets and pillowcases, played an important role in outbreak spread [36]. The anthropophilic dermatophyte Trichophyton tonsurans remains the major agent of dermatophytoses among American children. Using MLST, a T. tonsurans outbreak among health-care workers at a pediatric hospital was traced to a single genotype [37]. T. tonsurans is also a recognized cause of tinea gladiatorum in individuals practicing combat sports. An outbreak of T. tonsurans tinea capitis gladiatorum among wrestlers at a boarding school in Turkey was analyzed using a MLST scheme based on 24 sequence variations within 12 loci. The results revealed the clonal nature of the strains involved in the outbreak [5].

Aspergillosis

Aspergillosis is a serious threat for immunocompromised patients. Vonberg and Gastmeier reviewed a total of 53 invasive hospital-based aspergillosis outbreaks from 1966 to 2005. Construction work or renovation activities within the hospital or in surrounding areas were most commonly considered to be the probable cause of the Aspergillus outbreaks, followed by a contaminated or defective air supply system [38]. Many molecular typing techniques were used to investigate the source of Aspergillus spp. outbreaks. RAPD (R-108 and AP12h) analysis was performed on Aspergillus fumigatus strains isolated from patients in a hematology ward presenting with invasive aspergillosis. The study showed a substantial genetic variation among the strains, thereby indicating that the outbreak was not due to a common source within the hospital [39, 40]. In contrast, a separate study applying RAPD (R108 primer) typing of Aspergillus flavus isolates found identical genotypes in isolates from the environment and patients, which suggested that the clinical infection likely originated from the hospital environment [41]. RAPD (R108, CII, and P4) assays were also used to type Aspergillus terreus [42] and Aspergillus ustus isolates [43, 44].

Using IGS-PCR, Radford et al. identified up to three different DNA types in the tissues of patients with invasive A. fumigatus infection, and genetic relatedness suggested that some of these infections were acquired from the hospital environment [45].

The A. fumigatus short tandem repeats (STRAf) assay proved very efficient in investigating A. fumigatus invasive aspergillosis outbreaks in recent years [46, 47]. In a heart surgery unit, STRAf showed that patients were primarily infected by a unique genotype and/or colonized by a few genotypes, in contrast to the highly heterogeneous genetic background of the A. fumigatus environmental isolates [48]. To our knowledge, the A. fumigatus MLST scheme [49] was not yet used to investigate an outbreak.

A. flavus is only second to A. fumigatus as the major cause of human invasive aspergillosis worldwide. However, it represents the first etiological agent of human invasive aspergillosis in Saudi Arabia, Tunisia, Sudan, and other African countries [50, 51]. EcoRI- or PstI-RFLP was applied to analyze A. flavus isolates in two pediatric hematology-oncology patients. The probe pAF28 was used to trace two cases of cutaneous A. flavus infection among low-birth-weight infants in a neonatal intensive care unit to contaminated material (especially the tape used to fasten umbilical catheters) in the ambulance [52].

A 12-microsatellite marker STR typing scheme used to type clinical and environmental A. flavus isolates to reveal evidence of hospital-acquired colonization and infection in a hematology unit in Tunisia [53]. STR typing was used to investigate A. flavus endocarditis cases in a heart surgery unit. This study demonstrated the persistence of a single genotype that was involved in three endocarditis episodes over 6 six years in the ward, which contrasted with the highly heterogeneous genetic background of the environmental strains [54•].

A. terreus infection prognosis is relatively poor due to amphotericin B resistance. Inter-simple sequence repeat PCR analysis of A. terreus isolates from the USA and Europe demonstrated that one clade comprised exclusively of isolates from Europe and another was enriched with isolates from the USA [55].

Fusarium Outbreaks

Fusarium keratitis is a serious corneal infection most commonly associated with corneal injury [56]. A 66-case fungal keratitis outbreak caused by fungi of the Fusarium solani species complex was identified. Epidemiological investigations revealed improper contact lens wear and specific contact lens solutions as risk factors. The high genetic heterogeneity of the isolates confirmed that this was not a point source outbreak [57]. Similarly, multilocus sequence analysis identified up to 10 different species of Fusarium spp. involved in a keratitis epidemic [58].

Other Filamentous Fungi

RAPD typing of a rare and difficult to diagnose Rhizopus microsporus intestinal infection outbreak indicated that the primary source was likely to be the cornstarch used in the manufacturing of allopurinol tablets or ready-to-eat food [59]. To our knowledge, an MLST scheme devised to analyze Penicillium marneffei was never used for outbreak investigation [60]. RAPD typing traced an Exophiala jeanselmei outbreak to contaminated hospital water [61]. Exserohilum rostratum, a dematiaceous mold species rarely involved in human infection, caused an outbreak that affected 751 patients in the USA (http://www.cdc.gov/hai/outbreaks/meningitis.html; accessed 2015/08/12). This outbreak was traced to injections of contaminated methylprednisolone acetate. The clinical presentation of the cases included meningitis, stroke, paraspinal/spinal infections, and peripheral joint infections. WGST showed that the outbreak strains were highly clonal. Furthermore, isolates from the two different lots of methylprednisolone acetate were also indistinguishable from each other and from the isolates derived from the case patients, thereby suggesting that the source of the contamination was persistent [1•].

Pneumocystis jirovecii

RFLP analysis was used to describe three outbreaks of P. jirovecii pneumonia in renal transplant patients. The study showed that a single strain responsible for pneumonia in transplant patients in Switzerland and Germany was different from the strain that caused an outbreak in Japan [62]. Another study involving transplant recipients provided evidence of an outbreak profile, which was most likely associated with inter-human transmission [2]. Typing using mitochondrial large-subunit (mtLSU) rRNA gene sequence analysis demonstrated the presence of P. jirovecii in exhalation from colonized patients [63]. A MLST scheme was recently devised, based on the analysis of the upstream conserved sequence region, mtLSU rRNA, and dihydrofolate reductase genes [64]. MLST was used to highlight an outbreak of this infection in a nephrology unit [65•] and among renal transplant patients [66]. Additionally, a six-marker STR-based typing assay showed good discriminatory power [67•], but was not used for a P. jirovecii outbreak investigation.

General Discussion and Conclusion

Fungal outbreaks are usually traced to a common source. If the source involves an unusually high level of environmental fungi, the outbreak is likely to be caused by different species of fungi. However, because fungal species display very heterogeneous levels of virulence, only one or two of these species will cause invasive fungal disease in a person who was exposed to multiple fungal species.

A characteristic of yeast and dermatophyte infection is the potential to propagate via patient-to-patient, patient-to-health-care worker, or health-care worker-to-patient transmission routes. During dermatophytoses outbreak investigations in schools or health-care facilities, precise species identification is generally sufficient and typing of isolates is not always required. Molecular typing recently demonstrated airborne patient-to-patient transmission of the non-cultivable P. jirovecii fungus involved in pneumonia outbreaks [63]. In contrast, airborne patient-to-patient transmission was never documented in mold-associated respiratory tract infections. Mold infections are usually traced to an environmental source, which is primarily airborne contamination or, less frequently, waterborne or foodborne contamination. For example, in fusariosis, multiple species and multiple genotypes (within a particular species) were involved in the keratitis epidemic that was associated with the use of specific contact lens solutions. Similarly, typing was helpful to identify multiple strains involved in aspergillosis outbreaks resulting from increased airborne environmental fungal levels following construction work or renovation activities within the hospital or in surrounding areas. However, STR-based typing enabled tracing the cause of endocarditis cases to a unique A. flavus genotype found in a cardiac operating room, in which contamination episodes occurred 6 years apart [54•].

The origin of health care-associated fungal infections was often traced to contaminated medical devices. Even fungi that exhibit relatively low virulence and are rarely involved in human diseases can cause disease outbreaks if they are inoculated directly into the host body along with the contaminated medication. One such scenario includes the recent E. rostratum epidemic that was associated with injections of contaminated steroids [1•]. In any case, typing enables discrimination between a clonal or multiple-strain contamination and identification of the contamination source.

A wide range of fungi-typing techniques are available, which vary in throughput, cost, processing time and discriminatory power (Table 1). The method used depends on the epidemiological question and the available laboratory equipment. Fingerprinting techniques with lower reproducibility and lower discriminatory power can be useful to investigate outbreaks that are limited in space and time. In this regard, MALDI-TOF mass spectrometry was recently proposed for the protein fingerprinting of related isolates within Candida species. Exact typing methods that yield unambiguous results are highly advantageous as they produce reproducible and exchangeable data. Currently, the two popular exact typing methods, which are both reproducible and discriminatory, are MLST- and STR-based typing. WGST emerged as a new typing method that will be increasingly accessible in the near future. Exact typing methods are suited to investigate epidemics at a larger scale; in particular to compare isolates involved in outbreaks or cases that occurred at different places and/or time periods.

Table 1 Primary findings of the typing analyses used to investigate fungal outbreaks in various patients’ populations
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A thorough understanding of fungal diseases, including insight into the reservoir, contamination route, and diagnostic methods, is central to identify the cause and implement appropriate measures to control fungal outbreaks. Invasive fungal diseases are relatively rare compared to many epidemic viral or bacterial diseases. With some exceptions, fungal disease outbreaks usually affect a relatively limited number of cases in small human communities, e.g., at a scale of at-risk patients in a particular hospital ward.