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Morphological aspects of Paracoccidioides yeast. (a) Paracoccidioides lutzii showing multiple buds with a "steering wheel" shape in vitro (scanning electron microscopy); (b) The positive direct mycological examination of pus showing large yeasts (5-15 µm) that have a thick, birefringent cell wall with single or multiple buds; (c) Paracoccidioides spp. in tissue (arrows) stained by hematoxylin and eosin (HE); (d) Paracoccidioides spp. in tissue stained by Grocott's methenamine silver (GMS) showing a "Mickey Mouse" shape (arrow).
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Paracoccidioidomycosis (PCM) is a mycotic disease caused by the Paracoccidioides species, a group of thermally dimorphic fungi that grow in mycelial form at 25 °C and as budding yeasts when cultured at 37 °C or when parasitizing the host tissues. PCM occurs in a large area of Latin America, and the most critical regions of endemicity are in Brazil,...
Contexts in source publication
Context 1
... reference method for the diagnosis of PCM is established by the finding of fungal elements suggestive of Paracoccidioides spp. in microbiological examinations of sputum or other clinical specimens such as skin scrapings or lymph node aspiration material after 10% KOH clarification with or without calcofluor [37]. The positive direct mycological examination of large yeasts (5-15 µm) that have a thick, birefringent cell wall with single or multiple buds (Figure 4b). These multiple buds have a "steering wheel" or "Mickey Mouse" shape and are considered pathognomonic findings in the diagnosis of PCM [37] (Figure 4). ...
Context 2
... positive direct mycological examination of large yeasts (5-15 µm) that have a thick, birefringent cell wall with single or multiple buds (Figure 4b). These multiple buds have a "steering wheel" or "Mickey Mouse" shape and are considered pathognomonic findings in the diagnosis of PCM [37] (Figure 4). Culture-based and biochemical methods are available for the isolation and characterization of Paracoccidioides [37]. ...
Context 3
... examination is a useful tool for detecting the pathogen in tissues, as it allows us to identify the etiological agent down to the genus level in 95% of cases [38]. The fungus's pathognomonic structures can be visualized in its yeast-like phase by experienced observers and accurate observation after routine staining such as with hematoxylin and eosin (HE) (Figure 4c). Nevertheless, the use of specific stains, such as PAS (periodic acid-Schiff) and silver-based stains, such as Grocott's methenamine silver (GMS) (Figure 4d), must be included to improve detection. ...
Context 4
... fungus's pathognomonic structures can be visualized in its yeast-like phase by experienced observers and accurate observation after routine staining such as with hematoxylin and eosin (HE) (Figure 4c). Nevertheless, the use of specific stains, such as PAS (periodic acid-Schiff) and silver-based stains, such as Grocott's methenamine silver (GMS) (Figure 4d), must be included to improve detection. ...
Context 5
... reference method for the diagnosis of PCM is established by the finding of fungal elements suggestive of Paracoccidioides spp. in microbiological examinations of sputum or other clinical specimens such as skin scrapings or lymph node aspiration material after 10% KOH clarification with or without calcofluor [37]. The positive direct mycological examination of large yeasts (5-15 µm) that have a thick, birefringent cell wall with single or multiple buds (Figure 4b). These multiple buds have a "steering wheel" or "Mickey Mouse" shape and are considered pathognomonic findings in the diagnosis of PCM [37] (Figure 4). ...
Context 6
... positive direct mycological examination of large yeasts (5-15 µm) that have a thick, birefringent cell wall with single or multiple buds (Figure 4b). These multiple buds have a "steering wheel" or "Mickey Mouse" shape and are considered pathognomonic findings in the diagnosis of PCM [37] (Figure 4). Culture-based and biochemical methods are available for the isolation and characterization of Paracoccidioides [37]. ...
Context 7
... examination is a useful tool for detecting the pathogen in tissues, as it allows us to identify the etiological agent down to the genus level in 95% of cases [38]. The fungus's pathognomonic structures can be visualized in its yeast-like phase by experienced observers and accurate observation after routine staining such as with hematoxylin and eosin (HE) (Figure 4c). Nevertheless, the use of specific stains, such as PAS (periodic acid-Schiff) and silver-based stains, such as Grocott's methenamine silver (GMS) (Figure 4d), must be included to improve detection. ...
Context 8
... fungus's pathognomonic structures can be visualized in its yeast-like phase by experienced observers and accurate observation after routine staining such as with hematoxylin and eosin (HE) (Figure 4c). Nevertheless, the use of specific stains, such as PAS (periodic acid-Schiff) and silver-based stains, such as Grocott's methenamine silver (GMS) (Figure 4d), must be included to improve detection. ...
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Introduction:
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Methods:...
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Paracoccidioidomycosis (PCM) is an endemic fungal infection in Latin America, which manifests as an acute
or chronic form and is more frequent in adult males. It is caused by Paracoccidioides brasiliensis or
Paracoccidioides lutzii, which are thermodimorphic fungi. The disease can present as a severe and
disseminated form involving the lungs, skin,...
Citations
... Although classical laboratory techniques provide valuable information by visualizing the fungal pathogen Paracoccidioides spp, they do not have a signi cant discriminatory power on the type of species. Several PCR-based methods can detect polymorphisms in the DNA of Paracoccidioides and thus support species identi cation, mainly sequencing and taxonomic analysis, allowing the location of the strain as originating from the endemic region of Sao Paulo, P. brasiliensis complex [39][40]. The results of the phylogenetic reconstruction using gp43 sequences con rmed the diagnosis of the etiologic agent, and the grouping of the two sequences obtained from different tissues suggested a systemic infection. ...
Background
Paracoccidioidomycosis (PCM) is a neglected deep mycosis caused by Paracoccidioides sp. We describe a fatal PCM case, presenting as osteomyelitis, in a man having frequent contact with an endemic region of São Paulo, Brazil.
Case presentation
A 67-year-old man who lived in an urban area, had frequent fishing trips to an endemic region. He presented with osteomyelitis of the femur and iliac artery thrombosis at hospital admission. Thoracic CT revealed multiple cavitated lung nodules. The patient rapidly progressed to irreversible respiratory failure. The autopsy revealed disseminated PCM and thrombosis of the iliac artery. Laboratory investigation confirmed a P. brasiliensis infection with phylogenetic results revealing sequences recovered from patient samples grouped with sequences characterized as P. brasiliensis complex.
Conclusion
Atypical PCM remains a diagnostic challenge. Increased awareness of contagion sites and different clinical presentations will lead to improved patient management.
... El diagnóstico en el laboratorio está dirigido a la visualización del hongo en muestras clínicas o su aislamiento mediante cultivo; la cuantificación de anticuerpos o la detección de antígenos del hongo en el suero, pueden considerarse como criterios diagnósticos indirectos (10). ...
... We developed a triplex-probe assay targeting the Paracoccidioides rDNA region (ITS2/28S) to overcome this problem of detecting DNA from isolated cultures to clinical samples. Our qPCR assay is an important tool for understanding epidemiological patterns, mapping the affected areas, and guiding early treatment of patients affected by the disease [49]. ...
... The species boundaries in Paracoccidioides impact the development of diagnostic tests [49]. Thus, despite good PCR-based tools available in the literature [4,10,11,[50][51][52][53], our assay has the advantage of covering the recent taxonomic developments in the genus [1,4,17,18,54]. ...
... Moreover, judging from the clinical perspective, speciating members of the P. brasiliensis complex has no impact on patient management [5,6,8,[62][63][64]. Therefore, differentiating between members of the P. brasiliensis complex and P. lutzii seems to be, at this moment, the safest and most cost-effective strategy for diagnosing classic PCM [1,33,49]. ...
Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the “steering wheel” or “Mickey Mouse” shape in the direct mycological examination, fresh biopsied tissue in 10% KOH, histopathological analysis, and/or the isolation of the fungus in culture. However, these procedures are time-consuming and do not allow for the speciation of Paracoccidioides due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the P. brasiliensis complex and P. lutzii. Primers (Paracoco-F and Paracoco-R) and TaqMan probes (PbraCx-Fam, Plu-Ned, and Paracoco-Vic) were developed to target the rDNA (ITS2/28S) in the Paracoccidioides genome. A panel of 77 Paracoccidioides isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human and murine DNA. The lower limit of detection was 10 fg of gDNA and three copies of the partial rDNA amplicon. Speciation using qPCR was in perfect agreement with AFLP and TUB1-RFLP markers (kappa = 1.0). As a proof of concept, we assessed a panel of 16 formalin-fixed and paraffin-embedded specimens from histopathologically confirmed PCM patients to reveal a significant sensitivity of 81.25% and specificity of 100% (AUC = 0.906 ± 0.05, 95% CI = 0.756–0.979, p < 0.0001, Youden index J = 0.8125). Our assay achieved maximum sensitivity (100%) and specificity (100%) using fresh clinical samples (n = 9) such as sputum, bronchoalveolar lavage, and tissue fragments from PCM patients (AUC = 1.0, 95% CI 0.872–1.000, p < 0.0001, Youden index J = 1.0). Overall, our qPCR assay simplifies the molecular diagnosis of PCM and can be easily implemented in any routine laboratory, decreasing a critical bottleneck for the early treatment of PCM patients across a vast area of the Americas.
... Molecular techniques can detect biomarkers such as DNA, RNA, and gene products from etiologic agents of the most varied diseases. Considering Paracoccidioides spp., several molecular techniques are used for the detection of fungi from soil samples in urban and rural environments and aerosols, helping and making it easier for ecological studies and geographical tracking [37]. However, the same may not be valid for clinical routine, since many variables must be considered. ...
... Additionally, the taxonomy of PCM agents went through several changes since the discovery of the disease at the beginning of the last century, stimulated by the massive morphological diversity [38,39] and differences in the genetic features of some isolates. Such characterizations have intensified in recent years by introducing molecular methods for the genetic differentiation of fungi, such as multilocus sequencing analysis [8,[11][12][13] or whole-genome sequencing analysis [37,40,41]. Sequencing followed by phylogenetic analysis is considered the gold standard for the species identification of Paracoccidioides, but it may present limitations depending on the locus used [42]. ...
Paracoccidioidomycosis (PCM) is the main and most prevalent systemic mycosis in Latin America, that until recently, it was believed to be caused only by Paracoccidioides brasiliensis (P. brasiliensis). In 2006, researchers described three cryptic species: S1, PS2, PS3, and later, another one, PS4. In 2009, Paracoccidioides lutzii (Pb01-like) was described, and in 2017, a new nomenclature was proposed for the different agents: P. brasiliensis (S1), P. americana (PS2), P. restrepiensis (PS3), and P. venezuelensis (PS4). These species are not uniformly distributed throughout Latin America and, knowing that more than one cryptic species could coexist in some regions, we aimed to identify those species in patients’ biopsy samples for a better understanding of the distribution and occurrence of these recently described species in Botucatu region. The Hospital of Medical School of Botucatu—UNESP, which is a PCM study pole, is located in São Paulo State mid-west region and is classified as a PCM endemic area. Genotyping analyses of clinical specimens from these patients that have been diagnosed and treated in our Hospital could favor a possible correlation between genetic groups and mycological and clinical characteristics. For this, molecular techniques to differentiate Paracoccidioides species in these biopsies, such as DNA extraction, PCR, and sequencing of three target genes (ITS, CHS2, and ARF) were conducted. All the sequences were analyzed at BLAST to testify the presence of P. brasiliensis. The phylogenetic trees were constructed using Mega 7.0 software and showed that 100% of our positive samples were from S1 cryptic species, therefore P. brasiliensis. This is important data, demonstrating the predominance of this species in the São Paulo State region.
... Concerning the recognition of Paracoccidioides species, several "in-house" PCR-based methods have been developed for the recognition of Paracoccidioides species, including nested PCR [138], quantitative real-time PCR [64], PCR-RFLP (restriction fragment length polymorphism) [139], and multilocus microsatellite typing (MLMT) [63]. However, the best method for Paracoccidioides spp. ...
... In recent decades, advances have been made in the development of new molecular techniques for the detection of Paracoccidioides DNA in clinical samples, including amplification of ribosomal DNA region (rDNA), gene-expressing GP43 glycoprotein, and quantitative Chain Reaction Polymerase (qPCR) platform duplex PCR assay [63,138,151,152]. The implementation of new methodologies based on photonic techniques and machine learning algorithms has been suggested, such as Fourier Transform Infrared (FTIR) and Raman spectroscopy [148,153,154]. ...
Coccidioidomycosis (CM) and paracoccidioidomycosis (PCM) are systemic mycoses that are highly endemic in Latin America and have recently been included on the World Health Organization (WHO) Fungal Priority Pathogens List. Coccidioides immitis and Coccidioides posadasii are recognized as etiological agents of CM, with peculiarities in their geographic distribution. The genus Paracoccidioides now includes Paracoccidioides lutzii and the Paracoccidioides brasiliensis complex, which encompasses four phylogenetic species. In both diseases, pulmonary signs and symptoms are the main reasons for patients to seek medical assistance, and they are frequently misdiagnosed as tuberculosis. In this paper, we present a critical view of the strategies for diagnosis and clinical management of CM and PCM. Over the past few decades, there has been an increase in the number of reports of endemic fungal infections in areas previously thought to be “non-endemic” due to climate change and increased travel, among other factors. Learning to recognize their main epidemiological aspects and clinical manifestations is crucial so that clinicians can include them in the differential diagnosis of lung disease and avoid late diagnosis.
... From an epidemiological perspective, we can profit from distinguishing Paracoccidioides lineages (Fig. 1G) [11]. Paracoccidioides brasiliensis is the major agent recovered from Southeastern and Southern Brazil, Argentina, Bolivia, Paraguay, Peru, Uruguay, Venezuela, and Guadeloupe Islands [12]. ...
... While molecular diagnostic for PCM is still scarce [11], proteomics (MALDI-ToF) and genotyping tools like PCR-fingerprinting, mating-typing, AFLP fingerprinting, microsatellite typing, multi-locus sequence analysis (e.g., ITS, ARF and GP43, Fig. 1D-G), and whole-genome sequencing are appropriate for elucidating genetic relationships and resolving Paracoccidioides taxonomy [1,11,12,[20][21][22][23][24][25][26]. Recently, molecular diagnostics revealed mixed infection due to P. brasiliensis and P. lutzii in humans [20] and uncovered new genotypes embedded in the P. brasiliensis complex [26], supporting that it has become vital to speciate Paracoccidioides. ...
... While molecular diagnostic for PCM is still scarce [11], proteomics (MALDI-ToF) and genotyping tools like PCR-fingerprinting, mating-typing, AFLP fingerprinting, microsatellite typing, multi-locus sequence analysis (e.g., ITS, ARF and GP43, Fig. 1D-G), and whole-genome sequencing are appropriate for elucidating genetic relationships and resolving Paracoccidioides taxonomy [1,11,12,[20][21][22][23][24][25][26]. Recently, molecular diagnostics revealed mixed infection due to P. brasiliensis and P. lutzii in humans [20] and uncovered new genotypes embedded in the P. brasiliensis complex [26], supporting that it has become vital to speciate Paracoccidioides. ...
Paracoccidioidomycosis (PCM) defines a broad spectrum of human and animal diseases caused by Paracoccidioides species (Onygenales). In the twenty-first century, Paracoccidioides advanced from a monotypic taxon to a genus that harbors seven species, including P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, P. lutzii, P. loboi, and P. cetii. Classic PCM, acquired upon inhalation of propagules from P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii, affects the human lungs and may progress to systemic granulomatous disease with tegumentary and visceral involvement. On the other hand, PCM loboi and PCM ceti caused by the unculturable P. loboi and P. cetii are subcutaneous mycoses, typically observed as keloid lesions in humans and dolphins. Such heterogeneity highlights the importance of recognizing species boundaries in Paracoccidioides to gain insights into the ecology, evolution, clinical features, and mitigation strategies to tackle the advance of PCM.
... yeast forms [73]. Other tools, such as cultures, immunodiffusion assays, and polymerase chain reaction (PCR) tests [7][8][9]74,75], are also used. Different types of clinical samples may be collected for testing. ...
... Paracoccidioides cells have a thick mucopolysaccharide wall with a double-contour appearance that is birefringent under light microscopy [83,84]. Paracoccidioides structures resembling a "ship's wheel" or "Mickey Mouse" are deemed pathognomonic findings [9,79,82]. ...
... According to Hahn et al. [78], the successful recovery of Paracoccidioides from clinical specimens depends on several factors, including the culture media and the number of tubes or plates seeded, besides the decontamination with antibiotics of sputum and bronchial lavage fluid. Despite its slowness, culture is always recommended because it allows species identification using molecular biology [9,[94][95][96]. ...
Abstract: Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America caused by
thermodimorphic fungi of the genus Paracoccidioides. In the last two decades, enhanced understanding
of the phylogenetic species concept and molecular variations has led to changes in this genus’
taxonomic classification. Although the impact of the new species on clinical presentation and
treatment remains unclear, they can influence diagnosis when serological methods are employed.
Further, although the infection is usually acquired in rural areas, the symptoms may manifest years
or decades later when the patient might be living in the city or even in another country outside
the endemic region. Brazil accounts for 80% of PCM cases worldwide, and its incidence is rising
in the northern part of the country (Amazon region), owing to new settlements and deforestation,
whereas it is decreasing in the south, owing to agriculture mechanization and urbanization. Clusters
of the acute/subacute form are also emerging in areas with major human intervention and climate
change. Advances in diagnostic methods (molecular and immunological techniques and biomarkers)
remain scarce, and even the reference center’s diagnostics are based mainly on direct microscopic
examination. Classical imaging findings in the lungs include interstitial bilateral infiltrates, and
eventually, enlargement or calcification of adrenals and intraparenchymal central nervous system
lesions are also present. Besides itraconazole, cotrimoxazole, and amphotericin B, new azoles may be
an alternative when the previous ones are not tolerated, although few studies have investigated their
use in treating PCM.
... The lack of high-quality diagnostic tests for PCM infection/disease results in deficient diagnoses and a poor prognosis for this neglected mycosis. Due to the multiple and diverse clinical manifestations of PCM, confirmatory diagnostic tests are needed to identify humans infected or affected by this fungus (283,284). In this scenario, immunoproteomics studies provide a unique opportunity to refine diagnostic tools for this important neglected systemic mycosis by uncovering the significant antigens in human PCM. ...
... Most molecular assays developed for PCM diagnostics employ DNA extracted from cultures (8-10, 32, 291). Sample preparation is a critical step in the molecular detection of PCM, and only a few methods have been developed to detect Paracoccidioides directly from sputum; pus; biopsied fresh tissue or formalinfixed, paraffin-embedded (FFPE) tissue; bronchoalveolar lavage fluid; and cerebrospinal fluid (283,292) (Table 5). This subject has been reviewed by Pinheiro et al. (283). ...
... Sample preparation is a critical step in the molecular detection of PCM, and only a few methods have been developed to detect Paracoccidioides directly from sputum; pus; biopsied fresh tissue or formalinfixed, paraffin-embedded (FFPE) tissue; bronchoalveolar lavage fluid; and cerebrospinal fluid (283,292) (Table 5). This subject has been reviewed by Pinheiro et al. (283). ...
Paracoccidioidomycosis (PCM), initially reported in 1908 in the city of São Paulo, Brazil, by Adolpho Lutz, is primarily a systemic and neglected tropical mycosis that may affect individuals with certain risk factors around Latin America, especially Brazil. Paracoccidioides brasiliensis sensu stricto , a classical thermodimorphic fungus associated with PCM, was long considered to represent a monotypic taxon. However, advances in molecular taxonomy revealed several cryptic species, including Paracoccidioides americana , P. restrepiensis , P. venezuelensis , and P. lutzii , that show a preference for skin and mucous membranes, lymph nodes, and respiratory organs but can also affect many other organs.
... Molecular techniques consist of methods for detecting biomarkers such as DNA, RNA, and gene products of a microorganism [91]. The development of molecular diagnostic techniques requires scientists to use certain criteria for assay success, for example, minimal sample preparation to avoid contamination [92,93]. ...
... This technique quantifies the amount of DNA in the sample, and the results are graphically displayed in real-time as the amplification cycles proceed. The technique is a good alternative to DNA sequencing, as it is cheaper and faster to apply, helping to reduce the time to diagnosis [89,91]. ...
Sporotrichosis is the most important subcutaneous mycosis that affects humans and animals worldwide. The mycosis is caused after a traumatic inoculation of fungal propagules into the host and may follow an animal or environmental transmission route. The main culprits of sporotrichosis are thermodimorphic Sporothrix species embedded in a clinical clade, including S. brasiliensis, S. schenckii, S. globosa, and S. luriei. Although sporotrichosis occurs worldwide, the etiological agents are not evenly distributed, as exemplified by ongoing outbreaks in Brazil and China, caused by S. brasiliensis and S. globosa, respectively. The gold standard for diagnosing sporotrichosis has been the isolation of the fungus in vitro. However, with the advance in molecular techniques, molecular assays have complemented and gradually replaced the classical mycological tests to quickly and accurately detect and/or differentiate molecular siblings in Sporothrix. Nearly all techniques available for molecular diagnosis of sporotrichosis involve PCR amplification, which is currently moving towards detecting Sporothrix DNA directly from clinical samples in multiplex qPCR assays. From an epidemiological perspective, genotyping is key to tracing back sources of Sporothrix infections, detecting diversity in outbreak areas, and thus uncovering finer-scale epidemiological patterns. Over the past decades, molecular epidemiological studies have provided essential information to policymakers regarding outbreak management. From high-to-low throughput genotyping methods, MLSA, AFLP, SSR, RAPD, PCR-RFLP, and WGS are available to assess the transmission dynamics and sporotrichosis expansion. This review discusses the trends in the molecular diagnosis of sporotrichosis, genotyping techniques applied in molecular epidemiological studies, and perspectives for the near future.
... With the advance and improvement of molecular tools, it is possible to identify, reclassify, and recognize new species of fungi, allowing a greater understanding of the biology of these microorganisms [236,237]. Regarding the molecular identification of Sporothrix, some critical points should be considered; among them, we highlight the type of sample (e.g., soil, biopsy, isolated culture, exudate, vegetable), the cost of technique, the time that the process is executed, sensitivity/specificity, and DNA quality [238,239]. The advances in molecular diagnosis of sporotrichosis were recently reviewed by de Carvalho et al. [240]. ...
Sporotrichosis, a human and animal disease caused by Sporothrix species, is the most important implantation mycosis worldwide. Sporothrix taxonomy has improved in recent years, allowing important advances in diagnosis, epidemiology, and treatment. Molecular epidemiology reveals that S. brasiliensis remains highly prevalent during the cat-transmitted sporotrichosis outbreaks in South America and that the spread of S. brasiliensis occurs through founder effects. Sporothrix globosa and S. schenckii are cosmopolitan on the move, causing major sapronoses in Asia and the Americas, respectively. In this emerging scenario, one-health approaches are required to develop a creative, effective, and sustainable response to tackle the spread of sporotrichosis. In the 21st century, it has become vital to speciate Sporothrix, and PCR is the main pillar of molecular diagnosis, aiming at the detection of the pathogen DNA from clinical samples through multiplex assays, whose sensitivity reaches remarkably three copies of the target. The treatment of sporotrichosis can be challenging, especially after the emergence of resistance to azoles and polyenes. Alternative drugs arising from discoveries or repositioning have entered the radar of basic research over the last decade and point to several molecules with antifungal potential, especially the hydrazone derivatives with great in vitro and in vivo activities. There are many promising developments for the near future, and in this review, we discuss how these trends can be applied to the Sporothrix-sporotrichosis system to mitigate the advance of an emerging and re-emerging disease.
