Hanseníase como uma Zoonose - Dra. Patrícia Deps

Aula sobre hanseníase como uma zoonose ministrada pela Prof. Dra. Patrícia Deps durante o 11º Simpósio Brasileiros de Hansenologia

Harnessing Clinical Trial Capacity to Mitigate Zoonotic Diseases: The Role of Expert Scientists in Ethiopia.

Background: The emergence and resurgence of zoonotic diseases have continued to be a major threat to global health and the economy. Developing countries are particularly vulnerable due to agricultural expansions and domestication of animals with humans. Scientifically sound clinical trials are important to find better ways to prevent, diagnose, and treat zoonotic diseases, while there is a lack of evidence to inform the clinical trials' capacity and practice in countries highly affected with the diseases. This study aimed to investigate expert scientists' perceptions and experiences in conducting clinical trials toward zoonotic diseases in Ethiopia. Methods: This study employed a descriptive, qualitative study design. It included major academic and research institutions in Ethiopia that had active engagements in veterinary and public health researches. It included the National Veterinary Institute, the National Animal Health Diagnostic and Investigation Center, the College of Veterinary Medicine at Addis Ababa University, the Ethiopian Public Health Institute, the Armauer Hansen Research Institute, and the College of Health Sciences at Addis Ababa University. In-depth interviews were conducted with expert scientists. Data were collected from October 2019 to April 2020. Data analysis was undertaken using open code 4.03 for qualitative data analysis. Results: Five major themes, with 18 sub-themes, emerged from the in-depth interviews. These were: challenges in the prevention, control, and treatment of zoonotic diseases; One Health approach to mitigate zoonotic diseases; personal and institutional experiences in conducting clinical trials on zoonotic diseases; barriers in conducting clinical trials toward zoonotic diseases; and strategies that promote conducting clinical trials on zoonotic diseases. Conducting clinical trials on zoonotic diseases in Ethiopia is hampered by a lack of clearly articulated ethics and regulatory frameworks, trial experts, financial resources, and good governance. Conclusion: In Ethiopia, conducting clinical trials on zoonotic diseases deserves due attention. Strengthening institutional and human resources capacity is a pre-condition to harness effective implementation of clinical trials on zoonotic diseases in the country. In Ethiopia where skilled human resource is scarce, One Health approach has the potential to form multidisciplinary teams to systematically improve clinical trials capacity and outcomes in the country.

Zoonotic parasites infecting free-living armadillos from Brazil.

Armadillos are specialist diggers and their burrows are used to find food, seek shelter and protect their pups. These burrows can also be shared with dozens of vertebrate and invertebrate species and; consequently, their parasites including the zoonotics. The aim of this study was to diagnose the presence of zoonotic parasites in four wild-caught armadillo species from two different Brazilian ecosystems, the Cerrado (Brazilian savanna) and the Pantanal (wetland). The investigated parasites and their correspondent diseases were: Toxoplasma gondii (toxoplasmosis), Trypanosoma cruzi (Chagas disease), Leishmania spp., (leishmaniasis), Paracoccidioides brasiliensis (Paracoccidioidomicosis) and Mycobacterium leprae (Hansen's disease). Forty-three free-living armadillos from Pantanal and seven road-killed armadillos from the Cerrado were sampled. Trypanosoma cruzi DTU TcIII were isolated from 2 out of 43 (4.65%) armadillos, including one of them also infected with Trypanosoma rangeli. Antibodies anti-T. gondii were detected in 13 out of 43 (30.2%) armadillos. All seven armadillos from Cerrado tested positive for P. brasiliensis DNA, in the lungs, spleen, liver fragments. Also, by molecular analysis, all 43 individuals were negative for M. leprae and Leishmania spp. Armadillos were infected by T. cruzi, T. rangeli, P. brasiliensis and presented seric antibodies to T. gondii, highlighting the importance of those armadillos could have in the epidemiology of zoonotic parasites.

Zoonotic risk of Hansen's disease from community contact with wild armadillos: A systematic review and meta-analysis.

Understanding and quantifying the risk of Hansen's disease (HD) through zoonotic transmission of Mycobacterium leprae infection from wild armadillos is important because hunting, handling and consumption of these animals is widespread in communities where HD is endemic, posing a potential threat to the health of individuals and to HD elimination. We conducted a systematic review (PROSPERO CRD42019159891) of publications in MEDLINE, EMBASE, Global Health, Scopus, LILACS, Biblioteca Digital Brasileira de Teses e Dissertações, Catálogo de Teses e Dissertações de CAPES, and Biblioteca Virtual em Saúde up to 09/05/2020 using Mesh and text terms in English, Portuguese, Spanish and French. Random effects meta-analyses were performed including of subgroups by endemicity and type of exposure. Seven of the nine included studies were case-control, four from Brazil and three from the USA, comprising 1,124 cases and 2,023 controls in total. The other two studies, one from Brazil and one from Colombia, were cross-sectional. The overall summary estimate (odds ratio, OR) for the relative odds of HD comparing people who had direct contact with armadillos and/or had eaten armadillo meat with those who had not was OR = 2.60 (95% CI 1.78-3.80, p < .001) with a predictive interval of OR = 1.10-6.17. Summary odds ratios for specific exposures were as follows: indirect contact, OR = 1.39 (95% CI 1.02, 1.89) (p = .04); eating, OR = 2.29 (95% CI 1.13, 4.66) (p = .02); hunting, OR = 2.54 (95% CI 1.21, 5.33) (p = .01). Most of the included studies had moderate risk of bias. Crude estimates were reduced by up to 24% when adjusted for confounders (where reported). Direct contact with wild armadillos was strongly associated with an increased risk of HD, whilst evidence for an increased risk of HD from indirect contact was weaker. The fraction of HD in endemic countries attributable to zoonotic transmission from armadillos remains unknown, but the precautionary principle needs to be adopted to protect public health.

Prevalence of Mycobacterium leprae in armadillos in Brazil: A systematic review and meta-analysis.

Understanding the prevalence of M. leprae infection in armadillos is important because of evidence from Brazil and other countries of an association between contact with armadillos and the development of Hansen's Disease (leprosy). Our aim was to characterize studies which have investigated natural M. leprae infection in wild armadillos in Brazil, and to quantify and explore variability in the reported prevalence of infection. We conducted a systematic review (PROSPERO CRD42019155277) of publications in MEDLINE, EMBASE, Global Health, Scopus, LILACS, Biblioteca Digital Brasileira de Teses e Dissertações, Catálogo de Teses e Dissertações de CAPES, and Biblioteca Virtual em Saúde up to 10/2019 using Mesh and text search terms (in English, Portuguese, Spanish, and French). The 10 included studies represented a total sample of 302 armadillos comprising 207 (69%) Dasypus novemcinctus, 67 (22%) Euphractus sexcinctus, 16 (5%) Priodontes maximus, 10 (3%) Cabassous unicinctus, and 2 (1%) Cabassous tatouay from 7 different states. Methods used included histopathology (4 studies), PGL-1 and LID-1 antigen detection (4 studies) and examination for clinical signs of disease (4 studies). Eight studies used PCR of which 7 targeted the RLEP repetitive element and 3 tested for inhibitory substances. M. leprae prevalence by PCR ranged from 0% (in 3 studies) to 100% in one study, with a summary estimate of 9.4% (95% CI 0.4% to 73.1%) and a predictive interval of 0-100%. The average prevalence is equivalent to 1 in 10 armadillos in Brazil being infected with M. leprae, but wide variation in sample estimates means that the prevalence in any similar study would be entirely unpredictable. We propose instead that future studies aim to investigate transmission and persistence of M. leprae within and between armadillo populations, meanwhile adopting the precautionary principle to protect human health and an endangered species in Brazil.

Serological and molecular detection of infection with Mycobacterium leprae in Brazilian six banded armadillos (Euphractus sexcinctus).

Leprosy was recognized as a zoonotic disease, associated with nine-banded armadillos (Dasypus novemcinctus) in the Southern United States of America in 2011. In addition, there is growing evidence to support a role for armadillos in zoonotic leprosy in South America. The current study evaluated twenty specimens of the six-banded armadillo (Euphractus sexcinctus), collected from rural locations in the state of Rio Grande do Norte (RN), Brazil for evidence of infection with Mycobacterium leprae. Serum was examined using two "in-house" enzyme-linked immunosorbent assays (ELISAs) and via two commercially available (ML flow and NDO-LID®) immunochromatographic lateral flow (LF) tests, for detection of the PGL-I and/or LID-1 antigens of the bacterium. The presence of M. leprae DNA in liver tissue was examined using the multi-copy, M. leprae-specific repetitive element (RLEP), as target in conventional and nested PCR assays. Molecular and anti-PGL-I-ELISA data indicated that 20/20 (100 %) of the armadillos were infected with M. leprae. The corresponding detection levels recorded with the LF tests were 17/20 (85 %) and 16/20 (85 %), for the NDO-LID® and ML flow tests, respectively. Our results indicate that, in common with D. novemcinctus, six banded armadillos (a species hunted and reared as a food-source in some regions of Brazil, including RN), represent a potential reservoir of M. leprae and as such, their role in a possible zoonotic cycle of leprosy within Brazil warrants further investigation.

Lack of evidence for the presence of leprosy bacilli in red squirrels from North-West Europe.

Leprosy is a human infectious disease caused by Mycobacterium leprae or Mycobacterium lepromatosis that can also occur in animals and even manifest as zoonosis. Recently, both mycobacteria were detected in red squirrels (Sciurus vulgaris) from the British Isles. To further explore the presence of leprosy bacilli in North-West Europe, we screened Belgian and Dutch squirrels. Tissue samples from 115 animals tested by qPCR were negative for both pathogens. No molecular or pathological evidence was found of the presence of these zoonotic pathogens in North-West Europe.

Leprosy survey among rural communities and wild armadillos from Amazonas state, Northern Brazil.

There is evidence that in southern US, leprosy is a zoonosis infecting wild Dasypus novemcinctus armadillos but the extent of this finding is unknown. This ecological study investigated leprosy in rural communities and in wild armadillos from the Brazilian Amazon. The study area was the Mamiá Lake of Coari municipality, Amazonas State, Northern region, a hyper endemic leprosy area where residents live on subsistence farming, fishing and armadillo hunting and its meat intake are frequent. The leprosy survey was conducted in sixteen communities by a visiting team of specialists. Local partakers provided wild armadillos to investigate M. leprae infection. Volunteers had complete dermato-neurological examination by a dermatologist with expertise in leprosy diagnosis, suspect skin lesions were biopsied for histopathology (Hematoxylin-eosin/HE, Fite-Faraco/FF staining); slit skin smears were collected. Armadillos' tissue fragments (skins, spleens, livers, lymph nodes, adrenal glands, others) were prepared for histopathology (HE/FF) and for M. leprae repetitive element-RLEP-qPCR. Among 176 volunteers, six new indeterminate leprosy cases were identified (incidence = 3.4%). Suspect skin sections and slit skin smears were negative for bacilli. Twelve wild D. novemcinctus were investigated (48 specimens/96 slides) and histopathological features of M. leprae infection were not found, except for one skin presenting unspecific inflammatory infiltrate suggestive of indeterminate leprosy. Possible traumatic neuroma, granuloma with epithelioid and Langhans cells, foreign-body granuloma were also identified. Granulomatous/non-granulomatous dermatitides were periodic-acid-Schiff/PAS negative for fungus. M. leprae-RLEP-qPCR was negative in all armadillos' tissues; no bacillus was found in histopathology. Our survey in rural communities confirmed the high endemicity for leprosy while one armadillo was compatible with paucibacillary M. leprae infection. At least in the highly endemic rural area of Coari, in the Brazilian Amazon region where infectious sources from untreated multibacillary leprosy are abundant, M. leprae infected armadillos may not represent a major source of infection nor a significant public health concern.

Leprosy survey among rural communities and wild armadillos from Amazonas state, Northern Brazil

There is evidence that in southern US, leprosy is a zoonosis infecting wild Dasypus novemcinctus armadillos but the extent of this finding is unknown. This ecological study investigated leprosy in rural communities and in wild armadillos from the Brazilian Amazon. The study area was the Mamia´ Lake of Coari municipality, Amazonas State, Northern region, a hyper endemic leprosy area where residents live on subsistence farming, fishing and armadillo hunting and its meat intake are frequent. The leprosy survey was conducted in sixteen communities by a visiting team of specialists. Local partakers provided wild armadillos to investigate M. leprae infection. Volunteers had complete dermato-neurological examination by a dermatologist with expertise in leprosy diagnosis, suspect skin lesions were biopsied for histopathology (Hematoxylin-eosin/HE, Fite-Faraco/FF staining); slit skin smears were collected. Armadillos' tissue fragments (skins, spleens, livers, lymph nodes, adrenal glands, others) were prepared for histopathology (HE/FF) and for M. leprae repetitive elementRLEP-qPCR. Among 176 volunteers, six new indeterminate leprosy cases were identified (incidence = 3.4%). Suspect skin sections and slit skin smears were negative for bacilli. Twelve wild D. novemcinctus were investigated (48 specimens/96 slides) and histopathological features of M. leprae infection were not found, except for one skin presenting unspecific inflammatory infiltrate suggestive of indeterminate leprosy. Possible traumatic neuroma, granuloma with epithelioid and Langhans cells, foreign-body granuloma were also identified. Granulomatous/non-granulomatous dermatitides were periodic-acid-Schiff/ PAS negative for fungus. M. leprae-RLEP-qPCR was negative in all armadillos' tissues; no bacillus was found in histopathology. Our survey in rural communities confirmed the high endemicity for leprosy while one armadillo was compatible with paucibacillary M. leprae infection. At least in the highly endemic rural area of Coari, in the Brazilian Amazon region where infectious sources from untreated multibacillary leprosy are abundant, M. leprae infected armadillos may not represent a major source of infection nor a significant public health concern.

Evidence of zoonotic leprosy in Pará, Brazilian Amazon, and risks associated with human contact or consumption of armadillos.

Mycobacterium leprae (M. leprae) is a human pathogen and the causative agent for leprosy, a chronic disease characterized by lesions of the skin and peripheral nerve damage. Zoonotic transmission of M. leprae to humans by nine-banded armadillos (Dasypus novemcinctus) has been shown to occur in the southern United States, mainly in Texas, Louisiana, and Florida. Nine-banded armadillos are also common in South America, and residents living in some areas in Brazil hunt and kill armadillos as a dietary source of protein. This study examines the extent of M. leprae infection in wild armadillos and whether these New World mammals may be a natural reservoir for leprosy transmission in Brazil, similar to the situation in the southern states of the U.S. The presence of the M. leprae-specific repetitive sequence RLEP was detected by PCR amplification in purified DNA extracted from armadillo spleen and liver tissue samples. A positive RLEP signal was confirmed in 62% of the armadillos (10/16), indicating high rates of infection with M. leprae. Immunohistochemistry of sections of infected armadillo spleens revealed mycobacterial DNA and cell wall constituents in situ detected by SYBR Gold and auramine/rhodamine staining techniques, respectively. The M. leprae-specific antigen, phenolic glycolipid I (PGL-I) was detected in spleen sections using a rabbit polyclonal antibody specific for PGL-I. Anti-PGL-I titers were assessed by ELISA in sera from 146 inhabitants of Belterra, a hyperendemic city located in western Pará state in Brazil. A positive anti-PGL-I titer is a known biomarker for M. leprae infection in both humans and armadillos. Individuals who consumed armadillo meat most frequently (more than once per month) showed a significantly higher anti-PGL-I titer than those who did not eat or ate less frequently than once per month. Armadillos infected with M. leprae represent a potential environmental reservoir. Consequently, people who hunt, kill, or process or eat armadillo meat are at a higher risk for infection with M. leprae from these animals.

Differential growth of Mycobacterium leprae strains (SNP genotypes) in armadillos.

Leprosy (Hansen's Disease) has occurred throughout human history, and persists today at a low prevalence in most populations. Caused by Mycobacterium leprae, the infection primarily involves the skin, mucosa and peripheral nerves. The susceptible host range for Mycobacterium leprae is quite narrow. Besides humans, nine banded armadillos (Dasypus novemcinctus) and red squirrels (Sciurus vulgaris) are the only other natural hosts for M. leprae, but only armadillos recapitulate the disease as seen in humans. Armadillos across the Southern United States harbor a single predominant genotypic strain (SNP Type-3I) of M. leprae, which is also implicated in the zoonotic transmission of leprosy. We investigated, whether the zoonotic strain (3I) has any notable growth advantages in armadillos over another genetically distant strain-type (SNP Type-4P) of M. leprae, and if M. leprae strains manifest any notably different pathology among armadillos. We co-infected armadillos (n = 6) with 2 × 109 highly viable M. leprae of both strains and assessed the relative growth and dissemination of each strain in the animals. We also analyzed 12 additional armadillos, 6 each individually infected with the same quantity of either strain. The infections were allowed to fulminate and the clinical manifestations of the disease were noted. Animals were humanely sacrificed at the terminal stage of infection and the number of bacilli per gram of liver, spleen and lymph node tissue were enumerated by Q-PCR assay. The growth of M. leprae strain 4P was significantly higher (P < 0.05) than 3I when each strain was propagated individually in armadillos. Significantly (P < 0.0001) higher growth of the 4P strain also was confirmed among animals co-infected with both 3I and 4P strain types using whole genome sequencing. Interestingly, the zoonotic strain does not exhibit any growth advantage in these non-human hosts, but the varied proliferation of the two M. leprae strains within armadillos suggest there are notable pathological variations between M. leprae strain-types.

Zoonotic Leprosy in the Southeastern United States.

Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and have been implicated in zoonotic transmission of leprosy. Early studies found this disease mainly in Texas and Louisiana, but armadillos in the southeastern United States appeared to be free of infection. We screened 645 armadillos from 8 locations in the southeastern United States not known to harbor enzootic leprosy for M. leprae DNA and antibodies. We found M. leprae-infected armadillos at each location, and 106 (16.4%) animals had serologic/PCR evidence of infection. Using single-nucleotide polymorphism variable number tandem repeat genotyping/genome sequencing, we detected M. leprae genotype 3I-2-v1 among 35 armadillos. Seven armadillos harbored a newly identified genotype (3I-2-v15). In comparison, 52 human patients from the same region were infected with 31 M. leprae types. However, 42.3% (22/52) of patients were infected with 1 of the 2 M. leprae genotype strains associated with armadillos. The geographic range and complexity of zoonotic leprosy is expanding.

Current knowledge on Mycobacterium leprae transmission: a systematic literature review.

UNLABELLED: Summary BACKGROUND: The transmission pathways of Mycobacterium leprae are not fully understood. Solid evidence exists for an increased risk for individuals living in close contact with leprosy patients but the existence of zoonotic leprosy, environmental reservoirs and trauma-related transmission has also been established. PURPOSE: To assess the current state of knowledge on M. leprae transmission, we conducted a systematic review of the peer-reviewed literature pertaining to this topic. METHOD: Major electronic bibliographic databases were searched for relevant peer-reviewed articles published up to January 2014. No restrictions on study types, participants and location were applied, and all outcomes demonstrated to contribute to the transmission of M. leprae were considered. Included studies were grouped by mode of transmission, namely (i) human-to-human via aerosols or direct contact; (ii) direct inoculation (e.g. injury); and (iii) transmission to humans from environmental or zoonotic reservoirs, and by insects. The importance of the different transmission pathways and the strength of the evidence were assessed considering the number of publications describing similar findings, the consistency of the findings and the methodological quality of the studies. RESULTS: A total of 79 relevant articles were retained out of 3,805 hits resulting from the application of the search strategy. Solid evidence for transmission among contacts exists, and for zoonotic leprosy in the southern States of the USA. Based on the extant evidence, skin-to-skin contact, aerosols/droplets and shedding of bacteria into the environment and subsequent infection, e.g. through dust or small wounds, all remain possible options. CONCLUSION: No study has unequivocally demonstrated the mechanisms by which M. leprae bacteria travel from one case of leprosy to another.

El camino a la eliminación: un panorama de las enfermedades infecciosas desatendidas en América Latina y el Caribe / The road to elimination: an overview of neglected infectious diseases in Latin America and the Caribbean

Las enfermedades infecciosas desatendidas (EID) afectan, principalmente, a las poblaciones que viven en condiciones aisladas y socioeconómicas de pobreza. Estas enfermedades, por su naturaleza crónica y silenciosa, aquejan con frecuencia a comunidades con voz política débil. Lo anterior se traduce en muy poca atención o prioridad política; lo que se refleja en mínimas e insuficientes acciones de prevención, vigilancia y control. Sin embargo, hay evidencia de que la situación está cambiando favorablemente en algunos países de las Américas. En los últimos años, varias resoluciones (acuerdos oficiales de los países miembros de la Organización Panamericana de la Salud/Organización Mundial de la Salud- OPS/OMS), convenios regionales y mundiales en materia de salud pública, junto a un mayor compromiso por parte de la industria farmacéutica, y otros donantes y socios internacionales, en combinación con el desarrollo y uso de los planes de acción integrados, han permitido que los países intensifiquen las intervenciones públicas hacia el control de estas enfermedades y así alcanzar los objetivos de eliminación de las EID. La oncocercosis, la filariasis linfática, la enfermedad de Chagas, la lepra y la ceguera por tracoma, entre otras, han sido eliminadas en varios países o zonas endémicas, sin importar el nivel de desarrollo del país o área geográfica donde se localizan. La voluntad política reflejada en los recursos financieros suficientes en la próxima década, serán determinantes para lograr los objetivos regionales y nacionales de eliminación de las EID...

Neglected infectious diseases (NID) affect mainly isolated populations living in isolation and in poor socioeconomic conditions. These diseases, by their chronic and silent nature, often affect communities with a weak political voice. This translates into very little attention or political priority; which is reflected in minimal and insufficient preventive measures, monitoring and control. However, there is evidence this situation is changing favorably in some countries of the Americas. In recent years, several resolutions (official agreement of the member countries of the Pan American Health Organization / World Health Organization-PAHO / WHO), regional and global agreements on public health; with a greater commitment from the pharmaceutical industry, and other donors and international partners in combination with the development and use of integrated action plans have allowed countries to intensify public interventions to control these diseases and thus achieve target elimination of NID. Onchocerciasis, lymphatic filariasis, Chagas disease, leprosy and trachoma blindness, among others, have been eliminated in several countries and endemic areas, regardless of the level of development of the country or geographical area where they are located. The political decision reflected in adequate financial resources in the next decade will be crucial to achieving the goals of elimination of NID (regional and national)...

China's sustained drive to eliminate neglected tropical diseases.

Non-communicable diseases dominate the public health arena in China, yet neglected tropical diseases (NTDs) are still widespread and create a substantial burden. We review the geographical distribution, prevalence, and epidemic characteristics of NTDs identified in China caused by helminths, protozoa, bacteria, and viruses. Lymphatic filariasis was eliminated in 2007, but schistosomiasis still affects up to 5% of local village residents in some endemic counties with around 300 000 people infected. China harbours more than 90% of the world's burden of alveolar echinococcosis and food-borne zoonoses are emerging. In 2010, the overall prevalence of soil-transmitted helminth infections caused by Ascaris lumbricoides, Trichuris trichiura, and hookworm was 11·4%, with 6·8% of these infections caused by A lumbricoides. Corresponding figures for food-borne trematodiasis, echinococcosis, and cysticercosis are more than 5%. Dengue, leishmaniasis, leprosy, rabies, and trachoma exist in many areas and should not be overlooked. Transmission of vector-borne diseases can be interrupted; nevertheless, epidemics occur in remote areas, creating a challenge for surveillance and control. Rigorous surveillance, followed by immediate and integrated response packages tailored to specific social and ecological systems, is essential for progress towards the elimination of NTDs in China.

Lobomycosis: risk of zoonotic transmission from dolphins to humans.

Lobomycosis, a fungal disease of the skin and subcutaneous tissues caused by Lacazia loboi, is sometimes referred to as a zoonotic disease because it affects only specific delphinidae and humans; however, the evidence that it can be transferred directly to humans from dolphins is weak. Dolphins have also been postulated to be responsible for an apparent geographic expansion of the disease in humans. Morphological and molecular differences between the human and dolphin organisms, differences in geographic distribution of the diseases between dolphins and humans, the existence of only a single documented case of presumed zoonotic transmission, and anecdotal evidence of lack of transmission to humans following accidental inoculation of tissue from infected dolphins do not support the hypothesis that dolphins infected with L. loboi represent a zoonotic hazard for humans. In addition, the lack of human cases in communities adjacent to coastal estuaries with a high prevalence of lobomycosis in dolphins, such as the Indian River Lagoon in Florida (IRL), suggests that direct or indirect transmission of L. loboi from dolphins to humans occurs rarely, if at all. Nonetheless, attention to personal hygiene and general principals of infection control are always appropriate when handling tissues from an animal with a presumptive diagnosis of a mycotic or fungal disease.