Histoplasma capsulatum develops in bird excrement or bat guano in closed areas (caves, mines, tunnels) or in open spaces (parks, orchards, and abandoned homes, among others). The organic material found in these waste materials under humid conditions and optimal temperatures establishes the ecological niche for the mycelial phase of development, and microconidium constitutes the infectious form of the fungus 1 2 3 . There have been three distinct genotypes and varieties of H. capsulatum, and two have infected human beings: H. capsulatum var. capsulatum, and H. capsulatum var. duboisii; in addition, one has infected equines (H. capsulatum var. farciminosum). These exhibited different clinical manifestations and geographical distributions 4 5 6 . However, a phylogenetic study by Kasuga and colleagues 7 8 was conducted in an attempt to resolve the relationships among the major classes and three varieties of H. capsulatum. At least eight clades were identified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade, and (viii) African clade. Seven of the eight clades represented genetically isolated groups that may be recognized as phylogenetic species.

This fungus is the etiological agent of systemic mycosis histoplasmosis. The mycosis is an endemic disease of tropical, subtropical, and temperate areas throughout the world. Histoplasmosis has been described as present in all of the 32 states of the Mexican Republic, with highly variable prevalence 3 9 10 11 12 13 14 15 16 17 18 19 . Veracruz is one of the federal entities with the greatest number of cases reported 18 . In Mexico, histoplasmosis is considered an occupational disease that especially affects miners, farmers, guano collectors, cockfight breeders, geologists, speleologists, anthropologists, and biologists. Primary pulmonary histoplasmosis (PPH) is the most relevant clinical form of the disease with the highest mortality rate worldwide 10 11 18 20 . The clinical manifestations of this mycosis may range widely, from being clinically benign to severe, even fatal, depending upon the amount of propagules inhaled, the immunological state of the infected host, and the strain's virulence 3 . It is important to report outbreaks of histoplasmosis in order to know the current status of this disease, identify new endemic areas, and for molecular characterization of the causative agents. This study reports on an outbreak of histoplasmosis in treasure hunters in the endemic zone of Veracruz and the molecular characterization of two isolates from these patients.

In Mexico, the first probable outbreak of histoplasmosis was described and recorded in a Public Health Acts ledger from the state of Nuevo León at the end of the 19^th century (ca. 1885) when certain clinical scenarios were described that led to the suspicion that there was an epidemic of this mycosis in mine workers from areas containing a large amount of bat guano. In 1943, Martínez-Báez reported the first proven case of histoplasmosis in Mexico, while the first officially described outbreak was observed in subjects who had entered into "El Refugio", an abandoned mine located in Lampazos, Nuevo León; both data were referred by Aguirre-Pequeño 34 .

From studies by Aguirre-Pequeño 34 and González-Ochoa 9 10 11 12 13 , histoplasmosis in Mexico is considered an occupational disease because a relationship has been established between severe Primary pulmonary histoplasmosis (PPH) in miners exposed to high concentrations of H.-capsulatum propagules from bat guano. Histoplasmosis outbreaks have reported with a death rate as high as 100% in affected individuals 18 19 20 . Despite that the number of histoplasmosis outbreaks continues to rise, there are no official records of the majority of these. The most recent outbreak reported occurred in Spring-Break student tourists in Acapulco between March and May 2001 35 . During this outbreak, H. capsulatum was isolated from compost employed as fertilizers in ornamental plants at the hotel where the students stayed 17 .

The results of our study show that the patients had acquired histoplasmosis in the town of Tamarindos, Ciudad Cardel, Veracruz (Figure 1). Patients' signs and symptoms were clearly associated with chest x-ray images and chest CT scans. The etiological agent was clearly identified utilizing macro- and micromorphology and assessment of anti-Histoplasma antibodies, and IDR, and its identification was confirmed by PCR (Figure 2a). In addition, using RAPD-PCR, the two isolates were observed to possess the same polymorphic pattern based on their genetic material, indicating that the patients were infected with the same H. capsulatum strain prevalent at this site (Figures 2b and 2c).

In this study, five of the six patients developed disseminated histoplasmosis. The severity of the infection in these patients can be attributed to factors such as i) extreme physical effort, ii) inhalation of a large number of infectious propagules (when digging to find a treasure, the moving of the bat and bird excreta found at the infection site), iii) long exposure time (patients remained for 3 full days excavating and sleeping at the same site), iv) low socioeconomic status of the workers with severe nutritional and immunological deficiencies, and v) a probable high virulence of the strains present at the infection site. The EH-53 reference strain is highly virulent, proceeding from HG, Mexico, and isolated from a patient with fatally disseminated histoplasmosis. Although the molecular characterization of isolates was not carried out in full, 100% similarity (observed in the dendrogram) between the two VZ isolates and the EH-53 strain (Figure 3), and the severity of the patients' clinical problems, could suggest the presence of the strain in this geographic area. Several authors have reported a high virulence rate of H. capsulatum strains in Mexico 10 11 18 20 36 .

Molecular typification of the two isolates from this outbreak was compared with the polymorphic DNA patterns of strains collected from VZ-neighboring areas. EVP and LFD isolates presented 100% similarity to the EH-53 HG strain (Figure 3), which was isolated in 1977; therefore, the two isolates are considered as probably the same strain. The EH-53 strain is included in the Lam A clade (forming a monophyletic group for Latin America) according to Kasanuga et al. 8 . Due to that these isolates were not characterized by four genes as utilized by Kasuga because we did not possess the appropriate primers, it was not possible to perform a definitive classification.

The Ciudad Cardel, La Antigua, Municipality, Veracruz (Figure 1), endemic zone where patients acquired the infection is located in the central coastal zone of the state of Veracruz at coordinates 19°22' latitude North and 96° 22' longitude West, and at an altitude of 20 m above sea level. Its climate is warm with an average temperature of 25°C. Average annual rainfall is 1,500 mm. In this municipality, fauna is mainly comprised of rabbits, foxes, coyotes, armadillos, raccoons, and bats; reptiles such as rattlesnakes, migratory birds, and other birds are often found to be H. capsulatum reservoirs. The house where the patients slept was surrounded by trees in which bats and migratory birds roosted overnight. Maintenance of this strain in the environment, in the neighboring areas of both HG and VZ, could probably be explained by the very important role that bat guano plays in spreading H. capsulatum, as reported by Taylor et al. 16 , in which the authors mention that H. capsulatum propagules can be dispersed at different distances. In general, it appears that bats are the ideal candidates for spreading H. capsulatum in both short, as well as in long, distances. The cave-colonizing behavior of bats, their ability to fly, and their habit of remaining in the same caves for long periods are important factors that explain the dynamics behind the dispersion of the fungus in nature. For example, infected bats can act as dispersers of the parasite and incorporate the fungus into new, favorable environments, possibly through cadavers 15 . Additionally, the wide diversity of the fauna present, which is characteristic of the infectious site, also contributes importantly to maintenance of H. capsulatum in the environment.

The therapeutic scheme administered to three patients was amphotericin B deoxycholate (0.7 mg/kg/day every 7 days), and methylprednisone (1 mg/kg/day every 7 days), prior to administering amphotericin B, to avoid collateral effects; afterward, itraconazole 600 mg/day was administered for 3 days, and finally, itraconazole 400 mg/day was given every 12 weeks), while the remaining three patients were only administered itraconazole (200 mg/day every 12 weeks). Symptoms and lesions in all patients resolved. The symptomatology of the febrile respiratory disease is not specific for pulmonary histoplasmosis, and on occasion, a mistaken diagnosis is reached, with subsequent administration of the inappropriate treatment.

The two isolates were observed as possessing the same polymorphic pattern, indicating that the patients were infected with the same H. capsulatum strain prevalent at this site. It is important to remain updated on recent outbreaks of histoplasmosis and the manner of exposure to the fungi, as well as to emphasize molecular characterization of isolates located in this endemic zone. The severity of cases indicates that these strains are highly virulent, and it is probable that these isolates are prevalent in Hidalgo and Veracruz states. People residing in or visiting this endemic area should be aware of the health risks of histoplasmosis infection to which they might be subjected, to as well as health care personnel for them to exercise the necessary preventive measures.

The authors declare that they have no competing interests.

BM Participated in study design and coordination and helped to draft the manuscript. MAM Participated in sequence alignment and drafted the manuscript. GP Performed the serologic tests and obtained the samples. AR Participated in the clinical studies and patient data case studies. MGF Conducted the molecular studies. MRR Performed and interpreted the molecular studies. MLT Assisted in characterization of isolates with reference strains. ALH Participated in clinical and case studies and performed data analysis. AC Obtained the samples and performed the data analysis. MEM Conceived of the study, participated in its design, and drafted the manuscript.

All authors read and approved the final manuscript.