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ORIGINAL CONTRIBUTIONS

Received: August 27^th 2015

Accepted: February 18^th 2016

Frequency of invasive fungal infections in a Mexican High-Specialty Hospital. Experience of 21 years

Luis Javier Méndez-Tovar,^a Jessica Aline Mejía-Mercado,^b Patricia Manzano-Gayosso,^c Francisca Hernández-Hernández,^c Rubén López-Martínez,^c Israel Silva-González^d

^aLaboratorio de Investigación Médica en Dermatología y Micología, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social

^bMedical Communications Department, PPD México

^cUnidad de Micología, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México

^dLaboratorio Central, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social

Ciudad de México, México

Communication with: Luis Javier Méndez-Tovar

Telephone: 5627 6900, extensión 21480

Email: ljmt@unam.mx; ljmendez@alestra.net.mx

Background: Factors such as cancer, HIV infection, use of corticosteroids and antibiotics, favors the increase in the number of invasive fungal infections (IFI) worldwide.

Methods: To determine the frequency and epidemiological aspects of IFI at a mexican, a review of the proven cases diagnosed over the past 21 years (1993-2013) in the Laboratory of Medical Mycology was performed.

Results: A total of 472 cases were identified as: 261 candidiasis, 82 mucormycosis, 60 cryptococcosis, 43 aspergillosis and 16 histoplasmosis. A decrease in the frequency of candidiasis was observed, with 74 cases in the first 6 years and 48 in the last five. C. albicans was the most common agent and pulmonary infection the most prevalent. Cryptococcosis also declined from 24 to 10 cases, mainly caused by C. neoformans; two cases of C. laurentii and C. terreus and C unigutulatus were isolated once. Mucormycosis remained steady, but aspergillosis increased significantly, and from 2 cases found in the first studied period, it rose to 23 in the last one.

Conclusions: It is important that High Specialty Hospitals have well-equipped laboratories of Medical Mycology. We suggest the creation of a National Reference Center for Mycoses to collect all the data of these infections, in order to help to the development of strategies for health education, prevention, diagnosis and treatment of them. Keywords: Bacterial Infections and Mycoses; Mycoses; Infection Introduction: Factors such as cancer, HIV infection, and the use of steroids and antibiotics, increase the number of invasive fungal infections (IFI). Methods: To determine the frequency and epidemiological aspects of IFI at an IMSS hospital, proven cases diagnosed in the last 21 years (1993-2013) at the Laboratorio de Micología Médica were reviewed.

Keywords: Bacterial infections and mycoses; Mycoses; Infection

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The definition of invasive fungal infections (IFI) was developed in 2002 based on joint work between the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group, the National Institute of Allergy and Infectious Diseases, and the Mycoses Study Group (EORTC/MSG).^1,2 The term encompasses fungal infections caused by yeast, filamentous, or dimorphic fungi, which spread through the blood and affect various organs.^3,4 These infections are severe and sometimes fatal. Fever is one of the main clinical manifestations.^2,3,5 Its optimal management depends on successful clinical diagnosis, isolation and identification of the causative agent, and early, specific, and timely treatment.⁶

According to the criteria issued by EORTC/MSG, published in 2008, IFIs are classified as proven, probable, and possible.2 Proven infections require demonstration of fungal elements in infected tissues by: microscopic analysis (histopathology, cytopathology, or direct examination), culture of the etiologic agent, or immunological tests of high specificity and sensitivity. Probable infections require the host to present one or more of the most often associated predisposing factors (neutropenia, transplant recipients, prolonged use of corticosteroids or other immunosuppressive therapies and inherited immunodeficiency, clinical data suggestive of fungal infection, and mycological evidence). Finally, possible infections are considered cases that have risk factors for the host and sufficient and consistent with clinical evidence of IFI, even without mycological evidence.² 

IFIs also can be grouped into two clinical and mycological categories: opportunistic and endemic. The most common opportunistic fungal agents are Candida spp., Cryptococcus neoformans, Pneumocystis jirovecii, Aspergillus spp., and fungi of the order Mucorales. The main agents of endemic invasive fungal infections in North America are Histoplasma capsulatum, Coccidioides immitis, C. posadasii, and Blastomyces dermatitidis.^3,4

Risk factors for developing IFI are neutropenia, bone marrow or solid organ transplantation, hematologic malignancies, prolonged use of high doses of corticosteroids, chemotherapy, HIV infection, treatment with new immunosuppressive agents, malnutrition, extended stay in intensive care units, major surgery, advanced age, and comorbidities such as diabetes mellitus associated with ketoacidosis, cirrhosis, and prematurity.^3,4,6 In the case of aspergillosis, the number of cases of invasive infection increases in frequency when renovations are done in hospitals or in the vicinity, due to the increase in the number of suspended fungal particles.⁴  

In highly specialized hospitals with facilities for Transplant, Hematology, Intensive Care, Peritoneal Dialysis, Rheumatology, etc., the frequency of these infections has been steady increasing because patients simultaneously have several predisposing factors.^3,7 Patients in these units often develop fever of unknown origin. In a study published in 2012 conducted in patients of this hospital with febrile syndrome, this was caused by fungal infections in up to a third of cases.⁷

Mortality in patients with IFI is high and is associated with delayed diagnosis, treatment, or the severity of the disease.^5,8,9 In recent years there has been a new factor that increases mortality: therapeutic failure due to resistance to antimycotics. Pathogenic fungi or fungal contaminants can develop antifungal resistance from repeated exposure to antifungal compounds, as has been demonstrated in various studies on Candida, Cryptococcus, and Aspergillus;^4,10,11 however, some species of fungal contaminants such as Fusarium have primary resistance to antifungal agents.

The aim of this study was to determine the frequency of major IFI in patients at the Hospital de Especialidades, Centro Médico Nacional Siglo XXI of IMSS over 21 years, and to find out some of the epidemiological characteristics of cases and associated factors.

Methods

The registration of IFI diagnosed in the Laboratorio de Micología Médica between January 1993 and December 2013 were reviewed. 

The study included only IFI cases proven by: a) direct examination with KOH and smears of specimens stained with Gram or Ziehl-Neelsen techniques; b) cultures in Sabouraud dextrose agar with and without antibiotics; c) observation of fungal tissue fragments stained with hematoxylin-eosin, periodic acid-Schiff, or silver impregnation. In some cases tissues were macerated with isotonic saline for direct examination, smear, and culture.

The identification of filamentous fungi was determined by macro and microscopic morphology, and in some cases microcultures. The identification of yeasts was done by macro- and microscopic morphology, serum hyphal testing, chromogenic medium culture (CHROMagar), biochemical assays by automated system (Vitek, Biomerieux), or commercial kits (API-20 AUX® and APIC-32, Biomerieux).

The creation of this research did not include the participation of human subjects or animals. The review of records, lab results, and reports of biopsy studies maintained patient confidentiality at all times.

Results

472 IFI were diagnosed during the period 1993-2013; the most frequent were: candidiasis, mucormycosis, cryptococcosis, and aspergillosis (Figure 1).

The number of cases recorded in the study period is shown in Table I. Cases of candidiasis and cryptococcosis showed a significant decrease, from 74 and 24 cases respectively between 1993-1998, to only 48 candidosis and 10 cryptococcosis in the period 2009-2013. Mucormycosis remained at similar figures, while aspergillosis increased significantly: 2 cases were recorded in the period 1993-1998, and 23 in the later period.

Table II shows the location of the 261 cases of candidosis. The main sites of infection were the lung and peritoneum. The biological products with the most positivity in Candida infections were bronchoalveolar lavage (BAL) fluid, peritoneal dialysis fluid, and tissue fragments. Most patients had serious blood disorders (aplastic anemia, leukemia) or bone marrow transplantation. The most common causative agent was Candida albicans (164 cases, 63%); other less frequent species were C. tropicalis (21 cases, 7.9%), C. glabrata (18 patients, 7.1%), C. parapsilosis (10 cases, 3.9%), C. krusei (6 cases, 2.4%), C. famata (3 cases), C. lusitaniae and C. humicola with two cases each. In 35 patients only the genus of the causative agent was determined.

The most frequent filamentous fungal infection was mucormycosis (82 cases), of which 79 (96 3%) were rhinocerebral and three pulmonary (Figure 2). 85.3% of patients (70 cases) had diabetic ketoacidosis, eight had immunosuppression from various causes (renal disease, treatment with steroids, autoimmune diseases, etc.), and in four cases the only factor identified was old age (> 65 years). Of the 82 patients, 37 (45.12%) died, and of the survivors, 40 (88.9%) had permanent sequelae due to surgery or extensive surgical debridement. Direct examination and culture were positive in 42 patients (51.2%); in the remaining 40 patients the diagnosis was established by histological examination and/or direct examination. In cultures, the genus most frequently identified was Rhizopus sp. (26 cases, 31.7%), followed by Mucor sp. (7 cases, 8.53%). Moreover, an isolation was obtained that corresponded to Syncephalastrum sp. and another to Cunninghamella sp.; in 7 cases filamentous fungi with coenocytic mycelium were developed with no useful morphological structures to identify genus.

Another case of infection caused by yeast was cryptococcosis, which in the study period constituted 12.7% (60 cases) of the total IFI. Of the 60 isolates of Cryptococcus, 56 corresponded to C. neoformans, two to C. laurentii, one to C. terreus, and one to C. unigutulatus; these last two species were isolated in 1995 (Figure 3), the patients were male, the predisposing factor was AIDS, and the infection only spread to the central nervous system.

43 cases of aspergillosis were diagnosed, most patients were hospitalized in Hematology, Transplantation, and Intensive Care. Seven were recorded in 2009 and coincided with the remodeling of the hospital. Aspergillus fumigatus was identified in 10 patients (23.2%), A. flavus in 10, and A. niger in four cases (9.3%). In 19 isolates it was only possible to determine the genus.

The most frequent endemic mycosis was histoplasmosis (16 cases). Nine of the patients were infected with HIV; five had autoimmune diseases and received high-power corticosteroids, and two patients had a diagnosis of febrile syndrome in testing with no known predisposing factor. One patient with histoplasmosis simultaneously presented herpes simplex virus infection with extensive skin ulcers on the buttocks. In the histopathological examination of skin biopsy, few yeasts were observed, while bone marrow culture developed countless colonies of Histoplasma capsulatum (Figure 4).

The invasive fungal infections recorded least often were coccidioidomycosis, pneumocystis, alternariosis, and scedosporiosis. The four cases of coccidioidomycosis were positive for culture and histopathology; ED was positive in two cases (one lung and one hand abscess). Pneumocystis pneumonia cases were diagnosed by direct immunofluorescence using BAL; one of these patients had Crohn's disease and in addition to the steroids common in the treatment of this disease, was treated with adalimumab. The two cases of alternariosis and the case of scedosporiosis were located in the lungs. The diagnosis of alternariosis was based on pigmented hyphae in LBA ED and the growth of pigmented colonies, where microscopic examination showed the dichondra characteristic of the genus. Scedosporiosis was diagnosed by the presence of hyaline hyphae in the LBA ED and culture; this microscopically showed the abundant annelloconidia typical of the genus.  

Discussion

Worldwide the number of cases of IFI caused by opportunistic fungi such as Candida spp., Cryptococcus spp., Aspergillus spp., and Pneumocystis jirovecii has increased,^4,12-14 however, there is no accurate epidemiological data on the incidence or overall prevalence in the world; in Mexico, as in other developing countries, there are no records on the overall incidence of MI, since fungal infection does not have mandatory reporting.^12,14,15

The present study shows that this group of diseases is underestimated; in Mexico, IFI are not considered within the national health priorities. The number of cases in this casuistry is high, as evidenced by the 261 cases of candidosis; however, they do not correspond to reality, due to the lack of resources for innovative, specific, sensitive, and rapid diagnostic techniques. In many patients with suspected IFI, the culture was negative and no immunological tests were done to confirm the diagnosis. Among the techniques that have demonstrated good sensitivity and specificity are enzyme immunoassay (ELISA), useful for the diagnosis of aspergillosis and candidiasis, which detect biomarkers such as 1,3-β-D-glucan or galactomannan; direct immunofluorescence for Pneumocystis carinii, and in the last decade, molecular techniques whose usefulness has been demonstrated in patients with candidiasis and aspergillosis.^5,9,16 

Regarding candidosis agents, although C. albicans prevailed, there was a notable increase of other species such as C. tropicalis, C. glabrata, and C. parapsilosis, which caused 18.9% of these infections, similar to other reports.¹⁷ This change of agents is important because some species such as C. glabrata develop secondary resistance easily, while others like C. krusei are intrinsically resistant to fluconazole.^18,19

In patients with rhinocerebral mucormycosis, mortality similar to that reported in the literature was observed, close to 46%.²⁰ The prognosis of mucormycosis depends both on appropriate treatment and the speed with which patients are sent to a specialty care center to treat comorbidities. However, despite medical treatment, patients generally require extensive surgery on the face, mouth, eyes, and upper respiratory tract, which decreases the quality of life in approximately 90% of patients.

Reports from international literature tell that posaconazole, an antifungal in the azole group, is very effective against these agents and of low toxicity for patients, so it is a therapeutic alternative to amphotericin B.^21,22 However, in Mexico the availability of this azole is limited, even in highly specialized hospitals.

The cryptococcosis cases detected in this study reflect the global epidemiological trend. The number of patients remained high until 1998, gradually decreasing to account for only 10 cases in the period 2009-2013. This decrease was due to a better control of HIV infection using HAART, which was the main factor predisposing these patients.⁴  

Until the end of the last century, C. neoformans was believed worldwide to be the main and almost sole agent of this infection, with two recognized varieties: C. neoformans var. neoformans and C. neoformans var. gattii. Morphological, physiological, and genetic studies of both varieties showed that they are different species;¹⁴ this study detected no isolated C. gattii, but four cases of infection were identified by agents very seldom reported to cause infection, C. laurentii (two cases), C. terreus, and C. unigutulatus, always with AIDS as the predisposing factor. These agents can be considered emerging species along with others such as C. liquefaciens and C. albidus that have been reported in other studies.^23,24

Aspergillosis is a rare disease in the population of this hospital. The increase in cases observed in 2009 was due to remodeling work in various areas of the hospital, which has been shown in other research to generate dispersion of conidia, causing serious infections in patients with severe immunosuppression.⁴

In conclusion, this study makes clear the urgent need for all specialty hospitals that care for patients with severe immunosuppression of any etiology, to have medical mycology laboratories and the trained personnel and materials needed to perform microbiological, immunological, and molecular diagnosis, as well as antifungal susceptibility studies to indicate the best treatment. Finally, it is necessary to create a National Reference Center for Mycoses, to gather all data on these infections and help developing plans for health education, prevention, diagnosis, and treatment of fungal infections.

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