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MEDICINE

The silent attack of the fungi

Species of Candida and Aspergillus cause drug-resistant infections and are more deadly than malaria and tuberculosis

Candida sp. in a Petri dish: infecting almost three million people per year in Brazil

LÉO RAMOSCandida sp. in a Petri dish: infecting almost three million people per year in BrazilLÉO RAMOS

It’s not a freezer, it’s a cage full of dangerous creatures. In metal boxes inside, at negative 80 degrees Celsius, in a laboratory at the Federal University of São Paulo (Unifesp), infectologist Arnaldo Colombo maintains a collection of roughly 4,000 yeasts and 1,000 filamentous fungi sampled from patients treated in hospitals across Brazil. These fungi were considered harmless up to a few years ago, but became aggressive as people’s natural defenses declined due to illness or drugs. Now, they are silently spreading severe, antifungal-resistant, potentially fatal infections.

Almost four million people in Brazil are likely to get a fungal infection each year, according to a survey conducted by Juliana Giacomazzi at the Federal University of Health Sciences in the city of Porto Alegre. Out of that total, 2.8 million are caused by Candida and one million by Aspergillus, which take hold mainly in people whose bodily defenses have been weakened – usually by drugs to prevent rejection of a transplanted organ, treatments for cancer or AIDS, intensive antibiotic use or invasive procedures, such as catheterizations in intensive care units (ICUs).

Worldwide, reported cases of meningitis caused by Cryptococcus neoformans and C. gattii have grown from a few hundred in the 1950s to a million per year today, particularly among HIV/AIDS patients. Fungal pathogens cause an estimated 11.5 million severe infections and recurring superficial mycoses per year, 1.5 million of them fatal – more than the total annual death toll of malaria and tuberculosis.

“Do you know what happened in this case?” asked Colombo, pointing to a Petri dish where various drugs had been applied on samples of a fungus variety, which had recently arrived at his laboratory for identification and analysis. “None of the drugs worked and the patient died from the infection.” Many species of fungi have been proving resistant to the handful of drugs used to combat them. In 2013, the research team at Unifesp pinpointed Candida glabrata as a top concern among hospital-acquired infections because it has proven resistant to almost all antifungal drugs, starting with fluconazole, the most commonly used. This type of yeast infection now has a mortality rate of almost 50% for patients admitted into ICUs. Two species of fungi, Aspergillus fumigatus and Fusarium solani, were isolated in 36 out of 164 samples of water used in the pediatric oncology ward of a hospital in the city of São Paulo. This is an indication that the water supply itself could be a source of contamination, since fungal propagules or spores – reproductive structures similar to seeds – could be transmitted via faucet or shower use.

Aspergillus niger: in the laboratory for genetic identification and drug susceptibility testing

LÉO RAMOSAspergillus niger: in the laboratory for genetic identification and drug susceptibility testingLÉO RAMOS

The average person inhales an estimated 200 to 2,000 spores every day.  They are found not only dispersed in the air, but also inside the human body. “We have millions of colonies of Candida albicans in our mouths and intestines and on our skins, but they only grow and make trouble when our defenses are compromised,” said infectologist Márcio Nucci from the Federal University of Rio de Janeiro (UFRJ). Nucci participated in a study that assessed the occurrence of abdominal infections, caused mostly by C. albicans and C. glabrata, in 481 patients admitted to 13 hospitals in Italy, Spain, Greece and Brazil, from 2011 to 2013. Most of them (85%) had candidiasis. The mortality rate was 60%, which Nucci attributes to the fact that this type of infection normally only afflicts patients who are terminally ill or who have had a late diagnosis of the source of infection, delaying the start of adequate treatment.

In people who have had tuberculosis, fungal infections are easily mistaken for bacterial ones. “Little consideration is given to the possibility of chronic pulmonary aspergillosis, which appears in 10% to 15% of patients with pulmonary cavities or bronchial dilation as complications of tuberculosis, who are usually just given another course of antibiotics,” said Colombo. He then presented an X-ray of a 51-year-old man who had lost weight and suffered for months from a chronic cough and fever, and who was treated for tuberculosis until it was discovered he actually had chronic pneumonia associated with the fungus Histoplasma capsulatum, a frequent cause of hard-to-treat pneumonias. Another species, Paracoccidioides brasiliensis, causes pneumonia even in people whose defenses are functioning normally, if they live in areas where this type of mycosis is common.

To make matters worse, a single fungus can cause different diseases, depending on the defense capacity of the organism that it infects. Aspergillus causes two types of pneumonia: acute and chronic. It is almost an established fact that these are no longer as rare as they were a few decades ago – the acute form will manifest in up to 12% of people with acute myeloid leukemia, according to estimates from a survey conducted in eight Brazilian public hospitals. The International Society for Human and Animal Mycology (ISHAM, its.mycologylab.org), an international, open-access database, currently holds 3,200 fungal DNA sequences that permitted the molecular identification of 524 species that cause diseases in humans, among more than 500,000 described species of fungi.

Genetic analyses have shown that samples once believed to be composed of a single species can sometimes include several types of fungi, with different levels of drug resistance. This was the case of Candida parapsilosis, which has been reclassified into three species: C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. All three species can be found on the hands of medical personnel in hospitals and result in infections associated with handling catheters and other devices commonly used in intensive care units.

Fungi collection: fungi immersed in mineral oil after being collected from hospital patients

LÉO RAMOSFungi collection: fungi immersed in mineral oil after being collected from hospital patientsLÉO RAMOS

It is no simple task to find out how fungi acquire virulence (the ability to cause infection) and drug resistance. In his laboratory at the University of São Paulo (USP) School of Pharmaceutical Sciences in the city of Ribeirão Preto, Gustavo Goldman, a biologist by training, verified that Aspergillus fumigatus can use different strategies to proliferate in the lungs, due to its ability to evade the body’s defenses as well as the most widely used antifungal drugs, known as azoles. According to Goldman, resistance to azoles might be explained by the use of fungicides to eliminate agricultural pest species in areas near cities, favoring the selection and dissemination of varieties that can cause disease in humans.  “Fungi are essentially opportunistic organisms,” he says. “Varieties that are important in carbon recycling in nature can cause disease if they find a debilitated host. They also have high genotypic plasticity and strong adaptability to different environments.”

Groups of fungi can show increased resistance and virulence, says Maria José Giannini, professor of mycology at the São Paulo State University (Unesp) School of Pharmaceutical Sciences, in the city of Araraquara. Together with her team, she found that fungal agglomerations – or biofilms – of Cryptococcus are more virulent and drug-resistant, both in vitro and in vivo, than isolated forms, as had already been observed for species of other genera, including Paracoccidioides brasiliensis and Histoplasma capsulatum. Biofilm formation may be why the research team in Araraquara found it so hard to eliminate onichomycosis, or nail fungus, caused by Candida and Cryptococcus. It can also explain the resistance shown by Trichosporon to triazole and amphotericin B, two drugs widely used against skin or internal infections, in a study by the group at Unifesp.

The results achieved by public policies designed to measure and deter fungal infections are not as consistent as those observed in basic research, says Giannini. “As a result, we may face serious problems related to an increase in fungal diseases,” she says. “The at-risk population has grown – especially the immunosuppressed –, as has the use of invasive medical procedures like catheters, which can facilitate fungal transmission. But this has not led to increased response capacity in the healthcare system, which should be alert and ready to deter this situation,” says Colombo. To exemplify, he mentions that no specific system is in place to diagnose post-tuberculosis fungal pneumonias, a measure that would be relatively easy to implement.

Worried about the situation, the researchers offer recommendations to other professionals in the field (see sidebar) and try to act in unison to disseminate information about these health concerns. Unifesp has been working with a research team at the University of Manchester, in England, and with medical centers in the states of São Paulo, Paraná, Rio de Janeiro and Espírito Santo, to determine how far-reaching acute and chronic fungal pneumonias are among the population, and the best alternatives for their diagnosis and treatment. As an additional initiative, the Brazilian branch of the Global Action Fund for Fungal Infections (GAFFI, gaffi.org) was established in 2015 to provide constant updates to the healthcare professionals responsible for identifying or treating these diseases.

Fungi

Project
Pulmonary Aspergillosis and correlation between clinical forms and differential expression of virulence attributes in Aspergillus fumigatus (nº 2014/50294-3); Grant Mechanism: Regular Research Grant; Principal Investigator: Arnaldo L. Colombo (Unifesp); Investment: R$42,905.00

Scientific articles
BENADUCCI, T. et al. Virulence of Cryptococcus sp. biofilms in vitro and in vivo using Galleria mellonella as an alternative model. Frontiers in Microbiology. V. 7, p. 290. 2016.
COLOMBO, A.L. et al. Candida glabrata: An emerging pathogen in Brazilian tertiary care hospitals. Medical Mycology. V. 51, No. 1, pp. 38-44. 2013.
GIACOMAZZI, J. et al. The burden of serious human fungal infections in Brazil. Mycoses. V. 59, No. 3, pp. 145-50. 2016.

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