A 43-year-old man, Caucasian, underwent a transplant procedure secondary to the renal failure (for which he underwent peritoneal dialysis) and type I diabetes mellitus he was suffering from. He received a pancreas-kidney transplant from a healthy 26-year-old donor, the surgery being without any complications. After the procedure the patient received immunosuppressing agents (tacrolimus, mycophenolat mofetil, basiliximab and corticosteroids), as well as prophylactic antibiotics. A week after the transplant surgery, the patient began to develop acute pancreatitis. The cause of it was a thrombus at the level of the splenic artery. A new surgical intervention was done for removing the pancreas and the patient was given an empirical antibiotic treatment with piperacillin-tazobactam and fluconazole(1). A new complication appeared soon after when the right branch of the external iliac artery ruptured spontaneously, making an emergency surgery inevitable. Samples from the ruptured vessel were sent to the pathology laboratory where it was found that the arterial inflammation and consecutively rupture were caused by C. glabrata (fluconazole dose-dependent susceptible) arteritis.
Antifungal therapy was started with caspofungin. A week from the event, the patient was diagnosed with peritonitis and another surgical intervention was performed in an emergency setting. A sigmoid colon perforation was discovered and a tissue sample from the lesion revealed spores of C. glabrata. Even though the therapy course of caspofungin was of one week, the infection had not been eradicated. The patient was switched to fluconazole and his evolution was good afterwards(1).
Comments upon the case
It has been shown that candidiasis has a higher risk to develop in patients with an injured pancreas. In this case, it may come to mind that the pathogen was transmitted from the donor, along with the graft, or was acquired during the harvesting and transportation of the organs, but tissue samples from the grafts were sterile. Prior to the transplantation, the patient had sterile blood cultures and showed no symptoms of Candida infection. One of the important risk factors of the patient was the peritoneal dialysis. Given all these, how the infection took place remains unclear, the best hypothesis being that of the contaminated grafts(1). Although C. glabrata isolated from the artery sample was fluconazole susceptible, the infection developed under the therapy with fluconazole. It is known in literature that C. glabrata has become more and more resistant to fluconazole (the mechanisms for the resistance will be detailed later in the same article). The importance of this case is to draw attention towards fungal infections. They should not be a neglected pathology especially in the context of patients with chronic diseases such as diabetes mellitus or in those who are in an immunodeficient state. Fungal infections have become a real problem nowadays because of the high resistance to antifungal drugs, making the treatment spectra of such diseases to decrease more and more.
C. glabrata’s resistance to azoles
There are roughly 1.5 million of identified yeast species. Only a very small percentage of them actually cause infection in humans(2). Candida spp. comprises a large number of members of which Candida albicans is by far the most well-known. These yeasts are part of the normal oral, gut and vaginal flora and can cause infections only on special occasions. They are opportunistic pathogens and can cause nosocomial infections(3), being on the fourth place in the list of germs causing nosocomial hematogenous infections(4). In a comparative study made in the US during two-time intervals (1992-1993 and 2008-2011), it was revealed that the percentage of C. albicans isolated from blood infection has decreased (but still remained the most often encountered), whereas the percentage of C. glabrata increased(5). This remark draws attention to this apparently rare species of Candida identified in human infections, C. glabrata. Apart from the common characteristics of the species it belongs to, C. glabrata shows some particularities. Its genome is haploid, consisting of only 13 chromosomes(6). But by far, the most important peculiarity of this yeast is the intrinsic resistance to azole drugs(7). This category of antifungal therapy is probably the most widely used to cure fungal infections. They have an effective mechanism of action against fungi, their adverse effects are quite low and their price is mostly convenient. The target of the drug is the enzyme lanosterol demethylase which is involved in the process of ergosterol synthesis, an important component of the fungal plasma membrane(8).
The key factors for the azole resistance are represented by ABC transporters encoded by CDR1 and CDR2 genes. An upregulation of these genes makes the yeast resistant to fluconazole, whereas a knockout model of the yeast (without CDR1 and CDR2) transforms the fungus in a susceptible strain(9,10). These ABC transporters act like efflux pumps which remove the azole molecules from the cell, sparing the yeast from their effect and rendering the microorganism resistance to the drugs. These two genes are controlled by a transcription factor, Pdr1. Single-point mutations in the gene encoding for this protein are responsible for an upregulation of CDR1 and CDR2(11). There have been identified over 50 “hot spots” where the mutations occur. They are gain of function (GOF) mutations, inducing a higher activity of Pdr1 and an overexpression of the genes encoding for the ABC transporters(11).
Another mechanism by which C. glabrata increases its azole resistance is by altering the mitochondrial DNA. The resulting “petit mutants” upregulate ABC transporters in the yeast and confer azole resistance. These mitochondrial DNA mutants can appear spontaneously, in vivo, and induce the ABC transporter overexpression totally independent from the GOF mutations in PDR1(12). Apart from the azole resistance, C. glabrata strains containing the GOF PDR1 mutations show enhanced virulence demonstrated on mice models(11). In vitro studies have revealed another particular characteristic for PDR1 mutated strains. It seems that there is a difference in the adherence process of the mutated yeasts to epithelial cells and macrophages compared with the ones not bearing the mutation. A pattern of increased adherence to epithelial cells and decreased adherence to macrophages was observed in the GOF mutation strains in comparison with the wild-type strains(13). The proteins responsible for the adhesion process are part of the outer layer of the yeast wall and are called adhesins. One of the most important and most studied among them was Epa1 (Epithelial adhesin 1)(14). A certain fact is that using the azole resistance mechanisms which form a tandem with the increased virulence and the adhesion pattern, C. glabrata works its way out of the immune attack and causes severe and hard to manage infections.
So, if the azoles become inefficient in treating C. glabrata infections, what else can be of good use? The sad part is that there are not many options. The first option is to increase the dose of azoles, given the fact that the resistance is dose-dependent. Next comes amphotericin B(8), which is the drug of choice for severe systemic fungal infections, but it has some serious adverse effects. There are other drugs like echinocandins (caspofungin belongs to this family) which, like amphotericin B, have a broad antifungal spectrum. These are used as a backup medicine when the patient has a severe infection that does not heal with other antifungals.
Fungal infections, including those produced by C. glabrata, develop in certain conditions such as immune suppression (for example, in hosts with human immunodeficiency virus - HIV) or an unbalanced commensal flora during an antibiotic treatment(7). The infections that C. glabrata produces can be mucosal (mostly oral, pharyngeal, esophageal, urinary tract, vaginal) or systemic. Little is known about the interaction of the defense immune mechanisms and C. glabrata, they are considered to be similar with those against C. albicans. In a mouse model study of C. glabrata infection, it was observed that the most important immune response against the microorganism is a Th1 activation with subsequent production of interferon gamma and interleukin 2 (IL-2)(7). In the context of systemic infection, it is speculated that the increase in the percentage of C. glabrata as causative agent for candidemia is because of a higher use of antifungal drugs(7). A systemic candidemia (either with C. glabrata or C. albicans) has no specific signs or symptoms and is often suspected in a patient with a bad condition, with fever, having no improvement when given antibiotic therapy and with sterile blood cultures(7). It is a diagnostic that is made primarily on suspicion and secondarily by isolating the pathogen from the blood.
Returning to the case described at the beginning, some aspects can be underlined. In an immunocompromised patient, the likelihood of developing a fungal infection is increased. The immunodeficiency was acquired following the therapy with immunosuppressive agents and was accentuated by the chronic disease the patient was suffering from, diabetes mellitus. It is known that agents such as cortisone, tacrolimus or mycophenolat mofetil inhibit the immune response, but there is little information about the influence of chronic hyperglycemia on the immune system. Observational studies show that the persons suffering from diabetes mellitus show a higher infection rate, but there was no proof of a malfunction of the immune system components in these patients. A mechanism encountered in C. albicans which may be an explanation for C. glabrata as well is the increased adherence of the pathogens to the target cells which have a different carbohydrate composition of the surface proteins(15). So, this patient had two favorable conditions which made him susceptible for a fungal infection. The most intriguing question to answer is why did he develop the infection during the fluconazole therapy? And the answer can be explained by the unique characteristic of C. glabrata to remain resistant to azoles. This particularity allowed the yeast to reproduce in an environment poorly protected by the immune “soldiers”, not being influenced by the drug. Curiously enough, even after the treatment with caspofungin, the infection was not eradicated and it finally disappeared when using a higher dose of fluconazole as therapy. This clinical case reveals how important is to take into account fungal infections according to the patient’s status. Knowing about the epidemiology of the fungal infections and their characteristics regarding the treatment is also crucial for a better approach of the disease and for finding the perfect cure.
Conflict of interests: The authors declare no conflict of interests.