Although cutting edge modern technologies have advanced overall knowledge of microbial infections with consequent better outcome, what microorganisms will do when challenged with different surface active agents is still vague and incompletely understood. Disease diagnostic precision remain far below the minimum threshold needed to yield meaningful and reliable outcome especially in resource poor settings. Understanding the idiosyncrasy or characteristic uniqueness an organism when challenged with antimicrobial agents is a hug challenge to many antibiotic stewardship programs in resource limited settings. Perhaps the unpredictability of microbial response to surface active agent’s emphasis the need to embrace the use of tests that are more sensitive and specific for more result oriented outcome.
Oropharyngeal lesions remain significant health challenge (26) that HIV infected population must overcome for a better life, because there are many questions that needs hard-to-get answers about possible disease comorbidities that could delay or prolong the healing of such oropharyngeal lesions. Confirmatory investigations are needed to determine if prolong or delayed healing of oropharyngeal lesions could be linked to: drug-resistance (1), non-adherence to prescription guidelines (2)), abuse of antibiotics with sub-lethal doses (3-4), re-infection (5), progression of HIV disease to AIDS (1-2) or resistant opportunistic infections (27). With the increasing notorious nature of yeast as predominant opportunistic pathogens in oropharyngeal lesions of HIV/AIDS patients, it becomes necessary to determine the characteristic vulnerability of yeasts to routine prophylactic antifungal agents to achieve better intervention.
In this study, Candida albicans were sensitive to fluconazole, Itraconazole and Amphotericin B respectively (Table 1; Fig 1, 4-5). Dose dependent sensitivity to various antifungal agents used were also shown by Candida glabrata (MIC=12.0), Candida sake (MIC=0.5) and Candida norevegensis (MIC=8.0) respectively (Fig 2). This may indicate possible emergence of antifungal drug resistance. Again, it is not clear why yeast strains development of dose dependent sensitivity were different in different TASO centres because disease management policies are presumed to be the same in these centres. The observation that same yeast species from different centres under same prescription and TASO management policy yielded different MIC, shows unique idiosyncratic nature of yeasts response to treatment regimen and this makes it hard to extrapolate results across centres. Thus inter regional surveillance to monitor the emergence and re-emergence of yeast susceptibility in a locale antimicrobial stewardship program is urgently needed.
While C. albicans remain the leading aetiology of candidiasis (28-30), reports that infections caused by other non albicans yeast species are globally gaining more research attention with species varying between different geographic regions. This is problematic, because resistance to fluconazole may also mean resistance to other azoles and resistance are commonly controlled by point mutation and efflux pumps (31-32). The rates of azole resistance may differ between institutions due to prescribing patterns of clinicians for both the treatment of and prophylaxis against pathogenic candidiasis (33). In India, C. tropicalis is the predominant species, and rates of fluconazole resistance may vary significantly (34). The prevalence of C. parapsilosis was similar to C. albicans in some Chinese hospitals (35). Fluconazole susceptibility is also highly variable between institutions, with some reporting as high as 50% in intensive care units (35).
In Table 1 and Fig 1-5, all yeast isolates showed dose dependent sensitivity (DDS), susceptibility to both azoles and polyene antifungals, while C. glabrata, C. norvegensis and Zygosaccharomyces species exhibited reduced susceptibly (SDD) to fluconazole and were resistant to Itraconazoles in all studied districts. While the advantages of prophylaxis in HIV infected population cannot be denied, emergence of sub-population showing reduced sensitivity may be announcing the imperatives of a new prophylactic algorithms in immunocompromised HIV infected population living in resource limited countries like Uganda. Again, while prophylactic medication usually have clearly visible entry plan for infected patients, exit plan are often not stated, omitted or ignored by drug prescribers in Uganda. Patients on prophylactic drugs therefore continue to use the drugs even when the efficacy of the drugs decreases (36).
Non-albicans species, including C. glabrata have known innate resistance to antifungal drugs (4, 37-38). The discrepancy between this report (Table 1) of reduced susceptibility and known innate resistance of C. glabrata may be explained by a possibility of misdiagnoses with other phenotypically similar yeast strains. Thus, the phenotypic similarity between C. glabrata, C. nivariensis and C. bracarensis, could be misdiagnosed as C. glabrata in the absence of good confirmatory typing methods (39). There may be emerging generation of Candida glabrata which may not be highly resistant to conventional antifungals as previously anticipated because sensitivity to Candida glabrata reports are beginning to emerge from different geographical settings (37-38, 40-42). Reports of C. glabrata resistant from some African countries include Cameroon (43), Ethiopia (44) and Tanzania (45), while reduced or dose dependent sensitivity to C. glabrata has been reported in South Africa (43) and Nigeria (42). The result of these study is similar to most reported studies reporting that C. globate resistance/dose dependent sensitivity to fluconazole but selectively sensitive to other antifungal agents such as nystatin, and amphotericin B, which are similar to the result of this study. Despite this window of hope for non albicans C glabrata susceptibity, resistant strains has increased in patients presenting with candidiasis in recent years (46) with increased mortality rates (39).
To understand the emergence of this dose dependent sensitivity in Candida albicans and non-Candida albicans species cohabiting the microecology of the oropharyngeal cavity of HIV patients, Melo et al. (47), found high-frequency transfer of plasmid DNA when pathogenic and food-borne yeasts were grown together despite the fact that many pathogenic yeast species are asexual and therefore not involved in intra- or inter species mating. This high-frequency transfer of plasmid DNA could define the observed emergence of dose dependent sensitivity by oropharyngeal Candida albicans, Candida glabrata, Candida sake and Candida norevegensis respectively (Tables 1).
Micro evolutionary high-frequency transfer of plasmid DNA (through cell lyses, or cytoduction) has been proved between C. glabrata and Saccharomyces cerevesiae (48). Although factors that govern the evolutionary revolution of micro-organisms are not yet completely understood, it is thought that environmental and nutritional factors are key to this evolutionary revolution of micro-organisms that can make them change from the original characteristics of their ancestral genealogy (the wild type), to something new (48-49). Microbial evolution tends to remove with time what will not assist its bid to survive in a particular environment and acquire new characters that will assist the organism to manipulate its environment (50) thus accounting for the suggested emergence of dose dependent sensitivity in the reported yeast strains (51). In addition, despite the “asexual” nature of C. glabrata, a potential cryptic sexual life cycle could promote rare interspecies mating with C albicans and other non albicans species of Candida (52).
From (Table 1, fig 1-6), C. norvegensis, C glabrata, Saccharomyces cerevisiae and Zygosaccharomyces species isolated from different districts showed a resistant MIC to Itraconazole, and Amphotericin B respectively. Immunosuppression, previous exposure to oral azoles, Clinical, cellular and molecular factors are known to contribute to development of antifungal drug resistance (53-54). Clinical factors leading to treatment failure of refractory disease may be associated with the patient’s immune status (55), pharmacology of the drugs (13), or type of fungal infection (8). Cellular factors could be the result of replacement of a susceptible strain with a more resistant species or the alteration of an endogenous strain (by mutation or gene expression) to a resistant phenotype.
Molecular factors responsible for the resistance phenotype in the cell identified has been reported to include over expression of two types of efflux pumps, mutation of the target enzyme, and alteration of other enzymes in the same biosynthetic pathway as the target enzyme (53, 11). Generally, azoles and polyenes actions are mainly directed against ergosterol, the major sterol of the fungal plasma membrane, which is analogous to cholesterol in mammalian cells, (13-14). The dosing schedule may have an effect on the development of resistance (4), since patients treated with intermittent therapy were more likely to develop resistance than those treated continuously. Primary resistance and secondary resistance have been documented in HIV-infected patients (56). Although symptomatic candidiasis develops more commonly due to C. albicans, candidiasis are known to be caused also by non-albicans Candida sp. Fluconazole-resistant species, Candida norvegensis, have recently been reported as pathogens in patients receiving fluconazole (41).
The polyenes are known class of amphipathic antifungal drugs that target membranes containing ergosterol forming a channel through which cellular potassium ions, leak and thereby destroying the proton gradient within the membrane (11, 57). Amphotericin B has antioxidant effect in vivo, protecting fungal cells against oxidative attack from the host (11, 58). Reports of Amphotericin B resistance are limited, but it appears prolonged pre-exposure to azole antifungal drugs and severely immunocompromised patients, especially patients with cancer, are at the highest risk perhaps explained by an alteration of cellular membrane components (59). Amphotericin B-resistant clinical isolates of C. tropicalis have also been found (11, 58). Minimum inhibitory concentration (MIC) of tested antifungal agents (Table 1) was significantly (p<0.05) dependent on strains of Candida species isolated from oropharyngeal lesions of studied population. Thus, types of oropharyngeal yeast strains present in the oral lesions significantly (p<0.05) impacted on the MIC values as well as the efficacy of antifungal agents tested.
There is evidence to suggest that intermittent versus continuous dosing, the amount of drug administered, the length of treatment (27), the immune status of the patient and alterations in the membrane structure or in the sterol-to-phospholipid ratio in the membrane may be associated with resistance (53, 11, 58). The success or failure of antifungal drug therapy is dependent not only on the drug, but also on the immune status of the host and a combination of azoles and cytokines may be an important therapeutic strategy for resistant fungal infections in immunocompromised individuals.
Susceptibility of C. norvegensis were significantly (p<0.05) dependent on types of antifungal used. The depicted different levels of exposure to various antifungal agents tested may be a pointer to emergence of resistant clones of oropharyngeal yeast strains among the HIV infected patients studied. The frequency of diagnosed fungal infections is known to have risen due to increase in the number of immunosuppressed patients resulting from the AIDS epidemic and increase in the frequency of recalcitrant infections to standard antifungal therapy (13-14), Susceptibility of isolates to antifungals varied and MIC values were significantly (p<0.05) dependent on district of sample collection. Thus, despite being treated with same guidelines by clinicians in the same TASO centres, patient’s uptake and compliance to treatment guidelines were different, thus depicting different levels of exposure to antifungals agents and probable prescription pattern. Fluconazole-resistant Candida norvegensis, have been reported as pathogens in patients receiving fluconazole (41, 24, 60). It has been suggested that selection for resistant strains occurs with azole treatment but nosocomial transmission and transmission of resistant strains between partners can also occur (53). The polyene are amphipathic drugs which intercalate into membranes containing ergosterol, forming a channel through which cellular components, especially potassium ions, leak and thereby destroying the proton gradient within the membrane (57, 24, 60). Amphotericin B resistance in Candida spp. can develop in patients previously exposed to azoles (59). Several strains of fluconazole- and amphotericin B-resistant C. albicans have been found in HIV-infected patients who have received prolonged courses of azole developed resistance with azoles (59).
Underlying medical conditions of the immunocompromised HIV infected population studied together with type of antifungal used, district of yeast isolation, and strains of yeast isolates significantly (p<0.05) impacted on the MIC values obtained in this investigation (Table 1). To justify the role of underlying medical conditions in the healing of oral lesions in the immunocompromised HIV infected population (60), Nube et al (61) summarised specific risk factors of delayed wound healing to include but not limited to: infection, ischemia, ulcer size, depth, and duration as well as probing to bone (or osteomyelitis), location of ulcer, sensory loss, deformity (and high plantar pressure), advanced age, number of ulcers present and renal disease with ulcer depth (or size), infection, and ischemia listed as the most common risk factors. When there is high-quality treatment protocols and guidelines, 66%–77% of foot ulcers will heal and the remaining group are unlikely to heal, thus supporting the fact that quality of treatment and care impacts on wound healing rate (61). Non healing wounds are prevalent in resource poor settings due to poor treatment protocol while healing or delayed healing wound are more prevalence in adequate resources settings (62).