Characterization of Candida species isolated from the oral mucosa of HIV-positive African patients

Philosophiae Doctor - PhDOne of the most common HIV-associated opportunistic infections is candidiasis, caused by Candida albicans or other Candida species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of Candida species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant Candida species. The mechanisms of Candida drug resistance in the African continent have also not been described. In this study, 255 Candida species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. Candida cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in Candida species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of Candida infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant Candida strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of Candida colonization mechanisms in HIV-infected African patients

HIV/Candida co-infection in Sub-Saharan African women on ART

5th FIDSSA Congress 2013: Champagne Sports Resort Drakensberg, KwaZulu Natal, South Africa - Poster Presentations.INTRODUCTION: Sub-Saharan Africa has 23.5 million cases of HIV and is home to 92% of the world’s HIV-positive pregnant women of whom 24% die of pregnancy related complications. Oral candidiasis is a common condition in HIV-AIDS patients, caused by commensal yeasts which may colonise the mucous membranes of the mouth causing morbidity due to several factors including immunosuppression, smoking, poor nutrition and the use of antibiotics. Methods: One hundred and ninety-four South African and Cameroonian HIV-positive women participated in the study. Only subjects who had white pseudomembranous plaque on the tongue or visible oral candidiasis were included. Samples were collected by scraping the patient’s oral mucosa and tongue with a sterile swab. Candida species were differentiated using selective and chromogenic media and their susceptibility to antifungal drugs was tested using the TREK Sensititre system. Results and conclusion: One hundred and ninety-six isolates, representative of six Candida species were identified. C. albicans was the predominating species, with C. glabrata and C. dubliniensis being the more frequent of the non-albicans isolates. Azole drug resistance patterns were very high for C. albicans, while C. glabrata showed high resistance patterns to echinocandins drugs. The duration of ART could be associated with the presence of different Candida species but no concrete conclusions could be drawn concerning HIV/Candida co-infection when controlling for other risk factors such as HIV stage, pregnancy, age and treatment for tuberculosis. This may be a cause for concern, particularly in the case of pregnancy, where co-infection may pose a risk for maternal morbidity and mortality.National Research Foundation of South AfricaDepartment of HE and Training approved lis

Strengths and limitations of different Chromogenic Media for the identification of Candida species

The treatment of invasive candidiasis and other Candida infections with the appropriate antifungal agent is assisted by the identification of Candida isolates to the species level. Rapid and accurate methods of differentiation are therefore imperative if treatment is to be effective, particularly in HIV-positive patients and in pregnant mothers where intervention may be necessary to reduce the risk for preterm delivery. The time used for isolation, identification and detection of mixed cultures may be reduced with the help of available chromogenic media. In this study, five commercial chromogenic media were evaluated for the differentiation of Candida species. Six type-strains of Candida species were streaked onto each of five different chromogenic media and incubated for up to 4 days at the different temperatures recommended by the manufacturers. This comparative evaluation demonstrated the strengths and weaknesses of each medium employed and found CHROMagar™ Candida and Chromogenic Candida Agar to be the most effective for distinguishing between different Candida species.National Research Foundation of South Afric

Proteomic mechanisms of drug resistance in Candida cell membrane fractions

Poster presented at the International Association for Dental Research, 92nd General Session, Cape Town, South Africa, 25-28 June 2014.INTRODUCTION: The African continent has the highest burden of HIV infection, accounting for approximately 65% of new infections globally. Oral candidiasis is a major cause of increased morbidity in HIV-infected individuals and is usually treated with fluconazole, an antifungal drug which interferes with ergosterol synthesis in the fungal cell membrane. The increased resistance of Candida species to antifungal drugs leads to treatment failure and an increase in untreatable Candida infections. OBJECTIVES/METHODS: Drug susceptibility patterns were determined for Candida species isolated from oral swabs of 254 patients from two HIV-positive African populations. Forty cell membrane fractions isolated from the clinical isolates were analyzed using High Performance Liquid Chromatography – Mass Spectrometry (HPLC-MS) and the results compared to the UniProt protein database for the identification of Candida peptides known to be involved in colonization and drug resistance strategies within the host. RESULTS: Fluconazole-susceptible and –resistant C. albicans isolates were found to express oxidoreductases that bind mammalian estrogens with high affinity. Peptides that confer resistance to formaldehyde were found in C. albicans, C. glabrata and C. dubliniensis in both susceptible and resistant cell fractions. Multidrug resistance proteins (CDR1 and CDR2) were seen in C. albicans cell fractions. CONCLUSION: The combination of different drug resistance mechanisms and binding abilities to salivary histatins and estrogen seem to be instrumental in the colonisation and retention of Candida in these immunocompromised patients. The results also suggest the presence of differences in efflux transporter protein expression in fluconazole-susceptible and –resistant isolates within the HIV population.National Research Foundation of South Africa (NRF

Proteomics of drug-resistant HIV-associated candidiasis

Candidiasis and HIV co-infection may cause increased patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence, antifungal susceptibility profiles and drug resistance mechanisms of Candida species on the African continent, the highest HIV-affected region globally and home to new and emerging drug resistant Candida species. Candida species isolated from the oral mucosa of HIV-positive African patients were found to be resistant to many of the antifungals routinely used in HIV-associated candidiasis. Candida cell membrane fractions were examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography mass spectrometry (HPLC/MS) in order to elucidate the cell membrane proteins specifically expressed by antifungal drug resistant isolates. SDS-PAGE and HPLC/MS allowed for the identification of multi-drug resistance efflux transporter CDR2 proteins and the elucidation of Candida colonization mechanisms and pH-responsive proteins, with significant associations observed between specific drug resistance and the duration of antiretroviral (ARV) therapy. This study provided useful information on the mechanisms of antifungal resistance in Candida species. It also formed the basis for further studies to address the transfer of resistance between Candida species in an oral microbial biofilm

Measuring Streptococcus mutans, Streptococcus sanguinis and Candida albicans biofilm formation using a real-time impedance-based system

Candida albicans and streptococci are amongst the most common fungal and bacterial organisms present in the oral cavity, with a growing body of evidence implicating C. albicans in increased caries severity and in the formation of the cariogenic biofilm. However, the interactive mechanisms between cariogenic streptococci and Candida are yet to be elucidated. In this study, the real-time biofilm formation of C. albicans, S. mutans and S. sanguinis was assessed individually and in combination using the xCELLigence system, an impedance-based microbial biofilm monitoring system. The impedance signal was the highest for C. albicans, followed by S. mutans and S. sanguinis. Although the streptococcal mixed adhesion was found to follow a similar trend to that of S. sanguinis, the introduction of C. albicans resulted in higher adhesion patterns, with the combined growth of S. sanguinis and C. albicans and the combination of all three species resulting in higher biofilm formation than any of the individual organisms over time. This study, the first to use impedance for real-time monitoring of interkingdom biofilms, adds to the body of evidence that C. albicans and oral streptococcal adhesion are interlinked and suggests that interkingdom interactions induce changes in the oral biofilm dynamics over time.National Research Foundation of South Afric

Antimicrobial resistant Gram-positive cocci in pregnant mothers with aerobic vaginitis

The vaginal microbiota of a healthy asymptomatic woman consists of an extensive diversity of anaerobic and aerobic bacterial genera and species dominated by the microaerophilic genus Lactobacillus , known to inhibit the growth of potentially pathogenic non-acid tolerant microorganisms. An imbalance of species within this biofilm may result in endogenous opportunistic infections such as aerobic vaginitis (AV) caused by S. agalactiae and E. faecalis, which have been implicated in neonatal and obstetric sepsis. The prevalence and antimicrobial susceptibility of E. faecalis and S. agalactiae in pregnant women with AV in the Western Cape, South Africa was determined using standard microbiological culture methods and the Sensititre TREK system. AV was detected in 26.13% of the 199 tested pregnant women, with S. agalactiae and E. faecalis isolated from 32 and 20 mothers respectively. S. agalactiae and E. faecalis showed resistance to 12 of the 17 antibiotics tested, including those recommended for prophylaxis according to the CDC guidelines. The resistance of S. agalactiae and E. faecalis to commonly administered antimicrobials highlights the need for alternative treatment regimens for AV during pregnancy to reduce the risk of AV-associated negative pregnancy outcomes.National Research Foundation of South Afric

Real-time assessment of Candida biofilm formation

Background: Candida infections are responsible for increased morbidity and mortality in immunocompromised patients, particularly when the Candida biofilm is composed of drug-resistant species. Although the biofilm formation abilities of individual Candida species have been described, the real-time interactions between common and rarer Candida species are yet to be elucidated. Methods: In this study an impedance-based biofilm monitoring systemwas used in comparison with the conventional crystal violet (CV) staining method, for demonstrating the biofilm formation of commonly isolated and less common Candida species. Results: The maximum cell index increased in most mixed biofilms, with the exception of the C. glabrata/C. parapsilosis and C. albicans combinations. Bulk biofilm formation measured by CV stainingwas the highest in C. albicans and C. tropicalis combinations and was the lowest for the C. glabrata/C. parapsilosis combination. Extensive pseudohyphae, which have been associated with increased virulence, were observed in C. albicans and C. glabrata combinations with C. tropicalis or C. parapsilosis. Conclusion: This study appears to be the first to report on the realtime biofilm interactions of Candida species using the xCELLigence system and suggests that the presence of specific species influences the biofilm formation of commonly isolated Candida species. This is important since biofilms act as reservoirs for disseminated infection and as demonstrated in this study, mixed Candida species act in synergy resulting in an increase in biofilm mass and subsequent risk for drug resistance

Drug susceptibility profiles of Candida species isolated from the oral mucosa of HIV-positive West African patients using the TREK Sensititre system

Poster presented at ASM2013, 113th General Meeting, American Society of Microbiology, 18-21 May, Denver, ColoradoBACKGROUND: Candida infections are a common cause of death in immunocompromised patients. The prevalence and anti-mycotic drug susceptibility profiles of Candida species from Cameroon in Africa are unavailable. This study was prompted by an increasing incidence of treatment failure. Drug susceptibility profiles, necessary to improve treatment outcomes, is particularly important in countries where the sale of antimicrobials and antifungals is uncontrolled and resistance may emerge due to the indiscriminate use. OBJECTIVE: The goal of this study was to characterize and determine drug susceptibility of oral Candida species in Cameroonian patients with HIV/AIDS. MATERIALS AND METHODS: Candida species were isolated from the oral cavity of 126 HIV-positive patients attending a local HIV/AIDS clinic in the Cameroon. Drug susceptibility to azoles and echinocandins was determined using the commercial TREK Sensititre® YeastOne™ platform that provides the minimal inhibitory concentration of amphotericin B, 5-flucytosine, anidulafungin, caspofungin, micafungin, fluconazole, itraconazole, posaconazole, and voriconazole. RESULTS: Ninety two isolates identified were Candida albicans. Remaining isolates were C. glabrata (24), C. tropicalis (4), C. krusei (3), C. parapsilopsis/lusitanreae/keyfr (2), and one isolate was C. dubliniensis. More than 50% of C. albicans isolated were resistant to azoles but 115 Candida species (87%) were susceptible to amphotericin B. Twenty one of the twenty four C.glabrata identified (88%) were resistant to micafungin. The majority of Cameroonian Candida species were sensitive to flucytosine (5-FC) (95%) and echinocandins (79%). CONCLUSIONS: The report of azole resistance in all Candida species isolated from immunocompromised patients in Cameroon is a new and important observation. We found the approach using a broad screening platform an effective means to obtain data rapidly. We propose confirmation of these data and regional surveillance of Candida species in other areas in Cameroon and surrounding countries to develop an effective public health management and treatment strategy

Candida species carriage in diabetic patients in Misrata, Libya

Background: There is a paucity of studies describing the prevalence and antimicrobial profiles of Candida in Libya. Limited treatment choices in the antifungal armamentarium in public healthcare settings in Africa require a study of the prevalence and susceptibility of Candida species in Libya, where antifungals are not routinely prescribed in public healthcare settings. Methods: In this study, 170 diabetes mellitus type 2 (T2DM) patients were examined for Candida carriage in the oral mucosa, using differential Fluka and Oxoid chromogenic media and API 32 ID C biochemical testing. Fluconazole susceptibility was investigated by disk diffusion on YNBG agar. Isolates were graded as susceptible, intermediate or resistant according to their inhibition zone measurements and microcolony scores. Results: Thirteen species were identified from 182 isolates with a frequency of 68 C. albicans, 42 C. dubliniensis, 26 C. humicola, 20 C. glabrata , 5 isolates of each C. krusei, C. tropicalis and C. kefyr, 4 C. sake, 2 C. parapsilopsis, 2 C. magnoliae and 1 isolate each of C. guilliermondii, C. globosa and C. membranifaciens. Although largely susceptible to fluconazole, C. albicans, C. dubliniensis, C. humicola and C. sake demonstrated an emerging resistance with intermediate to total resistance observed in all the other species except for C. magnolia and C. globosa which were both susceptible to fluconazole. Conclusion: Early recognition and treatment of rare or resistant Candida species which may be contributing to patient morbidity and mortality in Libya is imperative