European Journal of Cardiovascular Medicine

HIV related Opportunistic Fungal Infections (OFIs) cause of morbidity and mortality. It has been estimated that 60% to 90% of people with HIV disease will present with at least one oral manifestation.1 The importance of oral lesions as clinical biomarker indicator of HIV infection and predictor of progression of HIV to AIDS. Oropharyngeal candidiasis (OPC) is the most common opportunistic infection observedinHIVseropositivepatients,occurringinanestimated80to95%ofthese patients when the CD4 T lymphocyte counts are below 200 cells /mm is an early indication to indicate that immune system is impaired in HIV-infected patients. The lowCD4 counts and high plasma HIV RNA levels, both significantly correlate with oral candidiasis in HIV patients. These observations suggest that decreasing the viral load and increasing the CD4 count by initiation of highly active antiretroviral therapy (HAART) would reduce the need for specific antifungal therapy. Increased retroviral replication and an associated decline in immune defences render these patients more susceptible to oropharyngeal candidiasis .2,3 Although the introduction of antiretroviral therapy has had a major impact on the infectious complications of AIDS, candidiasis still remains a common opportunistic infection in HIV infected patients.4 OPC is considered as one of the earliest indicator of HIV infection and is relatively reliable indicator marker of disease progression. Regardless of the CD4 count, OPC is predictive for the development of AIDS related illnesses if left untreated.5 Candida albicans is the most common species of yeast isolated from patients with OPC. The incidence of opportunistic infection due to Candida albicans and other species has been increasing .6 Antifungal drug resistance is fast becoming a major problem. The high incidence of mucosal and deep seated forms of candidiasis has resulted in the use of systemic antifungal agents, especially fluconazole and itraconazole .7 Many of these patients require long-term treatment to suppress oropharyngeal candidiasis. The widespread use of these antifungal agents have been followed by an increase in antifungal resistance and by a noticeable shift toward non albicans species with relative resistance to fluconazole and itraconazole and there have been reports of emergence of resistance to antifungal agents in HIV/AIDS patients with OPC.8,9 The increased reports of antifungal resistance and expanding drug therapy options prompted the need for clinically relevant antifungal susceptibility testing. Prompt use of antifungal drugs judiciously reduces the advent antifungal resistance. Further the emergence of other Candida species such as Candida krusei and Candida glabrata with innately reduced susceptibilities to fluconazole also results in treatment failure, emphasizing the need for speciation of the oral yeast isolates. 10The aim of the present study is identification of Candida species from the clinical isolates and detection of antifungal susceptibility testing by Kirby Bauer Disc diffusion method. The Primary objective is the detection of MIC’s using the E strip test. The secondary objective is the correlation of CD count by flow cytometry and Viral load.

The present study was a cross-sectional study conducted in the Department of Microbiology, Rangaraya Medical College, Kakinada, Andhra Pradesh, India, for a period of one year from September 22^nd 2023 to September 23^rd 2024. Ethics committee approval (IEC/RMC/2023/1055) for the study was obtained before initiating the study. Informed consent was obtained from each patient before sample collection. Blood was collected in EDTA vacutainer tubes from each patient for enumeration of their CD4 count using flow cytometry method. Oropharyngeal specimens were collected by firmly swabbing the lesion site with two sterile cotton swabs under strict aseptic precautions, taking care not to contaminate the swab with saliva.

Inclusion criteria: Seropositive patients, patients of age group 20-60years, patients willing to give an informed consent.

Exclusion criteria: Seronegative patients, patients who were on antifungals for the last 6 months, and the patients who are  not willing to give an informed consent.

Study Procedure

Specimen Collection:

Oral cavity of the patients was examined for white plaques signs of candidiasis. Two sterile swabs were collected from the site of the lesion under strict aseptic precautions. One swab was used for the direct smear by Gram stain and the other for culture. Specimens collected were subjected to standard mycological procedures. Five milliliter of whole blood was collected from the patient and blood was drawn into EDTA anti- coagulated bottle for CD4 count estimation.

CD4 cell count estimation:

The CD4 count T lymphocyte count of all the patients was determined by the Sysmex flow cytometer. The instrument works on the principle of Flow cytometry.

Microscopy:

The direct smear shows pus cells with gram positive budding yeast cells with/without pseudo hyphae.(Figure- 1)Gram stain of an oropharyngeal swab was a valuable initial diagnostic tool to suspect the presence of candida.

Culture:

The other swab was inoculated onto Sabouraud’s Dextrose Agar, incubated at 37°C for 24-48 hours and observed for Cream coloured, smooth, pasty colonies. Yeast like colonies were subjected to Gram stain for confirmation.(Figure-2)

Speciation of Candida: Candida isolates were identified and speciated based on colony morphology, germ tube production, colony colour on HiCrom agar, chlamydospore production on corn meal agar (Dalmau plate technique), growth at 45°C, carbohydrate fermentation and carbohydrate assimilation test.

Figure-1: Direct smear – Microscopy of Gram stain under 100x objective

Figure-2: Microscopic examination of yeasts on culture under 100x objective

A small proportion of isolated colony suspended in test tube containing 0.5ml of human serum. The test tube was incubated at 35⁰c for not more than two hours. A drop of yeast-serum  suspension was taken on microscope slide overlaid with coverslip and examined under 40x objective lens for presence or absence of germtubes.Germ tubes were positive for C.albicans and C.dubliensis, both were differentiated by growth at 45⁰c by C.albicans whereas C.dubliensis does not grow at 45⁰c.

A heavy inoculum of yeast was streakd across plate containing cornmeal agar with tween 80 and coverslip as placed over it. The streak should project beyond cover slip. Plates were incubated at 25⁰c for 24-48hours. Examination was done for presence of chlamydospores, arrangement of pseudohyphae and observed under the microscope 40x objective

 Hichrom Candida agar:

Streaking was done on Hichrom candida agar with an isolated colony of yeast. Colour and morphology of the colony was observed after 48 hours of incubation. Light green colour C.albicans, light pink colour C.krusei, cream colour C.glabrataand steel blue colour C.tropicalis were seen  (Figure- 3)

Figure-3: Hichrom Candida agar

Carbohydrate fermentation test:

Sugar fermentation test was done to detect acid & gas production from 2% Glucose, Maltose, Sucrose, Lactose, Trehalose and Galactose with Andrade’s indicator and Durham’s tube, incubated for 5-7days at 25⁰c. Pink colour in the test tube indicates acid production and bubble in Durham’s tube indicates gas production. (Table-1)

Table-1:Speciation of Candida by sugar Fermentation test

Note: AG indicates Acid & Gas; - Indicates No acid & gas

Table-2: Speciation of Candida by Sugar Assimilation test:

Note: + indicates sugar Assimilates; - indicates sugar not assimilates

Antifungal Susceptibility Testing :Antifungal susceptibility testing was done by disc diffusion method as per CLSI guidelines M44-A.The anti-fungal drugs tested were Fluconazole 25μg, Itraconazole 10μg, Voriconazole 1μg, Caspofungin 5μg, Amphotericin 100units. A total 100 candida isolates, antifungal susceptibility was done by disc diffusion method. Out of 100 candida isolates, 18 isolates were fluconazole resistant which were subjected to Minimum inhibitory concentration(MIC’s).Inoculum is prepared by picking five distinct colonies of about 1mm in diameter from a 24- hour-old culture of Candida species. Colonies are suspended in 5ml of sterile 0.145 mol/L saline (8.5g/L Nacl; 0.85% saline). Suspension is vortexed for 15 seconds and its turbidity is matched to 0.5McFarland standard. Lawn culture was made using a sterile cotton swab in three directions on Muller Hinton Agar plate supplemented with 2% glucose and 0.5µg/ml methylene blue, discs were placed on the surface of agar and incubated at 37ᵒC for 24hours. The diameter of zone of inhibition was measured and compared with standard zones interpretive breakpoints according to CLSI M44-A2.

Figure-4:Antifungal susceptibility testing by disc diffusion method

Figure-5:Antifungal susceptibility testing by E test

Statistical analysis:

Data collected was entered in Microsoft excel sheet. Tables with frequency and percentages are calculated.

A total of 100 patients were included in this study with 34 (34%) females and 66 (66%) males. The minimum and the maximum age of the patients were 22 and 65, respectively. C.albicans was the most frequently isolated species 84 (84%) and the remaining were non-albicans species 16 (16%), with the frequency of C.krusei 7(7%),C.glabrata 6(6%),C.tropicalis 3 (3%).

Table-3: Antifungal susceptibility for Candida isolates by Disc diffusion method (n=100)

Antifungal susceptibility testing of the Candida species, out of 84 isolates of C.albicans showing sensitive to Fluconazole 69(82.1%), SDD 15(17.8%) Resistant 3(3.5%), For Itraconazole, sensitive 54(64.2%) SDD 4(4.7%), Resistant 3(3.5%), For Voriconazole, Sensitivity is 84(100%), SDD & Resistance are nil, For Caspofungin, sensitivity is 67(79.7%), SDD 3(3.5%), Resistance 14(16.6%). For Amphotericin B sensitivity 78(92.8%), SDD 2(2.3%), Resistant 4(4.7%). (Table-3)

Antifungal susceptibility testing for candida isolates were done by ‘E’ (Epsilon) test for the detection of minimum inhibitory concentration (MICs).Out of 84 isolates of C.albicans, 18 were resistant to Fluconazole which were subjected to E test for MICs. Eighteen isolates are showing MICs were in the range of 6-7 μg/ml which are resistant. Total 6 isolates of C.glabrata, were resistant to Fluconazole and were subjected to E test for MICs where the isolates were in the range of 48μg/ml which are resistant.

Table-4: Distribution of Viral load and CD4 count among the study population

Out of 66 seropositive patients, viral load wherewith in the range of 1lakh above, 16 seropositive patients where the viral load were in the range of ten thousand and ninety-nine thousand nine hundred and nine followed by 11 seropositive patients were within the range of thousand and nine thousand and nine hundred and nine, followed by 7 seropositive patients where the viral load were within the range of less than thousand. (Table-4)

Table-5: Distribution of Candida species in correlation with CD4count

C.albicans where CD4 count < 200cells/mm³ are 40, followed by CD4count 201-500cells/mm³ are 21, >500cells/mm³ CD4count 20 in number. For C.krusei where CD4 count <200cells/mm³ are 4, followed by CD4count 201-500cells/mm³ are 2 and >500cells/mm³ CD4 count isolates are 1 in number. For C.glabrata where CD 4 count is <200cells/mm³ are 4, CD 4 count 201-500cells/mm³ are 2 in number and CD 4 count >500cells/mm³ were no isolates of C.glabrata. Finally for C.tropicalis single isolates for CD 4count <200cells/mm³, 201-500cells/mm3 and >500cells/mm³.(table-5)

Figure-6: Sugar assimilation test: Growth around the disc is positive, no growth around the disc is negative.

Figure-7: Glucose, sucrose, Maltose, Xylose sugars r positive and for lactose negative

Oral Candidiasis is the most common fungal infection in HIV infected patients and has been identified as a clinical predictor for progression to AIDS. Predominant isolate in the present study is the Candida albicans 84 (84%) correlated with other studies like Franker et al 11 where Candida albicans 84 (84%) is the predominant isolate. There is increasing incidence of non albican candida species  in oral Candidiasis and increasing rates of antifungal drug resistance particularly with the immunocompromised patients are seen in the present study, which are correlated with other studies like Champa Hemachandran et al12 and Shymala R et al13 shows an increasing incidence of non-albicans Candida. Hencerapid identification of candidiasis is important for the clinical management of immunocompromised patients.In the present study, male preponderance were seen which are correlated with studies done by Anwar et al14 and Maheswari et al15 who studied the opportunistic infections spectrum and candida profile in PLWHIV. Majority of the isolates in the present study were in the age group 31 – 49 yrs. Studies have also reported the disease common in the age 25 – 49 yrs  as HIV infection is most commonly seen in the sexually active age group. Sex preponderance in the present study are males 66(66%) and females 34(34%) correlated with Alexander patella et al 16. Among the non-albicans Candida spp, C.krusei was the most common species, a finding similar to Jayacharan et al 17 studies. The evolving importance of non-albicans Candida spp.in HIV patients with oral candidiasis requires incorporation of standard techniques for Candida speciation.

Azoles are considered the drug of choice for treating oral candidiasis associated with HIV / AIDS patients.18 Fluconazole is a triazole agent that has been widely used for the treatment of mucosal candidiasis because of its low toxicity and ease of administration.19 In the present study, 82.1% of the isolates were susceptible to fluconazole. This result is similar to that reported by Swetha et al19 & Talukdar et al 20studies. Itraconazole is used as an alternative to fluconazole for treating oral candidiasis. In this study the isolates of C.albicans, C.krusei, C.glabrata were 66%, 28%, 16% susceptible, and were resistant to itraconazole., 19%, 71%, 83%, 33% of C.albicans ,C.krusei ,C.glabrata and C.tropicalis accounted for more than half of azole resistance. Although C.krusei had a low prevalence, its intrinsic resistant activity against fluconazole may have therapeutic implications. Species other than C.albicans are generally less susceptible to therapy and arise mainly with low CD4 count and after repeated or prolonged antifungal treatment. This can be considered important since infections caused by C. albicans generally have the best prognosis in comparison to those caused by non-albicans species. C. glabrata and C.krusei remain the least susceptible species to fluconazole and because of cross resistance between azole drugs, they have high MICs to other azoles. The widespread use of fluconazole and itraconazole as therapeutic or prophylactic doses has increased recently and most often associated with the HIV infected with OPC. This has led to the increase of reports of resistance.21 Several authors have reported that apart from prolonged exposure, advanced immunosuppression is a major risk factor for azole resistance.22 In the present study, among the 100 HIV patients with oropharyngeal candidiasis, 70.7% had CD4 count statistically significant by proportional test (p=<0.001) This correlated well with the study conducted by Usha et al22 where 76.66% patients with oropharyngeal candidiasis had CD4 count <200 cells/ µl. Oral candidiasis can be used as a marker of immune status in field based settings where CD4 Count and viral RNA load estimation cannot be routinely done.

Limitations:

Variation in patient backgrounds and patient care is the major limitation of the present study, as it may influence the outcome of the results. However, a large sample size can help to mitigate this limitation and provide more comprehensive findings.

Candida albicansis the predominant isolate in the present study. C.krusei, C.glabrata, C.tropicalis are non albican candida species were isolated. There is increasing trend towards non albican Candida species, hence speciation is necessary. Low CD 4 count and high viral load suggests significantly correlate with oropharyngeal candidiasis in immunosuppressed patients especially in HIV. The significant relationship of oral candidiasis with severe immunosuppression suggests that when oral candidiasis is present it can be used as a surrogate marker for CD4 depletion. Increasing drug resistance among non- albican candida species indicating that there is need for speciation and antifungal susceptibility methods.

Acknowledgements

The authors would like to thank

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