Candida and other yeasts of clinical importance in Aseer region, southern Saudi Arabia. Presentation of isolates from the routine laboratory setting.

OBJECTIVES: To isolate, identify, and determine the prevalence of Candida and other yeasts of clinical importance in Aseer region, Saudi Arabia.
METHODS: This is a cross-sectional study involving retrospective analysis of 6100 samples submitted to the Microbiology Laboratory, Aseer Central Hospital, Abha, Saudi Arabia between 2011 and 2012, and prospective isolation and identification of 84 isolates recovered from various clinical specimens presented to the Microbiology Laboratory between 2012 and 2013 using the classic morphological schemes and the Vitek 2 automated system.
RESULTS: The results of the retrospective analysis (2011-2012) indicated that of the 6100 various clinical specimens submitted to the routine microbiology analysis, 143 (2.35%) revealed the presence of Candida spp. The distribution of the 143 Candida spp according to specimens was as follows: urine 72%, sputum 10.5%, endotracheal tube 7%, blood 4.2%, catheter tip 2.1%, throat swab 2.1%, eye swab 0.7%, wound exudates 0.7%, and cerebrospinal fluid 0.7%. The results of the prospective study (2012-2013), which involved the identification of yeast recovered from 84 specimens indicated that Candida albicans 28.6% was the predominant species, followed by Candida parapsilosis 21.4%, Candida tropicalis 14.3%, and Candida lusitaniae 9.5%.
CONCLUSIONS: Along with the commonly encountered Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida lusitaniae were detected with significant rates. Many other Candida species and some other pathogenic yeasts have been detected for the first time in the region. Urinary tract samples were the main source of Candida species.

Fungal diseases notably those due to candida have become an increasing risk to human health. This is particularly true among patients with immune compromised systems.1,2 Candida and Aspergillus species are the most common agents associated with invasive fungal infections.3 Candida infections like other fungal infections are believed to be opportunistic in nature, since some aspects of the host’s defense system is impaired in some way. On the contrary, Candida infections manifest in a variety of forms ranging from superficial skin conditions, onychomycosis, oral, vaginal infections to fatal invasive illnesses that involve vital body organs such as heart, lungs, and central nervous system.1,2 Candidiasis, notably candidemia continues to be a major cause of morbidity and mortality in the health care settings. Moreover, the epidemiology of Candida infection is changing.4,5 Candida species are frequently encountered as part of the human commensal flora. Colonization mostly paves the way to candidemia and is considered an independent risk factor for the development of candidemia.1,4 The frequency of nosocomial bloodstream infections by Candida species has risen dramatically in the past 2 decades. It has been found that more than two-thirds of patients with invasive candidiasis in ICUs have candidemia. Of these isolates, the non-albicans Candida species constituted about half of the isolates and death from these invasive ICU infections was notable.1 There is a lack of sufficient literature showing in a systematic way, the incidence of fungal infections in the Kingdom of Saudi Arabia. Available data indicated that fungal infections, generally, represent approximately 10% of reported laboratory diagnosed infections; whereas gram-positive organisms (10%), gram-negative organisms (32%), and the remaining 48% were polymicrobial.6,7 The aims of this study were to isolate, identify (prospectively), and to determine the prevalence (retrospectively) of Candida infections and other yeasts of clinical importance in Aseer region, Saudi Arabia.

Methods

This is a cross-sectional study involving firstly, a retrospective analysis of 6100 samples submitted to the Microbiology Laboratory, Aseer Central Hospital, Abha, Saudi Arabia between February 2011 and January 2012. Clinical and microbiology data of positive cases were collected. Secondly, a prospective analysis was undertaken, which included the isolation and identification of strains presented to the laboratory from October 2012 to November 2013. This was carried out using initial phenotypic identification based on morphological and culture characteristics8 followed by confirmation using the Vitek 2 automated system. Samples included in this study were the ones with complete clinical records, requests from the relevant wards, and samples that met the criteria of submission. Samples that did not meet the above mentioned criteria were excluded from the study.

Ethical approval

This research was approved by the Research Ethics Committee, College of Medicine, King Khalid University, Abha, Saudi Arabia.

Isolation of yeasts

Fungal cultures were carried out on sabouraud dextrose agar (SDA) and Brain Heart Infusion Agar + 5% sheep blood (BHIA) plates. Inoculated plates were incubated at 30°C and examined daily for up to 10 days for yeast growth.

Identification of yeasts

Identification of yeasts encountered during routine bacteriological cultures or from SDA and BHIA plates was performed using conventional growth and colonial morphology criteria.8

Confirmation of identification of Candida spp. by VITEK 2 system

The VITEK 2 automated system was used for confirming identities of Candida species following protocols described by the manufacturer (bioMérieux Inc., Durham, NC 27712, USA). The VITEK card consists of 64 wells that contain various fluorescent biochemical tests. Of these, 20 are carbohydrate assimilation; 4 are phosphatase, urea, nitrate, and actidione tests. When a test result is recorded as “low discrimination,” this means that the result is doubtful. In such cases, supplementary tests were carried out manually to resolve such uncertain findings. These supplementary tests were: microscopic detection of blastospores or arthrospores, apiculated cells, capsule, carotenoid pigment, convoluted colony, hyphae or pseudohyphae, sporangia, growth at 37°C, and growth without oil.

The VITEK 2 device handled card automatically from filling, sealing then transferring them into the connected incubator (35°C). The cards are filled automatically every 15 min by a fluorescence system. Each output profile is decoded as per a specific algorithm. The obtained results were equated to the ID-YST (identification of yeasts) database. This, in most of the known yeast with clear cut profile, led to a correct identification of the unknown yeast.

Results

Retrospective analysis of 6100 samples

Out of the 6100 various clinical specimens, 143 (2.35%) revealed the presence of Candida spp. When the observed Candida infections proportion was 2.35%, the confidence intervals for the sample size was ±0.38. The distribution of the 143 Candida spp. recovered from the 6100 clinical specimens are shown in .

Figure 2

Distribution of the 143 Candida spp. recovered from 6100 various clinical specimens analyzed retrospectively between 2011 and 2012 among patients from Aseer Central Hospital, Abha, Kingdom of Saudi Arabia.

Isolation and conventional identification of yeasts

Eighty-four yeasts were isolated in pure forms using SDA and BHIA. Yeasts were tentatively recognized on the basis of their morphologies, which are: colonies with white to cream colored, smooth, glabrous, and yeast-like in appearance (). Microscopically, they exhibit spherical to subspherical large yeast-like cells with budding, blastoconidia, and pseudohyphae, or both (). These were considered for confirmatory identification.

Figure 1

Growth of Candida albicans on sabouraud dextrose agar A) Colonies with white to cream colored, smooth, glabrous, and yeast-like in appearance. B) Microscopic appearance of Candida albicans showing spherical to subspherical budding yeast-like cells or blastoconidia (Safranin stain x 100).

Identification of yeasts using Vitek 2

Eighty-one of the totally isolated 84 yeasts were successfully identified using the Vitek 2 system. The results of the identification system using the Vitek 2 system were as follows: C. albicans was the predominant species (28.6%), C. parapsilosis (21.4%), C. tropicalis (14.3%), and C. lusitaniae (9.5%). Other species encountered are shown in .

Figure 3

Distribution of Candida spp. and other yeasts recovered from 84 clinical specimens among patients from Aseer Central Hospital, Abha, Kingdom of Saudi Arabia between October 2012 and November 2013 using the Vitek 2 automated system.

Discussion

Candida is an important opportunistic fungus, once barriers are broken down, infection and dissemination may occur with fatal consequences. The clinical signs and symptoms are nonspecific, and the routine laboratory methods are insufficient. This insufficiency is at least true when it comes to identifying the unknown “species level”. As we have seen this in 143 isolated strains (), all isolates were designated as “Candida spp.” Our current recognition rate is lower (2.35%) than rates published from other parts of Saudi Arabia7 or from other countries.9 The incidence of candidemia is growing with the increasing complexity of surgical procedures, the existence of populations at higher risk of infections, and the changes in patient demographic characteristics. Approximately 80% of fungal infections in health care settings are due to Candida spp. Nosocomial represents approximately 93.6% and the community acquired 6.4% with 5-71% mortalities, mainly from invasive candidemia.1,7,9 The overall incidence of candidiasis has a 5-fold increase in the past 10-years, and Candida spp.are currently between the fourth and the sixth most common nosocomial bloodstream isolates according to American10 and European studies.11 The present study recorded a prevalence of 2.35% for candidiasis in Aseer Central Hospital. This figure, though lower, it can be compared with the rates in other countries: Turkey 6.6%,9 10% in Scotland and Wales,5 1.7-10 episodes per 100,000 inhabitants in Spain,11 and 5.79% in India.12 In the present study, C. albicans was the predominant species (28.6%) (). The prevalence of Candida spp. according to previous studies: Kingdom of Saudi Arabia,13 Spain,14 Brazil,15 and France16 observed variably high rates of C. albicans followed by C. parapsilosis, C. glabrata, C. tropicalis, C. krusei, C. pelliculosa, C. guilliermondii, and other yeasts.

The distribution of Candida spp. according to specimen types are shown in . When data collected from Aseer Central hospital (2011-2012) was compared with the study from India17 with the same period, we observed that there are some variations. Urine was the main source of Candida infections (72%) in Aseer, followed by the respiratory tract (4.2%) compared with 10.8% (urine) and 30.6% (respiratory tract) in India.17 The respiratory tract (44.1%) followed by blood (30.6%) represented the highest body sites from which Candida was isolated in the Indian study compared with 17.5% and 4.2% in Aseer region in the present study.

The limitations of this study are represented by the fact that neither risk factors nor the in vitro sensitivity profiles of the isolates have been determined. However, in the retrospective study, the isolation of 143 Candida species was successful, but obviously our routine microscopic and colony morphology characterization practice were not sufficient to reveal species names other than C. albicans. This deficiency has been averted when the Vitek 2 identification system was applied in the prospective analysis of the 84 samples. The latter enabled us to uncover many species of the genus Candida and other yeasts as well.

The present study showed the implication of yeasts notably Candida in causing clinical diseases in different body systems especially the urinary tract. Future microbiological screening studies should consider these yeasts, the need for their early diagnosis, and determining their in vitro antimicrobial sensitivities to facilitate correct treatment. Clinicians are informed to consider empiric treatment in risk groups notably among those with the prolonged antibiotic (bacterial) therapy, frequent surgical interventions, frequent instrumentation, the immune-compromised patients, and the extensive use of intensive care facilities.1,6 In all these patients at risk, cultures should be rationally performed.

In conclusion, many Candida species; namely: C. parapsilosis, C. tropicalis, C. lusitaniae, C. sphaerica, C. glabrata, C. krusei, C. haemolunii, C. dubliniensis, C. famata; and some other yeasts such as Cryptococcus terreus have been recorded from patients admitted to Aseer Central Hospital. Unlike other studies, urine and not blood samples were the main source of Candida species. Aside from C. albicans, other species encountered with significant rates were: C. parapsilosis, C. tropicalis, and C. lusitaniae.