Distribution, antifungal susceptibility pattern and intra-Candida albicans species complex prevalence of Candida africana: A systematic review and meta-analysis

Candida africana is a pathogenic species within the Candida albicans species complex. Due to the limited knowledge concerning its prevalence and antifungal susceptibility profiles, a comprehensive study is overdue. Accordingly, we performed a search of the electronic databases for literature published in the English language between 1 January 2001 and 21 March 2020. Citations were screened, relevant articles were identified, and data were extracted to determine overall intra-C. albicans complex prevalence, geographical distribution, and antifungal susceptibility profiles for C. africana. From a total of 366 articles, 41 were eligible for inclusion in this study. Our results showed that C. africana has a worldwide distribution. The pooled intra-C. albicans complex prevalence of C. africana was 1.67% (95% CI 0.98–2.49). Prevalence data were available for 11 countries from 4 continents. Iran (3.02%, 95%CI 1.51–4.92) and Honduras (3.03%, 95% CI 0.83–10.39) had the highest values and Malaysia (0%) had the lowest prevalence. Vaginal specimens were the most common source of C. africana (92.81%; 155 out of 167 isolates with available data). However, this species has also been isolated from cases of balanitis, from patients with oral lesions, and from respiratory, urine, and cutaneous samples. Data concerning the susceptibility of C. africana to 16 antifungal drugs were available in the literature. Generally, the minimum inhibitory concentrations of antifungal drugs against this species were low. In conclusion, C. africana demonstrates geographical variation in prevalence and high susceptibility to antifungal drugs. However, due to the relative scarcity of existing data concerning this species, further studies will be required to establish more firm conclusions.


Introduction
The medically important polyphyletic genus Candida contains more than 300 different yeast species, around 20 of which are regularly reported from human infections ranging in spectrum from superficial mycoses to deep-seated and disseminated infections [1][2][3]. Candida albicans is widely accepted as the most virulent species in the genus, and is the etiological agent in approximately 50%, 95%, and 80-90% of cases of nosocomial bloodstream Candida infections, oropharyngeal and vulvovaginal candidiasis, respectively [4][5][6][7].
C. albicans is a complex of three closely-related species, C. albicans sensu stricto, C. dubliniensis, and C. africana [6,8]. C. africana, which was first isolated in Africa in 1995, was proposed as a new species within the C. albicans complex in 2001 [9,10]. With a worldwide distribution, C. africana has been isolated from diverse clinical specimens (mucous membranes, cutaneous samples, specimens from the urinary and respiratory tracts, blood) and has been reported to cause a wide variety of human infections including vulvovaginal candidiasis, oral thrush, and blood stream infections. [11][12][13][14][15].
Unlike the other members of C. albicans complex, C. africana is unable to form chlamydospores and cannot assimilate glucosamine, N-acetylglucosamine, trehalose, or DL-lactate. However, in common with C. albicans and C. dubliniensis it has retained the capacity to produce germ-tubes. Moreover, molecular studies have demonstrated high levels of genetic relatedness between C. africana and C. albicans [16][17][18]. Thus, differentiation of C. africana from the other members of C. albicans complex using conventional identification techniques is difficult [19,20].
Given these issues, molecular methods such as an end point PCR based on size polymorphism of the hwp1 gene (C. albicans: 941bp, C. dubliniensis: 569 bp, and C. africana: 700 bp) have been designed to discriminate between C. albicans, C. dubliniensis, and C. africana [21]. Using such approaches, the prevalence of C. africana within the C. albicans complex has been reported to vary significantly from 0 to 8.4% depending on the geographic regions in which analyses were performed [11,19,[22][23][24]. Furthermore, while some studies have suggested that the susceptibility profiles of C. africana to antifungal drugs are similar to those of C. albicans [25], others have reported different antifungal susceptibility patterns for these species [8,26]. In light of the above discrepancies concerning C. africana prevalence and antifungal susceptibility, the present review and meta-analysis was designed to summarize all of the available data concerning this recent addition to the C. albicans species complex.

Search strategy
Two independent researchers conducted bibliographic search in PubMed, Scopus, and Web of Science databases as well as in Google Scholar using keywords or phrases "Candida africana", "C. africana", "Candida albicans complex", "Candida albicans sibling species", and "Candida albicans cryptic species" and their combinations. Since Candida africana was first described as a novel species in 2001 [10], our search covered the literature published in the English language from 2001 to 21 st March 2020.

Study selection
Citations were included into EndNote software version X8, duplicates were deleted and the title and abstract of remaining citations were reviewed to exclude irrelevant articles. For the remaining citations, full texts were downloaded and evaluated. All English language articles with available full texts that reported data on antifungal susceptibility patterns of Candida africana and/or prevalence of Candida africana within the Candida albicans species complex using molecular methods met the inclusion criteria. Conference abstracts, review articles, and articles reporting data other than the susceptibility pattern and/or prevalence of Candida africana were excluded. The quality of the selected studies was checked using the STROBE checklist [27]. References cited in the eligible articles were also screened to guarantee the inclusion of all relevant studies.

Data extraction
Data including the name of the first author, publication year, country, number of Candida albicans complex isolates, number of identified Candida africana isolates, the source of Candida africana isolates, and the minimum inhibitory concentration (MIC) values of various drugs against Candida africana isolates were extracted into a pre-prepared excel file by two independent researchers. Corresponding authors of studies reporting only the summary data of antifungal susceptibility pattern such as MIC range, geometric mean (GM), and MIC 50 were contacted via email for the raw data. In the case of no response, the summarized data of antifungal susceptibility patterns were excluded from the final analysis.

Data analysis
The pooled estimated prevalence of C. africana within the C. albicans complex was calculated using Stata software version 14. Variances and their confidence intervals were calculated using exact method. The pooled estimate was between 0 to 1. For studies reporting a prevalence of 0%, Freeman-Tukey double arcsine transformation was used to stabilize variances. Heterogeneity was determined using the I 2 statistic which was calculated using the DerSimonian-Laird method. For quantification of heterogeneity, Cochrane Q test was used. In the presence of heterogeneity, random effect model provides better estimates [28,29], accordingly, we used this model in calculations when heterogeneity was proved to exist. Subgroup analysis was done to define the prevalence of C. africana within the C. albicans complex in different countries and continents. The presence of publication bias was checked by using the funnel plot and the Begg's test. In the case of asymmetric funnel plot, Trim and Fill method was used to define the number of missing studies and the imputed estimated prevalence. To check for changes in prevalence over time, meta regression was conducted where the year of publication was set as the independent variable. In all calculations p-values <0.05 were considered to be significant.  (Table 1). Due to the presence of heterogeneity (I 2 = 66.02%, 95% CI 44-77, p<001), random-effect model was used. The pooled prevalence of C. africana within the C. albicans complex was 1.67% (95% CI 0.98-2.49) (Fig 2). Data on prevalence were available for 11 countries from 4 continents. Iran Information on isolation source was available for a total of 167 isolates. Although the vast majority of isolates were from the female genital tract (vagina; n = 155, 92.81%), there were also isolates from patients with balanitis (n = 5, 2.99%), oral lesions (n = 4, 2.39%), and isolates from respiratory, urine, and skin samples (1 isolate each, 0.6%).

Results
MIC values were available for C. africana isolates and 16 antifungal drugs including azoles, echinocandins, polyenes, allylamine, and 5-flucytosine. As shown in Table 3, the MIC ranges, MIC 50 and MIC 90 and geometric mean values were generally low.

Discussion
C. africana, a member of C. albicans species complex, is genetically and phenotypically closely related to C. albicans. The pathogenicity of C. africana and its impact on the health of humans is poorly understood. Moreover, the global prevalence and antifungal susceptibility profiles of this species are not clearly defined [16,18,30]. In this study we tried to provide an overview of the available data published to date on both of these aspects of C. africana epidemiology/ biology.
C. africana appears to be globally distributed, with an intra-C. albicans complex prevalence that varies in different regions and countries [16,31,43]. To date, data concerning prevalence are available for 11 countries from 4 continents (Africa, America, Asia, and Europe), with a pooled intra-complex prevalence of 1.67% (95% CI 0.98-2.49). Based on the available  Table 1. Characteristics of 41 studies reporting data on intra-Candida albicans complex prevalence and/or antifungal susceptibility pattern of Candida africana which were eligible to be included in the current systematic review and meta-analysis.

Reference
Year Country No. of C. albicans complex/C. africana

Source of isolates (N) Data of antifungal drugs
Alonso-Vargas et al. [30] 2008 The intra-complex prevalence of C. africana appears to be constant over time. In recent decades, the prevalence of non-albicans Candida species has increased [49,50] and there are reports describing species other than C. albicans as being the most common etiologic agents of infection locally [51][52][53]. However, it seems that a similar scenario has not been occurring within the C. albicans species complex since the meta-regression analysis of our data indicates that there is no significant change in the intra-complex prevalence of C. africana with the passage of time. However, once again there are caveats to this suggestion. First, it is based on data from a limited number of countries. Moreover, the power of meta-regression analyses is low especially when the number of studies included is low, which is the case in the present study.
Female genital specimens are the most common source of isolation of C. africana. Of 167 C. africana isolates with available data, the majority (n = 155, 92.81%) were from the vagina. Vulvovaginal candidiasis due to C. africana has been reported in various countries [32]. This species was also isolated from cases of balanitis (n = 5, 2.99%) and oral lesions (n = 4, 2.39%), and from respiratory, urine, and skin samples (each 1 isolate, 0.6%), all of which could conceivably become contaminated with vaginal flora or pathogens. The apparent preponderance of C. africana for the female genital tract highlights the need for appropriate methods for discrimination of C. africana from C. albicans complex isolates, especially for vaginal specimens.
Data on antifungal susceptibility of C. africana to 16 antifungal drugs are available in the published literature (Table 3). It should be highlighted that the data presented in Table 3 are limited to articles in which detailed results of antifungal susceptibility testing are provided. Other articles that have reported their results as the number of resistant/susceptible isolates or as geometric mean and MIC range (and not the raw MICs) could not be included in Table 3. Similar patterns of susceptibility to various antifungal drugs has been reported for C. africana and C. albicans [25], while other studies have noted that C. africana exhibits a different

Reference
Year Country No. of C. albicans complex/C. africana

Source of isolates (N) Data of antifungal drugs
Yazdanpanah et al. [11] 2014 Malaysia 98/0 -NA Yazdanparast et al. [48] 2015 susceptibility pattern to C. albicans [8,26]. Since there are no specified clinical breakpoints or epidemiological cut-off values (ECVs) for antifungal drugs against C. africana, the interpretation of MICs as susceptible/resistant or wild-type/non wild-type is potentially controversial. However, there are reports in which isolates of C. africana have been categorized as resistant to itraconazole, 5-flucytosine, terbinafine, fluconazole, and clotrimazole [28,33,54]. By applying the clinical breakpoints for C. albicans (CLSI M60) [55], the species most closely related to C. africana, it could be inferred that almost all isolates of C. africana with available MICs for fluconazole, voriconazole, anidulafungin, caspofungin, and micafungin are susceptible to these antifungal drugs. For itraconazole, in contrast to CLSI (M60 supplement) [55] which no longer proposes breakpoints for Candida species, the European Committee on Antimicrobial   Susceptibility Testing recently published new breakpoints for itraconazole against C. albicans and C. dubliniensis [29]. Using those breakpoints (>0.06 μg/mL = resistance) 12 out of 43 (27.91%) C. africana isolates with available data would be itraconazole-resistant. Further studies will be required to generate MIC data for sufficient numbers of isolates of C. africana to allow the establishment of robust species-specific ECVs and clinical breakpoints for this species.

Conclusion
C. africana is a minor species within the C. albicans complex with a pooled prevalence of 1.67%. Reports of this species are available from a limited number of countries and further investigations are required internationally to fully address its global distribution. The vagina is the most common human source of C. africana and based on clinical breakpoints established for the related C. albicans, this species can be inferred to be generally susceptible to most currently available antifungal drugs.