Candida Coinfection among Patients with Pulmonary tuberculosis in the World; A Systematic Review and Meta-analysis of Cross-Sectional Studies

Background Diagnosis of fungal co-infections in patients with pulmonary tuberculosis has critical importance. In this review, we aimed to determine the prevalence of candida coinfection in patients with pulmonary tuberculosis.Methods The present systematic review of cross-sectional studies was conducted based on the Preferred Reporting Items for Systematic reviews and Meta-analysis (PRISMA) Protocol. Studies published online in English from January 2001 to March 2019 were assessed. Literature search was performed in Web of Science, MEDLINE/PubMed, and Scopus databases using keywords combinations of “pulmonary fungal”, “pulmonary coinfection”, OR “pulmonary mycosis”, “pulmonary fungal infections/agents”, OR “polymicrobial infection”, OR “secondary infection”, OR “mixed infections”, “pulmonary candidiasis”, “fungi coinfection”, “fungal co-colonization”, AND “pulmonary tuberculosis”, OR “pulmonary TB”. Data was analyzed using Comprehensive Meta-Analysis software. Heterogeneity between studies was evaluated by Cochran's Q, and I 2 tests.Results The pooled global prevalence of candida coinfection among patients with pulmonary tuberculosis was 25.7% (95% CI: 23.7-27.9). C. albicans was the most prevalent Candida spp. with a pooled prevalence of 65.8% (95% CI: 54.3-75.7). Risk factors of candida coinfection included smoking, diabetes, advanced age, and low body mass The present review showed the high rate of candida coinfection among patients suffering from pulmonary tuberculosis. Adequate measures are necessary to early diagnose and treat these infections.

Fungal co-infections are of critical importance in individuals receiving antibiotics and/or corticosteroids due to immunodeficiency disorders such as AIDS or severe pulmonary diseases such as tuberculosis [1]. Undiagnosed opportunistic fungal infections may even lead to death; particularly in patients with compromised immune system [2]. Patients with tuberculosis are immunocompromised and susceptible to fungal and lungs mycotic infections [3].
Generally, up to 1 million people recovered from tuberculosis develop concomitant fungal infections annually which are often misdiagnosed as relapsed TB [4]. Pulmonary tuberculosis is caused by Mycobacterium tuberculosis currently affecting about1/3 of human populations [5]. Furthermore, two million people die from TB complications annually [6].
Depending on the population studied and methods used for isolating fungi, candida species have been identified in 3 to 84% of sputum specimens. The prevalence of candida coinfection has been reported in 15-32% of patients with pulmonary tuberculosis [1].
The main risk factors of primary and secondary fungal pulmonary coinfection in TB patients include immunodeficiency due to mucosal or cutaneous barrier disruption, using broad-spectrum antibiotics and steroids, premature birth, hemodialysis, cytotoxic chemotherapy, tissue transplantation, chronic diseases, neutropenia, defects in the function of innate or cell-mediated immunity, metabolic syndrome, solid and hematologic malignancies, advanced age , recent radiation therapy, burns, prolonged hospitalization, severe trauma, bacterial infections, recent surgery, and implanted central intravascular access devices [7].
Unspecific clinical manifestations deter the diagnosis of fungal pulmonary co-infections imposing patients with tuberculosis at high risk of morbidity and mortality [8]. In particular, fungal infections caused by candida render relatively higher rates of morbidity and mortality [9]. Infections caused by fungi are responsible for death of about 1.5 million people annually. More than 90% of all fungal-associated deaths are caused by four main genera including Cryptococcus, Candida, Aspergillus, and Pneumocystis [10].
Candida albicans is a potentially pathogenic fungi causing very mild to severe invasive mucocutaneous infections, particularly in immunocompromised individuals [11]. Candida species comprise the fourth main causes of nosocomial bloodstream infections (BSI) and are responsible for 8% to 10% of all hospital acquired BSIs in the United States [12].
Therefore, it is important to focus on co-infections of these saprophytic organisms, especially in patients with pulmonary tuberculosis [13].
Recently, the incidence of infections caused by non-albicans Candida spp has increased [4]. Most of these organisms show resistance to routine antifungal drugs [14]. On the other hand, accurate identification of candida species is necessary to appropriately select antifungal agents and treat invasive candidiasis [15]. Concerning the significance of concurrent candidiasis in patients with tuberculosis and the lack of a comprehensive review on this issue, the present systematic review and meta-analysis aimed to determine the prevalence of candidiasis caused by different candida species in patients with pulmonary tuberculosis.
Only studies published in English were included. The literature search was conducted by A.K and A.S. Reference lists of recruited studies were checked to find additional relevant articles.

Inclusion and exclusion criteria
Titles, abstracts, and full texts of articles were read to find relevant studies and check eligibility criteria. Inclusion criteria were a) cross-sectional design; and b) assessing coinfection of candidiasis and pulmonary tuberculosis. Studies published before 2001, case reports, meeting reports, letters to editors, abstracts, case series, congress articles, editorials, literatures reporting inadequate data, articles published in languages other than English, duplicate publications, and narrative or meta-analysis/systematic reviews were excluded. Two reviewers (AK, AS) independently assessed the studies based on the inclusion and exclusion criteria. Disagreements on the inclusion or exclusion of studies were resolved by discussions between the two reviewers or by involving a third researcher.

Quality evaluation
Appraisal tool for Cross-Sectional Studies (AXIS) was used for assessing the quality of the included studies. Using this check, twenty items were evaluated in each study. If the related data was documented, a question was scored 'yes'. In case of any doubt or indistinct data, a question was marked 'no' or 'can't tell'. According to the scoring system and the number of questions scored 'yes', the quality of studies was finally categorized as either 'strong', 'intermediate', or 'weak' [16]. Studies with weak quality were excluded (File 2).

Data extraction
The following data was extracted and tabulated: first author's name, year of publication, location (country), sample size (TB + ), rate of candida coinfection, and detection method.

Statistical analysis
We used Comprehensive Meta-Analysis software (Version 3.3.070) for meta-analysis.
Statistical significance level was considered as P < 0.05. Random effects model was used to calculate overall effects. Sources of between-study heterogeneity were assessed using Cochran's Q and I square (I 2 ) tests and subgroup analysis. Egger's regression asymmetry test was used to detect publication bias. The correlations between pulmonary candidiasis coinfection and patients' characteristics such as age, gender, body mass index (BMI), and factors such as alcohol consumption, smoking, and diabetes mellitus (DM) were also scrutinized by Spearman correlation.

Study selection
In total, 984 records were identified in primary literature search (Figure1). Of these, 453 duplicates were removed. Then abstracts and titles of 533 remained records were screened which resulted in exclusion of 401 irrelevant articles. Next, 130 full-text articles were assessed for eligibility criteria which led to the exclusion of 105 additional studies that did not fulfill inclusion criteria. After that, 25 studies were selected for qualitative appraisal. From these, seven studies with weak qualities were excluded. Finally, 18 studies entered quantitative synthesis (Meta-analysis).

Features of the selected studies
All the 18 included cross-sectional studies had been published between January 2001 and March 2019. The studies were from countries in Asia and Africa, especially from India with 9 reports. No report was from developed countries. Overall, 2139 TB positive patients were analyzed. The rate of candidiasis coinfection varied from 2.8% to 55%. Only phenotypic methods (i.e. AFB analysis-Direct Microscopy-Culture, Gram staining, KOH Mount, Ziehl-Neelsen technique, CHROM agar Candida culture method, Sugar fermentation tests, and Germ tube test) had been used to detect tuberculosis and fungal infections in all the studies. The studies' characteristics have been described in Table 1 Overall effects The pooled global prevalence of candidiasis coinfection among patients with pulmonary tuberculosis was 25.7% (95% CI: 23.7-27.9, Q=177.5, Z=18.7) ( Table 2 (Table 3).

Risk factors of candidiasis coinfection and its correlation with patients' demographic characteristics
A significant correlation was reported between advanced age and candidiasis coinfection in two studies (p<0.05, Table 4). Also, three studies reported a significant correlation between male gender and candidiasis coinfection whereas only one study showed a correlation with female gender (p<0.05). Alcohol consumption was not associated with candidiasis coinfection in neither of the studies (p>0.05). One study reported low BMI as a potential risk factor for pulmonary candida coinfection, and two another studies described a correlation between candidiasis coinfection and smoking (p<0.05). Finally, two studies reported higher prevalence of pulmonary fungal coinfection in tuberculosis patients with diabetes mellitus (p<0.05).

Discussion
The global rate of candidiasis coinfection varies from 2.8% to 55% in patients with pulmonary tuberculosis. The present systematic review showed a combined global prevalence of 25.7% for candidiasis coinfection in patients with pulmonary tuberculosis.
The pooled frequencies of candidiasis coinfections were 27.9%, 9.7%, and 30.7% in India, Iran, and Nigeria respectively. The pooled prevalence of candida coinfection was also obtained as 45.5% in Cameroon. Nevertheless, as estimations in Cameroon, Iraq, and Kenya were based on only one study, the reported frequencies may not indicate the true incidence of candidiasis coinfection in these countries. Therefore, it is recommended to perform more studies on this issue in the mentioned regions to more accurately estimate the rate of coinfection. Variations in the frequency of candidiasis and pulmonary tuberculosis coinfection probably reflect the differences in the local prevalence of different candida species secondary to different environmental conditions. Also, the types of clinical samples and detection methods can affect the reported rate of the coinfection [2].
Several studies have described candida species as the most common fungal agents retrieved from sputum of patients with pulmonary tuberculosis. Nonetheless, because of the presence of these fungi in throats of 30% of healthy individuals and risk of crosscontamination during sputum collection, the importance of candidiasis in tuberculosis patients has been undermined [17]. Kahanpaa et al. showed that C. albicans was the most common organism recovered from 60-70% of sputum samples, 16.5% of bronchial secretions, and 20.4% of lung fragments. C. albicans has also been isolated from 53.0% of healthy subjects' oral samples [18].
Similarly, C. albicans comprised the most prevalent fungus coinfected patients with pulmonary tuberculosis followed by C. tropicalis and C. parapsilosis with respective frequencies of 65.8%, 16.9%, and 14.3%. In fact, all the studies included in present review reported C. albicans as the most common cause of fungal-tuberculosis coinfection. In line with our results, C. tropicalis has been noted as the second most prevalent cause of fungal coinfection in patients with tuberculosis. C. tropicalis is a new opportunistic pathogen representing higher rates of severe disease and deep tissue invasion than C. albicans in immunocompromised hosts [19,20]. According to our findings and results obtained by other studies, the high rate of coinfection with C. albicans and other Candida spp. may be due to cross-contamination or co-colonization and not necessarily reflect actual infection.
For obviating this problem, it is recommended to use bronchoscopy rather than sputum sampling to reduce the risk of cross-contamination of samples with upper respiratory secretions. In practice; however, bronchoscopy is not always used in developing countries such as India and Cameroon [21].
Because of insufficient clinical data and unavailability of radiologic images, a high ratio of pulmonary fungal and tuberculosis coinfections reported in the analyzed studies may merely indicate colonization rather than actual infections. Nevertheless, clinical and radiological studies alone are inadequate to differentiate pulmonary fungal from tuberculosis infections. Therefore, it is necessary to screen primary/secondary fungal infections using accurate detecting methods in patients with pulmonary tuberculosis [22]. This is of crucial importance considering that co-infections with opportunistic fungal and other non-tuberculosis pathogens can aggravate the clinical course of tuberculosis, accelerate disease recurrence, and increase mortality rate [10]. Nevertheless, no comprehensive epidemiological data is available about fungal coinfection as they frequently remain either misdiagnosed or undiagnosed. Except for Coccidioidomycosis, no fungal diseases have actually been reported to the Centers for Disease Control and Prevention (CDC) [10].
In the present review, three studies [3,23,24] showed a significant correlation between male gender and candidiasis coinfection (p<0.05). On the other hand, only one study [25] showed a correlation between female gender and candidiasis coinfection (p<0.05). Our findings agreed with the reports of WHO indicating male gender as a predisposing factor for tuberculosis infection [26]. This may possibly be related to higher environmental exposure of males than females to bacterial and fungal infections [27]. Nonetheless, Hidalgo et al. reported equal rates of Candida spp. colonization in both males and females [28]. Altogether, it is highly recommended to accurately identify all organisms recovered from clinical samples of patients with tuberculosis. The limitation of this study is that we only cite studies that existed in databases and other studies that were unpublished were not included in the study. We also did not contact to the authors of the articles in cases where further clarification was required.

Conclusion
The present review revealed high rate of candidiasis coinfection among patients with pulmonary tuberculosis. Considering that clinical and radiological evidences of fungal coinfections are unclear; accurate diagnostic procedures should be conducted to early diagnose and treat these opportunistic infections and decrease mortality and morbidity rates associated with fungal coinfection in tuberculosis patients.

Funding support
This study did not receive any financial support.    The Forest plot of the meta-analysis of the prevalence of Candida coinfection among patients with pulmonary tuberculosis