Candida Co-Infection with Mycobacterium tuberculosis in Tuberculosis Patients and Antifungal Susceptibility of the Isolates

It had been observed that tuberculosis (TB) subjects can be co-infected 
with Candida sp. which was previously 
assumed as normal flora of the oral cavity. Candida sp. might become an opportunistic pathogen in immune compromised 
individuals. Candida co-infection 
with Mycobacterium tuberculosis in TB 
patients might complicate underlying disease process in the lungs. Materials and Methods: A total of 400 sputum samples were collected from TB patients and 
examined using Ziehl-Neelsen staining technique and MDR/RIF Genexpert system 
for TB. Samples positive for Mycobacterium 
tuberculosis were cultured on Sabouraud 
Dextrose Agar with gentamicin and examined for the presence of budding 
yeast cells and pseudohyphae on Gram’s stain. Candida sp. isolated from TB positive sputa were cultured on 
CHROMager Candida for identification 
to species level and subjected to antifungal susceptibility testing. Results: Out of 400 sputum samples examined for TB 93 (23.3%) 
were positive and 32 (34.4%) out of 93 TB positive cases were co-infected with Candida sp. Candida albicans was the most predominant species with a prevalence of 23 (67.6%), C. tropicals 4 (11.8%), C. krusei 4 (11.8%) 
and C. parapsilosis 3 (8.8%). One sample had dual infection. Female 
subjects had high prevalence (19.4%) than the male (15.7%). Age 
group 31 - 40 years had both high prevalence of TB 
32.3% and Candida 25.0%. Antifungal 
susceptibility testing showed that 
isolated Candida sp. were more 
susceptible to vericonazole and fluconazole compared to nystatin. Conclusions: Tuberculosis weakens the immune systems of infected persons especially when 
prophylactic administration proves abortive or there is non-adherence 
to treatment prescriptions. This may 
cause the development of multidrug resistance TB. Candida sp. may utilize these opportunities to establish alongside M. tuberculosis and worsen treatment and patient condition. For good 
treatment of TB, Candida co-infection should be screened concomitantly with TB in TB suspected individuals.


Introduction
Co-infection is the simultaneous infection of a host with more than one disease causing agent. Worldwide, the prevalence or incidences of co-infection in the life of human were unknown but it is now known to be common [1] and even more common than single infection. A global common co-infection is that of tuberculosis and HIV and of recent, tuberculosis and Candida. In some countries up to about 80% of tuberculosis patients are co-infected with HIV [2] and about 15% -32% of tuberculosis patients are also co-infected with Candida sp. [3].
AIDS involves co-infection of end-stage HIV with opportunistic parasites and poly-microbial infections like Lyme disease with other diseases [4]. Candidiasis is a fungal infection that can be caused by any species of Candida. It can spread to other parts of the body apart from the mouth and vagina which can result in fever and other symptoms depending on the site of infection [5]. Despite all efforts made globally, tuberculosis as an infectious disease remains a global threat. About 9.6 million people were diagnosed of having new cases of tuberculosis infection and about 1.5 million deaths were recorded as a result of this disease in 2014. Approximately 1.2 million (12%) of these cases were related to HIV infection [6]. A total of 1.5 million people died from TB in 2018 including 251,000 people living with HIV. TB is the major cause of death from a single infectious agent (above HIV/AIDS) and one of the top 10 causes of death worldwide [7].
The estimate in 2018 was that 10 million people were sick with tuberculosis (TB) worldwide, out of these 5.7 million men, 3.2 million women and 1.1 million children. The largest number of new TB cases occurred in the South-East Asian region, with 44% of new cases, followed by the African region, with 24% of new cases and the Western Pacific with 18%. The 30 high TB burden countries accounted for 87% of new TB cases. Eight countries account for about two thirds of the total in decreasing order, India, China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh and South Africa [7].
Different kinds of pulmonary and extra-pulmonary infections might also aggravate factors associated with chronic obstructive pulmonary diseases such as diabetes in elderly patients, high cholesterol levels, lung cancer, immune deficiency and fungal infections of the lungs.
The rate of recurrence infections and the number of immunosuppressive disease cases are on the increase. It is of great importance that clinical research be carried out by medical health personnel into potential pathogenic secondary fungal diseases bearing in mind that it can influence the progression of tuberculosis infection which might be life threatening [8]. The occurrence of pulmonary tuberculosis co-infection cases with Candida albicans is about 15% -32% in dif-  [3]. The burden of active tuberculosis still continues at alarming rate especially amongst the low and middle income countries with estimated 580,000 new cases arising as a result of multiple drug resistance TB (MDR-TB) worldwide [6] and in 2018, 7.0 million new cases were notified [7]. The aim of this study were to determine the prevalence of tuberculosis, tuberculosis Candida co-infection, Candida species associated TB Candida co-infections and antifungal susceptibility of isolated Candida sp.

Study Area and Population
The study was conducted in the Model Primary Health Centre, Rumuigbo in

Sample Size
Sample size was determined using the formula of Araoye (2003)

Direct Smear Examination by Microscopy
A total of 400 sputum samples were obtained from patients attending the Model Primary Health Centre and examined for TB and TB Candida co-infection. The presence of budding yeast cells and pseudohyphae in direct Gram's stained smear using the ×100 objective may aid diagnosis [3]. The smears were fixed with alcohol to avoid over absorption of stains by Candida hyphae if present [9]. Candida sp. grown on Sabouraud dextrose agar were examined by microscopy (wet mount) with ×10 and ×40 objectives.

KOH Mount (KOH Preparation)
A drop of 20% w/v KOH solution was placed on a clean, grease free glass slide.
Sputum sample were added to the drop and covered with a cover slip. This was placed in petridish with a damp filter paper under to prevent drying. The prepa-

Ziehl-Neelsen Technique
A thin smear of each sputum sample was made on a clean grease free slide, allowed to air dry and heat-fixed by passing the slide 3 -4 times through a Bunsen flame. The slides were stained with strong carbol-fuschsin on a staining rack.
The slide was gently heated under by passing flame under the staining rack until steam rises without allowing it to boil. The slides were allowed to stand for 5 minutes, rinsed with water and decolourized with 20% H 2 S0 4 until the slide appeared light pink in colour. This was rinsed in water and then counterstained with methylene blue (0.5%) for 1 -2 minutes. Slides were rinsed with water and allowed to air dry after wiping the back of the slide and examined with oil immersion xl00 objective.

Germ Tube Production Test
A little portion of 18 -72 hours old colony of yeast was suspended in 0.5mL of human serum in a test-tube. Positive and negative controls were set up. All the test-tubes were incubated at 37˚C for 2 -3 hours. A drop of the mixture of serum and yeast suspension were placed on a clean glass slide and covered with clean cover slip and examined microscopically for presence or absence of germ tube production.

Cultivation of Candida Sp.
The sputum specimens were cultured on modified Sabouraud-dextrose gentamicin agar medium and incubated at 37˚C for up to 72 hours. Gentamicin inhibited bacterial growth [9]. Pure cultures of Candida sp. isolated were cultivated on CHROMagar Candida.

CHROMagar Candida
Culture on CHROMagar is used to identify Candida sp. Isolated yeasts sp. on Sabouraud-dextrose agar were also cultured on CHROMagar Candida and incubated at 37˚C for 48 hours. Identification of Candida sp. on this agar was based on colour production. Light green colonies was Candida albicans, blue Candida tropicalis, pink Candida krusei and cream Candida parapsilosis [10], CHOR-Magar candida is very useful in the rapid identification of Candida sp.

GeneXpert Test (Molecular Assay)
Sputum samples obtained from suspected TB patients were liquefied and inactivated with 2:1 sample reagent. Two (2)

Sugar Fermentation Reactions
Peptone water containing a single carbohydrate (1.0%) such as glucose, lactose, sucrose and the other carbohydrates were used. A p H indicator (phenol red) was added to the medium to detect change in pH as a result of acid production. Durham's tubes were also inserted to detect the production of gas. The isolated Candida sp. were inoculated into already prepared and sterilized set of sugars in test tubes. They were incubated at 37˚C for 18 -48 hours and examined.

Susceptibility Testing
Each pure culture of Candida sp. was inoculated onto already prepared and dried iso-sensitive agar plate. The inoculated plate was spread evenly by streaking with sterile wire loop to obtain uniform seeding on the entire plate. Commercial antifungal single discs of the three (3) antifungal agents were placed on the inoculated plate with a sterile forceps. The plates were incubated at 37˚C for 18 -48 hours and the zones of inhibitions were obtained.

Ethical Consideration and Sample Collection
Ethical approval was obtained from Rivers State Hospitals Management Board, participants were briefed on the objectives of the studies and confidentiality of each patient was assured.

Statistical Analysis
Statistical analysis was done using one-way Anova with the data generated.

Prevalence of TB and TB Candida Co-Infection
A total of 400 sputum samples collected from suspected TB patients were examined for TB and candidaisis. Out of these 93 (23.3%) were positive for tuberculosis and 32 (34.4%) were positive for tuberculosis Candida co-infection as shown on Table 1.

The Prevalence of Candida sp. among TB Patients
Four species of Candida were isolated co-infecting with M. tuberculosis. Namely, Candida albicans 21 (65.6%), C. tropicalis 5 (15.6%), C. krusei 5 (15.6%), C. parapsilosis 3 (9.4%). Out of the three species, C. parapsilosis was isolated with C. albicans in particular case. Candida albicans was the most predominant species associated with TB Candida co-infection as shown on Table 3.

Prevalence of Candida Species by Age Groups
The age groups with the high prevalence of Candida were between 31 -40 yrs with a prevalence of 8 (25.0%). Age groups 41 -50 and 51 -60 yrs were 5 (15.5%) each and age group 61 -70 yrs was 4 (12.5%). The other age groups were between 0.00% -6.3% as shown on Table 5.

Antifungal Susceptibility Patterns of Isolated Candida sp.
Three (

Carbohydrates Fermentation Reactions of Isolated Candida sp.
Carbohydrates fermentation reactions tests showed that C. albicans is a non-lactose and non-inositol fermenter but ferments glucose, maltose, sucrose, galactose and xylose and was positive for germ tube test. C. tropicalis and C. parapsilosis had the same biochemical characteristics as that of C. albicans in utilization of carbohydrates but are negative for germ tube test. C. Krusei fermented only glucose and was germ tube negative. C. parapsilosis fermented glucose, maltose, sucrose and galactose but not lactose and inositol and was also germ tube negative as showed on Table 7 below.

Discussion
Despite all global efforts to eradicate TB, tuberculosis (TB) as an infectious disease has remained a global threat. Co-infection of the respiratory tract by fungi and tuberculosis had been recognized for its wide range of clinical spectrum and chronicity. Among the respiratory fungal infections, infections by Candida sp.
were the commonest fungal infectious agents isolated from sputa of TB patients.
In this study, the prevalence of TB was 93 (23.3%) but the prevalence of TB Candida co-infection was 32 (34.4%). It may be assumed that some of the devastating effects of TB on infected persons might result from the synergistic actions of both organisms. A similar study [9] had a prevalence of 25.3% TB Candida co-infection and Candida albicans were the most prevalent species as also observed in this study.
Some researchers speculated that since Candida were part of the normal microbiota of the throat in about 32.5% of healthy individuals, the sputum produced may have been contaminated with Candida sp. Using Kahampaa criteria that three (3) or more repeated isolation of Candida or more than 30 colonies of Candida on sabouraud dextrose agar should be considered pathogenic and should not be treated as commensal [12]. The observations of pseudomycelial forms in sputum were also suggestive of fungal infection other than ordinary colonization.
The prevalence of Candida co-infection among tuberculosis subjects as observed by [3] was 40%. The prevalence of TB Candida co-infection was high among females (19.4%) than in males (15.1%) in this study. This result however is not in agreement with the results obtained by [13], they observed that colonization rate of Candida sp. were equal in both males and females, [14] in their study found Candida infection to be more in males than in females. Similarly, [9] observed that Candida infections were significantly high in males (26.1%) than in females (23.1%). In this study, the prevalence of TB Candida co-infection was high among females (19.4%) compared to males (15.1%) and this agrees with the study conducted by [3] who observed that TB Candida co-infections were significantly high among female patients (62.5%) compared to male patients (29.4%). The prevalence rates of Candida sp. among both sexes correlates with that of Mycobacterium tuberculosis in males and females. The high prevalence in the females could be attributed to the fact that more women seek for medical attention and treatment than males and due to increased out-Journal of Tuberculosis Research door and social activities observed among females. One-way Anova comparison of males and females did not show significant difference.
Among the Candida sp. isolated, C. albicans were the predominant species with a prevalence of 21 (61.8%). This was in line with the studies carried out by [3] [10] both reported similar prevalence of 50% and 66% respectively. The prevalence of TB/Candida co-infection ranged from 45% -92% in several Indian studies [15]. In this present study, the prevalence of Candida sp. isolated were 61.8%, 14.7%, 14.7% and 8.8% for C. albicans, C. tropicalis, C. krusei and C. parapsilosis respectively. On the contrary, [9] reported lower rates of C. albicans 15.1%, and C. tropicalis 8.4%. [15] observed C. tropicalis 3.25%, C. parasilosis 3.25%, [16] documented Candida tropicalis 19.95%, C. glabrata 16.54% C. parapsilosis 13.14% and C. krusei 5.10%. These variations may be attributed to differences in local prevalence of different species of Candida due to different environmental conditions as well as the isolation techniques employed. It was also observed in this study that among the non C. albicans sp., Candida tropicalis was the second predominant species which correlates with findings of similar studies conducted by [16] [17] [18]. Comparison of all species of Candida showed that there was significant variation (p = 0.0001) between the different species of Candida isolated.
The prevalence of TB Candida co-infection by age groups was high among age group 21 -30 years (18.8%) and 31 -40 years (25.0%). The high prevalence among the two age groups could be as a result of increased outdoor activities as they associate with one another, overcrowding in most of the settlements and poor standard of living. It could also be attributed to increased reproductive activities within the age groups that were described as the most active segment of any economy. This observation was in compliance with the work done by [19] with a prevalence of 81.6% among the age group 21 -40 years. The findings of this study were also in agreement with the work conducted by [20] and [21] both reported high prevalence of TB Candida co-infection among age group 21 -40 years. However, the finding of this study differs with the work conducted by [22] which showed a high prevalence of TB Candida co-infection among age groups 40 years and above. The corresponding high prevalence of TB and Candida in- tuberculosis (MDR-TB) were TB with bacilli resistance to at least isoniazide and rifampicin which are the two main first line anti-tuberculosis drugs which have become an important concern in the control of TB in many countries, especially in the sub-Sahara Africa where the burden of other competing diseases like malaria, enteric fever, HIV, etc. were also high [25]. The discovery of Genexpert MTB/RIF testing has made possible the detection of tuberculosis bacilli resistant to rifampicin [26]. Drug resistance emanating from inadequate treatment of active pulmonary tuberculosis could result from inappropriate therapy, poor prescription practices, in-sufficient treatment duration, irregular medication intake, poor drug selection, inadequate public health resources and socio-economic determinants of health [27]. The three antifungal agents used for susceptibility testing were voriconazole, fluconazole and nystatin. Voriconazole showed high susceptibility against the Candida sp. closely followed by fluconazole. In a similar research carried out [28] [29] it was clearly shown that the azoles are frequently used for pulmonary fungal infections and are preferred to intravenous amphotericin B. Though Candida albicans was the commonest pathogen associated with pulmonary candidiasis, there is also an increasing incidence of other Candida sp. Among the non C. albicans species isolated was Candida tropicalis which showed increased prevalence as a new emerging opportunistic pathogen causing severe invasive pulmonary disease. It had been shown to have great capacity of invasion than C. albicans into deep tissues of immunocompromised patients [14] [29] One-way analysis of variance of the susceptibility patterns showed significant variation (p = 0.0001) between Voriconazole, Fluconazole and Nystatin. Student t-test comparison of Voriconazole and Fluconazole did not show significant difference (p = 0.0705). Student t-test comparison of Voriconazole and Nystatin showed significant difference (p = 0.0003). Student t-test comparison of Fluconazole and Nystatin also showed significant difference (p = 0.0006).

Conclusion
Tuberculosis remains a global threat despite effort to eradicate the disease and TB co-infection with Candida sp. may complicate infection and treatment. The most predisposed age groups to both infections were ages 20 to 40 years, the most active segment of the economy. Screening for TB should be conducted concomitantly with candidiasis and TB treatments should be combined with the administration of anti-fungal agent confirmed by susceptibility testing in cases of TB Candida co-infections.

Limitation of Study
The limitation of this study was the non inclusion of the molecular aspect of Candida.