Fungi Associated With Persistent Cough In Immunocompromised Hosts In Jos And Environ

DOI: http://dx.doi.org/10.24018/ejmed.2020.2.3.325 Vol 2 | Issue 3 | June 2020 1 Abstract — This study was conducted to determine the fungi associated with persistent cough in immunocompromised patients attending Jos University Teaching Hospital (JUTH) and Vom Christian Hospital both in Plateau State. Hundred (100) immunocompromised patients with persistent cough were selected by random sampling. The sputum samples were collected and assayed for fungi using standard methods. The degree of disparity was analyzed using statistical method. Four different fungi were isolated from sputum of 37 (37%) immunocompromised patient with persistent cough, with Candida albicans been the highest 18 (48.6%) followed by Aspergillus niger 12 (32.4%), Cryptococcus neoformans 5 (13.5%) and Penicilium species 2(5.4%). Male subjects 22 (22%) were more susceptible to fungi associated cough than female 15(15%) with significant difference (P value 0.026). HIV, Tuberculosis and co-infection of both (HIV and TB) and prolong use of antibiotics were more predisposing factor associated with fungi related cough. These isolates were tested on 5 antifungal drugs. Ketoconazole and intraconazole were most susceptible to 83.8% of isolates, Amphotericin B had 54.1% susceptible while Griseofulvin and Vericonazole were 18.9% susceptible to the isolates. However, in managing the immunocompromised patient, there is a need for antifungal prophylaxis and sending of sample of immunocompromised host with persistent cough not only for bacteriological analysis but also mycological evaluation.


I. INTRODUCTION
Fungi are microscopic plant organisms that consist of cells such as mould, mildew and yeast. They cannot produce their own food; thus, they behave as either parasite or saprophytes, absorbing nutrients from organic matter, such as human and animals [1]. They are present everywhere indoor and outdoor environment. The most common symptoms of fungal exposure are runny nose, eye irritation, cough, congestion and aggravation of asthma [2].
Invasive fungal disease continues to be a significant problem among immunocompromised patients. Most fungal infections are caused by commonly recognized opportunistic fungi [3]. Opportunistic fungi are generally harmless in its normal environment but become pathogenic in immunocompromised host [4], [5]. A compromised host is a host seriously debilitated and has lowered resistance to infection [5]. There are many causes of this condition. They include: Malnutrition, Alcoholism, Cancer, Diabetes, Leukemia or some other infectious disease, trauma from surgery or injury, an altered microbiota with prolong use of antibiotics, immune suppression drugs, virus (HIV), hormones, genetic deficiency, old age [6]. Persistent cough can be debilitating, social distressing and adversely impair quality of life [7]. Among these opportunistic fungal infections are Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Mucor sp (Mucormycosis), and Pneumocystis carinii [8]. Fungal ball is one of leading cause of haemopytsis in Indian [9].
Incidence of systemic mycoses in patients with suspected tuberculosis, attending Infectious Diseases Hospital (IDH) and Murtala Mohammed Specialist Hospital (MMSH), Kano, Nigeria between May 2003 and May 2004 has been reported. The implication of the investigation revealed that some cases of tuberculosis may be compounded with mycosis caused by Aspergillus fumigatus [10].
However, persistent cough is often mis diagnosed by laboratory worker and even some clinicians are guilty of prescribing antibiotics to immunocompromised patients suffering from persistent cough; supposing, they have contracted bacteria infection (particularly TB). It has been observed that some patients that have low immunity suffers persistent cough which might not be bacterial, viral and parasitic origin but some opportunistic fungi. In line with this, the objectives are to: 1) isolate and identify fungi etiologic agents associated with persistent cough in immunocompromised host in the study population and location.
2) determine the prevalence of fungi associated with persistent cough in immunocompromised host.
3) carry out in vitro antifungal sensitivity on etiologic agents isolated. 4) evaluate the Demographic association with the persistent cough and the predisposing factor.

B. Study Population
The study population comprised HIV patient, TB patient and patient on prolong antibiotics that were diagnosed as immunocompromised with persistent cough.

C. Ethical Consideration, Questionnaire And Informed Consent
The ethical clearance for this research was gotten from Jos University Teaching Hospital (JUTH) ethical committee after due processes had been followed. Questionnaire to obtain the demographic characteristics and other relevant information to the study as well as an informed consent were administered to the participant.

D. Sample Collection
Sequel to the identification of the immunocompromised patient with persistent cough, sterile universal bottles were given to the patients, instructed on the type of sample and aseptic condition required. The sputum samples collected were brought to the laboratory as soon as possible.

E. Sample Processing
Direct Mount preparation was done with 10% KOH using method described by [11].
The sputum sample was mixed very well and a sterile applicator stick was used to pick small quantity which was seeded on the middle of plates of Sabouraud's dextrose agar and Potato dextrose agar and allowed to deep in. Duplicate cultures were made, and the plates were incubated aerobically at room temperature and at 37 ºC. Culture plates were then examined daily for fungal growth for period of up to 3 weeks. Cultures were held for 30 days before discarded as negative result. Colonies growing outside the inoculated area were regarded as contaminant [11].
F. Characterization And Identification 1. Macroscopy and Microscopy examinations were carried out using method described by [12].
2. Germ tube test. This test was used for identification of C. albicans. A known C. albicans was set as control [12], [13].
3. Negative staining method using Indian ink. This test was used for the identification of Cryptococcus neoformans. A known Cryptococcus neoformans was set as control [14]. 4. Lactophenol cotton blue Needle mounting method was used to demonstrate other fungi growth and this was observed microscopically for macro conidia, micro conidia, Chlamydosphore, hyphae which may appear spiral, pectinate, anther-like [12].

G. Biochemical Test
Fermentation and Assimilation test were done to further identify the yeast and yeast like organisms [13]. All stock solutions were prepared in 100% dimethyl sulfoxide at a concentration of 800µg/ml. They were labeled accordingly and frozen at -20ºC until when needed [14], [15].

I. Preparation Of Inoculums 1. Mould
Stock inoculums suspensions of the moulds were prepared from 15 day-old culture grown on culture plates. Mature colonies were covered with approximately 10ml of sterile normal saline (0.85%) by scrapping the surface with of pipette. The resulting mixtures of conidia and hyphae fragments were transferred to sterile tube. The heavy particles were then allowed to settle for 15 minutes at room temperature. The supernatant was decanted and adjusted with normal saline to about No 1 Mac farland turbidity. Each suspension was diluted 1:50 in nutrient broth to obtain final inoculums twice (1:100) [14], [15].
2. Yeast Stock inoculums of yeast were prepared by picking a colony of yeast emucified in normal saline. The turbidity was adjusted to 0.5 Mc Farland standards (1Mcfarland standard for C. neoformans) [14], [16].

J. Mould Susceptibility Testing
Working standard of antifungal drugs (16 µg/ml) [16], each were diluted 1:50 (1 volume of drugs to 49 volumes of medium) with medium cooled to 40 ºC and mixed thoroughly by shaking and the mixture were poured into a Petri dish. Control plates were prepared containing 1 volume of dimethyl super oxide to 49 volumes of medium and blank was also prepared as control by adding 1volume of distilled water to 49 volumes of medium. They were both allowed to solidify. A loopful of well homogenized inoculums prepared in above was spread over the surface of the plate and was then incubated at room temperature and was observed daily, the test plates were read after the growth has become visible on the blank control plates [15], [17].

K. Yeast Susceptibility Testing
Clinical Laboratory and Standard Institute methods is a broth micro dilution, but it is labour-intensive and cumbersome. However, disk or agar diffusion method most attractive alternative method so far investigated and it is also well suited for routine use in mycology laboratories [18]. Agar diffusion method was used for this research work.
The suspension of yeast inoculums prepared in above were poured over a dried Potato Dextrose Agar plate previously dried at 37 ºC for 10 minutes, excess suspension were decanted and the plate was allowed to dry. A sterile cork borer was then used to make 6 holes which were labeled according to the antifungal drugs under test. About 2 drops of antifungal drugs each was then dispensed into respective hole avoiding over floating. Distilled water was dispensed into the 6th hole which serves as control. It was then incubated as approved by CLSI (Candida sp for 48 hrs and C. neoformans 72 hours) [18].

III. RESULTS AND DISCUSSION
Response rate A total of 100 questionnaires and consent form were distributed to patient screened and out of 100 questionnaires distributed, 100 were returned indicating a 100% response rate. Table 1 shows the prevalence of fungi associated with persistent cough in immunocompromised patient. It reveals that 37 (37.0%) out of 100 immunocompromised patients tested were positive for fungi materials whereas 63 (63.0%) were negative showing absent of fungi growth. For this number of people to be infected, it is significant (P-value 0.009, chi square 6.760 df 1) since invasive fungal infection are associated with high morbidity and mortality, thereby increasing the challenge for management of immunocompromised patient [5]. This agree with previous work of [19] that had the frequency of fungi isolated in sputum of immunocompromised patient as 42.9%. The distribution of fungi in relation to the study population and location shows that 16 (32.0%) out of 50 samples collected from Vom Christian Hospital were positive for fungi growth while 21 (42.0%) out of 50 samples collected from Jos University Teaching Hospital were positive for fungi growth as shown in the table 2. Despite the prevalence of these organisms, there was no significant difference in their existence in study location (Table II) (P-value 0.300 chi square 1.073 df 1). This might be due to rural-urban migration for better medical treatment. The breakdown of the type of fungi isolated from the sputum of immunocompromised host is shown in table 3. It reveals that C. albicans had the highest with 18 (48.6%), followed by Aspergillus niger with 12 (32.4%), Cryptococcus neoformans 5 (13.5%) and Penicillium species were 2 (5.4%) in the sputum sample analyzed. However, the distribution of fungi in the study population (Table III) shows that C. albicans is the most prevalent with overall frequency of 18 (48.6%) which agree with previous work of [20]. This may be because C. albicans has possibility of oropharngeal colonization [19]. A. niger was next with overall frequency of 12 (32%), C. neoformans had overall frequency of 5 (13.5%) and Penicillium species 2 (5.4%) which are significant (P-value 0.01, chi square 0.716 df 3). This may be attributed to the fact that the fungi are distributed through the world and are the most common fungi causing life threatening infections in immunocompromised patient particularly-HIV/AIDS. It has also been documented that emergence of Candidiasis, Cryptococcosis, Aspergillosis and Penicillosis and other Pheolyphomycosis among HIV patient on steroid therapy, had a dual infection with A. niger and S. aureus [2].  Table IV shows the prevalence of fungi in relation to demographic data. The age group 21-30, 31-40, 41-50, and 61-70 years had 8 (8.0%) of the fungi positive sputum while age 11-20 and 51-60 had 0 (0%) and 5 (5.0%) respectively.
The prevalence to sex shows that male had 22 (22.0%) of fungi positive sputum while female had 15 (15.0%). Gender wise distribution of positive subject (Table IV) showed significant difference between male and female observed in study population which probable might cause by exposure or involving of male subject to area or occupation where they inhale the spore of these organism [9]. Prevalence to occupation reveals that civil servant had 5 (5.0%), farmer 27 (27.0%), Bussiness 5 (5.0%) fungi positive sputum. However, distribution of the fungi in the sputum of the study population shows that married had 37 (37.0%) while both single and Divorced had 0 (0.0%) positive for fungi growth. However, occupational distribution (Table IV) showed no significant difference (Pvalue 0.142) among the occupation listed, indicating that this prevalence does not really matter the occupation but probably due to the fact that most civil servant and Businessmen also involve themselves in farming activities in the study population. Other demographic data (Age, Education, Marital Status) were also linked with the prevalence of these fungi in the study population. It noticed that there was no significant difference as regards to age groups (Table IV) indicating that any age might be susceptible to the fungi, most especially when the individual is immunocompromised. Subjects that were married had the highest prevalence of 37 (37%) unlike single and divorced with 0 (0%) which agreed with previous work of [9].
The associated risk factor of patient studied is shown in Table V. HIV, Tuberculosis, HIV and Tuberculosis, prolong use of antibiotics and old age were the risk factor in this work. HIV patients had 11 (11.0%) positive for fungi growth, Tuberculosis patients and patient on prolong use of antibiotics had 2 (2.0%) and old age had 14 (14.0%) positive for fungi growth. Prevalence with respect to available risk factor were also demonstrated (Table V) with HIV, TB and Co-infection (HIV and TB), prolong use of antibiotics were significant risk factor (P-value 0.014, 0.000, 0.000, 0.007 respectively) unlike old age which did not appear to be significant. This agreed with previous work of [19] and [20].  The frequency of invasive mycoses has increase dramatically during the past two decades owing to the medical advances such as Broad-spectrum antimicrobial agent, invasive medical device, organ transplantation, HIV epidemic and expanding aging population [20]. Treatment of fungal infection can now be guided by in vitro susceptibility testing. However, susceptibility testing of fungi is not considered a routine testing procedure in many laboratories and is not promptly available. Nevertheless, susceptibility testing is especially helpful in deep infection [14].
However, there are limited but increasing number of antifungal that can be used to treat mycotic infection. Most have one or more limitation such as side effect, narrow antifungal spectrum and poor penetration of certain tissue [21].
The antifungal susceptibility of isolates was carried out. Some azole group (Ketoconazole and Intraconazole) were found out to be more susceptible to fungi isolated. This agreed with previous work of [15]. This is because some azoles antifungal are broad spectrum and have good penetration power of certain tissue [21]. For organisms that are resistant to these two azoles could be due to the variation of strain of fungi isolates which is supported by previous work of [16] that documented variation in response to drugs based on frequency of occurrence of species such as azoleresistant strain.
However, the resistant of Amphotericin B to some isolates might be due to ability of the organisms to prevent binding of the drugs to ergo sterol in the membrane which is the active site of the drugs [17]. On the other hand, Griseofulvin resistant to some isolates might be caused by the ability of organisms to prevent interfering with microtubule assembly. Since the active site of the drug is microtubule assembly [17]. Vericonazole that showed little susceptibility to the isolates could be due to poor penetration of tissue by some drugs which was earlier discussed by [21] and azole-resistant starin of Candida spp [16]. Table VII shows the susceptibility of fungi isolates to antifungal drugs with their MIC. Five different drugs were used against 37 fungi isolates. However, among the drugs used, Ketoconazole(MIC 0.20 ug/ml) was found out to have the highest susceptibility to C. albicans, followed by Intraconazole (MIC 0.16 ug/ml), Amphotericin B (MIC 0.16 ug/ml), Vericonazole (MIC 0.30 ug/ml) and the least susceptible was Griseofulvin (MIC 0.20 ug/ml). The antifungal biogram on A. niger reveals that Intraconazole (MIC 0.16 ug/ml) was found to be highest susceptible and Griseofulvin (MIC 0.25ug/ml) was the least susceptible. C. neoformans was found to have highest susceptibility to Intraconazole (MIC 0.16 ug/ml) and Amphotericin B (MIC 0.16 ug/ml), Vericonazole was resistant to all the neoformans isolates.