Prevalence of Pulmonary Mucormycosis among Poultry Farmers in Barkin Ladi Local Government Area of Plateau State

Objectives

The Objectives of this work are: To determine prevalence of pulmonary mucormycosis among Poultry Farmers.

To determine the relationship between duration of exposure and possible infection.

Hypothesis

H01: There is no relationship between duration of poultry farming to possible infection with pathological fungi. H02: There is no significant difference in the distribution of abnormal substances in sputum of males and females.

Signs and Symptoms of Pulmonary Mucormycosis

Most patients with the pulmonary form of the disease are being treated for leukemia. The fungus enters the patient’s lungs where it eventually invades a major blood artery or vessel causing the patient to cough up blood or hemorrhage into the lungs. Early symptoms include fever, difficulty in breathing with eventual bleeding from the lungs [8]. It is also the second most common form of mucormycosis next only to rhino cerebral disease and accounts for more than 30% of the disease [9]. Its clinical presentation in the lungs is defined as being acute if symptoms are present for less than 30 days, where it is usually localized. It is found among a wide age range but with male predominance. Invasion of the blood vessel by the fungi hyphae results in necrosis of tissue parenchyma which may ultimately lead to cavitation and hemoptysis. Most common predisposing conditions in mucormycosis are uncontrolled diabetes mellitus, malignancy, chronic illness and transplant [10]. For diagnosis of the infection swabs of tissue and discharge could be used. A biopsy specimen of the tissue could also be done.

If mucormycosis is suspected, prompt amphotericin B therapy is administered. Posaconazole has been shown to be effective against mucormycosis. After administration of either amphotericin B or Posaconazole, surgical removal of the "fungus ball" is indicated. The disease must be monitored carefully for any signs of reemergence [11].

Prognosis

In most cases, the prognosis of rnucormycosis is poor and has varied mortality rates depending on it's form and severity. In the rhino cerebral form, the mortality rate is between 30% and 70%, whereas the disseminated mucormycosis presents with the highest rate of mortality rate of up to 90%. Patients with AIDS have a mortality rate of almost 100% [11]. Possible complications of mucormycosis include the partial loss of neurological functions, blindness and clotting of brain or lung vessels [12].

Epidemiology

Pulmonary mucormycosis is a very rare infection and as such it is hard to note histories of patients and incidence of infection [13]. The disease affects both sex and is somewhat more common in men than in women, it may develop in any age group including the newborn.

Prevention

Prevention depends on protecting high-risk persons from contacts with sugary foods, decaying plants, moldy bread and other breeding grounds for fungi. In addition, healthcare professionals treating hospital inpatients should be careful to change dressings frequently and check the underlying skin for signs of possible fungal infection [6].

Sample Size

A total of 100 sputum samples were collected for this work from Poultry farmers. The sample size was calculated using the formula according to Daniel WW, et al [14].

p (1-P) Z2 n =

d2 Where n = sample size, Z = statistic for a level of confidence (1.96 at 95% level of confidence). P = expected prevalence or proportion based on a previous study at 7.2% [15]. d = precision (6%) Minimum sample is 72 but 100 samples were collected

Sample Collection

Consent was formally obtained from the participants and questionnaires used to obtain their Biodata. All sputum samples were collected into sterile screw caped plastic containers which were given out to participants a day prior to sample collection. Each sputum sample collected was shared into two portions; the first portion was cultured on Sabouraud Dextrose Agar (SDA) while the second portion was transferred into another plastic container having 2mls of 70% alcohol, these were transported to the Microbiology Laboratory of the Plateau State Polytechnic. Statistical Packages for the Social Sciences (SPSS) version-25 software was used for Chi- Square and t-test analyses of the research findings.

Cultural Studies

Sterile Sabouraud Dextrose Agar (SDA) was prepared (according to manufacturers instruction), sterilized by autoclaving at 121psi for 15mins, allowed to cool and while in molten form, modified by addition of 0.4g of chloramphenicol, 20,000 units of penicillin and 4,000 units of streptomycin sulphate each added to a litre of molten agar which was then dispensed into sterile Petri dishes, allowed to set and stored in the refrigerator. With the aid of a sterile wire loop, the sputum samples were inoculated on the SDA and streaked out to obtain discrete colonies. These plates were then incubated at both room temperature and at 37oC in the incubator to aid growth of dimorphic fungi i.e. those that grow as yeasts (parasitic phase) at 37oC and as moulds (saprophytic phase) at room temperature. Cultures were observed daily for growth for 2 weeks. Gross colonial morphology, colour and texture of the colony surface and underside which can be very distinctive were observed.

Direct Microscopy

A drop of sodium hydroxide (20% w/v) solution was placed on a slide. An aliquot of each sputum sample was transferred to the drop of sodium hydroxide on the side and this was covered with a glass cover slip. This was then allowed to stay for 5-10 mins to enable it to clear, after which it was examined microscopically using X40 objective. Characteristic morphological elements such as nature and form of microconidia, macrocondia, spherule, fruiting bodies, sporangium, encapsulated cells, budding cells and broad based cyst, spherical fish like yeast cells were observed. Direct microscopy of culture isolates was also done. A drop of lactophenol cotton blue was placed on grease free slide and a fraction of culture was transferred to drop of lactophenol cotton blue on the slide and covered with a glass cover slip.

This was then examined microscopically using the X10 and X40 objectives with the condenser iris diaphragm closed sufficiently to give good contrast. Structures such as spherules, microconidia, pseudohyphae, encapsulated cells, sporangia were noted.

Germ Tube Test

Human Serum (0.5ml) was pipetted into a small test tube. Using a sterile wire loop, the serum was inoculated with a yeast colony from the culture. The tube was placed in an incubator at 37oC for 2-3 hrs. With the aid of a Pasteur pipette, a drop of the serum yeast culture was transferred to a glass slide and covered with a cover slip. This was then examined using the X10 and X40 objectives.

Formation of Pseudomycelia and Chlamydospores

Certain yeasts develop special fruiting forms (called chlamydospores) when grown in glucose free medium like Rice Agar. Isolates identified as yeasts were inoculated onto prepared sterile rice agar plates and incubated at room temperature for 24-48 hours and observed for pseudomycelia and chlamydospore development.

To the second portions of sputum, equal volumes of 8% Hydrochloric acid was added to each sample and allowed to stand overnight. They were centrifuged at 5000 rpm for 5 minutes after which the supernatant was discarded. For each sample 2 smears were made on clean grease-free albuminized glass slides which were then placed in 70% alcohol while still wet. The smears were stained using the Papanicolaou staining technique according to Carleton, in Drury, [16]. These were then microscopically examined using x10 and x40 magnifications.