Patients and Samples
This was a laboratory based cross sectional study that recruited 113 patients clinically suspected for PTB. From these patients, an on-spot early morning sputum sample was collected aseptically in dry wide- necked, leak-proof containers from each of the suspected patients referred the TB clinic of Mbarara Regional Referral Hospital (MRRH) between 2019-2020. Sputum samples were then transferred to the Mycology Laboratory Unit at Microbiology Department, Faculty of Medicine, MUST. Other information collected from the patients’ also included demographic data and HIV sero status. The study included TB suspects with a negative TB diagnostic history and those who were not on antifungals whilst TB suspects on antifungal therapy that showed improved prognosis and samples of PTB suspects whose request forms lacked demographics were excluded.
Specimen collection / sampling procedure.
Sputum samples were collected from the Medical ward, TB ward and Outpatient department as instructed by the clinicians under sterile conditions in clean, dry, wide-naked, leak proof containers as per the Uganda national guidelines for tuberculosis infection control in health care facilities, congregate settings and households. Sputum samples were then received in the laboratory upon request by the clinicians for PTB test.
The biodata and laboratory results of HIV status of the study participants were recorded corresponding to each participant’s sample.
Bacteriological procedure and Mycological Profiling
Bacterial procedures were predominated by TB diagnosis using Gene xpert which was a diagnostic test for MTB. This was aimed at establishing Mycobacterium aetiology or PTB- fungal co-infection. Fungal examination protocols included a direct wet mount examination with 20% potassium hydroxide (KOH). Fungal isolation was through cultivation on basic saboruad dextrose agar (SDA) (-/+ antibiotics), incubated at 25°C and 37°C for 2-4 weeks, and evaluated every 2-3 days. Filamentous fungal isolates unable to vegetatively fruit on SDA were sub-cultured and reisolated on Potato Dextrose Agar (Formedium), incubated and observed as above. Identification was through microscopy by lacto phenol cotton blue staining of fruiting bodies. On the other hand, yeasts were identified biochemically using germ tube method. Definitive diagnosis of pulmonary mycosis was determined based on the presence or absence of fungal elements in a direct examination of culture growth.
Fungal culture
Sabouraud dextrose agar (SDA) containing antibiotic chloramphenicol and gentamicin was used to culture sputum samples. The specimens were streaked onto the medium in the Universal bottles with a sterile inoculating loop in order to obtain isolated colonies. The preparations were then incubated at 25 – 30°C in an inverted position (agar side up) in humidity conditions. Cultures were examined at least weekly for fungal growth and held for 4 weeks before being reported as negative. After sufficient incubation, the Universal bottles that showed isolated colonies in streaked areas and confluent growth in areas of heavy inoculation were identified and the growth was examined using other methods.
GeneXpert
The Cepheid GeneXpert ® System was used to detect Mycobacterium tuberculosis for the diagnosis of PTB.
This method was used as a gold standard to diagnosis TB in sputum samples. The sputum samples were first disinfected using Sodium
hypochlorite in a level 2 bio safety cabinet. The prepared samples were then run by the GeneXpert according to the manufacturer’s instructions. The GeneXpert MTB/RIF detects DNA sequences specific for Mycobacterium tuberculosis and rifampicin resistance by polymerase chain reaction. It is based on the Cepheid GeneXpert system, a platform for rapid and simple-to-use nucleic acid amplification tests (NAAT). The Xpert® MTB/RIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples, isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR. The process identifies most of the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons. Results are obtained from unprocessed sputum samples in 90 minutes, with minimal biohazard and very little technical training required operating.
Potassium hydroxide (KOH) mounts
A drop of 10% KOH solution was placed on a slide. A small portion of specimen (sputum) was transferred on to the drop of KOH and covered with glass. The slide was placed for 15 minutes on a damp cotton wool to prevent the preparation from drying out. Ensured that the material of the preparation was cleared.
The preparation was then examined microscopically using the 10X and 40X objectives with the condenser iris diaphragm closed sufficiently to give a good contrast to detect the fungal elements.
Lacto phenol cotton blue staining.
LPCB was used for microscopic identification and characterization of fruiting bodies such conidia, sporangia, rhizoids and hypha or mycelia of cultivated fungi on SDA. A drop of lactophenol cotton blue stain was placed on a clean grease-free glass slide. A small fragment of cottony, woolly, or powdery colony was picked from the midpoint of the culture using a sterile straight wire and placed on clean glass slides for the staining process. A clean coverslip was applied avoiding air bubbles. Excess stain was removed with blotting paper and the preparation examined using ×10 and ×40 objectives of the microscope. Fungal element features like microconidia, macroconidia, chlamydospores, and hyphae that appear spiral, pertinate, and antler-like structures were investigated. These features seen in the stained slide were compared with established characteristic fungal features using mycology atlases.
Germ tube test
Germ-tube test was a simple, reliable procedure for the identification of Candida albicans. 0.5 – 1 ml of human serum was put into a 12×75 mm test tube and the yeast colonies were suspended into the serum to obtain faintly turbid suspension. The preparation was then incubated in the tubes at 37 °C for 2-3 hours in an incubator. Using sterile Pasteur pipette, the suspension was removed and examined microscopically for the presence or absence of germ tubes. Positive test showed Germ tubes arising directly from the yeast cell and had parallel walls without any constriction at their point of origin which was diagnostic for C. albicans.
Safety and environment
All biological specimens, including used cartridges, are capable of transmitting infectious agents and thus, were treated with universal precautions. All laboratory procedures were done in a level 2 TB laboratory. Personal protective equipment such as disposable gloves, laboratory coats were used when handling specimens and reagents. Washing of hands were done thoroughly after handling specimens and test reagents. Disposing of used Xpert MTB/RIF cartridges was done according to the country’s safety guidelines for hazardous material.
Quality control measures
Only early morning sputum samples were accepted for analysis so as to easily detect Mycobacterium tuberculosis. Known standard fungal element morphologies and reference cultured fungal elements growth were used for reference. For example, known fungal atlases were used to confirm established characteristic fungal features.
Ethical Consideration.
The proposal was submitted to the Department of Microbiology, the Faculty of Medicine Review committee (FRC) and approved by the Institutional Review Committee (IRC) of Mbarara University of Science and Technology.