FUNGAL GENETICS, KENYA EPIDEMIOLOGY & LABORATORY BIOSAFETY --- A. FUNGAL GENETICS### Basic Concepts Genetics — the discipline that explains how information need
FUNGAL GENETICS, KENYA EPIDEMIOLOGY & LABORATORY BIOSAFETY --- A. FUNGAL GENETICS Basic Concepts Genetics — the discipline that explains how information needed to reproduce an organism is stored within it, and how that information may change and be reassorted before passing to the next generation. Genetic information (genome): - Maintained as long, linear sequences of nucleotide base pairs making up DNA molecules - Order of bases = genetic code → specifies amino acid sequences for all proteins - Long DNA molecules incorporating thousands of base pairs = chromosomes - Prokaryotes vs Eukaryotes: - Prokaryotes (bacteria) — genome contained in a single chromosome in the cytoplasm - Eukaryotes — genome contained in two or more chromosomes within a nucleus - Eukaryotic plants and animals are basically diploid — nuclei contain two matched sets of chromosomes (one from male gamete, one from female gamete) - Each chromosome has a paired counterpart; most genes have a counterpart called an allele on the paired chromosome — alleles affect the same characters but not necessarily in the same way - --- Fungal Genome — Haploidy- The vast majority of fungi are haploid — nuclei contain a single set of chromosomes - This is a key difference from most other eukaryotes (which are diploid) - Advantages of haploidy for genetic studies: - No competing alleles - Every gene is potentially capable of being expressed in the phenotype (physical manifestation) - Absence of masking (no dominant/recessive relationships) makes genetic analysis much easier - This is why fungi like Neurospora crassa and Saccharomyces cerevisiae are invaluable model organisms in genetics research - --- B. FUNGAL DISEASE EPIDEMIOLOGY IN KENYA Background- Kenya is a developing country with a high rate of tuberculosis (TB) and a moderate HIV infection burden - Both TB and HIV predispose patients to opportunistic fungal infections - Sub-Saharan Africa is the epicentre of HIV/AIDS — main fungal infections include: - - Respiratory fungal infections - Eye (ocular) fungal infections - Dermatological fungal infections - Cryptococcal meningitis — most significant Burden of Fungal Infections in KenyaTotal estimated HIV-positive population in Kenya: 1,400,000 (2012 data) Infection Total Burden Rate per 100,000 --- --- --- Oral candidiasis 306,000 769 Oesophageal candidiasis 114,000 286 Recurrent vaginal candidiasis 594,660 2,988 Candidaemia 1,990 5 ABPA 17,696 44 SAFS (Severe Asthma with Fungal Sensitization) 23,359 58 Chronic pulmonary aspergillosis 12,927 32 Invasive aspergillosis 11,900 29 Cryptococcal meningitis 17,000 43 Pneumocystis pneumonia 1,712,676 4,302 Fungal keratitis 80 0.2 Tinea capitis 239 0.6 Total burden estimated 3,186,766 — Key observations: - Pneumocystis pneumonia has the highest total burden - Oral and vaginal candidiasis are the most common mucosal infections - Cryptococcal meningitis remains a major killer in HIV/AIDS patients - Most infections are driven by the HIV/AIDS burden - --- Emerging and Re-emerging Fungal DiseasesKey factors driving emergence and re-emergence of fungal diseases: - HIV/AIDS pandemic — massive increase in immunocompromised population - Increased use of immunosuppressive therapy — transplants, cancer chemotherapy, biologics - Antifungal resistance — especially in Candida and Aspergillus species - Climate change — expanding geographic range of fungi like Coccidioides and Cryptococcus gattii - Increased travel and globalisation — spread of endemic mycoses to non-endemic regions - Candida auris — emerging multidrug-resistant yeast; nosocomial outbreaks worldwide; resistant to multiple antifungal classes; difficult to identify with standard methods - Mucormycosis — re-emerged significantly during COVID-19 pandemic particularly in diabetic patients on corticosteroids - --- C. MYCOLOGY LABORATORY BIOSAFETY Why Biosafety MattersSafety in the mycology laboratory is crucial because of: - Potentially pathogenic fungi being studied - Hazardous nature of reagents, flames, glassware and procedures - Risk of laboratory-acquired infections (LAIs) - Senior supervisory staff must develop biosafety guidelines for all workers. --- Key Terminology Term Definition --- --- Bio-risk Any harm where the source is a biological material Bio-risk assessment Analysis of the severity and frequency of exposure to harm Bio-risk management Identification of bio-risks and steps towards control and mitigation --- Five Steps of Risk Assessment- Identify potential hazards - Decide who is at risk - Assess likelihood of harm - Implement adequate precautions - Manage risks and hazardous situations - --- Pre-Analytical Phase Hazards Risk factors: - Patients with active, latent, chronic or undiagnosed infections are sources of disease - Sample collection, handling and transport must be done carefully - Best practices: - Only trained staff (phlebotomist, nurse, doctor) should collect samples — to avoid needlestick and prick injuries - Always perform skin scrapings and nail drilling in an isolated room — to avoid creating contaminated aerosols - Staff must wear PPE — masks, gloves, gowns - --- Analytical Phase Hazards Most common laboratory-acquired fungal infections (LAIs): - Caused by dimorphic fungi — Blastomyces , Histoplasma , Coccidioides - Most LAIs are secondary to inhalation of conidia - Common modes of lab-acquired infections: - Parenteral inoculations with syringes and sharp equipment - Splashes onto skin and mucous membranes - Inhalation of aerosols — most common - Ingestion during mouth pipetting or eating in laboratory - Animal bites or scratches during animal studies - Best practices: - All specimens must be processed in a Biosafety Cabinet (BSC) - Staff must wear PPE and be competent to handle specimens - Never sniff a fungal culture to determine its odour - Plates containing moulds must not be uncovered on open bench - Petri plates growing mould must be sealed with porous tape to prevent accidental opening and spread of spores - Yeast cultures can be read on open bench in BSL-2 lab - Slow-growing white or black moulds suspected to be dimorphic fungi → shift to Class II BSC immediately - Storage and transport of filamentous moulds must be done in screw-cap slants — petri dishes not recommended - --- Highly Pathogenic Fungi — BSL-3 RequiredMust be handled in a Biosafety Level 3 (BSL-3) facility: - Histoplasma capsulatum - Blastomyces dermatitidis - Coccidioides immitis and C. posadasii - Paracoccidioides brasiliensis - Penicillium marneffei - Cladophialophora bantiana - If BSL-3 unavailable: use BSC + respiratory precautions + good laboratory practices. Such pathogens can be sent to reference labs or identified using molecular techniques to avoid exposure. --- Biosafety Cabinet (BSC)- Class II BSC recommended for diagnostic mycology laboratory - Must be installed away from walking paths and doors - BSC filter can be damaged at glue joint or gasket if moved → dangerous leaks - Filter and cabinet integrity must be tested whenever equipment is shifted - BSCs must be certified by trained professionals - --- Reagent Safety Reagent Hazard Precautions --- --- --- KOH (10–15%) Corrosive — burns/irritation to skin, eyes, respiratory tract Gloves and eye protection; avoid inhalation and ingestion Calcofluor White Not hazardous alone; if combined with KOH — stringent precautions needed Good hand hygiene; rinse eyes if contact occurs LPCB (Lactophenol Cotton Blue) Acidic; toxic by inhalation and skin contact; especially hazardous if swallowed Handle large amounts in well-ventilated area; avoid skin/eye contact India Ink Yeast cells may remain viable Good hand hygiene; gloves when handling wet mounts Gomori Methenamine Silver Toxic and corrosive Avoid skin/eye contact; adequate ventilation for large amounts Giemsa Stain Toxic by inhalation, absorption and ingestion (methanol content) Handle with care; adequate ventilation --- Material Safety Data Sheet (MSDS)- Document containing information about chemic