Lecture 1 - Candida Infections

There are five main fungal phyla:

• Chrytidiomycetes

• Zygomycetes

• Glomeromycetes

• Ascomycetes (Saccharomyces cerevisiae, Candida albicans)

• Basidiomycetes (Cryptococcus neoformans)

There are about 1500 species of single celled fungi. Yeasts are eukaryotic organisms.

S. cerevisiae Life Cycle

Yeast cells can reproduce asexually via budding, there are two mating types for yeast; type a and type alpha. Two cells of different mating typed can fuse creating diploid cells which can then undergo meiosis.

Filamentation is a dimorphic transition related to the virulence of pathogenic fungi. There are two types of filamentation; pseudohyphal and hyphal. Pseudohyphal growth has a septum whereas hyphal has no septum. The transition from a budding yeast cell to pseudohyphal yeast cells results in pathogenicity to humans.

Candida

Candida is a common commensal yeast normally found on the skin, mouth, gastro-intestinal tract and the vagina. It only becomes pathogenic under certain circumstances.

Candida albicans is a dimorphic fungus which grows both as yeast and filamentous structures.

Poor nutrient density leads to elongation and filamentation in order to forage for nutrients and explore the surroundings.

There are over 20 species of Candida yeasts which can cause infection in humans known as Candidiasis. The most common of which is Candida albicans, it is responsible for 50-90% of all cases of candidiasis in humans.

Candida auris is an emerging fungus which is a serious global health threat as it is resistant to anti-fungals.

Candida auris

C. auris was first isolated in Japan in 2009. The first candidemia case caused by C. auris was in Korea in 2012, it took three years to identify pathogenicity.

The vast majority of 270 isolates of C. auris detected in the UK have been recovered from patients in the South of England with no cases in Northern Ireland or Scotland. There are 4 major clades where infectious strains seem to originate from. (South Asia, East Asia, South Africa and South America).

In 2020 the CDC classified C. auris as one of the biggest drug resistance threats in America. 7% of all candida blood samples tested at CDC are resistant to fluconazole. C. albicans is the most common cause of severe infection however resistance is most common in C. auris.

C. auris is often resistant to multiple antifungal drugs, mortality when in the blood stream us between 28 and 70%. It is dependent on location in the world and the healthcare facilities.

Candida albicans

Candida normally reside in the intestinal tract and can be found on mucous membranes and skin without causing infection however certain circumstances lead to overgrowth which causes symptoms to develop.

This organism is opportunistic, long courses of antibiotics will kill good bacteria leaving candida free to grow. In women antibiotic use can modify the vaginal flora leading to overgrowth of candida. It causes disease by invading human tissue by means of pseudohyphae which can penetrate intracellular cracks.

Common causes of this infection are:

• Overuse of antibiotics

• Steroid hormone medication (corticosteroids - stop once no longer needed)

• Supressed immune systems (diabetes, HIV, transplant patients, radiation/chemotherapy patients)

• High sugar/starch diets (yeast live off sugar - starches are converted to sugar)

Candidiasis

Symptoms of candidiasis vary depending on where in the body is infected.

Thrush is a type of candidiasis infecting the oropharynx or oesophagus. Yeast infections are candidiasis infections in the genitals.

Invasive candidiasis is when Candida species enter the blood stream and spread throughout the body. This is a serious infection which can affect the blood, heart, brain, eyes, bones and other parts of the body. This type of infection is most common in hospitalised patients.

In healthy people candidiasis is usually a localised infection of the skin, fingernails or mucosal membranes including the oral cavity and pharynx, oesophagus, and the genitalia. It is less common for the gastrointestinal tract, urinary tract and respiratory tract to be sites of infection in healthy individuals.

In immunocompromised individuals candida infections in the oesophagus occur more frequently than in healthy individuals and have a higher potential of becoming systemic causing candidemia (type of fungemia (septic fungal infection)).

Most infection will result in minimal complications such as redness, itching and discomfort however complications may be severe or fatal if left untreated in certain populations.

Thrush is commonly seen in infants, it is not considered abnormal unless it lasts longer than a few weeks. Most people who get candidiasis in the oesophagus have weakened immune systems. Thrush presents as white patches on the inner cheek, tongue, roof of the mouth, redness or soreness of the throat, cottony feelings in the mouth, loss of taste, pain while eating or swallowing, and cracking and redness at the corners of the mouth.

Onychomycosis is a nail infection categorised under cutaneous candidiasis. It is characterised by red swelling around the nails, destruction of nail tissue and loss of nails, It occurs more often in toenails than fingernails because toenails are often confined to warm moist environments.

Yeast infections can be asymptomatic with 20% of women having candida in the vagina without any symptoms however potential symptoms include vaginal itching or soreness, pain during sex, pain or discomfort when urinating, and abnormal vaginal discharge. Most vaginal candidiasis is mild, come women can develop severe infections involving redness, swelling and cracks in the wall of the vagina. Sometimes candida can multiply and cause and infection in the environment inside the vagina, this happens because of hormones, medicines, or changes in the immune system.

Symptoms of infection of the male genitalia include red skin around the head of the penis, swelling, irritation, itchiness and soreness around the head of the penis and when passing urine or during sex.

Ocular candidiasis is characterised by cloudy vision and lesions within the eye. It is caused through the spread of candida in the blood stream, indwelling catheters, IV drug abuse, or ocular trauma/surgery.

Candidemia

This is a bloodstream infection which is most common in hospitalised patients. Systemic fungal infections including C. albicans have emerged as important causes of morbidity (infection) and mortality in immunocompromised patients.

People with candidiasis are often already sick so it is difficult to identify which symptoms are related to a candida infection. Most common symptoms are fever and chills that dont improve after antibiotic treatment for suspected bacterial infections. Other symptoms can develop if the infection spreads to other parts of the body such as the heart, brain, eyes, bones, or joints.

Antifungals

Antifungals will either target the pathway for ergosterol integration into the membrane, ergosterol biosynthesis or membrane integrity via ergosterol. Nystatin will bind ergosterol so it cannot be inserted into the membrane. Clotrimazole will inhibit ergosterol biosynthesis. Septic infections will require a cocktail of antifungals in treatment.

Prevention

Good personal hygiene is key to prevention, keeping the skin clean, dry and free from abrasions or cuts will prevent fungi entering the body.

Avoiding high sugar, starch and carbohydrates in the diet will deprive the fungi of nutrients preventing growth.

Taking sufficient amounts of probiotics will repopulate the normal bacterial flora.

Lecture 2 - Bacterial Urinary Tract Infections

Healthcare Associated Infections

This is a local or systemic infection which has been acquired at a healthcare facility. They are also known as nosocomial infections. Some HAIs will be acquired from other patients and others will be acquired from hospital personnel.

UTIs

UTIs are come of the most common bacterial infections, they affect 150 million people each year worldwide. In 2007, in USA there were around 10.5 million cases for UTI symptoms (they make up 0.9% of all GP visits and 2-3million of emergency department visits).

The societal costs of UTIs in the US is around $3.5 billion per year. They are a significant cause of morbidity in infant boys, older men and women of all ages.

The serious consequences include:

• frequent recurrence

• pyelonephritis with sepsis

• renal damage (young children)

• pre-term birth

• complications due to frequent antimicrobial use (high levels of ABR and C. diff)

Clinically UTIs are categorised as uncomplicated or complicated, the most common causative agent for both uncomplicated and complicated UTIs is uropathogenic Escherichia coli.

Uncomplicated UTIs

These typically affect individuals who are otherwise healthy and have no structural or neurological urinary tract abnormalities. They are differentiated into lower UTIs (cystitis) and upper UTIs (pyelonephritis).

There are several risk factors associated with cystitis:

• female sex

• prior UTIs

• sexual activity

• vaginal infection

• diabetes

• obesity

• genetic susceptibility

Complicated UTIs

These are UTIs which are associated with factors which compromise the urinary tract or host defence such as:

• urinary obstruction

• urinary retention caused by neurological disease

• immunosuppression

• renal failure

• renal transplantation

• pregnancy

• foreign bodies (calculi, indwelling catheters)

In the USA 70-80% of complicated UTIs are attributable to indwelling catheters accounting for 1 million cases per year. UTIs are the second most common gram negative antibiotic resistant bacteria.

Catheter associated UTIs are associated with increased morbidity and mortality. Collectively CAUTIs are the most common cause of secondary bloodstream infections.

Risk factors for developing CAUTIs include:

• prolonged catheterisation

• catheterisation

• female sex

• older age

• diabetes

Adherence and Colonisation

UTIs start with periurethral contamination by a gut uropathogen, colonisation of urethra and migration to the bladder which requires flagella and pili.

Multiple bacterial adhesins recognise receptors on the bladder epithelium and mediate colonisation.

UPEC survives by invading the bladder epithelium , producing toxins and proteases to release nutrients from host cells and synthesising siderophores to obtain iron.

Uropathogens can ascend to the kidneys by multiplying and overcoming host immune surveillance, again by attaching via adhesins or pili to colonise renal epithelium and producing toxins. They can also cross the tubular epithelial barrier to access the blood stream initiating bacteraemia.

Enterobacteriales (Gamma Proteobacteria)

This is the largest and most heterogeneous collection of gram negative rods with 40 genera and 150 species with less than 20 species being responsible for 95% of infections. They are ubiquitous in soil and water as well as being part of the intestinal microflora of most animals.

They cause 1/3 of septicaemias and 2/3 of urinary tract infections. Some species are always pathogenic where as some are opportunistic and some are commensal organisms which become pathogenic on obtaining virulence factors (temperate bacteriophages, plasmids).

Infections can originate from a human carrier or animal reservoir.

Key genera include Enterobacter, Escherichia, Klebsiella, Proteus, Salmonella, Serratia and Shigella.

Enteric bacteria are a phylogenetic group within the Gamma proteobacteria which are facultative aerobes. They are motile or non-motile nonsporulating rods which possess relative simple nutritional requirements. They ferment sugars to a variety of end products and there are many diagnostic tests used to separate the various genera but distinguishing genera is difficult due to close relationships. Identification can be done based on computer analysis of a large number of diagnostic tests.

Mixed Acid Fermenters

Escherichia are universal inhabitants of the intestinal tract of humans and warm-blooded animals which synthesise vitamins for the host. Some strains are pathogenic.

Salmonella and Shigella are closely related to Escherichia and are usually pathogenic, Salmonella is characterised immunologically by surface antigens.

Proteus is a genus containing rapidly motile cells capable of swarming. They are a frequent cause of urinary tract infections in humans.

Direct Detection

Urine culture can be used for detection. UTIs are common especially in women and disease causing agents are often in normal flora. Urinary tract pathogens can be cultured using general purpose media (blood agar), selective media (MacConkey) or additional differential media.

Virulence Factors

Enterobacteria have endotoxins which depend on the lipid A component of LPS:

• O polysaccharides

• Capsular K antigens

• Flagellar H proteins

The capsule is usually composed of polysaccharides and sometimes proteins to protect the cell from phagocytosis. Antigenic phase variation is important in immune evasion and occurs when K and H antigens varies. They also have resistance to serum killing. They are also often resistant to antibiotics and can sequester growth factors such as iron by siderophores. Most of these were developed for survival in the environment not pathogenicity.

Enterobacterial Antigen Structure

Heat stable lipopolysaccharide is a major cell wall antigen and has three components:

• Outermost somatic O polysaccharide

• Core polysaccharide (enterobacterial common antigen)

• Lipid A

The serological classification of the Enterobacteriaceae is based on 3 major groups of antigens:

• O polysaccharides

• Capsular K antigens

• Flagellar H proteins

Specific O antigens present in each genus although cross reaction can occur. Genera outside the family can also possess K antigens.

Flagella and Pilli

Most enteric bacteria are motile with the exception for Klebsiella, Shigella and Yersinia. Most strains possess peritrichous flagella. There are two classes of pilli:

• Chromosomally encodes fimbriae required for host cell attachment

• Plasmid encoded pilli required for conjugation

Biofilms

Biofilms allow nutrient enrichment, no detachment from surface in flowing systems such as rocks, heart valves and the urethra. They provide protection from chemicals such as antibiotics as well as protection from predation, parasitism and phagocytosis. They facilitate intracellular communication via quorum sensing and sex.

Siderophores

These are required for iron sequestration. Siderophores are important potential targets for vaccine development and for designing small molecules which interfere with their function.

UPEC siderophores are aerobactin which is highly expressed, stable at low pHs and display higher levels of iron binding, and yersiniabactin which is responsible for biofilm formation in urine and play a protective role against intracellular killing by copper stress as it sequesters host derived copper.

Bladder Colonisation

One UPEC strain can encode 12 different pili, there are 38 in the E. coli pangenome. Type 1 pili are essential for UPEC infection. FimH recognises uroplankins and inegrins on epithelial cells, pili binding triggers actin rearrangement and membrane engulfment of UPEC. It can then multiply to form intracellular bacteria communities, these then subvert host defences and avoid antibiotic treatments. LPS are sensed by TLR4 resulting in exocytosis of UPEC. Maturation of IBCs causes bacterial dispersal and allows the invasion of other host cells. UPEC survives in the bladder by secreting several factors important for nutrient acquisition:

• Toxin alpha haemolysin (HlyA) promotes host cell lysis through pore formation facilitating iron release and nutrient acquisition.

• HlyA also triggers epithelial exfoliation to promote UPEC spread or to expose deeper layers of the uroepithelium

• Siderophores allow iron scavenging

Kidney Colonisation

This is dependent on pyelonephritis-associated pili which require additional genes. they bind globoside containing glycolipids lining the renal tissue. The P pilus adhesin PapG interacts with TLR4 and reduces the expression of polymeric immunoglobulin receptor (PICR). This impairs IgA transport across the epithelium to reduce immune efficacy, modulating local secretory antibody immune response and preventing UPEC opsonisation and clearance.

Pathogenesis During CAUTIs

These are usually mediated by Proteus mirabilis. Mannose resistant Proteus like pili are required for attachment, biofilm formation on the catheter and bladder. Urease production induces formation of calcium crystals and magnesium ammonium phosphate precipitates in urine through hydrolysis of urea to form CO2 and ammonia resulting in high pH.

Extracellular polymeric substance production by bacteria attached to catheters traps crystals allowing the formation of crystalline biofilms which protects the community from the host immune system. This will prevent proper urine drainage resulting in reflux and promoting progression to pyelonephritis, septicaemia and shock.

The production of bacterial toxins haemolysin (HmpA) and Proteus toxin agglutination (Pta) destroys tissue and allows dissemination to the kidneys. Hp,A induces pore formation in cell membranes destabilising the host cell, causing tissue damage, exfoliation and nutrient release. Pta punctures the host cell membrane causing cytosol leakage and resulting in osmotic stress and depolymerisation of actin filaments compromising the structural integrity of the cell. Nutrients released via these toxins also allows the bacteria to scavenge iron using siderophores.

UTI Antibiotic Resistance

Extended spectrum beta lactamases are plasmid or chromosomally encoded beta lactamases with broad activity against penicillins and cephalosporins. These are encoded on plasmids that typically carry other resistance genes which provide activity against aminoglycosides, sulfonamides and quinolones making the bacteria that acquire these plasmids multi drug resistance:

• cefotaximases are the most prevalent beta lactamases in community associated isolates and are typically encoded on plasmids with other resistance genes.

• oxacillinases are ESBLs that are typically encoded by plasmids and mediate resistance to ampicillin, cephalothin, oxacillin and cloxacillin.

• Amp-C type beta lactamases hydrolyse penicillins, third generation and extended specturm cephalosporins and cephamycins and are resistant to beta lactamase inhibitors including clavulanate.

• carbapenemases (NDM-1) inactivate carbapenems in addition to penicillins and extended spectrum cephalosporins.

Lecture 3 - HPV

Papillomaviruses

There are over 200 types of human papillomaviruses known and over 80 which infect other animals. HPVs exist in alpha, beta, gamma, mu and nu genera. They can cause benign or malignant tumours (oncovirus) but are often asymptomatic infections. They are epitheliotropic meaning that they infect epithelial cells and are transmitted via direct contact.

Virion Structure

The HPV virion is a non enveloped icosahedron which has major capsid proteins L1-72 which are pentameric capsomers and minor capsid proteins L2-12-72. The viral genome is supercoiled and associated with cellular histones which are hijacked from the host. The virion is stable in the environment, resistant to acid, heat and desiccation.

Genome Structure

The genome is a circular dsDNA making them part of Group I under Baltimore classification. Early genes are E1-E7/8 and late genes L1 and L2. There are regulatory regions such as the long control region which contains the origin of replication. Genes are all transcribed from the same DNA strand.

HPV Proteins

E1 and E2 are involved in viral replication, E5-E7 have roles in transformation and L1 and L2 are capsid proteins.

HPV Replication

This is difficult to study in vitro but the virion binds receptors (likely heparin sulfate proteoglycans) on the epithelial cell membrane. The virion then undergoes endocytosis and uncoating. The viral genome is trafficked to the nucleus which requires L2. This then allows transcription, DNA replication and virion assembly in the nucleus.

Viral replication depends on the host cell differentiation state, only early genes are expressed in the basal layer of epidermis. There are a single promoter (P97) for early gene expression, there is then differential splicing to generate mRNAs for early proteins. E1 and E2 are required for viral genome replication. In differentiated cells, late genes are expressed from a second promoter (P742) - L1 and L2 are expressed, post-translationally modified then undergo nuclear localisation and virion assembly.

Diseases Caused by HPVs

Alpha papillomaviruses preferentially infect the mucosa of the mouth and the anogenital region. This leads to benign or malignant tumours and some species also cause cutaneous legions.

Beta papillomaviruses preferentially infect the skins causing epidermodysplasia verruciformis in immunocompromised patients.

Gamma papillomaviruses infect the skin and cause benign lesions.

Few mu and nu papilomaviruses are characterised and some are known to cause benign skin warts.

Cervical Cancers

HPV is transmitted through sexual contact and cervical cancer is the 4th most common cancer in women globally with around 500,000 cases per year. There are two types, HPV-16 and 18 which cause 70% of cervical cancers and pre-cancerous cervical legions. Certain cofactors affect the risk of developing malignant tumours such as:

• hormonal contraceptives

• smoking

• parity (multiparity)

incidence of cervical carcinoma/100000 population. increased incidence correlates to poorer healthcare.

In addition to cervical cancer HPV can also cause oropharyngeal cancer, cancers of the vulva, vagina, penis, and anus.

HPV Classification

Risk is assessed based on the transformative potential of a strain E proteins, some strains are more likely to cause malignancies than others. Other factor such as the host immune system may affect whether malignant legions develop.

Low risk types (60, 11, 42-44) are found in benign lesions only. Intermediate risk types (31, 33, 35, 51, 52, 58) are found in benign legions are invasive cancers. High risk types (16, 18, 45, 56) are usually found in carcinomas and occasionally seen in benign lesions.

Development of Malignant Lesions

Infections can be transient and cleared however development of a persistent infection can progress to malignancy. Cervical intraepithelial neoplasia is observeed in disease progression involving new abnormal and disorganised growth of the cervix epithelium. Gynaecological CIN diagnosis involves an atypical pap smear however this is not definitive so a further culposcopy will be done to definitely determine if CIN is present by examining the specimen under culposcopes.

Early Proteins and Oncogenesis

E6 and E7 are pleiotropic with E6 interacting with p53 and E7 interacting with Rb, together these override the G1/S phase checkpoint.

E5 is thought to have a role in oncogenesis but not as well understood. It is likely to bind to epidermal growth factor receptors and activate signalling cascades.

p53 has a key role in preventing tumours and is mutated in many human cancers. E6 along with the cellular protein E6AP can bind p53 leading to p53 ubiquitination and degradation. E6 and E6AP may also be involved in degradation of other cellular proteins with PDZ domains. E6 also upregulates telomerase and leads to extension of telomeres which is a hallmark of cancer cells.

Rb proteins are tumour suppressor proteins which are usually active during mitosis. In infected cells E7 will bind the Rb protein leading to the release of E2F which is a transactivator which leads to premature entry into the S phase. Low risk HPV strains have E7 proteins with a lower affinity for Rb.

Increased E6 and E7 activity leads to tumorgenesis. In most cases changes in oncoprotein expression results from integration of the viral genome into the host genome which is not a normal part of the viral replication. Disruption of E2 ORF leads to overexpression of E6 and E7, it can also disrupt a host genome potentially leading to oncogenesis. Other mechanisms of E6 and 7 overexpression such as epigenetics also exist.

Diagnosis and Treatment

A pap smear will detect malignant cells. HPV testing via PCR is useful as an alternative primary screening tool for cervical cancer, clinical trials have indicated HPV testing as a way to solve cases where cytology based screening results are ambiguous. Determination of strain characterises associated carcinogenic risk. Early detection is key.

Treatment options include:

• Cryotherapy/laser therapy which involves freezing off or cutting away or abnormal cervical epithelial cells

• Partial/full hysterectomy which involves removal of the uterus and cervix and sometimes the ovaries and fallopian tubes

• Radiation therapy/chemotherapy

Cervical Cancer Control

Primary prevention in girls aged 9-14 includes the HPV vaccination and providing health information and warnings about tobacco use, sex education and condom promotion and provision as well as male circumcision for girls and boys where appropriate.

Secondary prevention in women older than 30 involves a screen and treat single visit approach with point of care rapid HPV testing for high risk HPV types. This is followed by immediate treatment and on site treatment.

Tertiary prevention is provided to all women as needed and involves treatment of invasive cancer at any age and palliative care surgery, radiotherapy, chemotherapy and palliative care.

HPV Vaccines

L1 is a major capsid protein the purified protein self assembles to form virus like pathogens. It activates humoral immune responses in the host. The vaccine does not contain viral genetic material and are not infectious.

There are two prophylactic vaccines available, both are L1 VLPs which prevent development of CIN from high risk HPV types 16 and 18. A bivalent vaccine comprised of HPV types 16 and 18 proteins expressed and purified from insect cells infected with recombinant baculovirus. This vaccine is formulated with a novel adjuvant AS04 which contains aluminium hydroxide and monophosphoryl lipid A. The second vaccine is comprised of HPV types 6, 11, 16 and 18 expressed and purified from yeast cells containing the specific L1 expression plasmids.