LABS 4-6 Flashcards
why is ziehl Nelson (acid-fast) stain useful in lab diagnosis of cryptosporidium diarrhoea?
crypto cysts are 4-6um and so hard to distinguish from plant cells, yeasts and pollen grains in faecal sample. The usual stains used for stool diagnosis of parasites (trichrome, iron hematoxylin) don’t work for crypto oocysts. Several widely used techniques which do work include acid fast staining and negative staining. Acid-fast staining procedures are effective cause they stain the oocyst wall red but yeast cells take on the background stain
how does catalase production enhance bacterial resistance to killing by phagocytic leukocytes?
catalase breaks down hydrogen peroxide which is a toxic oxygen species produced by phagocytic cells in order to kill ingested bacteria
which cells of the immune system are attacked by HIV?
cells that express CD4 cell surface protein, including CD4+ T cells, macrophages, dendritic cells and monocytes
which receptors must be present for HIV to infect a host cell?
as well as CD4, co-receptors must be present. These are the chemokine receptors CCR5 (macrophages and T cells) and CXCR4 (T cells)
what impact does HIV infection have on the ability to mount an immune response?
progressive decline in CD4 cell numbers results in increased susceptibility to infection. Opportunistic infections occur (e.g. pneumocystis pneumonia, oral candidiasis etc) due to loss of T helper cells which are a major source of cytokines that are essential for support of adaptive (T and B cell) immune responses. (Also affects antigen presentation due to infection of DC’s and macrophages)
what is cryptosporidium, candida albicans, pneumocystis jiroveci and aspergillus fumigatus examples of and what are they important in?
eukaryotic pathogens which are important in immunocompromised and immune deficient patients causing diseases which range in severity from mild to deadly based on the host defences
what does Candida albicans cause?
superficial infections e.g. oral/vaginal thrush and deep infections of the blood and major organs
what is pneumocystis jiroveci particularly associated with?
respiratory pathogen particularly associated with acquired immune deficiency syndrome (AIDS)
what is aspergillus fumigatus and what does it cause?
saprophytic environmental fungus which causes either allergy or infection in susceptible host
how do we identify CD4 and CD8 cells in a patient by flow cytometry?
using antibodies conjugated with fluorescent dyes (APC and PE)
why are HIV infected individuals vulnerable to opportunistic infection?
loss of Th1 cells (stimulate CMI) and Th2 cells (stimulate production of neutralising antibody)
cytotoxic T lymphocytes (CD8+) target HIV infected macrophages and Th cells and lack of these cells increase susceptibility
at what point does HIV transition into AIDS?
when CD4 T cell count is <200/ul
what is the most common opportunistic infection in AIDS patients?
pneumonia caused by pneumocystis jiroveci, an unusual fungus which in many respects resembles a protozoan
at what point does aspergillus generally infect the lungs of AIDS patients?
when CD4 count has fallen to or below 50/ul
what is the difference between infection by P. jiroveci and A. fumigatus?
P. jiroveci infects lung tissue (interstitial pneumonia) and A. fumigatus infection tends to begin in the alveoli (usually forming a fungal ball called an aspergilloma but can spread to blood causing invasive aspergillosis)
what are the main morphological differences between aspergillus and pneumocystis?
aspergillus is a hyphae-forming, multicellular fungus (mould) well pneumocystis is a single celled fungus (yeast)
what are the main differences in the X-ray presentations of fungal diseases caused by aspergillus and pneumocystis?
opacity in a lung x-ray indicates there is consolidation, the healthy lung tissue is black as x-ray passes through alveoli filled with air
aspergillus usually causes discrete area of opacity in one lung (unilateral). infection is localised beginning with formation of a fungal ball in alveoli (aspergilloma). If untreated fungal hyphae may invade lung tissue (aspergillosis). If erodes into blood vessel infection may become systemic
pneumocystis in HIV/AIDS patients usually in both lungs (bilateral). Patchy, diffuse opacity occurs throughout lungs. It is an interstitial form of pneumonia often affecting the tissues between alveoli rather than infection of alveoli themselves. Causes more scarring
what is the dihydrorhodamine (DHR) test for phagocyte function?
test for the production of reactive oxygen species such as hydrogen peroxide and toxic radicals produced by phagocytic leukocytes
what is required for intracellular killing of microorganisms ingested by phagocytes?
reactive oxygen species produced by an enzyme during an oxidative burst
what is chronic granulomatous disease (CGD)?
an immune deficiency disease where mutations in the X-linked gene encoding the PHOX (phagocyte NADPH oxidase) enzyme result in deficiencies in intracellular killing. This makes the patient highly susceptible to infections by catalase-positive bacteria and fungi e.g. the yeast candida
what does the DHR test measure and assess?
measures oxidative burst and used to assess phagocyte function
how is the DHR test done?
whole blood is incubated with dihydrorhodoamine 123 and then stimulated. When the neutrophils are stimulated and undergo oxidative burst DHR is oxidised to rhodamine. Rhodamine is fluorescent and detected and measured by flow cytometry
what does less fluorescence indicate in the DHR test?
indicates phagocytic oxidative burst is deficient e.g. in people with CGD
what does a bimodal distribution on a woman’s flow cytometry plot mean?
she has a mixture of normal and deficient phagocytes indicating she is a carrier however she still has enough phagocyte function to prevent chronic infection. As a female she has two X chromosomes and 2 copies of the PHOX gene (one good with normal function one not with decreased function)
why is it important to identify yeasts in the laboratory?
because innate susceptibility susceptibility to anti-fungal agents varies among different species
what are chromogenic agars commonly used for?
culture and presumptive identification of bacteria and yeasts
what is CHROMagar candida?
a selective medium containing cosmogenic dyes linked to different substrates and when metabolised the substrates develop colour meaning C. albicans can be identified by colony colour
identify can be further confirmed by the gram stain and ‘germ tube test’ where C. albicans grows either as an oval shaped yeast with budding (gram stain) or mycelial form producing hyphae (also called germ tubes). True hyphae have parallel sides and aren’t constricted at the point they emerge from the yeast cell meaning it is C. albicans
what is the colony colour of C. albicans on CHROMagar candida plates?
green
what is the colony colour of C. tropicalis on CHROMagar candida plates?
metallic blue
what is the colony colour of C. krusei on CHROMagar candida plates?
rose
what should you see in a gram stain to confirm a microbe as a yeast?
unicellular, larger than bacteria, budding may be apparent (how many yeasts divide), gram stain colour may be purple to purple/pink (yeasts cannot be said to be gram pos or neg as they have different cell wall structure to bacteria)
what is catalase?
an enzyme that breaks down hydrogen peroxide to oxygen and water
what are biofilms?
consist of cells growing on a surface (typically a solid-liquid interface) embedded in a slimy matrix of polymers (sugar polymers important component). This matrix is called the EPS (extracellular-polymeric substance). Biofilms are associated with 50-80% of all infections
why do bacterial biofilms form?
as a form of defence by helping with adherence to surfaces so the bacteria can’t get swept away, helps with resistance to immune system cells e.g neutrophils and macrophages and other immune factors e.g. antibodies, complement. Helps with resistance to antimicrobials which can’t diffuse easily through the matrix and also cause bacteria in matrix are often less metabolically active (antimicrobials often target cellular processes such as peptidoglycan synthesis). Also waste products from a microbe might act as nutrients for another and also opportunity for genetic exchange (e.g of resistance genes)
what are super antigens?
proteins that activate large numbers of T cells non-specifically. They form a bridge between class II MHC molecules on APCs to TCRs on T cells without specific antigen in the MHC binding site. Up to 30% of T cells can be activated and they produce INF-gamma
what does INF-gamma induce?
pro-inflammatory cytokines such as TNF-alpha and IL-1 and IL-6 from macrophages, resulting in endothelial damage, circulatory shock (DIC, thrombosis, haemorrhage) and multi-organ failure
what is toxic shock syndrome (TSS)?
caused by S. areas super antigen TSST-1 which leads to cytokine overproduction resulting in circulatory collapse and multi-organ failure. TSS has a high mortality rate and high morbidity due to surgical debridement and amputation
where does S. epidermis inhabit?
it is a universal commensal of the skin and mucous membranes
where does S. aureus inhabit?
carried in the nasopharynx of approximately one third of the population and sometimes also colonises the skin
what does washing the biofilm do in the crystal violet assay for biofilm forming ability?
removes floating (planktonic) bacteria so that the adherent (sessile) population can be stained with crystal violet
what is the amount of stain present in the wells at the end of the crystal violet assay directly proportional to?
the amount of bacteria in the biofilm
of the two staphylococcal species we studied in lab 5, which had the best biofilm forming ability?
S. epidermis (good) (well S. aureus was intermediate)
what does staphylocoagulase do?
is an extracellular molecule that causes plasma to clot through the conversion of fibrinogen to fibrin. Fibrin-coated staphylococci tend to aggregate thus localising infection and the fibrin layer protects the bacteria against phagocytic cells
tested for in tube coagulase test
what is protein A?
a staphylococcal cell wall protein which binds firmly to immunoglobulins (antibodies). The Fc portion adheres to protein A so that the active site (Fab) is facing away from the bacterial cell. This can be referred to as ‘upside down’ antibody
how do we test for protein A?
immunoglobulin binding test
the ‘upside down’ antibody can be detected with a secondary antibody labelled with a fluorescent dye. After washing away unbound antibody, fluorescence can be measured using a fluorescent plate reader. If protein A present then the primary antibody (human IgG) will be bound to the staphylococcal cells and the secondary fluorescent antibody (mouse anti-human antibody) will be bound to the primary antibody
why was a high fluorescence reading obtained from S. aureus during the immunoglobulin binding test?
because protein A was present
why did S. epidermis give a reading similar to the negative control in the immunoglobulin binding test?
because protein A was not present
which staphylococcal species was positive for the tube coagulase test?
S. aureus
which staphylococcal species was positive for the catalase test?
both!
which tests cannot be used to distinguish S. aureus and S. epidermis?
gram stain (both gram-pos cocci in clusters) and catalase test (both catalase positive)
which test would be most useful in distinguishing the two staphylococcal species?
tube coagulase test (easiest to perform and gives clear result)
protein A test good specificity but takes way longer so not as good
biofilm forming test has little diagnostic value and is hard to standardise
how does protein A help staphylococci to overcome the host defences?
by binding the non-reactive Fc portion of the antibody molecule. The active binding sites are on the Fab portion but the antibody is upside down inhibiting opsonisation where the Fc region is recognised by a receptor on the phagocyte. No opsonisation means phagocytosis is inhibited
how does catalase production assist staphylococci to overcome host defences?
catalase allows bacteria to resist breakdown by ROS in phagolysosome by converting hydrogen peroxide to water and oxygen
why can S. aureus cause disease in a less susceptible host than S. epidermis?
it has more virulence factors and so is more virulent
what cells does S. aureus TSS toxin 1 activate?
cells with the V beta 2 domain
which cytokines are produced in response to S. aureus TSST-1 protein and what do they do?
INF-gamma is produced by activated CD4+ T cells which activates macrophages to make TNF alpha (upregulates inflammatory response including fever and mediates septic shock), IL-1 (pro-inflammatory including fever), IL-6 (upregulates acute phase response, fever, neutrophil differentiation), IL-8 (recruitment of neutrophils)
IL-2 also produced by activated CD4+ T cells resulting in proliferation of more T cells and further production of INF-gamma which activates more macrophages to produce cytokines worsening the response
what are the consequences for patient of systemic inflammation in TSS?
fever, diarrhoea, vomiting, desquamation. Vasodilation leading to decreased blood volume and cardiac insufficiency (low BP and higher HR). Damage to endothelial cells results in intravascular coagulation with thrombosis, embolism, ischaemia and necrosis of organ systems (MODS). Generalised inflammatory response similar to septic shock which can be fatal due to overproduction of cytokine (cytokine shock)
what is latent TB infection?
an asymptomatic, contained (non-infectious) infection that can reactivate into active disease. Latent TB occurs in 90-95% of people infected with M. tuberculosis
why is it important to identify TB infected patients that are co-infected with HIV?
HIV infection most important known risk factor for progression from latent TB infection to TB disease. Progression to TB disease is often rapid among HIV infected people and can be deadly. TB outbreaks can rapidly expand in groups infected with HIV
how is latent TB distinguished from active TB?
clinical symptoms, chest radiograph (x-ray) and diagnostic microbiology (positive culture, PCR, sputum staining). In active disease M. tuberculosis must be recovered from the sputum
why is diagnosis of latent TB important?
latent TB can develop into active disease, especially if the person is HIV-infected or has other risk factors decreasing immune function. Preventative treatment can be given. Active disease also a risk for transmission
how does an IGRA test work?
a persons white blood cells are mixed with antigens from M. tuberculosis. If they have been infected with M. tuberculosis the white blood cells will activate and produce cytokine (IFN-gamma) which can be measured (by a method like ELISA)
Both tests for diagnosis of TB measure immune reactivity to TB antigens. How is diagnosis of TB likely to be affected by HIV co-infection?
TST - reaction may be smaller so a reaction of 5mm is considered positive (may increase chance of false positives, but in patients with low CD4 T cell counts more likely to provide false negative reading)
IGRA - in individuals with low CD4 T cells likely to get a false negative reading
how is diagnosis of latent TB infection likely to be affected by prior BCG vaccination and why?
TST - false positive
IGRA - no effect from prior BCG vaccination
there is a strong overlap between antigens present in the BCG vaccine and antigens in TB
M. tuberculosis is the main disease-causing species in humans, what is it in animals?
M. bovis
TB from M. bovis can also be transmitted to humans through shit like milk
what is BCG?
the only available TB vaccine
is an attenuated form of M. bovis
M. bovis and M. tuberculosis share many antigens such as…
Ag85
what are some of the major challenges that need to be addressed in order to control and reduce the incidence of TB?
a new more effective vaccine
drugs that are effective against multi-drug resistant (MDR) and extensively drug-resistant (XDR) TB
improved diagnostics (distinguish between latent and active TB)
development of biomarkers for the advancement and testing of new therapeutics and vaccines
what can biomarkers provide?
info about disease status, risk of progression and protective immunity against vaccination. T cell responses to TB antigens may have potential as biomarkers
what is an enzyme-linked immunosorbent assay (ELISA)?
a way of measuring the levels of IFN-gamma produced by T cells in response to M. tuberculosis antigens not found in BCG vaccine
high production of IFN-gamma by T cells stimulated with M. tuberculosis antigens indicated infection with TB
can the ELISA test distinguish latent TB infection from active TB disease?
nope
what does an ELISA measure?
how much of a specific protein is present in a biological sample e.g. IFN-gamma in blood
what do TB specific T cells present in an infected patient produce?
IFN-gamma
why is a capture antibody used in an ELISA?
antibody is specific for IFN-gamma and so they will bind
what is Ki67?
a nuclear protein that can be used as a cellular marker for proliferation as it is actively expressed in dividing cells
tuberculin is largely Ag85, what does this mean for the Mantoux tuberculin skin test (TST)?
can’t accurately distinguish between a response to BCG or tuberculosis in a vaccinated individual
in TB infection, which subset of white blood cells increases the most?
monocytes and lymphocytes
