1. Discuss the microbiological and/or immunological basis of the following, 1.1 Herpes simplex virus causes recurrent disease in some patients 1.2 Acute renal failure in leptospirosis
1.1 First encounter with HSV gives rise to primary infection(formation of vesicles which will give rise to ulcers) Usually primary infection is asymptomatic. After primary HSV remains latent, Virus entering via skin or mucus membrane reaches sensory ganglia via sensory nerves and remains dormant without replicating. HSV expresses latent associated transcript (LAT) RNA which regulates the host cell genome and interferes with the natural cell death mechanism thereby maintains host cell and preserve the reservoir of virus. HSV1 and HSV2 establish latency in the trigeminal and sacral ganglia respectively. Virus can get reactivated by illnesses such as cold and influenza, eczema, stress or exposure to bright sunlight. Virus reaches skin via sensory nerves and causes recurrent infection. In HSV1 and HSV2 recurrent infection occurs in the distribution of maxillary or mandibular branches of trigeminal nerve and sacral nerves respectively.
1.2 Leptospirosis is a zoonosis caused mainly by Leptospira interrogans icterohaemorrhagica. Its a biphasic disease; leptospiraemic phase and leptospiriuremic (immune) phase. Leptospira enter via abraded skin or intact mucus membrane. Virlulent organisms access to blood stream via lymphatics, resulting in leptospiremia and spread to all organs. If the host survives the acute infection septicemia remains until the development of antibodies followed by rapid immune clearance. After clearance, leptospira tend to remain in immunologically privileged sites sanctuary sites; liver kidney brain Leptospira contains antigenic lipoprotein, lipopolysaccharides and endotoxins that may account for kidney injury leading to acute renal failure. LipL32 is specifically important in renal failure. They act on toll like receptors(TLR 2) in proximal tubular cells and activate nuclear factor NF-k This stimulates synthesis of inflammatory mediators (interleukins,TNF,CCL2/MCP1) and causes interstitial nephritis, tubular necrosis and impaired capillary permeability results in renal failure. Leptospirosis associated with renal and liver failure is known as Weils disease
2. A male school teacher having fever for 1 weekend found to be anemic with enlarged liver. List 3 parasitic infections in order of priority in the differential diagnosis of the above patient who has never been abroad. Malaria Leishmaniasis Ameobiasis
3. Briefly describe the pathological changes and pathogenic mechanisms associated with cerebral malaria. Cerebral malaria is caused by plasmodium falciparum infection In its life cycle, during blood meal malaria infected female anopheles inoculate sporozoites into human host. Then sporozoites infect liver cells and in liver cells they mature into schizonts Schizonts rupture and release merozoites Merozoites invade any stage of RBC and develop, after ring stage they leave peripheral circulation and enter vessels of the deep oragans 2
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It is called sequestration And due to infected RBC s knobby appearance they can adhere to other RBC and endothelial cells and this is called as rosette formation These rosette can block small vessels in the brain and cause microvascular obstruction So due to decreased blood flow to brain cells, cells undergo anaerobic respiration due to hypoxia and result in increased production of lactic acid At the same time there will be releasing of merozoites from ruptured schizonts Those released merozoites and parasitic products will stimulate endothelial cells and mononuclear cells to produce reactive nitrogen species which cause endothelial damage And also it will stimulate monocytes and lymphocytes to produce cytokines like TNF Cytokines also cause endothelial damage resulting fluid leakage Due to rosette formation and microvascular obstruction, capillaries are dilated and cerebral tissue is markedly congested Due to increased fluid leakage cerebral tissue becomes oedematous Due to malaraial pigment deposition, cerebral cortex is dusky gray or brown in colour Due to capillary obstruction and endothelial damage there will be petechial hemorrhage in perivascular tissue
4.1 Describe the physiological basis of fever in infections. Toxins from bacteria such as endotoxins act on monocytes, macrophages and kupffer cells to produce cytokines Cytokines act as endogenous pyrogens So cytokines such as IL-1, IL-6, TNF can act independently to produce fever These cytokines act on vascular receptors in the thermoregulatory center of the hypothalamus Stimulate production of prostaglandin E Prostaglandin act on vasomotor centre and activate sympathetic nervous system Increased sympathetic stimulation causes cause vasoconstriction in skin vessels Reduced heat dissipation occurs. Prostaglandin also raise temperature set point above normal stimulating body heat generating mechanisms and increased heat production Increased heat generation and reduced dissipation causes fever
4.2 Name 1 bacterial and 1 parasitic infection that presents as acute fever in Sri Lanka. Bacterial- Typhoid fever Parasitic- Malaria
4.3 For each of the infection listed above name the specimen/s, appropriate microbiological and parasitological investigations for definitive diagnosis. Blood culture- 1 st week urine culture- 2 nd week stool culture- 3 rd week Blood smear stained with giemsa stain both thick and thin smears
4.4 For one of the infections listed in 4.2 1. List the therapeutic objectives To destroy bacteria To relieve symptoms and complications To reduce transmission Prevent emergence of carrier stages Reduce occurrence of endemics 3
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2. Explain how you would select an antimicrobial based on efficacy and safety Antimicrobial must cover the spectrum of infectious agents Side effects has to be low and benefits must be outweighed than side effects Drugs which are not toxic and high therapeutic index drugs are preferred Infectious agent must be sensitive to the antimicrobial So in typhoid now 3 rd generation cephalosporin can be used 3. Describe the mechanism of action Cephalosporins inhibit cell wall synthesis of bacteria by competitively inhibiting the penicillin binding proteins crosslinking of peptidoglycan Therefore it is an bactericidal drug
5.1 What do you understand by the term sepsis syndrome?
Sepsis syndrome or SIRS (Systemic Inflammatory Response Syndrome) is systemic level of acute inflammation, that may or may not be due to infection, and is generally manifested as a combination of vital sign abnormalities, such as fever/hypothermia, tachycardia and tachypnea. It is a systemic response to a wide variety of clinical insults. In sepsis syndrome there is also evidence of altered organ perfusion with at least one of the following features : hypoxaemia, elevated lactate, oliguria, altered mental status. Risk factors for SIRS would be extremes of age, indwelling lines/catheters, immunocompromised states, malnutrition, alcoholism, malignancy, diabetes, cirrhosis and genetic predisposition. Overall mortality from SIRS is high and the mortality rate is proportionate to the no. of failed organs. Sever sepsis is SIRS associated with organ dysfunction, hypoperfusion or hypotension and the marker is acute organ dysfunction.
5.2 What are the characteristic features of this condition?
2 of the following 4 features should be present : Temp>38.3 or <36.0 C Tachypnea (RR > 20 or Minute Volume > 10L ) / PaCO 2 < 32mmHg Tachycardia (HR > 90, in the absence of intrinsic heart disease) WBC > 10,000/mm3 or <4000/mm3 OR > 10% band forms on differential count.
In severe Sepsis, 1 of the following should also be there : Altered mental status Systolic BP<90mmHg or fall of >40mmHg from baseline Impaired gas exchange Metabolic acidosis Oliguria Hyperbilirubinemia Coagulopathy
5.3 List 2 important cytokines that are responsible for the pathophysiology of this condition
TNF IL 1
5.4 What is the primary reason for the above cytokines in the sepsis syndrome? The above cytokines are produced in response to the microbial signals. About 70% of cases are due to endotoxins produced by gram (-) bacteria. These endotoxins are bacterial wall lipopolysaccharides consisting of a toxic fatty acid core (lipid A), and a complex polysaccharide coat (O antigen) unique for each species. 4
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Peptidoglycan/lipoechoic acids in gram (+) bacteria can also elicit this. Toxins such as TSST 1 (Toxic Shock Syndrome Toxin 1) produced by S. aureus (gram (+) cocci), Group B Streptococci Toxin can also cause this. The free lipopolysaccharides bind to CD14 receptor in monocytes, macrophages, neutrophils. It circulates in blood bound to a lipopolysaccharide binding plasma protein. Engagement of CD14 results in intracellular signaling pathways being activated (TLR -4) resulting in activation of mono nuclear cells. The macrophages present the antigen to the T helper cells which activates the T helper cells. These in turn activates the macrophage again which triggers them to secrete TNF and IL 1 Both of these cytokines which are the early mediators, act on endothelial cells and other cells to produce additional cytokines. (IL6, IL8) These are the late mediators. These induce adhesion molecules and other factors such as prostaglandins, coagulation factors are also induced. These result in systemic vasodilatation, intravascular volume depletion and endothelial injury.
6.1 List 2 lab diagnostic methods that could be used for Plasmodium vivax malaria Light microscopic diagnosis 1. PCR technique
6.2 Write advantages and disadvantages of each of the above methods.
Light Microscopy Advantages : o Can be performed under field conditions. o Requires inexpensive equipment (microscope, glass slides and reagents) o Results available in 15 20 minutes useful in OPD setting o Detection of any species of Plasmodia are possible in a single test o Detects all parasitic stages present in peripheral blood. o Sensitivity is high (If analyst is trained) o Can monitor the response of a patient to therapy.
Disadvantages o May give false negatives if the parasite density is very low or if parasites are absent in peripheral blood. o Required trained personnel to identify parasites o Sensitivity and specificity are high but varies : At low incidence rates At very low parasite densities On the skill of the analyst Lab conditions o Relies on electricity PCR technique Advantages o Highly sensitive o Highly specific o Can be diagnosed even with a very low parasite density
Disadvantages o Requires expensive lab equipment and reagents o Requires good laboratory conditions o Might give false positives even after cure of the patient, because parasite DNA might remain in circulation for several days to a week after successful treatment and termination of infection.
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7. A 55 year old male who underwent an emergency bowel surgery developed fever on the 3 rd post-operative day. He has an indwelling urinary catheter and an IV cannula in situ.
7.1 List 4 likely infections causing fever in this patient.(8) Surgical site infection Urinary tract infection IV line associated infection and bacteremia Hospital acquired pneumonia
7.2 Name 2 likely microorganisms responsible for each of the infections listed above in 7.1 (32) Surgical site infection Staphylococcus aureus, Escherichia coli Urinary tract infection Escherichia coli, Pseudomonas aeruginosa IV line associated infection & bacteremia Staphylococcus aureus, Staphylococcus epidermidis Hospital acquired pneumonia Staphylococcus aureus, Pseudomonas aeruginosa
7.3 Name the relevant specimen required for microbiological diagnosis of each of the infections listed in 7.1 (20) Surgical site infection blood for culture and ABST Urinary tract infection urine for culture and ABST IV line associated infection & bacteremia blood for culture and ABST Hospital acquired pneumonia expectorated sputum for culture, ABST, gram stain
7.4.1 List the therapeutic objectives (10) Elimination of the causative organism with a suitable antimicrobial drug or a combination Symptomatic relief (antipyretics, bronchodilators) Enhancing patients immunity (nutrition, deficient vitamins) Prevent recurrences (surgical wound hygiene)
7.4.2 State the principles on which you would select the appropriate antimicrobial/s (20) Specificity activity of the drug should match the infecting organisms.(narrow spectrum) Empirical therapy before identification of causative organism must be reasonably broad spectrum. Pharmacokinetic factors chosen drug must reach the site of infection in adequate amounts. (Eg. crossing the blood brain barrier) Patient factors previous allergy to the drug, impaired routes of elimination (renal disease)
7.5 2 days later, the patient develops tachycardia (130 beats/min) and an increased respiratory rate (40/min). His white cell count was 15x10 9 /L with 90% neutrophils. His urine output was decreased. What is the complication the patient has developed? (10) Systemic inflammatory response syndrome secondary to infection (or sepsis)
8. A 6 year old girl presents with anemia.
8.1 Name 2 parasitic infections that could be the cause of anemia in this patient. (10)
Malaria Hook worm infestation
8.2 Explain the pathophysiological basis for the anemia in each of the parasitic infections listed in 8.1 (30)
Malaria The released merozoites from infected hepatocytes infect the RBC. The intraerythrocytic parasites either continue asexual reproduction, producing more merozoites infecting more RBC or give rise to gametocytes. The new merozoites escape to the blood stream by lysing the RBC. 6
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This destroys large numbers of red cells causing hemolytic anemia.
Hook worm infestation Adult worms of Necator americanus live in the duodenum and upper jejunum, where they are found in large numbers in severe disease. They attach firmly to the mucosa using their buccal plate and feed on host blood. They secrete molecular inhibitors of coagulation factors causing bleeding from the attachment site. They also degrade host haemoglobin. The resulting massive loss of blood from the body causes an iron deficiency anemia.
9.1 List 5 infectious diseases where blood culture is useful in the diagnosis. (5)
in any infection with bacteraemia Meningitis Enteric fever Pneumonia Pyelonephritis Bacterial endocarditis Osteomyelitis
9.2 Describe in detail how you would obtain blood for culture & transport it to laboratory including universal precautions. (30)
first obtain the necessary equipment to perform the blood culture - Blood culture bottles - Hypodermic syringes and needles - Sterile gloves - Tourniquet - Alcohol - Sterile gauze pads - 10% povidone iodine (if allergic 4% chlorhexidine) Then check the identity of the patient and explain the procedure to the patient Next check the blood culture bottles for any contamination (see whether they are clear) Wash hands using soap and water thoroughly and dry on a sterile towel Wear the sterile gloves taking care not to contaminate Select an appropriate venepuncture site Clean the site by applying 70% alcohol concentrically rubbing from inwards to outwards. Allow this to dry Next clean the site using 10% povidone iodine in a concentric fashion as above from inwards to outwards Allow at least 2 minutes to dry While waiting for this site to dry, remove the protective covering from the bottle top and wipe the exposed diaphragm using an alcohol based disinfectant and allow it to dry Then perform the venepuncture using a dry, sterile needle and syringe and draw at least 20 ml of blood While drawing blood take precautions to prevent sharps injuries such as avoiding hand to hand passage and maintaining a safe distance from others when drawing blood Inoculate 5 ml of blood into each blood culture Do not change needles for this purpose and always place the culture bottle on a flat surface while inoculating blood Once the bottle has been inoculated, gently shake the contents so that it has been mixed Carefully dispose of the used needles and syringes into the sharps bin (Do not recap or break needles in the process) Then remove the gloves and wash hands Label the bottle with the relevant patient details Fill the laboratory request forms Keep the samples at room temperature before transporting them as soon as possible to the lab
9.3 From the steps you have mentioned in 9.2, select & write the steps that are taken to ensure asepsis.(aseptic precautions) (10)
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Wash hands using soap and water thoroughly and dry on a sterile towel Wear the sterile gloves taking care not to contaminate Disinfecting the skin over the venepuncture site by applying 70% alcohol concentrically rubbing from inwards to outwards and allowing this to dry Cleaning the site using 10% povidone iodine in a concentric fashion as above from inwards to outwards and allowing at least 2 minutes to dry Performing venepuncture using a dry sterile needle
9.4 From the steps you have mentioned in 9.2, select & write the steps that are taken to prevent occupational exposure to blood borne pathogens. (universal precautions) (10)
Wash hands using soap and water Wearing sterile gloves Taking precautions to avoid needle stick injuries such as avoiding hand to hand passage, maintaining safe distance from colleagues while using sharps Not changing needles to inoculate the blood Placing the culture bottles on a flat surface before inoculating Disposing used needles and sharps into sharps bins without recapping or breaking needle Washing hands after removing the gloves
9.5 Outline the principles of antimicrobial therapy for one of the infectious diseases you have listed in 9.1. (25)
MENINGITIS Before initiating any antimicrobial therapy, it is necessary to make a microbial diagnosis of the condition as precisely as possible For this purpose it is necessary to obtain a CSF sample by performing a lumbar puncture The sample is sent to the microbiological department for gram staining and culture Also blood samples must be taken for blood cultures (and to determine plasma glucose) Next it is necessary to consider whether antimicrobial therapy is really necessary For this purpose criteria such as diagnosis, severity of the disease, onset of the disese and the natural history of the disease are considered Since a meningitis is a serious life threatening disease of acute onset with many neurological sequale if left untreated, a diagnosis of meningitis usually necessitates treatement Removal of any barriers to the cure must be taken in order to ensure that antimicrobial drugs can reach the site of infection to exert their action Once all the above has been performed, it is necessary to select a drug/drugs to use in this situation In the case of meningitis, empirical/best guess therapy is given soon after the relevant samples have been taken The drugs used for empirical therapy are selected based on factors such as the site of infection, the likely organisms to be causing the infection as well the resistance patterns of these organisms to various antibiotics Once the microbial diagnosis arrives, it is necessary to narrow the spectrum of the antibiotic to target the identified agent The selection drugs in this case should be based on - Pharmacokinetic factors - Whether the selected drug can reach the site of infection in adequate amounts to exert its action - Patient factors Age Renal and Hepatic Function Pregnancy and Lactation History of allergy History of recent antibiotic use - Microbial factors - Resistance to various antimicrobials
Once a drug is selected based on the above factors, it should be administered at the - Optimal dosage (to prevent development of resistant organisms) 8
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- In the appropriate dosage forms - At the optimal frequency (intermittent dosing is preferred) - Via the appropriate route (parenteral considering the severity of meninigitis) - Correct duration (to prevent the development of resistant organisms Clinical and plasma monitoring must be done while the drug is being delivered Finally the resolving of the infection must be confirmed clinically or microbiologically before the patient is discharged
9.6 Based on the principles you have mentioned in 1.5, 9.6.1Choose an antimicrobial/s that is effective in the infectious disease discussed in 9.5, giving reasons for your choice. (15) 9.6.2Mention, 9.6.2.1Route of administration 9.6.2.2.Approximate duration of therapy (5)
9.6.1) Cefotaxime This is a 3 rd generation cephalosporin Which acts as an inhibitor of bacterial cell wall synthesis and therefore acts as a bactericidal drug It has a very good gram negative cover and is effective against common bacterial agents causing meningitis such as H.influenzae type B (noncapsulated) and Neisseria meningitides as well as being effective against gram positive organisms such as Streptococcus pneumonie It has a good distribution throughout the body tissues and passes through the blood brain barrier, meaning that it can reach the meninges in order to exert its antimicrobial action It is available in the parenteral dosage form which is the dosage form of drugs used in meningitis due to its severity It is safe to use in children and its excretion is not affected by liver failure (but it is affected by renal failure) It can also be used during pregnancy since it does not cause any harmful effects to the foetus even though it crosses the placenta In addition to the above, cefotaxime does not have many significant side effects except for beta lactam hypersensitivity which must be tested for before administration of the drug
9.6.2.1 Intravenous Route 9.6.2.2 14 days
10. A 34 year old patient presents to the OPD of the NHSL with the history blood & mucus diarrhoea of 2 weeks duration. He has vague abdominal pain & anorexia.
10.1 List 2 likely parasites that cause blood & mucous diarrhea. (5)
Entamoeba histolytica Trichuris trichiura
10.2 Describe the pathogenic mechanism for the blood & mucus diarrhoea for each parasite mentioned in 10.1
Entamoeba histolytica
The trophozoite form of the organism present in the intestinal lumen penetrates the colonic mucosa in between the glandular epithelium by the production of tissue lytic substances which degrade the protein and polysaccharide components of the mucus layer They then attach to the epithelial cells of the surface of the gut and induce apoptosis in these cells. (this results in the production of ulcers with pin head centre and raised edges) Sometimes the invasion is superficial and results in the formation of flask shaped ulcers The trophozoites then penetrate the submucosal layer of the intestine and multiply rapidly causing lysis of the surrounding tissue and blood vessels Causing the leaking of blood into the lumen 9
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Trophozoites also alter the mucus composition of the mucous secreted In goblet cells, thereby increasing the mucus secretion This results in the production of a blood and mucus diarrhoea
Trichuris trichiura
Once the embryonated egg of the organism is ingested, the activated larvae hatches out from the weakened egg shell in the upper small intestine and penetrates through the intestinal villus A spear like projection in the anterior extremity allows the worm to penetrate and embed its whip like anterior portion into the intestinal mucosa of the host In this process it releases various chemicals to liquefy the adjacent mucosal cells As a result of penetration of blood vessels at the site of attachment of these worms, blood leaks into the intestinal lumen giving rise to blood and mucus diarrhoea This occurs especially in heavy infections of the parasite
10.3 Taking one of the parasites mentioned above as an example, 10.3.1 Describe the collection of stool sample & its transport to the laboratory. (20) 10.3.2. Outline the laboratory procedures you would carry out on the stool sample collected in 10.3.1, and the expected findings to arrive at a definitive diagnosis. (20)
10.3.1 Entamoeba histolytica
The patient should defecate into a clean and dry bed pan without any disinfectant If this is not possible the patient should defecate into a clean bowl or even a banana leaf A selected portion of the stool from the mucoid and bloody area is transferred into a clean and wide mouthed disposable container with a lid (Eg : Ice cream container) This should be transported as soon as possible to the laboratory If any delay occurs a suitable transport medium should be used
10.3.2 Light microscopy using a wet smear for demonstration of trophozoites or cysts must be performed within 30 min Iron haematoxylin stains are used for better visualization If negative, three repeat samples are viewed under the microscope under high concentrations If a trophozoite is present it would consist of outer ectoplasm which is wide and clear and inner endoplasm which is granular. It also contains a round nucleus with a cartwheel appearance and also contains ingested RBCs If a cyst is present it is round and surrounded by a wall and consists of 2 -4 nuclei with undifferentiated cytoplasm containing sausage shaped chromatoid bodies
10.4 For each of the parasites listed in 10.1, list one anti microbial drug effective for the treatment & give reasons for your selection. (20)
Entamoeba histolytica - Metronidazole
Metranidazole can kill the trophozoites of Entamoeba histolytica and can therefore act against the active organisms present within the lumen of the gut It is well distributed after oral and rectal administration and reaches effective concentrations within the gut and liver where trophzoites are usually found It has only a few unwanted effects at therapeutic doses including gastrointestinal disturbances, metallic taste, dizziness, headache and disulfuram reaction
Trichuris trichiura - Mebendazole Mebendazole acts on both the larvae and the adult worm as well as on the ova of the organism It causes inhibition of mitochondrial enzymes and inhibits glucose uptake by the parasites and therefore causes hypoglycaemia in them 10
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It is poorly absorbed in the gut meaning that it can remain there in order to exert its action It does not cause any significant systemic toxicity in active clinical use even in the presence of anaemia and malnutrition which are common in patients infected with these parasites
10.5 What advice would you give to the patient in order to, 10.5.1 prevent re-infection in this patient 10.5.2 prevent spread of this infection from the patient to the community. (10)
10.5.1 Since these infections are mainly transmitted by the faeco oral route, reinfection can be prevented by 1) Food safety This involves proper cleaning of hands before preparing or consuming food and after toilet use, washing vegetables whose edible portions grow within the soil and covering food to prevent contamination by flies and cockroaches which can be used as mechanical vectors. Children should not be allowed to play in contaminated soil as they may accidentally ingest the eggs of the parasite
2) Water safety Only boiled water or water that has been sterilized properly using iodine salts or other suitable agents should be taken for consumption
10.5.2 Spread of infection to the community can be prevented by 1) Preventing soil contamination Proper sanitary facilities should be used and there should be sanitary disposal of waste in places where eggs do not come into contact with humans. Drainage systems and water lines should be cleaned and maintained in a proper manner so as to prevent contamination with infected waste 2) Screening and treatment It is important that individuals who have contact with foodstuffs prior to consumption such as chefs and restaurant carriers are screened for the condition since they may be an active source for the spread of infection. Once infection has been confirmed in these patients they should be treated as soon as possible
3) Supportive measures Health education regarding these diseases can be given to the general public to create awareness which would reduce spread. In addition to this public health measures such as providing decontaminated drinking water and sanitary facilities would also prevent the spread of the disease
Insect control is also important and would prevent spread of this infection since they act as mechanical vectors