Lecture 14 - Diagnosis of Parasitic infections: Protozoa Flashcards
What is leishmaniasis?
Leishmaniasis is a group of diseases the main three types are:
* Visceral leishmaniasis or kala-azar
* Cutaneous leishmaniasis or oriental sore
* Mucocutaneous leishmaniasis or espundia
It is transmitted during biting by infected female sand flies: Phlebotomus and Lutzomyia species.
Co-infection with HIV is known
Causative organisms: a complex genus with many (>20) species. Various species seen in Asia, Africa, Americas, Southern Europe.
Discuss the diagnosis and lifecycle of leishmaniasis
Diagnosis of Leishmaniasis and the life cycle
Amastigotes can be seen in blood smears and this is the main diagnostic stage - in humans. Diagnosis can occur in the human stage of the life cycle.
Promastigotes are motile in the sand fly and are the source of the antigen used in diagnostic tests.
Discuss the epidemiology of leishmaniasis.
- 350 million people at risk of infection in 98 countries
- The global estimated incidence of the disease
○ Visceral (VL) - 200-400K cases/year
○ Cutaneous (CL) 0.7-1.2M cases/year - VL is fatal if untreated (95% case fatality)
- 90% of VL in 4 countries - India, Bangladesh, Brazil and Sudan.
- Animal reservoirs
- Sand flies transmit disease
- The global estimated incidence of the disease
Describe diagnosis of visceral leishmaniasis.
- Clinical (indirect)
Anaemia (low haemoglobin), leucopenia (low white blood cell count), thrombocytopenia (low blood platelet count)- Antibody detection (indirect: diagnosis relies heavily on these tests)
○ DAT, IFAT
○ ELISA, ICT - Parasitological (direct)
Seeing amastigotes in aspirates from spleen, lymph node, bone marrow, or liver = current gold standard.
Promastigotes in cultures derived from aspirates - Antigen detection (direct)
KAtex urine test - Molecular biology methods (direct)
PCR, NASBA
- Antibody detection (indirect: diagnosis relies heavily on these tests)
Clinical diagnosis of visceral leishmaniasis.
- Incubation: 1-2 months to >10 years
- Different phases: asymptomatic - subclinical acute (severe) - chronic (persistent)
- Onset usually gradual with low grade fever, progressive splenomegaly, hepatomegaly, anaemia, wasting, pigmentation, swollen lymph nodes, and susceptibility to other infections.
- Onset sometimes abrupt with high and variable fever
Post kala-azar dermal leishmaniasis can occur later (PKDL) = skin rash with parasite rich nodules.
How is splenic aspirate used as a method of diagnosis of leishmaniasis.
- Large needle through the abdomen into the spleen
- Highly invasive
- Risk of infection
- Painful
- Requires great skill
- Appropriate hospital setting
The fining of amastigotes in the stained smear is still the best confirmatory test
Problems:
Amastigotes are difficult to find, when level of infection low (early stages of disease
A highly skilled microscopist is needed.
Promastigote culture
Although considered the gold standard, the sensitivity of microscopy alone is fare from 100%, even when performed in optimal conditions
In some reference centres, improved sensitivity is achieved by the cultivation of the biopsy specimens this requires specialised aseptic conditions: fume hood, culture expertise.
Describe the KAtex urine test
- Latex agglutination test using latex particles coated with antibodies against Leishmania antigen found in urine of VL patients
- Non-invasive method
- Sensitivity 47-95%
- Specificity 83-100%
Depends on circumstances.
Discuss the serological tests for Leishmaniasis
Direct Agglutination test (DAT)
DAT test relies on the agglutination of human antibodies and parasite antigen
During infection with VL
1. During infection with VL, antibodies are produced against the surface antigens of the invading parasites.
2. The DAT detects antibodies to the parasite in the blood or serum of patients.
3. The DAT uses a freeze-dried suspension of trypsin-treated, fixed and stained L. donovani promastigotes (from a culture).
4. In the absence of antibodies to Leishmania the DAT antigen accumulates at the bottom of the plate to form a dark blue spot a negative result.
5. If antibodies to Leishmania are present, then the antigen remains a pale blue solution a positive result
* Easy to read, does not require any equipment, is considered by many an ideal field test (cf malaria) * DAT was found 96·5–100% sensitive and 91–95% specific * Reproducibility problems have been observed under field conditions due to thermal instability of the antigen and reading problems * Freeze-dried antigen has helped to solve stability problems * However, tendency to false positives and problems with antigen batch variability
Indirect Fluorescent Antibody Test (indirect = looking for Abs)
IFAT slides are prepared by fixing Leishmania promastigotes (Ag) onto glass slides
The human serum of interest (Ab) is then, serially diluted to obtain quantitative results
Revelation is done using an d anti-human immunoglobulin -labelled with a fluorescent dye as secondary antibody.
The results correspond to a titre (i.e. last serum dilution yielding a fluorescence signal) evaluated by microscopic examination
* IFAT is very reliable, but requires a fluorescence microscope and is technically demanding
* Sensitive and specific (96% Se and 98% Sp) but not suited to field conditions
* IFAT has been widely used for the diagnosis of VL since the 1970’s.
Enzyme-linked Immunosorbent Assay
ELISA is a commonly used method for VL serological screenings. 96 well plates
1. The well bottoms are coated with the antigens of interest
2. The reaction between the patient antibodies and the antigens are quantified using enzyme labelled anti-human immunoglobulins.
This is an old assay - widely used for diagnostic purposes since 1972.
For VL, several ELISA assays based on different antigens have been developed.
Immunochromatographic strip test (indirect: looks for Ab)
A rapid serodiagnostic test with nitrocellulose strips impregnated with recombinant rK39 antigen which detects antibodies against L. donovani and L. infantum.
How helpful the test is depends on the location as different species will be better detected.
The latest version has added another antigen, rK26, to improve performance but still needs improvement.
What molecular biology techniques are used to diagnose leishmaniasis
Molecular biology methods
* Wide variety of PCR-based tests developed but mainly used in research or specialist centres
* Useful when combined with species-specific primers and/or sequencing to identify the species – as treatment varies depending on parasite involved
* Shows high sensitivity and specificity (close to 100% on both), but requires specialist equipment so not field friendly
* Loop-mediated isothermal amplification (LAMP) is a new version of PCR that does not require a thermocycler (uses constant temperature) and is being developed for field use
* Nucleic acid sequence-based assay (NASBA) detects Leishmania RNA and can be used in quantitative version to assess parasite load
Discuss the issues associated with leishmaniasis in Brazil.
Transmission of cycle of visceral leishmaniasis in Brazil
* Dogs are the sole reservoir
* Humans are incidental hosts
* Vector is abundant in animal sheds
The Brazilian MoH response
In Brazil they kill any dogs infected with leishmaniasis
By 2016 the number of DALYs had increased to c. 38K. VL in brasil is particularly severe for children under 4 and with that cohort for children up to 1 years old.
There are 4 strands to VL control strategy in Brazil
1. Vector control - Reactive insecticide spraying after human case
2. Reservoir (dog) control - proactive, removing infected dogs
3. Therapeutic drugs -
a. Antimonal based
b. No human vaccine
c. New drugs expensive and seem to generate resistance more quickly
4. Education (people can avoid conditions)
Detection of differences in odours could be a new method of diagnosis.
What is Giardiasis and give an overview of its diagnosis.
Giardiasis
Caused by Giardia duodenalis a flagellated enteric protozoan
Infection occurs by the ingestion of cysts in contaminated water, food, or by the faecal-oral route
IN the small intestine, excystation releases trophozoites (each cyst produces two trophozoites) that multiply by longitudinal binary fission
They can be free in the gut lumen or attached to the mucosa using a sucking disk
Encystation occurs as the parasites move towards the colon
Cysts passed in the faeces.
Giardiasis epidemiology
There is worldwide distribution however it is more prevalent in tropical countries
Cysts can survive for several months in cold water and are responsible for transmission
Diagnosis of Giardia
Diagnostic stages of Giardia are the trophozoites and cysts
* Clinical diagnosis: signs and symptoms
* Parasitological diagnosis: trophozoites & cysts in stools; direct immunofluorescence
* Direct antigen tests: lateral flow and ELISA
Molecular methods: real time PCR
Descuss the clinical diagnosis of giardiasis.
- Diarrhoea lasting over 1 week, but can be asymptomatic
- Typical incubation period of 7-10 days
- Early symptoms of clinical disease include belching with ‘eggy’ taste, abdominal distension, nausea, and foul-smelling bulky, explosive, often watery, diarrhoea.
- Chronic infection associated with non-bloody diarrhoea, cramps, bloating , nausea, weight loss, failure to thrive
- Severe diarrhoea, malabsorption and malnutrition if patient has low immunity
Highly contagious - clusters of cases e.g. in a family
Describe the paracitological diagnosis fo giardiasis.
Parasitological diagnosis of Giardia
* Microscopy of faecal samples for cysts and trophozoites
○ Cysts are 10-14um and can be detected with an iodine stain, detected in saline with a good microscope
○ Trophozoites 12-15um have a characteristic falling leaf motility.
How is direct flourecence assay used to diagnoses giardiasis.
- DFA can be used to detect Giardia cysts (and Cryptosporidium oocysts) e.g. commercial Merifluor test
- Uses fluorescently labelled antibodies against cyst wall antigens followed by fluorescence microscopy
- Sensitivity and specificity very good, 96-100%
- Can be combined with immunomagnetic separation to concentrate the cysts/oocysts – magnetic beads coated with anti-Giardia or anti-Cryptosporidium antibodies – but this is too complex and expensive for clinical diagnosis (mainly used for environmental sampling)
What is the entero test and how is lateral flow used in the diagnosis of giardiasis.
- Entero test - string test
- Lateral flow assay for detection of Giardia and or cryptosporidium
○ One step lateral flow immunoassay for the detection of Cryptosporidium and Giardia antigens in stool specimens
○ Sample mixed with reagents and added to sample well - results in 10 mins
○ All or nothing - doesn’t indicate level of infection
- Lateral flow assay for detection of Giardia and or cryptosporidium
