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Urine Filtration Part 3: Session Activities Urine Filtration The urine filtration test is used to identify and quantify S. haematobium eggs in urine; this tool is commonly used in SCH control programmes as it is one of the few methods available in the field. The tool is a fast and easy technique which uses 10mL of urine, the sample is passed through a nucleopore filter which can then be viewed under a microscope at 40x with iodine to determine the intensity of the S. haematobium infection. It is important to note that the reading should be recorded as the number of eggs per the volume used; this will usually be number of eggs/10mL. Part 3: Session Activities Activity 1: Sharing experiences Participants will form into groups of 4-6 individuals and will discuss their experiences with the current laboratory and diagnostic tools that they have used in the past and are familiar with. Groups should highlight the following: The tools they have used The challenges associated with each tool The gaps that they feel exist in NTD laboratory and diagnostic tools After 10 minutes each group will discuss the key points that they have highlighted with the facilitators and other participants. The aim of this activity is for participants to become familiar with the challenges that other individuals in the NTD community have faced in regard to laboratory and diagnostic tools. Activity 2: Case study In DRC, in the provinces of Equateur and Bas Congo, most of the districts are co-endemic to LF, Loa loa and Onchocerciasis. While completing mapping for LF, Loa loa and Onchocerciasis, could you describe the diagnostic methods that should be used to confirm endemicity to LF as we know that MDA with Ivermectin could not be implemented in districts where LF and Loa loa are endemic? Which methods will you carry out to map LF? Which diagnostic methods will you use to map Loa loa? Which additional laboratory and diagnostic methods will you use to confirm endemicity of LF and Loa loa in most of the districts? For each diagnostic and lab methods please explain your choice. Part 4: Summary Job aide related to this module (include where applicable) Key words (maximum Key action points for district level personnel 10) Part 5: References and Additional Resources References: CDC Onchocerciasis website (http://www.cdc.gov/parasites/onchocerciasis/) ICT bench aid (http://www.ntdsupport.org/resources/immunochromatographic-card-test-ict- bench-aid) LF test strip bench aid (http://www.ntdsupport.org/resources/filariasis-test-strip-bench-aid) WHO LF M&E Handbook (http://whqlibdoc.who.int/publications/2011/9789241501484_eng.pdf) WHO LF Entomology Handbook (http://apps.who.int/iris/bitstream/10665/87989/1/9789241505642_eng.pdf?ua=1) WHO Bench aid for the diagnosis of filarial infections (http://whqlibdoc.who.int/publications/1997/9241544899_eng.pdf) WHO Intestinal Helminth Bench Aids for the diagnosis of Intestinal Parasites (http://apps.who.int/iris/bitstream/10665/37323/1/9789241544764_eng.pdf?ua=1) Annexes and additional resources: Annexes 1. ICT SOP 2. LF Test Strip SOP 3. Brugia SOP 4. Thick blood film SOP 5. Sedgwick Counting Chamber SOP 6. Skin Snip SOP 7. Kato Katz SOP 8. Flotac SOP 9. Hemastix SOP 10. Urine filtration SOP Additional Resources: 1. WHO Bench aid for the diagnosis of filarial infections 2. CDC Onchocerciasis Website 3. WHO LF M&E Handbook 4. WHO LF Entomology Handbook 5. WHO Intestinal Helminth Bench aid Annex 1: Immuno chromatographic test (ICT ) card standard operating procedures (SOP) Guidelines Storage and Transportation 1. Cards have an approximate shelf life of 3 (WHO, 2011) - 6 months when stored at 30° and up to 9 months when stored at 4°. 2. Test 2 cards from each lot using a positive control, if they appear negative do not use the lot. 3. When transporting cards to the study locations do not expose them to extreme heat for prolonged periods of time. Sample Collection 1. Put on gloves. 2. Clean the site of the finger prick using a disinfectant wipe. 3. Draw a small amount of blood using a lancet to prick the participant’s finger. 4. From this collect 100uL of blood using a capillary tube (supplied with ICT card). 5. Add blood sample to the white portion of the sample pad. Record the time the blood was added to the card. DO NOT add blood directly to the pink portion. DO NOT close the card before the sample migrates to the pink portion; takes roughly 30 seconds. 6. Results are ready to read after 10 minutes; record results by marking the card as positive or negative DO NOT read the results at any other time as it can increase the chance of false positives 7. Safely dispose of the ICT card, capillary tube and any remaining blood found in the capillary tube Reference: World Health Organization, 2011; Global Programme to Eliminate Lymphatic Filariasis: Monitoring and Epidemiological Assessment of Mass Drug Administration Annex 2: LF strip test SOP Storage and Transportation 1. The kits should be storied at temperatures between 2° and 37°; they should NOT be frozen. The expiry date that is located on the outer packaging must be followed; once the data has passed the kits should NOT be used as they are no longer stable. 2. Test 2 strips from each shipment using a positive control, if they appear negative do not use the lot. 3. When transporting cards to the study locations do not expose them to extreme heat for prolonged periods of time. Sample Collection and Strip Usage Annex 3: Brugia rapid SOP The Brugia RapidTM test is an immunochromatographic antibody assay in a cassette format. These tests are used in LF control programmes to detect the presence of recombinant protein (BmR1) and specific human IgG4 to both Brugia malayi and Brugia timori infections. Guidelines Storage and Transportation 1. The test has a shelf life of 18 months when stored between 20°-25°C and can be extended in stored at 4°C. They should NOT be frozen. 2. When transporting cards to the study locations do not expose them to extreme heat for prolonged periods of time. Sample Collection 1. Put on gloves. 2. Clean the site of the finger prick using a disinfectant wipe. 3. Draw a small amount of blood using a lancet to prick the participant’s finger. 4. From this collect 35uL of blood using a capillary tube (or 25uL of serum/plasma). 5. Once collected the sample and one drop of the chase buffer should be added to the #1 well on the test kit. (see step 1 on below diagram) 6. Carefully add 3 drops of the provided chase buffer the #2 well. (see step 2 on below diagram) 7. Pull on the clear tab until resistance is felt and add 1 drop of the chase buffer to the square well. 8. Results are ready to read after 25 minutes for blood samples (or 15 minutes for serum or plasma samples); record results by marking the card as positive or negative DO NOT read the results at any other time as it can increase the chance of false positives 9. Cards must be read in a well-lit location, faint lines can be difficult to read if lighting is poor. 10. Safely dispose of the Brugia Rapid card, capillary tube and any remaining blood found in the capillary tube Reference: World Health Organization, 2011; Global Programme to Eliminate Lymphatic Filariasis: Monitoring and Epidemiological Assessment of Mass Drug Administration Annex 4: Thick blood film SOP TITLE: PREPARATION OF BLOOD FILM FOR EXAMINING MICROFILARIA DURING NIGHT BLOOD SURVEYS Introduction Night blood surveys of lymphatic filariasis sentinel site targets population aged >5 years and is used to determine the prevalence and density of microfilariae. Prior to night collection, the local community should be adequately sensitized and encouraged to assemble at a designated area where the collection will be carried out. Adequate provision should be made by the technical staff to keep the participants awake by showing movies and other visuals relevant to the disease on a projector. Purpose The purpose of this SOP is to use a properly stained blood smear to detect W. bancrofti microfilariae. Principle Night blood survey is normally achieved by collecting night blood, preparing blood films, staining and microscopic examination of the slides. A properly stained blood slide is an inexpensive method used for detecting whether a person has microfilariae in the peripheral blood. Wucheraria bancrofti microfilariae appears in the blood with a marked nocturnal periodicity so blood collection has to be done between 10.00pm and 2.00 am. Handling precautions Personal Protection Equipment (laboratory coat and gloves) should be worn during this procedure. Discard all sharps in the sharps boxes. Materials Slides Cotton wool (or lint) Lancet Collection tube Calibrated capillary tube Micropipettor Micropipette and tips Giemsa Microscope Labeling pens and pencils Gloves Lab coat Slide racks Sharp containers Waste container 70% ethanol Distilled water Methanol Recording form for results Storage conditions All dry slides should be stored in the slide rack for transportation to the laboratory for further processing. Procedure/ methods Note: Put on PPE (lab coat and gloves) 1. Reassure the client and make him/her comfortable 2. Clean slide with an alcohol swab to remove lint and oil residue by wiping the slides gently. 3. Label the edge of the slide with a pencil stating the clients identification details. 4. With the client’s left hand palm upwards, select the third or fourth finger. IMPORTANT -The big toe can be used with infants. The thumb should never be used for adults or children. Use cotton wool lightly soaked in ethanol to clean the finger – using firm strokes to remove dirt and grease from the ball of the finger (Figure 1).
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