District NTD Training module 9 Learners Guide Session 3: Diagnostic and laboratory tools for filarial worms, Soil- Transmitted Helminths and Schistosomiasis Key message addressed to communities: Community sensitization and mobilization is key for getting the sample size required to have the accurate prevalence rate of each filarial worm, intestinal and urinary parasite which has implication for MDA implementation in the districts. So it is important that people are informed correctly about the fact that their participation could help to decide if mass drug administration should be implemented or not in their areas of residency. Key message addressed to NTD District personnel During implementation of a mapping or follow up survey for LF, Onchocerciasis, SCH and STH personnel from district health are involved. NTD laboratory spaces within the district are also used. So it is important that laboratory personnel from district health are well trained and follow good laboratory practices and standard operating procedures for each method or technique so accurate results are given and used to guide on decision or policy to be taken by national authority regarding MDA implementation. Key message for this Unit Elimination of LF, Onchocerciasis, STH and SCH by 2020 requires implication and mobilization of targeted communities and well trained NTD personnel including NTD laboratories following Good Laboratory Practices and Standard Operating Procedures for each diagnostic method or technique. Thus, it is important that district personnel and NTD personnel from endemic countries are familiar with this book comporting preparation and advices for implementing successfully a Monitoring and Evaluation survey targeting LF and Onchocerciasis elimination.
Part I: Introduction Session Purpose: The purpose of this session is to provide individuals with an understanding of laboratory and diagnostic techniques that are used to detect and identify filarial worms, urinary and intestinal parasites. This session provides greater detail of each diagnostic test, building on session 1 of module 9/ For all diagnostic test described, more details of the methods can be found in the SOPs relevant to each test in the additional resources Prerequisite modules/sessions: Though the material covered in this module is self-contained a strong working knowledge of the PCT NTDs, their epidemiology and prevention measures as well as the monitoring and evaluation practices used for each disease is required. The general information on the PCT NTDs will be covered in Module 1 practical implementation and description of WHO recommended Monitoring and Evaluation (M&E) activities aimed to measure impact of MDA within communities will be covered in module 9. Learning Objectives: The objectives for unit 3 are as follows: 1. All participants will have knowledge of diagnostic tools used in the field for the detection, identification and quantification of filarial worms related to lymphatic filariasis and onchocerciasis, schistosomaisis and soil-transmitted helminths 2. All participants will understand the importance of following SOPs and quality control for detecting filarial worms Abbreviations and acronyms: The following abbreviations and acronyms are used in this session: ICT - Immunochromatographic Card Test CFA - Circulating Filarial Antigen FEC - Formalin-ether Concentration
KK Kato Katz test SCH Schistosomiasis STH Soil-transmitted Helminths Part 2: Key Concepts Concept 1: Rapid Diagnostic Field tests Rapid diagnostic tests are commonly used during PCT programmes; during mapping activities, sentinel site surveys and transmission assessment surveys. Sub-concept 1: Microfilarial tests Microfilarial tests are used to detect, identify and quantify any of the filarial species as long as the blood is collected at the correct time to account for the periodicity demonstrated by the species. Concept 2: Tools used to detect Intestinal Parasites Fecal egg count techniques are widely used for the detection, identification and quantification of intestinal parasites such as soil transmitted helminthes (Trichuris trichiura, Ascaris lumbricoides, Ancylostoma duodenale and Necator americanus) and schistosomiasis (Schistosoma mansoni). These tools require the collection of fecal samples followed by preparation and finally reviewed by microscopy. Powerpoint slides: Slide 4: For the tests described in this unit, the following information applies: - WHO will be tested? Diagnostics for filarial worms are conducted as part of community-based surveys - WHAT samples will be taken? The diagnostics described for filarial worms require either blood samples or small tissue samples - WHY are these diagnostic tests performed? The results of the diagnostic tests will enable the prevalence of each disease to be calculated, either as part of baseline surveys, or as part of M&E activities to determine whether PCT is needed Slide 5: the lymphatic filariasis RDT (ICT)
The Immunochromatographic Card Test or ICT card is used to detect the Wucheria bancrofti antigen. The test is a sensitive tool that is widely used by LF control programmes globally where W. bancrofti is the causative agent. During mapping activities ICT cards are the primary tool recommended by the WHO to determine the prevalence of the disease and whether MDA is required. The test is a relatively simple and user friendly test which requires a small volume of blood (100uL) which can be collected at any point of the day. Though simple, this test requires adequate training to reduce observe based variability and misreading of cards which can result in false positives. The results of the ICT card are ready after 10 minutes; these results must be read at the 10 minute mark as any delays can result in an increase in false positives. When the tests are
ready to be read the user should examine the test strip; on here a strong control line should be visible next to the C while another line will appear beside the T if the test is positive, if there is no line next to the T the test is negative. Please see below for further information: Slide 6: a new LF strip test The new LF test strips are designed to replace the ICT card use during LF control programmes targeting W. bancrofti. The new tests show a slightly higher sensitivity than the traditional ICTs as well as no need for a cold chain. Though the test is relatively simple and requires only small volume of blood (70uL), which can be collected at any point of the day, it can be more difficult to use due to the size of the strip itself. In addition to this adequate training to reduce observe based variability and misreading of cards which can result in false positives is required.
The results of the test strip card are ready after 10 minutes; these results must be read at the 10 minute mark as any delays can result in an increase in false positives. When the tests are ready to be read on here a strong control line should be visible next to the C while another line will appear beside the T if the test is positive, if there is no line next to the T the test is negative. Please see image below for further information: Slide 7: Brugia rapid test Similar to the ICT card the Brugia Rapid TM test is used to detect the presence of Brugia malayi and Brugia timori infections. These cards are used in LF control programmes were the causative agents are either B. malayi or B. timori. Though simple these tests are slightly more technical when compared to the ICT cards as they require a chase buffer.
The results are ready on the test after 15 minutes for serum/plasma samples and 25 minutes for whole blood samples. When the tests are ready to be read the user should examine the test strip; a strong control line should be visible next to the A while another line will appear beside the B if the test is positive, if there is no line next to the B the test is negative. Slide 8: Microfilarial diagnostic tests Microfilarial tests are used to detect, identify and quantify any of the filarial species as long as the blood is collected at the correct time to account for the periodicity demonstrated by the species as described below. Species Wucheria bancrofti Brugia malayi Brugia timori Mansonella spp. Loa loa* Periodicity Nocturnal periodicity Nocturnal periodicity Nocturnal periodicity Aperiodic Diurnal periodicity *Note: Loa loa is included here as the microfilaria can be detected in a blood film and should be recorded if found due to the implications it has to control programmes in central Africa. Slide 9: Microfilarial diagnostic tests Thick Blood Film The night blood film is the traditional method used for the detection, identification and quantification of microfilaria from capillary blood. To conduct the thick blood film a small volume of blood (60uL) needs to be collected and placed on a microscope slide in 3 parallel stripes amounting to approximately 20uL of blood each.
Once this has been done the slide needs to be left to dry before it can be dehaemoglobinzed and staining can take place, this can take between 12-24 hours depending on the humidity. Once the slide is dried the final preparations can be conducted. Sedgwick Counting Chamber An alternative to the thick blood film, the counting chamber is a fast, quantitative, cheap and reliable technique that allows for the microfilaria collecting samples to be measured. The counting chamber method is a very simple technique which requires the collection of 100uL of blood during the species specific periodicity pattern. This blood is then added to a collection tube with 900uL of 3% acetic acid and gently mixed. Once the samples have been collected they can be viewed using a microscope and counting chamber at any time and the number of microfilaria counted. Slide 10: interpreting results Prevalence and intensity of infection are calculated as follow: Prevalence: = no. individuals whose slides are positive for mf x100
Total no. of individuals examined for microfilariae Intensity: mf per ml of blood (presuming 60microl per slide) = Total count microfilariae in the slides found pos. x16.7 Total no. of slides found positive Slide 10: skin snip biopsy The skin snip biopsy method is used to detect the presence of the parasite Onchocerca volvulus which is responsible for onchocerciasis also known as river blindness. The skin snip biopsy remains the gold standard for the diagnosis of onchocerciasis as it is a highly specific though it can be affected by the intensity and stage of the infection. To perform the biopsy a small amount of tissue needs to be collected, ideally 2 to 6 samples from the iliac crest, buttocks, scapula or lower extremities while provide the best sensitivity. These samples can be collected by either using a sclerocorneal biopsy punch or by raising a small section of skin (approximately 3mm in diameter) and removing it with a scalpel. (Image from Dr Tekle s) Biopsies will be individually place in a well of a microtiter plate and incubated in 100 µl phosphate buffer at ambient temperature for 24 hours so that O. volvulus microfilaria could emerge and then be identified by microscopy. Wells will be checked for emerging MF after 30 min and after overnight incubation. Slide 12 For the tests described in this unit, the following information applies: - WHO will be tested? Diagnostics for STH and SCH are conducted as part of school-based programmes (where school enrolment is high) or community-based programmes (where school enrolment is low) in line with MDA delivery
- WHAT samples will be taken? The diagnostics described for STH and SCH require either urine or stool samples to be collected - WHY are these diagnostic tests performed? The results of the diagnostic tests will enable the prevalence of each disease to be calculated, either as part of baseline surveys, or as part of M&E activities to determine whether PCT is needed Slides 13-14: Kato katz The kato katz (KK) test is used to detect the presence of helminth eggs in fecal samples. This tool is widely used globally in STH and SCH control programmes as it is cheap and relatively easy to use and can detect the presence of Trichuris trichiura, Ascaris lumbricoides, Schistosoma mansoni, Ancylostoma duodenale and Necator americanus. During mapping the KK test kits are the primary tool used to determine the prevalence of infection and if PCT with Albendazole/Mebendazole or Praziquantel is required. After MDA implementation the test is used to monitor the changes in the prevalence and intensity of the infections. Stool samples are collected from individuals and once returned to the laboratory, a small portion of the sample is pushed through a sieve to remove large debris before being applied to the KK template that is placed on a labelled slide (this will amount to 41.7mg). Once the template has been removed, a piece of reagent soaked cellophane is placed on the sample before the second slide is placed on top. Within an hour of the preparation the slide can be examined for hookworm ova and after 24 hours it should be examined for the ova of Trichuris trichiura, Ascaris lumbricoides, Schistosoma mansoni. Slide 15: Flotac FLOTAC The FLOTAC apparatus is also very useful in order to recover parasitic elements after flotation. It is a valid, sensitive and potentially low-cost alternative technique that could be used in resource-limited settings - particularly for helminthic diagnosis. The technique
involves the spinning of the sample which will allow for the enumeration of the parasites to an accuracy of one egg per gram. The main limiting factor with the FLOTAC technique is that it requires centrifugation of the sample with a specific device; in many cases this equipment is not available in developing countries. This has been overcome by the mini-flotac which does not require the use of a centrifuge and can be transferred and carried out in laboratories easily. The mini-flotac requires a small amount of the stool sample to be added to the chamber (Xg of stool added = XmL 5% formalin added) with an equal amount of formalin. Once added and the chamber sealed it should be shaken to ensure that the sample and formalin is homogenized before adding the floatation fluid and re-shaking. Once this is done the two floatation chambers that are provided should be filled with the filtered sample which can be viewed after 5-10 minutes using a standard microscope.
Images of the intestinal Parasites
Slide 16: CCA The CCA test is a presumptive qualitative test which detects parasite circulating cathodic antigen (CCA) in patient urine. The test is good for detection of S. mansoni and can potentially replace the traditional Kato-Katz test. A drop of urine is filled into a CCA test well and a drop of buffer added to it. If CCA antigen is present, it will attach to labeled monoclonal antibody of the parasite and react with it to form a second pink line. CCA test is fast, very sensitive and easy to perform Slide 17: Tools used to detect Urinary Parasites Hemastix The hemastix tool, commonly referred to as the dip stick test cannot detect the presence of a parasitic infection but is used to detect the presence of blood in urine. This test is widely used in schistosomiasis programmes where Schistosoma haematobium is present as the release of eggs will result in haematuria. The individual strips can be used on a single urine sample; though to double the number of tests per kit the strips can be cut in half, lengthwise. Once urine has been collected it should be tested with 2 hours to ensure an accurate result. To do this a single test strip should be completely immersed in the sample for a few seconds before being removed and any excess urine removed by running the strip along the edge of the sample container. After waiting 1-2 minutes the reactive strip at the end of the test should be compared to the colour chart on the container to determine the level of haemolysis in the sample.