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 RapidTM 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 Periodicity

Wucheria bancrofti Nocturnal periodicity

Brugia malayi Nocturnal periodicity

Brugia timori Nocturnal periodicity

Mansonella spp. Aperiodic

Loa loa* 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.