Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Summary

Chronic infection with soil-transmitted helminths (STHs) causes malabsorption, stunting, and wasting in the growing child. Hence, it is plausible that these infections also reduce the physical fitness of children. Here, we visualize two techniques for the diagnosis of STHs and the 20-meter shuttle run test for assessing children's physical fitness.

Abstract

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty prevails and hygiene behavior, water supply, and sanitation are often deficient^1,2. Ascaris lumbricoides, Trichuris trichiura, and the two hookworm species, Ancylostoma duodenale and Necator americanus, are the most prevalent STHs³. The estimated global burden due to hookworm disease, ascariasis, and trichuriasis is 22.1, 10.5, and 6.4 million disability-adjusted life years (DALYs), respectively⁴. Furthermore, an estimated 30-100 million people are infected with Strongyloides stercoralis, the most neglected STH species of global significance which arguably also causes a considerable public health impact^5,6. Multiple-species infections (i.e., different STHs harbored in a single individual) are common, and infections have been linked to lowered productivity and thus economic outlook of developing countries^1,3.

For the diagnosis of common STHs, the World Health Organization (WHO) recommends the Kato-Katz technique^7,8, which is a relatively straightforward method for determining the prevalence and intensity of such infections. It facilitates the detection of parasite eggs that infected subjects pass in their feces.

With regard to the diagnosis of S.stercoralis, there is currently no simple and accurate tool available. The Baermann technique is the most widely employed method for its diagnosis. The principle behind the Baermann technique is that active S.stercoralis larvae migrate out of an illuminated fresh fecal sample as the larvae are phototactic⁹. It requires less sophisticated laboratory materials and is less time consuming than culture and immunological methods⁵.

Morbidities associated with STH infections range from acute but common symptoms, such as abdominal pain, diarrhea, and pruritus, to chronic symptoms, such as anemia, under- and malnutrition, and cognitive impairment¹⁰. Since the symptoms are generally unspecific and subtle, they often go unnoticed, are considered a normal condition by affected individuals, or are treated as symptoms of other diseases that might be more common in a given setting. Hence, it is conceivable that the true burden of STH infections is underestimated by assessment tools relying on self-declared signs and symptoms as is usually the case in population-based surveys.

In the late 1980s and early 1990s, Stephenson and colleagues highlighted the possibility of STH infections lowering the physical fitness of boys aged 6-12 years^11,12. This line of scientific inquiry gained new momentum recently^13,14,15. The 20-meter (m) shuttle run test was developed and validated by Léger et al.¹⁶ and is used worldwide to measure the aerobic fitness of children¹⁷. The test is easy to standardize and can be performed wherever a 20-m long and flat running course and an audio source are available, making its use attractive in resource-constrained settings¹³. To facilitate and standardize attempts at assessing whether STH infections have an effect on the physical fitness of school-aged children, we present methodologies that diagnose STH infections or measure physical fitness that are simple to execute and yet, provide accurate and reproducible outcomes. This will help to generate new evidence regarding the health impact of STH infections.

Protocol

1. Kato-Katz Technique

1. Place a standard Kato-Katz template with a hole for holding 41.7 milligrams (mg) of stool on a microscope slide.
2. Scoop 2-3 grams (g) of a fresh fecal sample onto a piece of newspaper or aluminum foil, and press a piece of wire or plastic mesh on top to sieve it.
3. Using a small plastic spatula, scrape the sieved material off the mesh and completely fill the hole in the Kato-Katz template with it. To remove excess fecal material, level the content of the hole with the spatula.
4. Vertically remove the template without disturbing the fecal material now adhering to the microscope slide. The template and spatula can be cleaned in water with detergent, and reused.
5. Place a piece of cellophane, pre-soaked in glycerine-malachite green solution for at least 24 hours (hr), over the fecal sample on the microscope slide.
6. To spread the fecal material into a thick smear, gently press a clean microscope slide against the sample slide, evenly distributing the material within a circle of a diameter slightly smaller than the breadth of the microscope slide.
7. Allow the slide to clear for 30-60 minutes (min), during which the slides must be kept away from direct sunlight. Then, systematically examine the thick smear under a light microscope (40-100x magnification). Count the number of STH eggs and stratify them by species (i.e., A.lumbricoides, T.trichiura, and hookworm). Of note, other helminth eggs (e.g. Schistosoma mansoni) can also be detected by this method and should be counted and recorded separately.
8. To get a standardized estimate of the intensity of infection, conventionally expressed as number of eggs per gram of stool (EPG), multiply the egg count by 24 (24 x 41.7 mg ≈ 1 g).

2. Baermann Technique

1. Set up a stand that can hold a glass funnel and attach a rubber tube, closed with a clip, to the bottom of the funnel.
2. Place a sieve or a piece of wire screen shaped like a cone, in the funnel and layer it with a piece of medical gauze.
3. Fill up the funnel with tap water and ensure that the whole set-up does not leak.
4. Scoop 20-30 g of fresh stool onto the middle of the medical gauze and ensure that it is completely submerged in water. Add more water if necessary. Fold the medical gauze over the sample.
5. From below, shine artificial light at the funnel for 2 hr.
6. Drain 50 milliliters (ml) of the lower portion of the water in the funnel into a centrifuge tube by gently releasing the clip on the rubber tube.
7. Centrifuge the collected water at 600 x g (relative centrifugal force) for 5 min and carefully pour out the supernatant without disturbing the sediment. Retain the last few drops in the tube.
8. Re-suspend the sediment and pipette 2 drops of the solution onto a microscope slide and examine the slide under a light microscope (40x magnification for detection and 100-400x magnification for species confirmation). First-stage larvae (L1) of S. stercoralis are alive and actively move under the microscope.

3. 20-m Shuttle Run Test

1. Measure a 20-m flat and straight running course and mark it with cones.
2. Help the test participant wear a heart rate monitor on the wrist and the corresponding transmitter around the chest¹⁸. This equipment allows the heart rate of the participant to be measured before and after the test and thus monitors whether the participant has exerted maximal effort.
3. Instruct the participant to run back and forth on the course by following the pace of pre-recorded sound signals delimiting 20-m laps. Starting with a running speed of 8.5 kilometers per hour (km/hr), the frequency of the signals indicating 20-m intervals increases by an equivalent of 0.5 km/hr every min.
4. Ensure that the participant starts a new 20-m interval each time a signal is sounded and finishes it before the next signal arrives.
5. Stop the participant if he/she fails to follow the pace for two consecutive 20-m intervals.
6. Record the highest running speed indicated for the last interval which the participant completed fully, along with the age and sex of the participant.
7. Verify that the heart rate achieved at the end of the test is the child's maximal heart rate¹⁹. If this heart rate is not reached, the child might not have exerted maximal effort, and hence the test should be repeated.
8. Estimate the maximum aerobic capacity (VO₂ max) of the participant, using an equation put forth by Léger and co-workers: VO₂ max = 31.025 + 3.238 * speed (in km/hr) - 3.248 * age (in years) + 0.1536 * speed (in km/hr) * age (in years).

4. Representative Results

In terms of STH diagnosis with the Kato-Katz technique, unique characteristics of the different helminth eggs allow for the identification of most species²⁰. The eggs of A.lumbricoides (approximately 45-75 micrometer (μm) long and 35-50 μm wide, Figure 1) have thicker shell walls than the eggs of T.trichiura (Figure 2) and hookworm (Figure 3). A fertilized egg of A.lumbricoides (Figure 1) has an outer albuminous coat, which makes it look golden brown, while a non-fertilized egg lacks this outer coat and is slightly elongated and larger overall. The eggs of T.trichiura (approximately 50 μm long and 22 μm wide, Figure 2) are elongated and have distinct polar plugs. Hookworm eggs (approximately 64-76 μm long and 36-40 μm wide, Figure 3), on the other hand, have a very thin wall, followed by a clear ring around a dense cluster of cells in the center of the egg.

S.stercoralis L₁ larvae (Figure 4) are approximately 180-380 μm long and have a very short buccal cavity. A distinct genital primordium (highlighted with a blue arrow in Figure 4) can be observed and the tail of the larvae (highlighted with a red arrow in Figure 4) has a sharp end.

As the true impact of STH infections on the physical fitness of children is still being debated^13,14,15, there is no representative result for their impact at this point. Interpretation of 20-m shuttle run test results can be performed in various ways. The physical fitness of children can be compared between infected and non-infected subjects, who are similar in terms of age and sex, other potentially relevant health conditions, socioeconomic status, and cultural habits. This can be done in a cross-sectional survey^13,14,15 where the STH infection status and physical fitness of infected and non-infected children are assessed at a particular time point. The strength of the evidence will be higher when following a randomized controlled trial design where the physical fitness of school-aged children is assessed before and after anthelminthic/placebo treatment, allowing for both short- and long-term evaluation. One can further try to identify a possible dose-response relationship between the intensity of STH infections and physical fitness and compare different levels of multiparasitism¹ with physical fitness. In the work presented by Yap and co-workers¹⁵, physical fitness, as depicted by VO₂ max, was found to be significantly reduced in children infected with T.trichiura compared to children who were not infected with T.trichiura (Figure 5).

Figure 1. Microscopic images of A.lumbricoides eggs: fertilized (A) and non-fertilized (B) under 500x magnification. Scale bar = 50 μm.

Figure 2. Microscopic image of a T.trichiura egg under 500x magnification. Scale bar = 50 μm.

Figure 3. Microscopic image of a hookworm egg under 500x magnification. Scale bar = 50 μm.

Figure 4. Microscopic image of a S.stercoralis L₁ larva under 250x magnification. Arrows indicate tail (red) and genital primordium (blue). Scale bar = 100 μm.

Figure 5. Graph showing the physical fitness level of children infected with T.trichiura compared to the physical fitness level of children not infected with T. trichiura.

Discussion

The three protocols described in this paper have been tested and executed in the south-western part of Yunnan province, People's Republic of China among members of the Bulang ethnic minority²¹ and in different parts of Africa^6,13,14.

There are several templates available for the Kato-Katz technique. Each distinct hole-size will deliver a distinct amount of fecal material. For the calculation of EPGs, it is therefore necessary to know the unit of the template, so as to mul...

Materials

Name	Company	Catalog Number	Comments
Note: Materials in this list are generic and can be obtained from different sources.
Kato-Katz kit [400 plastic templates with a hole of 6 millimeter (mm) (diameter) in a 1.5 mm thick template; 400 plastic spatula; a 20-m role of nylon screen of 80 mesh size; a 20-m role of hydrophilic cellophane, 34 μm thick.]	Vestergaard Frandsen		Please note that the glycerin-malachite green solution is not included in this kit but can be bought from any chemical supplier.
Glass funnel [about 8 centimeter (cm) wide and 6 cm deep]			The glass funnel for the Baermann technique should be able to hold about 60 mL of water. The size of the rubber hose and gauze is dependent on the type of glass funnel used.
Biological microscope	Olympus	CX21LED/CX21
Centrifuge	Shanghai Medical Instruments Group	80 - 2T
Pre-recorded signals	Bitworks Design	Team Bleep Test Version 1.3.1	iPhone application
Heart rate monitor	POLAR	FT1	Watch and transmitter