Diptera (Insecta: Pterygota) larvae as predators of Strongyloides stercoralis causing false negative stool cultures.

A stool sample of a five-year-old boy with suspected STH infection arrived at the Laboratory of the Instituto de Investigaciones de Enfermedades Tropicales (IIET), National University of Salta in Oran, province of Salta, Argentina in 2017. Three Harada Mori were prepared, of which only one showed the presence of S. stercoralis. In the other two, the presence of an unknown larva was observed, which was later identified as an insect larva of the Diptera order. PCR analysis of the liquid medium of Harada Mori and Diptera larvae revealed presence of S. stercoralis DNA. These results, added to the predatory characteristics of the dipteran larvae, indicate that the S. stercoralis larvae were prey for these organisms, resulting in a negative diagnosis for S. stercoralis in the Harada Mori.

Introduction

Strongyloides stercoralis is a soil transmitted helminth (STH) infection belonging to the group of neglected tropical diseases. S. stercoralis affects approximately 386 million people worldwide, [1] however, the global prevalence of S. stercoralis is underestimated due to the relatively low sensitivity of diagnostic methods. The successful diagnosis of S. stercoralis in stool depends on fresh samples and concentration methods [2]. This is because the diagnosis requires the identification of larvae in feces, which are excreted intermittently and in most cases in small quantities. Due to the absence of a gold standard, the most common approach for the detection of S. stercoralis is the combination of several techniques, [3] being the recommended techniques Baermann, Harada Mori and Koga agar plate culture [4]. Additionally, PCR from stool samples and serology have improved the diagnosis of S. stercoralis infections [5], [6].

The Harada Mori technique has lower diagnostic sensitivity than the Koga agar plate and Baermann techniques [7], [8]. However, a smaller amount of stool is required, several tubes with individual samples can be prepared and the use of charcoal or culture medium is not required [9]. This makes Harada Mori a simple technique to perform in any laboratory; although an incubator is needed.

Case

A stool sample of a five-year-old boy with suspected STH infection arrived at the Laboratory of the Instituto de Investigaciones de Enfermedades Tropicales (IIET), National University of Salta in Oran, province of Salta, Argentina in 2017. The stool sample was examined by Telemann concentration and McMaster´s methods. The sample was insufficient to include an aliquot for the Baermman technique, instead three Harada Mori culture tubes were prepared. For each Harada Mori test, one gram of fecal matter was weighed, it was smeared onto the lower third of a strip of filter paper folded in an accordion shape, the paper was placed in a test tube with five milliliters of water and covered the tube with pre-drilled paraffin paper. The tube was then incubated in an upright position at 28 °C for three days.The sample was positive for Strongyloides stercoralis by Telemann concentration technique. Two Harada Mori tubes were negative for S. stercoralis, but in both tubes the presence of larvae of an unknown species were observed. The third Harada Mori tube was positive for S. stercoralis, with presence of many rhabditiform larvae, and absence of the unknown larvae observed in the other tubes. Although this case report describes the event that occurred with the stool sample of a five-year-old child, it should be mentioned that two more cases were found in 2020 in children under ten years of age, where the same type of larvae were observed but no infection with helminths were found.

A video recording was made of the larvae of S. stercoralis and the unknown larvae found in the harada tubes [10].

Nine specimens of the unknown larvae were retrieved for identification under Zeiss optical microscope using taxonomic keys, [11], [12], [13], [14] and following the system proposed by Wiegmann et al. [15]. A Summary through the Past 3.23 software, [16] was carried out to obtain the mean and standard deviation (SD) of larval body length. The unknown larvae found in the stool samples all belong to the same morphospecies and correspond to insect larvae of the Diptera order. Specifically, they are Diptera from the Calyptrarae subsection and, according to their characteristics, they are closer to the Oestroidea superfamily. The Diptera larvae had a mean body length of 0.54 mm (SD=0.12). Regarding morphology (Fig. 1A), the dipteran larvae present two antennas in the anterior part (Fig. 1B), the cephalopharyngeal skeleton is compact with the dorsal cornua shorter than the ventral cornua, both ending with sharp edges (Fig. 2A). The mouthhooks are bifid and curved (Fig. 2A–B). The cuticular spines are rose-thorn-shaped, laterally flattened and unicuspid with sharply pointed tips (Fig. 2C), varying in length along the body, being longer at the anterior and posterior ends. The posterior spiracles are elevated over short caudal processes (Fig. 1A–B), which are distinctly separated, each bearing one of the two spiracles.

Fig. 1

Full view of dipteran larva under the light microscope at 20X: A. Alive larva. B. Mounted larva.

Fig. 2

Morphology of the most distinctive features under the light microscope at 40X: A. cephalopharyngeal skeleton. B. Mouthhooks. C. Cuticular spines.

PCR for S. stercoralis was performed on the Diptera larvae to verify if it had the ability to feed on S. stercoralis larvae and give a false negative result in the Harada Mori technique. For this, a Diptera larva was recovered from the first Harada Mori tube, that had been negative for S. stercoralis, washed ten times with sterile PBS 1X solution and finally placed in a tube with 100 μl of sterile 1X PBS. This tube was divided into two samples of 50 μl each, one including the unknown larva that was called sample 1 (S1) and another sample with 50 μl of 1X PBS without the unknown larva, wash control, that was named sample 2 (S2). Prior to the collection of the Diptera larvae, a 50 μl sample was taken from one of the negative Harada Mori tubes and it was called sample 3 (S3). S3 had no larvae of any kind. DNA was extracted from the three sample (S1, S2, and S3) using the FastPrep® Spin Kit for Soil (MP Biomedicals, Santa Ana, CA) according to manufacturer’s instructions [18]. All PCRs were performed in 25 μl volume containing 10 μl DNA sample, 0.125 μl Go Taq polymerase (Promega), 5 μl 5x GoTaq reaction buffer (Promega), 0.5 μl diethyl-nitrophenyl thiophosphate (dNTP) (Promega) mixture (10 mM), 5 μl of 0.45 μM of specific primers for S. stercoralis, [5] and 4.375 μl MiliQ water. The thermocycler program consisted of 95ºC for 3 min, followed by 35 cycles of 95 ºC for 45 s, 57 °C for 30 s, 72 ºC for 45 s, and a final extension step at 72 ºC for 5 min. For each PCR, negative control (H2O) was run together. PCR products were analyzed by electrophoresis on 1.5% agarose gels, stained with GelRed 1X (Biotium), and visualized on a UV transilluminator.

The PCR for S. stercoralis was positive for samples S1 and S3 (Fig. 3). The positive result of sample S3 demonstrates that the Harada Mori tube had, at some time prior to the microscopic observation, S. stercoralis larvae. In addition, the fact that sample S1 tested positive proved that the dipteran larvae had the ability to feed on the S. stercoralis larvae. Sample S2 showed a slight signal, indicating that although the dipteran washout was not optimal, it can be assumed that the signal observed in S1 is due to S. stercoralis DNA ingested by the dipteran.

Fig. 3

PCR for the detection of S. stercoralis DNA in Diptera larvae and in the liquid of Harada Mori negative for S. stercoralis. MM: molecular marked, B: reagent blank.

Discussion

As shown in this case report, contamination of the stool sample with Diptera larvae led to a false negative result that could result in an overall negative diagnosis if other diagnostic techniques had not been performed. The presence of the Diptera larva in the stool sample can be explained by two hypotheses. The first, is the deposition of eggs/larvae in fresh stool prior to collection in the sterile container. This means that the stool was exposed for a period of time to the outside environment, which facilitated contact with different insects. This can be common in homes with latrines, and when the fecal sample is not immediately collected into a closed container. The second hypothesis consists of the occurrence of a gastrointestinal myiasis. This type of myiasis occurs accidentally through facultative myiasis, caused by saprophage or necrophage diptera (or even another type of insect) that are found in contaminated food, either in the egg or larval stages. [17] Following this hypothesis, the larva/egg should have the capacity to remain viable until it was expelled through the feces; or a high resistance to the digestive juices of the gastrointestinal tract and to the low amount of oxygen present in it. However, the latter possibility is unlikely since the larva did not have the specialized structures of an obligatory parasite.

The Diptera larvae identified in this case seems not yet described or its appearance in studies is very scarce, so it was not possible to identify it at the family level following the available keys consulted for both North America, [12] and South America [11], [13], [14]. Another source of uncertainity for the identification of the Diptera is the presence of mixed distinctive characters that belong to several families so tracking of the key is truncated, especially when these focuses exclusively on the spiracles, either anterior or posterior. Even so, it could be observed that this dipteran larva has a predatory character, which is hinted at by its well-developed mandibular hooks. Added to this, is the larger size of the dipteran larvae, which makes rhabditiform S. stercoralis larvae a plausible prey, which was confirmed by PCR analyzes. This implies that greater considerations in the collection of stool samples for STH analysis should be taken. However, it is possible that although Diptera larvae may produce false negatives in microscopy techniques, this is not the case for PCR techniques. Since as it could be observed, the PCR detected the presence of S. stercoralis DNA in the liquid of the Harada Mori that was negative by microscopy (sample S3).

Ethical approval

This case does not require an ethics committee because it is limited to describing what was found in a stool sample that was voluntarily given to the IIET for the diagnosis of helminths.

Consent

Ethical approval was obtained from the Bioethics Committee of the Faculty of Health Sciences (#417/15) from the Universidad Nacional de Salta. Participation was voluntary and an informed consent was obtained from all patients involved in the study.

Funding sources

This work was supported by a research grant from Consejo de Investigacion de la Universidad Nacional de Salta (CIUNSa), Salta, Argentina. Grant number 2478/0.

CRediT authorship contribution statement

Pedro E. Fleitas: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing. Ximena Acosta: Investigation, Methodology, Writing – original draft, Writing – review & editing. Paola A. Vargas: Investigation, Methodology, Writing – review & editing. Nicolas Caro: Investigation, Writing – review & editing. Alejandro J. Krolewiecki: Conceptualization, Writing – original draft. Writing – review & editing. Julio R. Nasser: Methodology, Writing – review & editing. Rubén O. Cimino: Supervision, Project administration, Funding acquisition, Writing – review & editing.

Conflict of interest

The authors have no conflicts of interest to declare.