# Real-time multiplex PCR for human echinococcosis and differential diagnosis

Jenny Knapp, Séverine Lallemand, Franck Monnien, Sophie Felix, Sandra Courquet, Gérald Umhang, Laurence Millon

► To cite this version:

Jenny Knapp, Séverine Lallemand, Franck Monnien, Sophie Felix, Sandra Courquet, et al. Real-time multiplex PCR for human echinococcosis and differential diagnosis. Parasite, 2023, 30, pp.3. 10.1051/parasite/2023003. hal-04095822

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# Real-time multiplex PCR for human echinococcosis and differential diagnosis

Jenny Knapp, Séverine Lallemand, Franck Monnien, Sophie Felix, Sandra Courquet, Gérald Umhang, Laurence Millon

<figure><img src="image_1.png" /></figure>

To cite this version:

Jenny Knapp, Séverine Lallemand, Franck Monnien, Sophie Felix, Sandra Courquet, et al.. Real-time multiplex PCR for human echinococcosis and differential diagnosis. Parasite, 2023, 30, pp.3. 10.1051/parasite/2023003. hal-04095822

HAL Id: hal-04095822

https://hal.science/hal-04095822v1

Submitted on 21 Feb 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Real-time multiplex PCR for human echinococcosis and differential diagnosis

Jenny Knapp<fnref n="1" />,<fnref n="2" />*, Séverine Lallemand<fnref n="2" />, Franck Monnien<fnref n="3" />, Sophie Felix<fnref n="3" />, Sandra Courquet<fnref n="1" />,<fnref n="2" />, Gérald Umhang<fnref n="4" />, and Laurence Millon<fnref n="1" />,<fnref n="2" />

Received 25 August 2022, Accepted 06 January 2023, Published online 25 January 2023

**Abstract** – Molecular identification of rare human infectious pathogens appears to be one of the most relevant current methods for rapid diagnosis and management of patients. PCR techniques, in particular real-time quantitative PCR, are best suited for the detection of DNA from the pathogens, even at low concentrations. Echinococcosis infections are due to helminths of the *Echinococcus* genus, with closely related species involved in parasitic lesions affecting animals and, accidentally, humans. We developed a multiplex qPCR (MLX qPCR) assay allowing for the detection of four *Echinococcus* species involved in Europe in alveolar echinococcosis (AE) and cystic echinococcosis (CE) (*Echinococcus multilocularis*, *E. granulosus* sensu stricto, *E. ortleppi*, and *E. canadensis*), based on short mitochondrial targets. A collection of 81 fresh and formalin-fixed paraffin-embedded tissues (FFPE) of AE and CE lesions was assembled. The qPCR assays were performed in triplex for *Echinococcus* spp. detection, associated with a qPCR inhibitor control. A duplex qPCR was also designed to enable diagnosis of two other dead-end helminthiases (cysticercosis (*Taenia solium*), and toxocariasis (*Toxocara cati* and *T. canis*)). The sensitivity of the qPCR was assessed and ranged from 1 to 5 × 10⁻<fnref n="4" /> ng/µL (seven PCR assays positive), corresponding to 37–42 cycles for quantifiable DNA. The specificity was 100% for all the targets. This multiplex qPCR, adapted to low amounts of DNA can be implemented in the laboratory for the rapid molecular diagnosis of *Echinococcosis* species.

**Key words:** Human echinococcosis, Molecular diagnosis, Fresh material, FFPE, Quantitative real-time PCR, Multiplexing.

**Résumé** – PCR multiplex en temps-réel pour le diagnostic de l'échinococcose humaine et diagnostic différentiel. L'identification moléculaire des pathogènes infectieux humains rares semble être l'une des méthodes actuelles les plus pertinentes pour un diagnostic et une prise en charge rapides des patients. Les techniques de PCR, en particulier la PCR quantitative en temps réel, sont bien adaptées à la détection d'ADN de pathogènes, même pour de faibles concentrations. Les infections à échinocoque sont dues à des helminthes du genre *Echinococcus*, des espèces étroitement apparentées, impliquées dans des lésions parasitaires affectant les animaux et accidentellement l'homme. Une qPCR multiplex (MLX qPCR), permettant la détection de quatre espèces d'Echinococcus impliquées en Europe dans l'échinococcose alvéolaire (EA) et kystique (EK) (*Echinococcus multilocularis*, *E. granulosus* sensu stricto, *E. ortleppi* et *E. canadensis*), basée sur de courtes cibles mitochondriales a été développée ici. Une collection a été constituée de 81 tissus frais ou fixés en paraffine (FFPE) de lésions d'EA et EK. Les essais de qPCR ont été réalisées en triplex pour la détection d'Echinococcus spp., associés à une qPCR de contrôle d'inhibition. Une PCR duplex a été développée pour le diagnostic de deux autres helminthiases en impasse chez l'Homme (cysticercose (*Taenia solium*), et toxocarose (*Toxocara cati* et *T. canis*). La sensibilité de la qPCR a été évaluée et s'échelonne de 1 à 5 × 10⁻<fnref n="4" /> ng/µl (sept essais de qPCR positifs), correspondant à 37 à 42 cycles pour l'ADN quantifiable. La spécificité était de 100 % pour toutes les cibles. Cette qPCR multiplex, adaptée à de faibles quantités d'ADN peut être mise en œuvre au laboratoire pour un diagnostic moléculaire rapide des espèces d'Echinococcus.

<footnote n="1">¹ Department of Parasitology-Mycology, National Reference Centre for Echinococcoses, University Hospital of Besançon, 25030 Besançon, France</footnote>

<footnote n="2">² UMR CNRS 6249 Laboratoire Chrono-environnement, University of Franche-Comté, 16 Route de Gray, 25030 Besançon, France</footnote>

<footnote n="3">³ Department of Pathology, University Hospital of Besançon, 25030 Besançon, France</footnote>

<footnote n="4">⁴ ANSES Nancy laboratory for Rabies and Wildlife, National Reference Laboratory for *Echinococcus* spp., Wildlife Surveillance and Eco-epidemiology Unit, Technopole Agricole et Vétérinaire, 54220 Malzéville, France</footnote>
<figure><img src="image_3.png" /></figure>

RESEARCH ARTICLE

OPEN ACCESS

# Real-time multiplex PCR for human echinococcosis and differential diagnosis

Jenny Knapp<fnref n="1" />,<fnref n="2" />*, Séverine Lallemand<fnref n="2" />, Franck Monnien<fnref n="3" />, Sophie Felix<fnref n="3" />, Sandra Courquet<fnref n="1" />,<fnref n="2" />, Gérald Umhang<fnref n="4" />, and Laurence Millon<fnref n="1" />,<fnref n="2" />

Received 25 August 2022, Accepted 06 January 2023, Published online 25 January 2023

**Abstract** – Molecular identification of rare human infectious pathogens appears to be one of the most relevant current methods for rapid diagnosis and management of patients. PCR techniques, in particular real-time quantitative PCR, are best suited for the detection of DNA from the pathogens, even at low concentrations. Echinococcosis infections are due to helminths of the *Echinococcus* genus, with closely related species involved in parasitic lesions affecting animals and, accidentally, humans. We developed a multiplex qPCR (MLX qPCR) assay allowing for the detection of four *Echinococcus* species involved in Europe in alveolar echinococcosis (AE) and cystic echinococcosis (CE) (*Echinococcus multilocularis*, *E. granulosus* sensu stricto, *E. ortleppi*, and *E. canadensis*), based on short mitochondrial targets. A collection of 81 fresh and formalin-fixed paraffin-embedded tissues (FFPE) of AE and CE lesions was assembled. The qPCR assays were performed in triplex for *Echinococcus* spp. detection, associated with a qPCR inhibitor control. A duplex qPCR was also designed to enable diagnosis of two other dead-end helminthiases (cysticercosis (*Taenia solium*), and toxocariasis (*Toxocara cati* and *T. canis*)). The sensitivity of the qPCR was assessed and ranged from 1 to 5 × 10⁻<fnref n="4" /> ng/µL (seven PCR assays positive), corresponding to 37–42 cycles for quantifiable DNA. The specificity was 100% for all the targets. This multiplex qPCR, adapted to low amounts of DNA can be implemented in the laboratory for the rapid molecular diagnosis of *Echinococcosis* species.

**Key words:** Human echinococcosis, Molecular diagnosis, Fresh material, FFPE, Quantitative real-time PCR, Multiplexing.

**Résumé** – PCR multiplex en temps-réel pour le diagnostic de l'échinococcose humaine et diagnostic différentiel. L'identification moléculaire des pathogènes infectieux humains rares semble être l'une des méthodes actuelles les plus pertinentes pour un diagnostic et une prise en charge rapides des patients. Les techniques de PCR, en particulier la PCR quantitative en temps réel, sont bien adaptées à la détection d'ADN de pathogènes, même pour de faibles concentrations. Les infections à échinocoque sont dues à des helminthes du genre *Echinococcus*, des espèces étroitement apparentées, impliquées dans des lésions parasitaires affectant les animaux et accidentellement l'homme. Une qPCR multiplex (MLX qPCR), permettant la détection de quatre espèces d'Echinococcus impliquées en Europe dans l'échinococcose alvéolaire (EA) et kystique (EK) (*Echinococcus multilocularis*, *E. granulosus* sensu stricto, *E. ortleppi* et *E. canadensis*), basée sur de courtes cibles mitochondriales a été développée ici. Une collection a été constituée de 81 tissus frais ou fixés en paraffine (FFPE) de lésions d'EA et EK. Les essais de qPCR ont été réalisées en triplex pour la détection d'Echinococcus spp., associés à une qPCR de contrôle d'inhibition. Une PCR duplex a été développée pour le diagnostic de deux autres helminthiases en impasse chez l'Homme (cysticercose (*Taenia solium*), et toxocarose (*Toxocara cati* et *T. canis*). La sensibilité de la qPCR a été évaluée et s'échelonne de 1 à 5 × 10⁻<fnref n="4" /> ng/µl (sept essais de qPCR positifs), correspondant à 37 à 42 cycles pour l'ADN quantifiable. La spécificité était de 100 % pour toutes les cibles. Cette qPCR multiplex, adaptée à de faibles quantités d'ADN peut être mise en œuvre au laboratoire pour un diagnostic moléculaire rapide des espèces d'Echinococcus.

<footnote n="1">¹ Department of Parasitology-Mycology, National Reference Centre for Echinococcoses, University Hospital of Besançon, 25030 Besançon, France</footnote>

<footnote n="2">² UMR CNRS 6249 Laboratoire Chrono-environnement, University of Franche-Comté, 16 Route de Gray, 25030 Besançon, France</footnote>

<footnote n="3">³ Department of Pathology, University Hospital of Besançon, 25030 Besançon, France</footnote>

<footnote n="4">⁴ ANSES Nancy laboratory for Rabies and Wildlife, National Reference Laboratory for *Echinococcus* spp., Wildlife Surveillance and Eco-epidemiology Unit, Technopole Agricole et Vétérinaire, 54220 Malzéville, France</footnote>
## Introduction

In medical biology, molecular diagnosis based on non-cultivable endoparasites is an accurate method to confirm an infection for a suggestive clinical and epidemiological presentation due to its sensitivity and specificity, and because DNA is an unforgeable carrier of identity. Molecular diagnosis is particularly relevant in case of dead-end helminthiases, such as echinococcosis, toxocariasis, or cysticercosis. Indeed, no exit of eggs or adult stages is observed by direct examination. In such cases, besides imaging which is often the first step in the diagnosis, detection of specific antibodies is the main diagnosis tool, but this type of approach is subject to problems concerning specificity and sensitivity. Moreover, serological assays can lead to negative results for immunosuppressed patients because of their immune status [6, 7]. In this context, molecular diagnosis provides incontrovertible species identification on surgery or needle biopsy samples.

Alveolar and cystic echinococcosis are two serious diseases caused by cestodes from the genus *Echinococcus* Rudolphi (1801), often with years between the first contact with the parasite and diagnosis. Human alveolar echinococcosis (AE) is due to *Echinococcus multilocularis* Leuckart (1863), whereas cystic echinococcosis (CE) is caused by several species, grouped under the taxon name *Echinococcus granulosus* sensu lato (s.l.) [40], known to circulate in Europe, with human cases reported [5]. In Europe, the complex comprises *E. granulosus* sensu stricto (s.s.) Batsch (1786), first described in sheep as an intermediate host, *E. canadensis* (Webster and Cameron, 1961), described in camels (previous G6), pigs (G7), and deer (G8, G10), depending on the geographical occurrence, and *E. ortleppi* Lopez-Neyra and Soler Planas (1943), described in cattle. *Echinococcus multilocularis* is endemic only in the northern hemisphere and essentially maintained by a sylvatic lifecycle between red foxes and small mammals in Europe, whereas *E. granulosus* s.l. has a worldwide distribution, mainly supported by a domestic lifecycle between dogs and livestock. CE is classified as the second and AE as the third most relevant food-borne parasitic diseases in the world [11]. However, due to its greater mortality, AE is classified as the most severe helminthic zoonosis in the northern hemisphere [34, 35]. The diagnoses of human AE and CE require multidisciplinary consensus before implementing chemical treatment and surgery, when possible, which is the only curative treatment for echinococcosis infections. AE occurs mostly in the liver and CE in the liver and lungs [33]. For molecular diagnosis in humans, various matrices can be analysed, such as tissues from various organs, liquid punctures, and formalin-fixed paraffin-embedded (FFPE) tissues. In addition to *Echinococcus* spp. infections, molecular diagnosis could help in the diagnosis of “dead-end” helminthiases. Toxocariasis, a worldwide parasitosis, can also involve the same organs as *Echinococcus* spp., especially the liver and the brain. In humans, this disease is due to the ingestion of embryonated eggs of *Toxocara cati*, a nematode of cats, or *T. canis*, a nematode of dogs and foxes [8]. Humans are an aberrant host for *Toxocara* spp. because of the non-development of the L3 larvae stage in adults, as in dogs and cats, resulting in *larva migrans*. In patients, the liver is the most frequent organ involved, with abscess presentation

[16]. Human cysticercosis is a parasitosis due to the tapeworm *Taenia solium*, with the larval stage described in humans in muscles, the brain, the eyes, or, indeed, any organ, and is still a problem in a number of farming communities in developing countries of Africa, Asia, and Latin America [29]. Humans act as an intermediate host after the ingestion of tapeworm eggs present on contaminated vegetables or by self-contamination due to the faecal-oral cycle (cysticercus larva).

Quantitative PCR (qPCR) is a promising tool to detect and quantify DNA from infectious agents in different specimens (tissue, fluids, blood) and has allowed a step forward in the diagnosis of many infectious diseases, and especially parasitic diseases in the last decade [15, 31]. The advantages of qPCR-based techniques are higher sensitivity than end-point PCR, a reduction in PCR product contamination because of the limited manipulation of amplicons, and the possibility to quantify the targeted DNA. In addition, multiplexing in qPCR, permitted by multiple fluorescent channel dyed-amplicon detection, allows the detection of large panels of targets using the same DNA extract. Multiplex qPCR can be used in screening for first-line molecular diagnosis, with reduction in the quantity of DNA and PCR reagents required, and a reduced handling time.

For detection of the parasite *Echinococcus* sp., several qPCR assays have been developed. Some of them are able to detect the parasite *E. multilocularis* and hosts in environmental samples [9, 21]. Other qPCR assays are able to detect co-occurrence of parasites (for example *E. multilocularis* and *E. canadensis* in definitive hosts [30,43], *E. multilocularis* and *Toxoplasma gondii* [32], or *Toxocara* spp. [21] in environmental samples. Some qPCR assays were also developed for the diagnosis of echinococcosis in humans (*E. multilocularis* and *E. granulosus* s.l. in tissue sample [4] or in plasma [24]). However, none of the published qPCR assays is able to detect all the species potentially involved in human echinococcosis from tissue samples, and especially from FFPE specimens.

Here, we aimed to develop and assess a test for the accurate and sensitive molecular diagnosis of echinococcosis in humans from tissue samples. We developed a multiplex real-time quantitative PCR assay (MLX qPCR) targeting the four species that can be encountered in Europe (*E. multilocularis*, *E. granulosus* ss, *E. canadensis*, and *E. ortleppi*), combined with an external control for the detection of inhibitors. The MLX qPCR was also designed to offer diagnosis for human toxocariasis and cysticercosis, which could help to avoid diagnostic wandering in certain atypical clinical situations.

## Materials and methods

### Sample panel

A panel of 81 samples was established. The panel was composed of 46 AE (15 frozen tissues, 31 FFPE specimens) and 35 CE lesions (19 frozen tissues, 16 FFPE specimens), collected from 1997 to 2021, with the geographical origin of the patients when available (Table 1). The *Echinococcus* species of the sample material have been confirmed by either species-specific PCRs, qPCR or via sequencing in previous studies [19] (Table 1). The species of the AE lesions were
## Introduction

In medical biology, molecular diagnosis based on non-cultivable endoparasites is an accurate method to confirm an infection for a suggestive clinical and epidemiological presentation due to its sensitivity and specificity, and because DNA is an unforgeable carrier of identity. Molecular diagnosis is particularly relevant in case of dead-end helminthiases, such as echinococcosis, toxocariasis, or cysticercosis. Indeed, no exit of eggs or adult stages is observed by direct examination. In such cases, besides imaging which is often the first step in the diagnosis, detection of specific antibodies is the main diagnosis tool, but this type of approach is subject to problems concerning specificity and sensitivity. Moreover, serological assays can lead to negative results for immunosuppressed patients because of their immune status [6, 7]. In this context, molecular diagnosis provides incontrovertible species identification on surgery or needle biopsy samples.

Alveolar and cystic echinococcosis are two serious diseases caused by cestodes from the genus *Echinococcus* Rudolphi (1801), often with years between the first contact with the parasite and diagnosis. Human alveolar echinococcosis (AE) is due to *Echinococcus multilocularis* Leuckart (1863), whereas cystic echinococcosis (CE) is caused by several species, grouped under the taxon name *Echinococcus granulosus* sensu lato (s.l.) [40], known to circulate in Europe, with human cases reported [5]. In Europe, the complex comprises *E. granulosus* sensu stricto (s.s.) Batsch (1786), first described in sheep as an intermediate host, *E. canadensis* (Webster and Cameron, 1961), described in camels (previous G6), pigs (G7), and deer (G8, G10), depending on the geographical occurrence, and *E. ortleppi* Lopez-Neyra and Soler Planas (1943), described in cattle. *Echinococcus multilocularis* is endemic only in the northern hemisphere and essentially maintained by a sylvatic lifecycle between red foxes and small mammals in Europe, whereas *E. granulosus* s.l. has a worldwide distribution, mainly supported by a domestic lifecycle between dogs and livestock. CE is classified as the second and AE as the third most relevant food-borne parasitic diseases in the world [11]. However, due to its greater mortality, AE is classified as the most severe helminthic zoonosis in the northern hemisphere [34, 35]. The diagnoses of human AE and CE require multidisciplinary consensus before implementing chemical treatment and surgery, when possible, which is the only curative treatment for echinococcosis infections. AE occurs mostly in the liver and CE in the liver and lungs [33]. For molecular diagnosis in humans, various matrices can be analysed, such as tissues from various organs, liquid punctures, and formalin-fixed paraffin-embedded (FFPE) tissues. In addition to *Echinococcus* spp. infections, molecular diagnosis could help in the diagnosis of “dead-end” helminthiases. Toxocariasis, a worldwide parasitosis, can also involve the same organs as *Echinococcus* spp., especially the liver and the brain. In humans, this disease is due to the ingestion of embryonated eggs of *Toxocara cati*, a nematode of cats, or *T. canis*, a nematode of dogs and foxes [8]. Humans are an aberrant host for *Toxocara* spp. because of the non-development of the L3 larvae stage in adults, as in dogs and cats, resulting in *larva migrans*. In patients, the liver is the most frequent organ involved, with abscess presentation

[16]. Human cysticercosis is a parasitosis due to the tapeworm *Taenia solium*, with the larval stage described in humans in muscles, the brain, the eyes, or, indeed, any organ, and is still a problem in a number of farming communities in developing countries of Africa, Asia, and Latin America [29]. Humans act as an intermediate host after the ingestion of tapeworm eggs present on contaminated vegetables or by self-contamination due to the faecal-oral cycle (cysticercus larva).

Quantitative PCR (qPCR) is a promising tool to detect and quantify DNA from infectious agents in different specimens (tissue, fluids, blood) and has allowed a step forward in the diagnosis of many infectious diseases, and especially parasitic diseases in the last decade [15, 31]. The advantages of qPCR-based techniques are higher sensitivity than end-point PCR, a reduction in PCR product contamination because of the limited manipulation of amplicons, and the possibility to quantify the targeted DNA. In addition, multiplexing in qPCR, permitted by multiple fluorescent channel dyed-amplicon detection, allows the detection of large panels of targets using the same DNA extract. Multiplex qPCR can be used in screening for first-line molecular diagnosis, with reduction in the quantity of DNA and PCR reagents required, and a reduced handling time.

For detection of the parasite *Echinococcus* sp., several qPCR assays have been developed. Some of them are able to detect the parasite *E. multilocularis* and hosts in environmental samples [9, 21]. Other qPCR assays are able to detect co-occurrence of parasites (for example *E. multilocularis* and *E. canadensis* in definitive hosts [30,43], *E. multilocularis* and *Toxoplasma gondii* [32], or *Toxocara* spp. [21] in environmental samples. Some qPCR assays were also developed for the diagnosis of echinococcosis in humans (*E. multilocularis* and *E. granulosus* s.l. in tissue sample [4] or in plasma [24]). However, none of the published qPCR assays is able to detect all the species potentially involved in human echinococcosis from tissue samples, and especially from FFPE specimens.

Here, we aimed to develop and assess a test for the accurate and sensitive molecular diagnosis of echinococcosis in humans from tissue samples. We developed a multiplex real-time quantitative PCR assay (MLX qPCR) targeting the four species that can be encountered in Europe (*E. multilocularis*, *E. granulosus* ss, *E. canadensis*, and *E. ortleppi*), combined with an external control for the detection of inhibitors. The MLX qPCR was also designed to offer diagnosis for human toxocariasis and cysticercosis, which could help to avoid diagnostic wandering in certain atypical clinical situations.

## Materials and methods

### Sample panel

A panel of 81 samples was established. The panel was composed of 46 AE (15 frozen tissues, 31 FFPE specimens) and 35 CE lesions (19 frozen tissues, 16 FFPE specimens), collected from 1997 to 2021, with the geographical origin of the patients when available (Table 1). The *Echinococcus* species of the sample material have been confirmed by either species-specific PCRs, qPCR or via sequencing in previous studies [19] (Table 1). The species of the AE lesions were
identified as *E. multilocularis* and those of the CE samples as *E. granulosus* s.s. (n = 29), *E. canadensis* (n = 4), and *E. ortleppi* (n = 2). For frozen tissues, DNA was purified using a High Pure PCR Template Preparation kit (Roche Diagnostics, Mannheim, Germany) from cubic millimetre pieces of the surgical sample, following the manufacturer's protocol, and the DNA eluted in a volume of 200 µL of the provided buffer. For FFPE samples, DNA was purified and extracted immediately after de-paraffining the FFPE shavings using a QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany), following the manufacturer's protocol, and the DNA eluted in a volume of 50 µL of the provided buffer. All DNA extracts were stored at -20 °C until use. The DNA concentration from total DNA extracts was determined from 2 µL using a nanophotometer apparatus (Implen, Munich, Germany).

## Real-time quantitative PCR design for *Echinococcus* spp. DNA detection and differential diagnosis

Four pairs of primers and associated TaqMan probes were designed for the present study to detect human echinococcosis from surgical specimens: *E. multilocularis*, *E. granulosus* s.s., *E. canadensis*, and *E. ortleppi*. The TaqMan hydrolysis probes and primers were designed from mitochondrial sequence data available in the NCBI genetic database for each species (Table 2) using Primer Express software v3.0 (Applied Biosystems) and were designed to specifically amplify the sequences targeted by the qPCR. For *E. canadensis*, primers and probe were designed from G6 and G7 genomes, in order to amplify the species *E. canadensis*. In addition, an external qPCR control was used, called the Alea target and designed in a previous study, to test the presence of PCR inhibitors [21].

The PCR assays for differential diagnosis were designed to also amplify the DNA of other parasites likely to be encountered in the laboratory, such as *Taenia solium* and *Toxocara* spp. The *T. solium* PCR assay was designed in the present study, whereas that for *Toxocara* spp. was modified from a previous PCR assay designed for *T. cati* [21]. The present qPCR assay allows amplification of both *T. cati* and *T. canis* DNA because of degenerate nucleotides included in the primers (Table 2).

Each PCR assay was performed in a final volume of 20 µL containing 10 µL 2X TaqMan Gene Expression master mix (Life Technologies, Foster City, CA, USA), 5 pmol of each primer, 0.4 pmol of the hydrolysis probes with compatible fluorochromes, and 1 µL total DNA extracted from the studied specimens. Three multiplex PCR assays were designed to combine DNA detection of (1) *E. multilocularis*, *E. granulosus* s.s., and the external control (Alea) (MLX qPCR Em-Eg-Alea) and (2) *E. canadensis*, *E. ortleppi*, and Alea (MLX qPCR Ec-Eo-Alea), in combination or not with that of (3) *T. solium* and *Toxocara* spp. (MLX qPCR Tsol-Toxo).

Depending on the epidemiological and clinical context, MLX qPCR assays 1, 2, and 3 can be performed independently. The qPCR was run on a QuantStudio 5 real-time PCR model system (Life Technologies, Foster City, CA, USA). The PCR program was comprised of three steps: a first step at 50 °C for 2 min, a second step with denaturation at 95 °C for 10 min, and a third step with 45 cycles of denaturation at

95 °C for 15 s, followed by annealing and elongation at 60 °C for 1 min. In order to permit the late amplifications around 40 cycles to be complete, the number of 45 cycles was applied. All PCRs were performed in duplicate and the results are expressed as the mean of the quantitative cycle (Cq) number. A qPCR assay was considered positive when at least one reaction of the duplicate provided a positive result (Cq < 45 cycles).

## Real-time quantitative PCR design for differential diagnosis

### Sensitivity of the assays

A dilution series was prepared to obtain a DNA range with eight concentration points to test the method detection limits (MDLs) or the probability of successfully detecting n positive results out of N trials for each qPCR assay. The technical limits of each individual qPCR assay were tested for each target by performing the PCR seven times on each of the eight points of the DNA concentration range. The last point of the DNA range giving 7/7 positive PCR trials was considered to be the MDL for the targeted DNA, meaning that the DNA was quantifiable under this threshold, whereas detection beyond the limit indicated that DNA was detectable but not quantifiable [17]. To perform the MDL tests, DNA from adult worms for *E. multilocularis* (15 ng/µL of DNA concentration), *T. cati*, and *T. canis*, provided by the CERFE laboratory (Boult-aux-Bois, France), was used (4 and 3 ng/µL of DNA, respectively), as well as the laminated layer from metacestodes isolated from animal lesions for *E. granulosus* s.s. (Anses code 5872, 11 ng/µL), *E. ortleppi* (Anses code 6697, 46 ng/µL) [38], and *E. canadensis* (Anses code 3734, 5 ng/µL) [39], provided by the ANSES laboratory (Malzéville, France), and the cysticercus stage of *Taenia solium*, provided by the Pasteur Institute (Paris, France) (3 ng/µL). The MDL tests were performed for each individual qPCR in duplex assays and included the external control Alea.

### Specificity of the assays

The generated primers were first tested for specificity using the NCBI Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), which used the NCBI sequence databases for sequence alignment [42]. A mismatch threshold to ignore targets was applied from 6 differences. Second, a panel of samples (Table 1) or parasite DNA obtained from larval or adult specimens (for *T. solium*, *T. cati*, and *T. canis*) was tested for all qPCR assays to detect putative cross-reactions. These amplification reactions were performed in simplex PCR and in duplicate.

## Results

### Sensitivity of the assays

We assessed the sensitivity of the assays, which is presented through the MDL results (Table 3). The MDL for *Echinococcus* spp. (37-42 cycles), *T. solium* (37 cycles), *T. cati* (36 cycles), and *T. canis* (38 cycles) corresponds to the detection in 1 µL
identified as *E. multilocularis* and those of the CE samples as *E. granulosus* s.s. (n = 29), *E. canadensis* (n = 4), and *E. ortleppi* (n = 2). For frozen tissues, DNA was purified using a High Pure PCR Template Preparation kit (Roche Diagnostics, Mannheim, Germany) from cubic millimetre pieces of the surgical sample, following the manufacturer's protocol, and the DNA eluted in a volume of 200 µL of the provided buffer. For FFPE samples, DNA was purified and extracted immediately after de-paraffining the FFPE shavings using a QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany), following the manufacturer's protocol, and the DNA eluted in a volume of 50 µL of the provided buffer. All DNA extracts were stored at -20 °C until use. The DNA concentration from total DNA extracts was determined from 2 µL using a nanophotometer apparatus (Implen, Munich, Germany).

## Real-time quantitative PCR design for *Echinococcus* spp. DNA detection and differential diagnosis

Four pairs of primers and associated TaqMan probes were designed for the present study to detect human echinococcosis from surgical specimens: *E. multilocularis*, *E. granulosus* s.s., *E. canadensis*, and *E. ortleppi*. The TaqMan hydrolysis probes and primers were designed from mitochondrial sequence data available in the NCBI genetic database for each species (Table 2) using Primer Express software v3.0 (Applied Biosystems) and were designed to specifically amplify the sequences targeted by the qPCR. For *E. canadensis*, primers and probe were designed from G6 and G7 genomes, in order to amplify the species *E. canadensis*. In addition, an external qPCR control was used, called the Alea target and designed in a previous study, to test the presence of PCR inhibitors [21].

The PCR assays for differential diagnosis were designed to also amplify the DNA of other parasites likely to be encountered in the laboratory, such as *Taenia solium* and *Toxocara* spp. The *T. solium* PCR assay was designed in the present study, whereas that for *Toxocara* spp. was modified from a previous PCR assay designed for *T. cati* [21]. The present qPCR assay allows amplification of both *T. cati* and *T. canis* DNA because of degenerate nucleotides included in the primers (Table 2).

Each PCR assay was performed in a final volume of 20 µL containing 10 µL 2X TaqMan Gene Expression master mix (Life Technologies, Foster City, CA, USA), 5 pmol of each primer, 0.4 pmol of the hydrolysis probes with compatible fluorochromes, and 1 µL total DNA extracted from the studied specimens. Three multiplex PCR assays were designed to combine DNA detection of (1) *E. multilocularis*, *E. granulosus* s.s., and the external control (Alea) (MLX qPCR Em-Eg-Alea) and (2) *E. canadensis*, *E. ortleppi*, and Alea (MLX qPCR Ec-Eo-Alea), in combination or not with that of (3) *T. solium* and *Toxocara* spp. (MLX qPCR Tsol-Toxo).

Depending on the epidemiological and clinical context, MLX qPCR assays 1, 2, and 3 can be performed independently. The qPCR was run on a QuantStudio 5 real-time PCR model system (Life Technologies, Foster City, CA, USA). The PCR program was comprised of three steps: a first step at 50 °C for 2 min, a second step with denaturation at 95 °C for 10 min, and a third step with 45 cycles of denaturation at

95 °C for 15 s, followed by annealing and elongation at 60 ° C for 1 min. In order to permit the late amplifications around 40 cycles to be complete, the number of 45 cycles was applied. All PCRs were performed in duplicate and the results are expressed as the mean of the quantitative cycle (Cq) number. A qPCR assay was considered positive when at least one reaction of the duplicate provided a positive result (Cq < 45 cycles).

## Real-time quantitative PCR design for differential diagnosis

### Sensitivity of the assays

A dilution series was prepared to obtain a DNA range with eight concentration points to test the method detection limits (MDLs) or the probability of successfully detecting n positive results out of N trials for each qPCR assay. The technical limits of each individual qPCR assay were tested for each target by performing the PCR seven times on each of the eight points of the DNA concentration range. The last point of the DNA range giving 7/7 positive PCR trials was considered to be the MDL for the targeted DNA, meaning that the DNA was quantifiable under this threshold, whereas detection beyond the limit indicated that DNA was detectable but not quantifiable [17]. To perform the MDL tests, DNA from adult worms for *E. multilocularis* (15 ng/µL of DNA concentration), *T. cati*, and *T. canis*, provided by the CERFE laboratory (Boult-aux-Bois, France), was used (4 and 3 ng/µL of DNA, respectively), as well as the laminated layer from metacestodes isolated from animal lesions for *E. granulosus* s.s. (Anses code 5872, 11 ng/µL), *E. ortleppi* (Anses code 6697, 46 ng/µL) [38], and *E. canadensis* (Anses code 3734, 5 ng/µL) [39], provided by the ANSES laboratory (Malzéville, France), and the cysticercus stage of *Taenia solium*, provided by the Pasteur Institute (Paris, France) (3 ng/µL). The MDL tests were performed for each individual qPCR in duplex assays and included the external control Alea.

### Specificity of the assays

The generated primers were first tested for specificity using the NCBI Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), which used the NCBI sequence databases for sequence alignment [42]. A mismatch threshold to ignore targets was applied from 6 differences. Second, a panel of samples (Table 1) or parasite DNA obtained from larval or adult specimens (for *T. solium*, *T. cati*, and *T. canis*) was tested for all qPCR assays to detect putative cross-reactions. These amplification reactions were performed in simplex PCR and in duplicate.

## Results

### Sensitivity of the assays

We assessed the sensitivity of the assays, which is presented through the MDL results (Table 3). The MDL for *Echinococcus* spp. (37-42 cycles), *T. solium* (37 cycles), *T. cati* (36 cycles), and *T. canis* (38 cycles) corresponds to the detection in 1 µL
<table><caption>**Table 1.** Collection of alveolar and cystic echinococcosis lesion samples stored fresh or frozen or as formalin-fixed paraffin-embedded (FFPE) samples and qPCR results (Cq value and DNA concentration assessment) for the samples.</caption><thead><tr><th rowspan="2">Sample code</th><th rowspan="2">Geographical origin of patient</th><th rowspan="2">Disease</th><th rowspan="2">Species</th><th rowspan="2">Year of sampling</th><th rowspan="2">Tissue type</th><th rowspan="2">Lesion location/ origin</th><th rowspan="2">MLX qPCR tested</th><th colspan="8">(qPCR results in mean cycle number)</th><th rowspan="2">DNA quantif (ng/IL)</th></tr><tr><th>Alea</th><th>rrn</th><th>Em</th><th>Eg</th><th>Ec</th><th>Eo</th><th>Tsol</th><th>Toxo</th></tr></thead><tbody><tr><td>FE-577</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>33.4</td><td>ND</td><td>27.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.9</td></tr><tr><td>FE-842</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1</td><td>32.6</td><td>ND</td><td>25.1</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>5.0</td></tr><tr><td>FE-875</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>31.3</td><td>ND</td><td>25.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>3.9</td></tr><tr><td>FE-878</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>34.3</td><td>ND</td><td>24.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>4.6</td></tr><tr><td>FE-907</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>32.7</td><td>ND</td><td>23.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>12.4</td></tr><tr><td>FE-917</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Peritoneal</td><td>1-2-3</td><td>31.6</td><td>ND</td><td>36.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.002</td></tr><tr><td>FE-929</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>32.9</td><td>ND</td><td>33.4</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.01</td></tr><tr><td>LU-O</td><td>Luxemburg</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>32.3</td><td>ND</td><td>27.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.9</td></tr><tr><td>FE-755</td><td>France</td><td>AE</td><td>Em</td><td>2020</td><td>FFPE</td><td>Peri-duodenal</td><td>1-2-3</td><td>34.3</td><td>ND</td><td>29.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.3</td></tr><tr><td>FE-861</td><td>France</td><td>AE</td><td>Em</td><td>2020</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>31.6</td><td>ND</td><td>27.1</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>1.2</td></tr><tr><td>FE-908</td><td>France</td><td>AE</td><td>Em</td><td>2020</td><td>FFPE</td><td>Brain</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>25.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>3.0</td></tr><tr><td>FE-918</td><td>France</td><td>AE</td><td>Em</td><td>2020</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>34.8</td><td>ND</td><td>36.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.001</td></tr><tr><td>FE-836</td><td>France</td><td>AE</td><td>Em</td><td>2019</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>31.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.1</td></tr><tr><td>FE-652</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.7</td><td>ND</td><td>29.4</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.2</td></tr><tr><td>FE-653</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.8</td><td>ND</td><td>25.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>3.7</td></tr><tr><td>FE-766</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Foie</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>26.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>1.8</td></tr><tr><td>FE-792</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.8</td><td>ND</td><td>26.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>1.8</td></tr><tr><td>FE-800</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Spleen</td><td>1-2-3</td><td>34.0</td><td>ND</td><td>25.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>3.0</td></tr><tr><td>FE-805</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Brain</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>23.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>14.5</td></tr><tr><td>FE-806</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>FFPE</td><td>Adrenal</td><td>1-2-3</td><td>34.7</td><td>ND</td><td>22.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>25.0</td></tr><tr><td>FE-829</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.0</td><td>ND</td><td>26.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>2.2</td></tr><tr><td>FE-768</td><td>France</td><td>AE</td><td>Em</td><td>2017</td><td>FFPE</td><td>Peritoneal</td><td>1</td><td>34.5</td><td>ND</td><td>28.4</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.5</td></tr><tr><td>FE-782</td><td>France</td><td>AE</td><td>Em</td><td>2017</td><td>FFPE</td><td>Liver</td><td>1</td><td>36.4</td><td>ND</td><td>22.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>26.2</td></tr><tr><td>P16-1535</td><td>France</td><td>AE</td><td>Em</td><td>2016</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.5</td><td>35.5</td><td>30.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.08</td></tr><tr><td>P16-2314</td><td>France</td><td>AE</td><td>Em</td><td>2016</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.0</td><td>39.2</td><td>36.3</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.002</td></tr><tr><td>P16-5377</td><td>France</td><td>AE</td><td>Em</td><td>2016</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>29.3</td><td>29.3</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.25</td></tr><tr><td>FE-769</td><td>France</td><td>AE</td><td>Em</td><td>2015</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.5</td><td>ND</td><td>28.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.3</td></tr><tr><td>P15-11460</td><td>France</td><td>AE</td><td>Em</td><td>2015</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.4</td><td>40.8</td><td>39.2</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.0002</td></tr><tr><td>P1515434<sup>50</sup></td><td>France</td><td>AE</td><td>Em</td><td>2015</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.8</td><td>31.7</td><td>31.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.04</td></tr><tr><td>P15-15434</td><td>France</td><td>AE</td><td>Em</td><td>2015</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.4</td><td>38.9</td><td>34.8</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.005</td></tr><tr><td>P14-3252</td><td>France</td><td>AE</td><td>Em</td><td>2014</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.1</td><td>36.1</td><td>31.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.04</td></tr><tr><td>P14-8880</td><td>France</td><td>AE</td><td>Em</td><td>2014</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.0</td><td>31.5</td><td>31.5</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.05</td></tr><tr><td>P1315022<sup>50</sup></td><td>France</td><td>AE</td><td>Em</td><td>2013</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.8</td><td>27.4</td><td>27.3</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>1.05</td></tr><tr><td>P13-4653IIC</td><td>France</td><td>AE</td><td>Em</td><td>2013</td><td>FFPE</td><td>Liver</td><td>1-2</td><td>35.1</td><td>41.1</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>&gt;45</td></tr><tr><td>FE-904</td><td>Poland</td><td>AE</td><td>Em</td><td>2012</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>29.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>0.2</td></tr><tr><td>P12-16504ES</td><td>France</td><td>AE</td><td>Em</td><td>2012</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>38.4</td><td>34.6</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.01</td></tr></tbody></table>

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<table><caption>**Table 1.** Collection of alveolar and cystic echinococcosis lesion samples stored fresh or frozen or as formalin-fixed paraffin-embedded (FFPE) samples and qPCR results (Cq value and DNA concentration assessment) for the samples.</caption><thead><tr><th rowspan="2">Sample code</th><th rowspan="2">Geographical origin of patient</th><th rowspan="2">Disease</th><th rowspan="2">Species</th><th rowspan="2">Year of sampling</th><th rowspan="2">Tissue type</th><th rowspan="2">Lesion location/ origin</th><th rowspan="2">MLX qPCR tested</th><th colspan="8">(qPCR results in mean cycle number)</th><th rowspan="2">DNA quantif (ng/IL)</th></tr><tr><th>Alea</th><th>rrn</th><th>Em</th><th>Eg</th><th>Ec</th><th>Eo</th><th>Tsol</th><th>Toxo</th></tr></thead><tbody><tr><td>FE-577</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>33.4</td><td>ND</td><td>27.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.9</td></tr><tr><td>FE-842</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1</td><td>32.6</td><td>ND</td><td>25.1</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>5.0</td></tr><tr><td>FE-875</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>31.3</td><td>ND</td><td>25.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>3.9</td></tr><tr><td>FE-878</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>34.3</td><td>ND</td><td>24.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>4.6</td></tr><tr><td>FE-907</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>32.7</td><td>ND</td><td>23.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>12.4</td></tr><tr><td>FE-917</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Peritoneal</td><td>1-2-3</td><td>31.6</td><td>ND</td><td>36.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.002</td></tr><tr><td>FE-929</td><td>France</td><td>AE</td><td>Em</td><td>2021</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>32.9</td><td>ND</td><td>33.4</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.01</td></tr><tr><td>LU-O</td><td>Luxemburg</td><td>AE</td><td>Em</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>32.3</td><td>ND</td><td>27.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.9</td></tr><tr><td>FE-755</td><td>France</td><td>AE</td><td>Em</td><td>2020</td><td>FFPE</td><td>Peri-duodenal</td><td>1-2-3</td><td>34.3</td><td>ND</td><td>29.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.3</td></tr><tr><td>FE-861</td><td>France</td><td>AE</td><td>Em</td><td>2020</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>31.6</td><td>ND</td><td>27.1</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>1.2</td></tr><tr><td>FE-908</td><td>France</td><td>AE</td><td>Em</td><td>2020</td><td>FFPE</td><td>Brain</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>25.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>3.0</td></tr><tr><td>FE-918</td><td>France</td><td>AE</td><td>Em</td><td>2020</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>34.8</td><td>ND</td><td>36.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.001</td></tr><tr><td>FE-836</td><td>France</td><td>AE</td><td>Em</td><td>2019</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>31.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.1</td></tr><tr><td>FE-652</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.7</td><td>ND</td><td>29.4</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.2</td></tr><tr><td>FE-653</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.8</td><td>ND</td><td>25.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>3.7</td></tr><tr><td>FE-766</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Foie</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>26.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>1.8</td></tr><tr><td>FE-792</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.8</td><td>ND</td><td>26.5</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>1.8</td></tr><tr><td>FE-800</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Spleen</td><td>1-2-3</td><td>34.0</td><td>ND</td><td>25.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>3.0</td></tr><tr><td>FE-805</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Brain</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>23.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>14.5</td></tr><tr><td>FE-806</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>FFPE</td><td>Adrenal</td><td>1-2-3</td><td>34.7</td><td>ND</td><td>22.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>25.0</td></tr><tr><td>FE-829</td><td>France</td><td>AE</td><td>Em</td><td>2018</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.0</td><td>ND</td><td>26.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>2.2</td></tr><tr><td>FE-768</td><td>France</td><td>AE</td><td>Em</td><td>2017</td><td>FFPE</td><td>Peritoneal</td><td>1</td><td>34.5</td><td>ND</td><td>28.4</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.5</td></tr><tr><td>FE-782</td><td>France</td><td>AE</td><td>Em</td><td>2017</td><td>FFPE</td><td>Liver</td><td>1</td><td>36.4</td><td>ND</td><td>22.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>26.2</td></tr><tr><td>P16-1535</td><td>France</td><td>AE</td><td>Em</td><td>2016</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.5</td><td>35.5</td><td>30.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.08</td></tr><tr><td>P16-2314</td><td>France</td><td>AE</td><td>Em</td><td>2016</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.0</td><td>39.2</td><td>36.3</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.002</td></tr><tr><td>P16-5377</td><td>France</td><td>AE</td><td>Em</td><td>2016</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>29.3</td><td>29.3</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.25</td></tr><tr><td>FE-769</td><td>France</td><td>AE</td><td>Em</td><td>2015</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.5</td><td>ND</td><td>28.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.3</td></tr><tr><td>P15-11460</td><td>France</td><td>AE</td><td>Em</td><td>2015</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.4</td><td>40.8</td><td>39.2</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.0002</td></tr><tr><td>P1515434<sup>50</sup></td><td>France</td><td>AE</td><td>Em</td><td>2015</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.8</td><td>31.7</td><td>31.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.04</td></tr><tr><td>P15-15434</td><td>France</td><td>AE</td><td>Em</td><td>2015</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.4</td><td>38.9</td><td>34.8</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.005</td></tr><tr><td>P14-3252</td><td>France</td><td>AE</td><td>Em</td><td>2014</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.1</td><td>36.1</td><td>31.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.04</td></tr><tr><td>P14-8880</td><td>France</td><td>AE</td><td>Em</td><td>2014</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.0</td><td>31.5</td><td>31.5</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.05</td></tr><tr><td>P1315022<sup>50</sup></td><td>France</td><td>AE</td><td>Em</td><td>2013</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.8</td><td>27.4</td><td>27.3</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>1.05</td></tr><tr><td>P13-4653IIC</td><td>France</td><td>AE</td><td>Em</td><td>2013</td><td>FFPE</td><td>Liver</td><td>1-2</td><td>35.1</td><td>41.1</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>&gt;45</td></tr><tr><td>FE-904</td><td>Poland</td><td>AE</td><td>Em</td><td>2012</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>29.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>0.2</td></tr><tr><td>P12-16504ES</td><td>France</td><td>AE</td><td>Em</td><td>2012</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>38.4</td><td>34.6</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.01</td></tr></tbody></table>

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<table><caption>**Table 1. (Continued)**</caption><thead><tr><th rowspan="2">Sample code</th><th rowspan="2">Geographical origin of patient</th><th rowspan="2">Disease</th><th rowspan="2">Species</th><th rowspan="2">Year of sampling</th><th rowspan="2">Tissue type</th><th rowspan="2">Lesion location/ origin</th><th rowspan="2">MLX qPCR tested</th><th colspan="8">(qPCR results in mean cycle number)</th><th rowspan="2">DNA quantif (ng/IL)</th></tr><tr><th>Alea</th><th>rrn</th><th>Em</th><th>Eg</th><th>Ec</th><th>Eo</th><th>Tsol</th><th>Toxo</th></tr></thead><tbody><tr><td>P1215706III</td><td>France</td><td>AE</td><td>Em</td><td>2012</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.8</td><td>36.4</td><td>32.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.02</td></tr><tr><td>P11-12405</td><td>France</td><td>AE</td><td>Em</td><td>2011</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>40.8</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>&gt;45</td></tr><tr><td>P1112967<sup>50</sup></td><td>France</td><td>AE</td><td>Em</td><td>2011</td><td>FFPE</td><td>Liver</td><td>1-2</td><td>35.1</td><td>35.0</td><td>35.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.002</td></tr><tr><td>P08-1905I</td><td>France</td><td>AE</td><td>Em</td><td>2008</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>38.6</td><td>35.5</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.003</td></tr><tr><td>P06-11605I</td><td>France</td><td>AE</td><td>Em</td><td>2006</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>35.9</td><td>38.7</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.0003</td></tr><tr><td>P06-4509III</td><td>France</td><td>AE</td><td>Em</td><td>2006</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>35.6</td><td>34.6</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.01</td></tr><tr><td>P05-14960II</td><td>France</td><td>AE</td><td>Em</td><td>2005</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.1</td><td>33.9</td><td>33.2</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.01</td></tr><tr><td>S00-15720II</td><td>France</td><td>AE</td><td>Em</td><td>2000</td><td>FFPE</td><td>Sub cutan.</td><td>1</td><td>35.2</td><td>32.2</td><td>31.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.04</td></tr><tr><td>S99-6011IIA</td><td>France</td><td>AE</td><td>Em</td><td>1999</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.7</td><td>36.3</td><td>39.0</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.0002</td></tr><tr><td>S97-9110IV</td><td>France</td><td>AE</td><td>Em</td><td>1997</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.0</td><td>28.0</td><td>27.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.68</td></tr><tr><td>EK-A20</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2021</td><td>frozen</td><td>Liver</td><td>1-2-3</td><td>31.6</td><td>ND</td><td>&gt;45</td><td>21.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>139.5</td></tr><tr><td>EK-B21</td><td>ND</td><td>CE</td><td>Eg</td><td>2021</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>34.4</td><td>ND</td><td>&gt;45</td><td>37.1</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.003</td></tr><tr><td>EK-B22</td><td>France</td><td>CE</td><td>Eg</td><td>2021</td><td>FFPE</td><td>Lung</td><td>1-2-3</td><td>32.7</td><td>ND</td><td>&gt;45</td><td>39.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.001</td></tr><tr><td>EK-G23</td><td>France</td><td>CE</td><td>Eg</td><td>2021</td><td>FFPE</td><td>Lung</td><td>1</td><td>38.4<sup>#</sup></td><td>ND</td><td>&gt;45</td><td>27.1</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>3.3</td></tr><tr><td>EK-G24</td><td>Romania</td><td>CE</td><td>Eg</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>32.9</td><td>ND</td><td>&gt;45</td><td>25.4</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>10.4</td></tr><tr><td>EK-K25</td><td>Turkey</td><td>CE</td><td>Eg</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>32.9</td><td>ND</td><td>&gt;45</td><td>22.3</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>94.6</td></tr><tr><td>EK-H13</td><td>Algeria</td><td>CE</td><td>Eg</td><td>2020</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.8<sup>**</sup></td><td>ND</td><td>&gt;45</td><td>23.4</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>42.6</td></tr><tr><td>EK-L14</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2020</td><td>FFPE</td><td>Intra-abdominal</td><td>1-2-3</td><td>31.6</td><td>ND</td><td>&gt;45</td><td>36.6</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.004</td></tr><tr><td>EK-L15</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2020</td><td>Frozen</td><td>Kidney</td><td>1-2-3</td><td>31.9</td><td>ND</td><td>&gt;45</td><td>22.3</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>94.2</td></tr><tr><td>EK-M16</td><td>Algeria</td><td>CE</td><td>Eg</td><td>2020</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>33.5</td><td>ND</td><td>&gt;45</td><td>35.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.01</td></tr><tr><td>EK-M17</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2020</td><td>Frozen</td><td>Bottom</td><td>1-2-3</td><td>34.2<sup>**</sup></td><td>ND</td><td>&gt;45</td><td>25.6</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>9.0</td></tr><tr><td>EK-M18</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2020</td><td>FFPE</td><td>Bone</td><td>1</td><td>34.8</td><td>ND</td><td>&gt;45</td><td>33.0</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.05</td></tr><tr><td>EK-S19</td><td>France</td><td>CE</td><td>Eg</td><td>2020</td><td>Frozen</td><td>Spleen</td><td>1-2-3</td><td>34.2<sup>**</sup></td><td>ND</td><td>&gt;45</td><td>21.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>239.9</td></tr><tr><td>EK-M11</td><td>Tunisia</td><td>CE</td><td>Eg</td><td>2019</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>&gt;45</td><td>ND</td><td>&gt;45</td><td>16.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>8300.4</td></tr><tr><td>EK-M12</td><td>Tunisia</td><td>CE</td><td>Eg</td><td>2019</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>34.1</td><td>ND</td><td>&gt;45</td><td>21.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>134.1</td></tr><tr><td>EK-D28</td><td>Mali</td><td>CE</td><td>Ec</td><td>2019</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>32.2</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>14.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>2617.2</td></tr><tr><td>EK-H29</td><td>ND</td><td>CE</td><td>Ec</td><td>2019</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>33.2</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>28.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.2</td></tr><tr><td>EK-B1</td><td>Algeria</td><td>CE</td><td>Eg</td><td>2018</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>34.7</td><td>ND</td><td>&gt;45</td><td>27.6</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>2.3</td></tr><tr><td>EK-C2</td><td>Moldova</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Bone</td><td>1-2-3</td><td>33.9</td><td>ND</td><td>&gt;45</td><td>33.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.04</td></tr><tr><td>EK-C3</td><td>Moldova</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Bone</td><td>1-2-3</td><td>33.3</td><td>ND</td><td>&gt;45</td><td>32.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.06</td></tr><tr><td>EK-H4</td><td>France</td><td>CE</td><td>Eg</td><td>2018</td><td>FFPE</td><td>NR</td><td>1-2-3</td><td>34.4</td><td>ND</td><td>&gt;45</td><td>32.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.05</td></tr><tr><td>EK-H5</td><td>France</td><td>CE</td><td>Eg</td><td>2018</td><td>FFPE</td><td>NR</td><td>1-2-3</td><td>34.1</td><td>ND</td><td>&gt;45</td><td>35.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.01</td></tr><tr><td>EK-K6</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Bedsore</td><td>1-2-3</td><td>33.9</td><td>ND</td><td>&gt;45</td><td>31.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.1</td></tr><tr><td>EK-K7</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Bedsore</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>&gt;45</td><td>31.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.1</td></tr><tr><td>EK-L8</td><td>France</td><td>CE</td><td>Eg</td><td>2018</td><td>FFPE</td><td>Lung</td><td>1-2-3</td><td>34.9</td><td>ND</td><td>&gt;45</td><td>37.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.002</td></tr></tbody></table>

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<table><caption>**Table 1.** (Continued)</caption><thead><tr><th rowspan="2">Sample code</th><th rowspan="2">Geographical origin of patient</th><th rowspan="2">Disease</th><th rowspan="2">Species</th><th rowspan="2">Year of sampling</th><th rowspan="2">Tissue type</th><th rowspan="2">Lesion location/ origin</th><th rowspan="2">MLX qPCR tested</th><th colspan="8">(qPCR results in mean cycle number)</th><th rowspan="2">DNA quantif (ng/IL)</th></tr><tr><th>Alea</th><th>rrn</th><th>Em</th><th>Eg</th><th>Ec</th><th>Eo</th><th>Tsol</th><th>Toxo</th></tr></thead><tbody><tr><td>P1215706III</td><td>France</td><td>AE</td><td>Em</td><td>2012</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.8</td><td>36.4</td><td>32.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.02</td></tr><tr><td>P11-12405</td><td>France</td><td>AE</td><td>Em</td><td>2011</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>40.8</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>&gt;45</td></tr><tr><td>P1112967<sup>50</sup></td><td>France</td><td>AE</td><td>Em</td><td>2011</td><td>FFPE</td><td>Liver</td><td>1-2</td><td>35.1</td><td>35.0</td><td>35.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.002</td></tr><tr><td>P08-1905I</td><td>France</td><td>AE</td><td>Em</td><td>2008</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>38.6</td><td>35.5</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.003</td></tr><tr><td>P06-11605I</td><td>France</td><td>AE</td><td>Em</td><td>2006</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>35.9</td><td>38.7</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.0003</td></tr><tr><td>P06-4509III</td><td>France</td><td>AE</td><td>Em</td><td>2006</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.9</td><td>35.6</td><td>34.6</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.01</td></tr><tr><td>P05-14960II</td><td>France</td><td>AE</td><td>Em</td><td>2005</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.1</td><td>33.9</td><td>33.2</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.01</td></tr><tr><td>S00-15720II</td><td>France</td><td>AE</td><td>Em</td><td>2000</td><td>FFPE</td><td>Sub cutan.</td><td>1</td><td>35.2</td><td>32.2</td><td>31.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.04</td></tr><tr><td>S99-6011IIA</td><td>France</td><td>AE</td><td>Em</td><td>1999</td><td>FFPE</td><td>Liver</td><td>1</td><td>34.7</td><td>36.3</td><td>39.0</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.0002</td></tr><tr><td>S97-9110IV</td><td>France</td><td>AE</td><td>Em</td><td>1997</td><td>FFPE</td><td>Liver</td><td>1</td><td>35.0</td><td>28.0</td><td>27.9</td><td>&gt;45</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.68</td></tr><tr><td>EK-A20</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2021</td><td>frozen</td><td>Liver</td><td>1-2-3</td><td>31.6</td><td>ND</td><td>&gt;45</td><td>21.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>139.5</td></tr><tr><td>EK-B21</td><td>ND</td><td>CE</td><td>Eg</td><td>2021</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>34.4</td><td>ND</td><td>&gt;45</td><td>37.1</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.003</td></tr><tr><td>EK-B22</td><td>France</td><td>CE</td><td>Eg</td><td>2021</td><td>FFPE</td><td>Lung</td><td>1-2-3</td><td>32.7</td><td>ND</td><td>&gt;45</td><td>39.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.001</td></tr><tr><td>EK-G23</td><td>France</td><td>CE</td><td>Eg</td><td>2021</td><td>FFPE</td><td>Lung</td><td>1</td><td>38.4<sup>#</sup></td><td>ND</td><td>&gt;45</td><td>27.1</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>3.3</td></tr><tr><td>EK-G24</td><td>Romania</td><td>CE</td><td>Eg</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>32.9</td><td>ND</td><td>&gt;45</td><td>25.4</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>10.4</td></tr><tr><td>EK-K25</td><td>Turkey</td><td>CE</td><td>Eg</td><td>2021</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>32.9</td><td>ND</td><td>&gt;45</td><td>22.3</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>94.6</td></tr><tr><td>EK-H13</td><td>Algeria</td><td>CE</td><td>Eg</td><td>2020</td><td>Frozen</td><td>Liver</td><td>1-2-3</td><td>33.8<sup>**</sup></td><td>ND</td><td>&gt;45</td><td>23.4</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>42.6</td></tr><tr><td>EK-L14</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2020</td><td>FFPE</td><td>Intra-abdominal</td><td>1-2-3</td><td>31.6</td><td>ND</td><td>&gt;45</td><td>36.6</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.004</td></tr><tr><td>EK-L15</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2020</td><td>Frozen</td><td>Kidney</td><td>1-2-3</td><td>31.9</td><td>ND</td><td>&gt;45</td><td>22.3</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>94.2</td></tr><tr><td>EK-M16</td><td>Algeria</td><td>CE</td><td>Eg</td><td>2020</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>33.5</td><td>ND</td><td>&gt;45</td><td>35.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.01</td></tr><tr><td>EK-M17</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2020</td><td>Frozen</td><td>Bottom</td><td>1-2-3</td><td>34.2<sup>**</sup></td><td>ND</td><td>&gt;45</td><td>25.6</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>9.0</td></tr><tr><td>EK-M18</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2020</td><td>FFPE</td><td>Bone</td><td>1</td><td>34.8</td><td>ND</td><td>&gt;45</td><td>33.0</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>0.05</td></tr><tr><td>EK-S19</td><td>France</td><td>CE</td><td>Eg</td><td>2020</td><td>Frozen</td><td>Spleen</td><td>1-2-3</td><td>34.2<sup>**</sup></td><td>ND</td><td>&gt;45</td><td>21.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>239.9</td></tr><tr><td>EK-M11</td><td>Tunisia</td><td>CE</td><td>Eg</td><td>2019</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>&gt;45</td><td>ND</td><td>&gt;45</td><td>16.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>8300.4</td></tr><tr><td>EK-M12</td><td>Tunisia</td><td>CE</td><td>Eg</td><td>2019</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>34.1</td><td>ND</td><td>&gt;45</td><td>21.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>134.1</td></tr><tr><td>EK-D28</td><td>Mali</td><td>CE</td><td>Ec</td><td>2019</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>32.2</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>14.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>2617.2</td></tr><tr><td>EK-H29</td><td>ND</td><td>CE</td><td>Ec</td><td>2019</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>33.2</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>28.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.2</td></tr><tr><td>EK-B1</td><td>Algeria</td><td>CE</td><td>Eg</td><td>2018</td><td>FFPE</td><td>Liver</td><td>1-2-3</td><td>34.7</td><td>ND</td><td>&gt;45</td><td>27.6</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>2.3</td></tr><tr><td>EK-C2</td><td>Moldova</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Bone</td><td>1-2-3</td><td>33.9</td><td>ND</td><td>&gt;45</td><td>33.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.04</td></tr><tr><td>EK-C3</td><td>Moldova</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Bone</td><td>1-2-3</td><td>33.3</td><td>ND</td><td>&gt;45</td><td>32.8</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.06</td></tr><tr><td>EK-H4</td><td>France</td><td>CE</td><td>Eg</td><td>2018</td><td>FFPE</td><td>NR</td><td>1-2-3</td><td>34.4</td><td>ND</td><td>&gt;45</td><td>32.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.05</td></tr><tr><td>EK-H5</td><td>France</td><td>CE</td><td>Eg</td><td>2018</td><td>FFPE</td><td>NR</td><td>1-2-3</td><td>34.1</td><td>ND</td><td>&gt;45</td><td>35.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.01</td></tr><tr><td>EK-K6</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Bedsore</td><td>1-2-3</td><td>33.9</td><td>ND</td><td>&gt;45</td><td>31.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.1</td></tr><tr><td>EK-K7</td><td>Morocco</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Bedsore</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>&gt;45</td><td>31.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.1</td></tr><tr><td>EK-L8</td><td>France</td><td>CE</td><td>Eg</td><td>2018</td><td>FFPE</td><td>Lung</td><td>1-2-3</td><td>34.9</td><td>ND</td><td>&gt;45</td><td>37.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.002</td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 1.** (Continued)</caption><thead><tr><th rowspan="2">Sample code</th><th rowspan="2">Geographical origin of patient</th><th rowspan="2">Disease</th><th rowspan="2">Species</th><th rowspan="2">Year of sampling</th><th rowspan="2">Tissue type</th><th rowspan="2">Lesion location/ origin</th><th rowspan="2">MLX qPCR tested</th><th colspan="8">(qPCR results in mean cycle number)</th><th rowspan="2">DNA quantif (ng/µL)</th></tr><tr><th>Alea</th><th>rrn</th><th>Em</th><th>Eg</th><th>Ec</th><th>Eo</th><th>Tsol</th><th>Toxo</th></tr></thead><tbody><tr><td>EK-M9</td><td>Moldova</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Intraperitoneal</td><td>1-2-3</td><td>34.1</td><td>ND</td><td>&gt;45</td><td>32.1</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.09</td></tr><tr><td>EK-T10</td><td>Tibet</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>NR</td><td>1-2-3</td><td>33.9</td><td>ND</td><td>&gt;45</td><td>24.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>15.6</td></tr><tr><td>EK-S27</td><td>Mauritania</td><td>CE</td><td>Ec</td><td>2018</td><td>Frozen</td><td>NR</td><td>1-2-3</td><td>31.9</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>26.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.6</td></tr><tr><td>EK-S26</td><td>ND</td><td>CE</td><td>Ec</td><td>2017</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>37.5</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>21.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>34.2</td></tr><tr><td>EK-M30*</td><td>France</td><td>CE</td><td>Eo</td><td>2017</td><td>FFPE</td><td>Bone</td><td>1-2-3</td><td>34.4</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>34.7</td><td>&gt;45</td><td>&gt;45</td><td>0.03</td></tr><tr><td>EK-R31*</td><td>France</td><td>CE</td><td>Eo</td><td>2017</td><td>FFPE</td><td>Bone</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>25.8</td><td>&gt;45</td><td>&gt;45</td><td>59.4</td></tr><tr><td>P10-8854†,§</td><td>Turkey</td><td>CE</td><td>Eg</td><td>2010</td><td>FFPE</td><td>NA</td><td>1-2</td><td>35.2</td><td>&gt;45</td><td>&gt;45</td><td>39.9#</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.0004</td></tr><tr><td>P06-5181<sup>50</sup></td><td>ND</td><td>CE</td><td>Eg</td><td>2006</td><td>FFPE</td><td>NA</td><td>1-2</td><td>35.4</td><td>&gt;45</td><td>&gt;45</td><td>38.8</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.001</td></tr><tr><td>P0517231<sup>50</sup></td><td>ND</td><td>CE</td><td>Eg</td><td>2005</td><td>FFPE</td><td>Liver</td><td>1-2</td><td>35.1</td><td>&gt;45</td><td>&gt;45</td><td>36.9</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.003</td></tr><tr><td>P05-17231</td><td>ND</td><td>CE</td><td>Eg</td><td>2005</td><td>FFPE</td><td>Liver</td><td>1-2</td><td>35.4</td><td>&gt;45</td><td>&gt;45</td><td>37.0</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.003</td></tr><tr><td>Em</td><td>France</td><td>Control</td><td>Em</td><td>2005</td><td>Frozen</td><td>Fox</td><td>1-2-3</td><td>35.1</td><td>ND</td><td>22.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>25.8</td></tr><tr><td>5872</td><td>France</td><td>Control</td><td>Eg</td><td>2012</td><td>Frozen</td><td>Ovine</td><td>1-2-3</td><td>33.0</td><td>ND</td><td>&gt;45</td><td>24.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>17.5</td></tr><tr><td>3734</td><td>France</td><td>Control</td><td>Ec</td><td>2010</td><td>Frozen</td><td>Pig</td><td>1-2-3</td><td>34.7</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>23.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>8.0</td></tr><tr><td>6697</td><td>France</td><td>Control</td><td>Eo</td><td>2012</td><td>Frozen</td><td>Bovine</td><td>1-2-3</td><td>34.1</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>23.8</td><td>&gt;45</td><td>&gt;45</td><td>337.5</td></tr><tr><td>Ts-Pasteur</td><td>France</td><td>Control</td><td>Tsol</td><td>ND</td><td>Frozen</td><td>ND</td><td>1-2-3</td><td>34.8</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>15.9</td><td>&gt;45</td><td>86.2</td></tr><tr><td>Tcati</td><td>France</td><td>Control</td><td>Tcat</td><td>ND</td><td>Frozen</td><td>Cat</td><td>1-2-3</td><td>34.3</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.4</td></tr><tr><td>Tcanis</td><td>France</td><td>Control</td><td>Tcan</td><td>ND</td><td>Frozen</td><td>Fox</td><td>1-2-3</td><td>34.8</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>1.9</td></tr></tbody></table>

AE, alveolar echinococcosis; CE, cystic echinococcosis; rrn, qPCR targeting fragment of the mitochondrial 16S-*rrnL* gene [20]; Em, *Echinococcus multilocularis*; Eg, *E. granulosus*; Ec, *E. canadensis*; Eo, *E. ortleppi*; Tsol, *Taenia solium*; Tcat, *Toxocara cati*; Tcan, *T. canis*. qPCR tested: T for 3 multiplex qPCR tested on the sample, qPCR MLX 1, Em-Eg-Alea, 2, Ec-Eo-Alea, 3, Tsol-Toxo.

ND for no data.

<footnote>* Sample tested on 1:100 dilution.</footnote>

<footnote>** Alea qPCR negative on the triplex Em/Eg s.s./Alea and positive on the triplex Ec/Eo/Alea.</footnote>

<footnote># qPCR positive once from the duplicate.</footnote>

<footnote>† Sample not described in Knapp et al. [19] but tested with the same PCR techniques.</footnote>

<footnote>§ Sample positive for the multiplex PCR from Trachsel et al. [37].</footnote>

<footnote n="50">⁵⁰ For elution volume in 50 µL of elution buffer instead of 200 µL for the other FFPE samples.</footnote>
<table><caption>**Table 1.** (Continued)</caption><thead><tr><th rowspan="2">Sample code</th><th rowspan="2">Geographical origin of patient</th><th rowspan="2">Disease</th><th rowspan="2">Species</th><th rowspan="2">Year of sampling</th><th rowspan="2">Tissue type</th><th rowspan="2">Lesion location/ origin</th><th rowspan="2">MLX qPCR tested</th><th colspan="8">(qPCR results in mean cycle number)</th><th rowspan="2">DNA quantif (ng/µL)</th></tr><tr><th>Alea</th><th>rrn</th><th>Em</th><th>Eg</th><th>Ec</th><th>Eo</th><th>Tsol</th><th>Toxo</th></tr></thead><tbody><tr><td>EK-M9</td><td>Moldova</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>Intraperitoneal</td><td>1-2-3</td><td>34.1</td><td>ND</td><td>&gt;45</td><td>32.1</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.09</td></tr><tr><td>EK-T10</td><td>Tibet</td><td>CE</td><td>Eg</td><td>2018</td><td>Frozen</td><td>NR</td><td>1-2-3</td><td>33.9</td><td>ND</td><td>&gt;45</td><td>24.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>15.6</td></tr><tr><td>EK-S27</td><td>Mauritania</td><td>CE</td><td>Ec</td><td>2018</td><td>Frozen</td><td>NR</td><td>1-2-3</td><td>31.9</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>26.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.6</td></tr><tr><td>EK-S26</td><td>ND</td><td>CE</td><td>Ec</td><td>2017</td><td>Frozen</td><td>Lung</td><td>1-2-3</td><td>37.5</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>21.0</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>34.2</td></tr><tr><td>EK-M30*</td><td>France</td><td>CE</td><td>Eo</td><td>2017</td><td>FFPE</td><td>Bone</td><td>1-2-3</td><td>34.4</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>34.7</td><td>&gt;45</td><td>&gt;45</td><td>0.03</td></tr><tr><td>EK-R31*</td><td>France</td><td>CE</td><td>Eo</td><td>2017</td><td>FFPE</td><td>Bone</td><td>1-2-3</td><td>34.2</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>25.8</td><td>&gt;45</td><td>&gt;45</td><td>59.4</td></tr><tr><td>P10-8854†,§</td><td>Turkey</td><td>CE</td><td>Eg</td><td>2010</td><td>FFPE</td><td>NA</td><td>1-2</td><td>35.2</td><td>&gt;45</td><td>&gt;45</td><td>39.9#</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.0004</td></tr><tr><td>P06-5181<sup>50</sup></td><td>ND</td><td>CE</td><td>Eg</td><td>2006</td><td>FFPE</td><td>NA</td><td>1-2</td><td>35.4</td><td>&gt;45</td><td>&gt;45</td><td>38.8</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.001</td></tr><tr><td>P0517231<sup>50</sup></td><td>ND</td><td>CE</td><td>Eg</td><td>2005</td><td>FFPE</td><td>Liver</td><td>1-2</td><td>35.1</td><td>&gt;45</td><td>&gt;45</td><td>36.9</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.003</td></tr><tr><td>P05-17231</td><td>ND</td><td>CE</td><td>Eg</td><td>2005</td><td>FFPE</td><td>Liver</td><td>1-2</td><td>35.4</td><td>&gt;45</td><td>&gt;45</td><td>37.0</td><td>&gt;45</td><td>&gt;45</td><td>ND</td><td>ND</td><td>0.003</td></tr><tr><td>Em</td><td>France</td><td>Control</td><td>Em</td><td>2005</td><td>Frozen</td><td>Fox</td><td>1-2-3</td><td>35.1</td><td>ND</td><td>22.9</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>25.8</td></tr><tr><td>5872</td><td>France</td><td>Control</td><td>Eg</td><td>2012</td><td>Frozen</td><td>Ovine</td><td>1-2-3</td><td>33.0</td><td>ND</td><td>&gt;45</td><td>24.7</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>17.5</td></tr><tr><td>3734</td><td>France</td><td>Control</td><td>Ec</td><td>2010</td><td>Frozen</td><td>Pig</td><td>1-2-3</td><td>34.7</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>23.2</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>8.0</td></tr><tr><td>6697</td><td>France</td><td>Control</td><td>Eo</td><td>2012</td><td>Frozen</td><td>Bovine</td><td>1-2-3</td><td>34.1</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>23.8</td><td>&gt;45</td><td>&gt;45</td><td>337.5</td></tr><tr><td>Ts-Pasteur</td><td>France</td><td>Control</td><td>Tsol</td><td>ND</td><td>Frozen</td><td>ND</td><td>1-2-3</td><td>34.8</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>15.9</td><td>&gt;45</td><td>86.2</td></tr><tr><td>Tcati</td><td>France</td><td>Control</td><td>Tcat</td><td>ND</td><td>Frozen</td><td>Cat</td><td>1-2-3</td><td>34.3</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>0.4</td></tr><tr><td>Tcanis</td><td>France</td><td>Control</td><td>Tcan</td><td>ND</td><td>Frozen</td><td>Fox</td><td>1-2-3</td><td>34.8</td><td>ND</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>&gt;45</td><td>1.9</td></tr></tbody></table>

AE, alveolar echinococcosis; CE, cystic echinococcosis; rrn, qPCR targeting fragment of the mitochondrial 16S-*rrnL* gene [20]; Em, *Echinococcus multilocularis*; Eg, *E. granulosus*; Ec, *E. canadensis*; Eo, *E. ortleppi*; Tsol, *Taenia solium*; Tcat, *Toxocara cati*; Tcan, *T. canis*. qPCR tested: T for 3 multiplex qPCR tested on the sample, qPCR MLX 1, Em-Eg-Alea, 2, Ec-Eo-Alea, 3, Tsol-Toxo.

ND for no data.

<footnote>* Sample tested on 1:100 dilution.</footnote>

<footnote>** Alea qPCR negative on the triplex Em/Eg s.s./Alea and positive on the triplex Ec/Eo/Alea.</footnote>

<footnote># qPCR positive once from the duplicate.</footnote>

<footnote>† Sample not described in Knapp et al. [19] but tested with the same PCR techniques.</footnote>

<footnote>§ Sample positive for the multiplex PCR from Trachsel et al. [37].</footnote>

<footnote n="50">⁵⁰ For elution volume in 50 µL of elution buffer instead of 200 µL for the other FFPE samples.</footnote>
<table><caption>**Table 2.** Primers and hydrolysis probes designed based on GenBank reference sequences for the targeted parasite detection by qPCR.</caption><thead><tr><th>Target species</th><th>Target gene</th><th>Primer and probe</th><th>GenBank reference No (country of origin of the genotyped specimen)</th><th>Oligonucleotide sequence (5'-3')</th><th>Probe label</th><th>Target size (bp)</th><th>References</th></tr></thead><tbody><tr><td>Echinococcus multilocularis</td><td>Cytochrome b</td><td>Em_cytb_Fwd<br>Em_cytb_Rev<br>Em_cytb_Probe</td><td>AB018440 (Japan) [28]</td><td>CGAAAATCCACCAACCACATACT<br>GCTGCCACTGTCCTTACTTCAA</td><td></td><td>84</td><td>This study</td></tr><tr><td>Echinococcus granulosus s.s.</td><td>Cytochrome oxidase<br>subunit III</td><td>Eg_cox3_Fwd<br>Eg_cox3_Rev<br>Eg_cox3_Probe</td><td>AF297617 (for G1)<br>(United Kingdom) [22]</td><td>ACCATAGAACCAACCAACGGCAAACTATCA<br>TATCTGTAACACCACAAAAACTCAAACC</td><td>FAM-TAMRA</td><td>149</td><td>This study</td></tr><tr><td>Echinococcus canadensis</td><td>NADH<br>dehydrogenase<br>subunit V</td><td>Ec_nad5_Fwd<br>Ec_nad5_Rev</td><td>AB208063 (for G6)<br>(Kazakhstan) [26]<br>AB235847 (for G7)<br>(Poland) [26]</td><td>CGTTGGAGATTCCGTTTGTGTG<br>AACAAAAGCAAATCACAACAACGTCAACCC<br>ATACGCCATGACTTATCAACTGAAAT<br>AGATTGTGGCTTTGTCAACTTGTAA</td><td>CY5-TAMRA</td><td>105</td><td>This study</td></tr><tr><td>Echinococcus ortleppi</td><td>ATP synthase<br>membrane<br>subunit VI</td><td>Ec_nad5_Probe<br>Eo_atp6_Fwd</td><td>AB235846<br>(Argentina) [26]</td><td>TCACCAAAAATCAAGTACAAAACGCACCAA<br>CTTTTTAGTGTGTATAGCTGAGTCCATTAGT</td><td>FAM-TAMRA</td><td>145</td><td>This study</td></tr><tr><td>Taenia solium</td><td>NADH<br>dehydrogenase<br>subunit iV</td><td>Eo_atp6_Rev<br>Eo_atp6_Probe<br>Tsol_nad4_Fwd</td><td>AB086256<br>(China) [27]</td><td>CAACGCCCACCAACAACTTA<br>CCCCATAGTGTTGATTTTGCGTCCTTTCA<br>CCCAAAAACGGAACGACAA</td><td>CY5-TAMRA</td><td>106</td><td>This study</td></tr><tr><td>Toxocara spp.</td><td>Cytochrome oxidase<br>subunit I</td><td>Tsol_nad4_Rev<br>Tsol_nad4_Probe<br>Toxo_spp_cox1_Fwd</td><td>AM411108<br>(for T. canis)<br>(China) [23]</td><td>GCTGTGCATTATCTGTATCTTTTTTAATTG<br>ATGCTAATCAACGCTTCCCATCTAACTCGC<br>AAAATAGCCAARTCCACWCTMCTACCA</td><td>HEX-TAMRA</td><td>79</td><td>This study</td></tr><tr><td>Alea - PCR<br>inhibitor test</td><td>Random<br>sequence</td><td>Toxo_spp_cox1_Rev<br>Toxo_spp_cox1_Probe<br>Alea_Fwd<br>Alea_Rev<br>Alea_probe</td><td>[21]</td><td>GGTGTGGKACTAGTTGAACTGTGTA<br>CCCCATAGTCCTCAAAG<br>CCTAAAAATGTCTATGATTGGTCCACTA<br>GGGAGTACCTTGCCATACAAAATT<br>TTAAATCAACTCCTAAATCCGCGCGATAGG</td><td>FAM-MGB<br>VIC-TAMRA</td><td>167</td><td>Knapp<br>et al. [21]</td></tr></tbody></table>
<table><caption>**Table 2.** Primers and hydrolysis probes designed based on GenBank reference sequences for the targeted parasite detection by qPCR.</caption><thead><tr><th>Target species</th><th>Target gene</th><th>Primer and probe</th><th>GenBank reference No (country of origin of the genotyped specimen)</th><th>Oligonucleotide sequence (5'-3')</th><th>Probe label</th><th>Target size (bp)</th><th>References</th></tr></thead><tbody><tr><td>Echinococcus multilocularis</td><td>Cytochrome b</td><td>Em_cytb_Fwd<br>Em_cytb_Rev<br>Em_cytb_Probe</td><td>AB018440 (Japan) [28]</td><td>CGAAAATCCACCAACCACATACT<br>GCTGCCACTGTCCTTACTTCAA</td><td></td><td>84</td><td>This study</td></tr><tr><td>Echinococcus granulosus s.s.</td><td>Cytochrome oxidase<br>subunit III</td><td>Eg_cox3_Fwd<br>Eg_cox3_Rev<br>Eg_cox3_Probe</td><td>AF297617 (for G1)<br>(United Kingdom) [22]</td><td>ACCATAGAACCAACCAACGGCAAACTATCA<br>TATCTGTAACACCACAAAAACTCAAACC</td><td>FAM-TAMRA</td><td>149</td><td>This study</td></tr><tr><td>Echinococcus canadensis</td><td>NADH<br>dehydrogenase<br>subunit V</td><td>Ec_nad5_Fwd<br>Ec_nad5_Rev</td><td>AB208063 (for G6)<br>(Kazakhstan) [26]<br>AB235847 (for G7)<br>(Poland) [26]</td><td>CGTTGGAGATTCCGTTTGTGTG<br>AACAAAAGCAAATCACAACAACGTCAACCC<br>ATACGCCATGACTTATCAACTGAAAT<br>AGATTGTGGCTTTGTCAACTTGTAA</td><td>CY5-TAMRA</td><td>105</td><td>This study</td></tr><tr><td>Echinococcus ortleppi</td><td>ATP synthase<br>membrane<br>subunit VI</td><td>Ec_nad5_Probe<br>Eo_atp6_Fwd</td><td>AB235846<br>(Argentina) [26]</td><td>TCACCAAAAATCAAGTACAAAACGCACCAA<br>CTTTTTAGTGTGTATAGCTGAGTCCATTAGT</td><td>FAM-TAMRA</td><td>145</td><td>This study</td></tr><tr><td>Taenia solium</td><td>NADH<br>dehydrogenase<br>subunit iV</td><td>Eo_atp6_Rev<br>Eo_atp6_Probe<br>Tsol_nad4_Fwd</td><td>AB086256<br>(China) [27]</td><td>CAACGCCCACCAACAACTTA<br>CCCCATAGTGTTGATTTTGCGTCCTTTCA<br>CCCAAAAACGGAACGACAA</td><td>CY5-TAMRA</td><td>106</td><td>This study</td></tr><tr><td>Toxocara spp.</td><td>Cytochrome oxidase<br>subunit I</td><td>Tsol_nad4_Rev<br>Tsol_nad4_Probe<br>Toxo_spp_cox1_Fwd</td><td>AM411108<br>(for T. canis)<br>(China) [23]</td><td>GCTGTGCATTATCTGTATCTTTTTTAATTG<br>ATGCTAATCAACGCTTCCCATCTAACTCGC<br>AAAATAGCCAARTCCACWCTMCTACCA</td><td>HEX-TAMRA</td><td>79</td><td>This study</td></tr><tr><td>Alea - PCR<br>inhibitor test</td><td>Random<br>sequence</td><td>Toxo_spp_cox1_Rev<br>Toxo_spp_cox1_Probe<br>Alea_Fwd<br>Alea_Rev<br>Alea_probe</td><td>[21]</td><td>GGTGTGGKACTAGTTGAACTGTGTA<br>CCCCATAGTCCTCAAAG<br>CCTAAAAATGTCTATGATTGGTCCACTA<br>GGGAGTACCTTGCCATACAAAATT<br>TTAAATCAACTCCTAAATCCGCGCGATAGG</td><td>FAM-MGB<br>VIC-TAMRA</td><td>167</td><td>Knapp<br>et al. [21]</td></tr></tbody></table>
<table><caption>**Table 3.** Method detection limits performed for each targeted parasite, with DNA dilution series tested on seven PCR assays.</caption><thead><tr><th>Species</th><th>Last dilution<br>7 PCR(+) (ng/IL)</th><th>Mean Cq<br>MDL (cycle)</th><th>SD</th><th>Y-inter</th><th>Slope</th><th>r<sup>2</sup></th><th>Efficiency (%)</th><th>n dilutions</th></tr></thead><tbody><tr><td><i>E. multilocularis</i></td><td>5 × 10<sup>-4</sup></td><td>37.97</td><td>0.84</td><td>27.37</td><td>-3.18</td><td>0.97</td><td>106.5</td><td>4</td></tr><tr><td><i>E. granulosus</i> s.s.</td><td>1 × 10<sup>-4</sup></td><td>42.21</td><td>1.00</td><td>28.76</td><td>-3.27</td><td>0.99</td><td>102.3</td><td>4</td></tr><tr><td><i>E. canadensis</i></td><td>5 × 10<sup>-4</sup></td><td>36.82</td><td>0.87</td><td>26.20</td><td>-3.36</td><td>0.99</td><td>98.3</td><td>3</td></tr><tr><td><i>E. ortleppi</i></td><td>5 × 10<sup>-4</sup></td><td>41.00</td><td>1.46</td><td>29.40</td><td>-2.67</td><td>0.99</td><td>136.9</td><td>3</td></tr><tr><td><i>Taenia solium</i></td><td>3 × 10<sup>-5</sup></td><td>37.35</td><td>0.48</td><td>22.27</td><td>-3.31</td><td>0.99</td><td>100.5</td><td>4</td></tr><tr><td><i>Toxocara cati</i></td><td>4 × 10<sup>-4</sup></td><td>36.15</td><td>0.78</td><td>23.71</td><td>-3.76</td><td>0.93</td><td>84.6</td><td>4</td></tr><tr><td><i>Toxocara canis</i></td><td>3 × 10<sup>-5</sup></td><td>38.55</td><td>0.58</td><td>21.89</td><td>-3.55</td><td>0.99</td><td>91.2</td><td>5</td></tr></tbody></table>

Cq, cycle threshold; MDL, method detection limit; SD, standard deviation; Y-inter, Y-intercept; r², correlation coefficient.

% Efficiency calculated by qPCR efficiency calculator available on ThermoFisher website.

n dilutions: number of dilution points with 7 positive qPCR used to calculate the standard curve plot.

of the tested samples of 0.6 pg to 1 fg of DNA for *Echinococcus* spp., 0.3 fg for *T. solium*, 1 pg for *T. cati*, and 0.3 fg for *T. canis*. Standard curves were plotted for each PCR assay based on seven positive replicates from the DNA concentration range to obtain the slope and Y-intercept (from three to five dilution points with seven positive results used, Table 3). The efficiency of the TaqMan qPCR assays on DNA dilutions ranged from 84.6% to 136.9%.

## Specificity of the assays

The test of specificity performed using the online NCBI Primer-BLAST software showed good matching between designed primer sequences and referenced data for *E. multilocularis* (exact matching with 94 reference sequences versus 16 with one mismatch), *E. granulosus* s.s. (exact matching with three reference sequences versus two with one mismatch), *E. ortleppi* (exact matching with seven reference sequences), *Taenia solium* (exact matching with one reference sequence versus three with two mismatches), *Toxocara* spp. (for *T. cati*, exact matching with one reference sequence versus one with one mismatch and *T. canis*, exact matching with eight reference sequences versus one with two mismatches) and *E. canadensis* (exact matching with 127 reference sequences from the G6, G7 and G10 genotypes versus two mismatches with the G8 genotype). According to the Primer-BLAST tool, the current primer pair and probe designed for *E. canadensis* should correctly amplify at least the G6, G7 and G10 genotypes.

Specificity was tested on 50 DNA samples from echinococcosis lesions (21 AE and 29 CE samples) and DNA from the larval and adult specimens used for the sensitivity assessment (Table 1). The specificity was 100% for each qPCR assay on fresh and FFPE tissues from patients, as well as for larval and adult specimens from animals.

## Tested samples and the presence of PCR inhibitors

All 81 samples were analysed using one or three MLX qPCR assays. Each DNA sample was amplified by the appropriate specific assay, in agreement with the previous molecular or pathology-based identification (Table 1).

For the 34 fresh samples, *E. multilocularis* was detected by the specific qPCR assay with a mean Cq of 27.0 (95% CI

25.2–28.7), ranging from 23.7 to 36 cycles (14 ng to 2 pg of DNA detected, respectively). *Echinococcus granulosus* s.s. was detected by the specific qPCR assay with a mean Cq of 25.7 (95% CI 22.8–28.7), ranging from 16.0 to 33.2 cycles (8.0 µg to 10 pg of DNA detected, respectively). *Echinococcus canadensis* was detected by the specific qPCR assay with a mean Cq of 22.7 (95% CI 12.9–32.5), ranging from 14.7 to 28.2 cycles (2.6 µg to 0.2 ng of DNA detected, respectively). From the qPCR inhibitor test, one CE sample showed the presence of PCR inhibitors, but was positive for the qPCR Eg cox3 (Cq = 16). For the other samples the mean Alea Cq was 34.7 cycles (95% CI 32.9–33.8).

From among the 47 FFPE samples, 45 were amplified (Table 1). *Echinococcus multilocularis* was detected by the specific qPCR assay with a mean Cq of 31.8 (95% CI 30.1–33.5), ranging from 22.9 to 39.2 cycles (25 ng to 0.2 pg of DNA detected, respectively). *Echinococcus granulosus* s.s. was detected by the specific qPCR assay with a mean Cq of 35.4 (95% CI 33.1–37.7), ranging from 27.1 to 39.9 cycles (3 ng and 0.4 pg of DNA). *Echinococcus ortleppi* was detected by the specific qPCR assay with a mean Cq of 25.8 and 34.7 cycles, (59.4 and 0.03 ng of DNA detected respectively). Two AE samples from 2011 to 2013 were not amplified and showed a Cq of approximately 41 cycles in the previous study with the rrn qPCR applied to 5 µL of extracted DNA, the other PCR techniques being negative. One CE sample presented in the previous study was not amplified and showed only a positive result for the multiplex PCR developed by Trachsel et al. [37] in the previous study. The mean Alea Cq was 34.7 (95% CI 34.4–35.0).

A Welch *t*-test was performed on the parasite qPCR Cq values obtained between fresh and FFPE samples, highlighting a significant difference (*p* < 0.001). FFPE samples with molecular confirmation first (MB) were sampled between 2012 and 2021 and FFPE samples with a pathology exam first (PATH) were sampled between 1997 and 2016. A Welch *t*-test was performed on the parasite qPCR Cq values obtained between the two groups and showed a significant difference (*p* = 0.027), with Cq values lower in samples with molecular confirmation first, being isolated more recently.

## Discussion

*Echinococcus* species typing is a key point in the diagnosis of echinococcosis in humans to differentiate alveolar from
<table><caption>**Table 3.** Method detection limits performed for each targeted parasite, with DNA dilution series tested on seven PCR assays.</caption><thead><tr><th>Species</th><th>Last dilution<br>7 PCR(+) (ng/IL)</th><th>Mean Cq<br>MDL (cycle)</th><th>SD</th><th>Y-inter</th><th>Slope</th><th>r<sup>2</sup></th><th>Efficiency (%)</th><th>n dilutions</th></tr></thead><tbody><tr><td><i>E. multilocularis</i></td><td>5 × 10<sup>-4</sup></td><td>37.97</td><td>0.84</td><td>27.37</td><td>-3.18</td><td>0.97</td><td>106.5</td><td>4</td></tr><tr><td><i>E. granulosus</i> s.s.</td><td>1 × 10<sup>-4</sup></td><td>42.21</td><td>1.00</td><td>28.76</td><td>-3.27</td><td>0.99</td><td>102.3</td><td>4</td></tr><tr><td><i>E. canadensis</i></td><td>5 × 10<sup>-4</sup></td><td>36.82</td><td>0.87</td><td>26.20</td><td>-3.36</td><td>0.99</td><td>98.3</td><td>3</td></tr><tr><td><i>E. ortleppi</i></td><td>5 × 10<sup>-4</sup></td><td>41.00</td><td>1.46</td><td>29.40</td><td>-2.67</td><td>0.99</td><td>136.9</td><td>3</td></tr><tr><td><i>Taenia solium</i></td><td>3 × 10<sup>-5</sup></td><td>37.35</td><td>0.48</td><td>22.27</td><td>-3.31</td><td>0.99</td><td>100.5</td><td>4</td></tr><tr><td><i>Toxocara cati</i></td><td>4 × 10<sup>-4</sup></td><td>36.15</td><td>0.78</td><td>23.71</td><td>-3.76</td><td>0.93</td><td>84.6</td><td>4</td></tr><tr><td><i>Toxocara canis</i></td><td>3 × 10<sup>-5</sup></td><td>38.55</td><td>0.58</td><td>21.89</td><td>-3.55</td><td>0.99</td><td>91.2</td><td>5</td></tr></tbody></table>

Cq, cycle threshold; MDL, method detection limit; SD, standard deviation; Y-inter, Y-intercept; r², correlation coefficient.

% Efficiency calculated by qPCR efficiency calculator available on ThermoFisher website.

n dilutions: number of dilution points with 7 positive qPCR used to calculate the standard curve plot.

of the tested samples of 0.6 pg to 1 fg of DNA for *Echinococcus* spp., 0.3 fg for *T. solium*, 1 pg for *T. cati*, and 0.3 fg for *T. canis*. Standard curves were plotted for each PCR assay based on seven positive replicates from the DNA concentration range to obtain the slope and Y-intercept (from three to five dilution points with seven positive results used, Table 3). The efficiency of the TaqMan qPCR assays on DNA dilutions ranged from 84.6% to 136.9%.

## Specificity of the assays

The test of specificity performed using the online NCBI Primer-BLAST software showed good matching between designed primer sequences and referenced data for *E. multilocularis* (exact matching with 94 reference sequences versus 16 with one mismatch), *E. granulosus* s.s. (exact matching with three reference sequences versus two with one mismatch), *E. ortleppi* (exact matching with seven reference sequences), *Taenia solium* (exact matching with one reference sequence versus three with two mismatches), *Toxocara* spp. (for *T. cati*, exact matching with one reference sequence versus one with one mismatch and *T. canis*, exact matching with eight reference sequences versus one with two mismatches) and *E. canadensis* (exact matching with 127 reference sequences from the G6, G7 and G10 genotypes versus two mismatches with the G8 genotype). According to the Primer-BLAST tool, the current primer pair and probe designed for *E. canadensis* should correctly amplify at least the G6, G7 and G10 genotypes.

Specificity was tested on 50 DNA samples from echinococcosis lesions (21 AE and 29 CE samples) and DNA from the larval and adult specimens used for the sensitivity assessment (Table 1). The specificity was 100% for each qPCR assay on fresh and FFPE tissues from patients, as well as for larval and adult specimens from animals.

## Tested samples and the presence of PCR inhibitors

All 81 samples were analysed using one or three MLX qPCR assays. Each DNA sample was amplified by the appropriate specific assay, in agreement with the previous molecular or pathology-based identification (Table 1).

For the 34 fresh samples, *E. multilocularis* was detected by the specific qPCR assay with a mean Cq of 27.0 (95% CI

25.2–28.7), ranging from 23.7 to 36 cycles (14 ng to 2 pg of DNA detected, respectively). *Echinococcus granulosus* s.s. was detected by the specific qPCR assay with a mean Cq of 25.7 (95% CI 22.8–28.7), ranging from 16.0 to 33.2 cycles (8.0 µg to 10 pg of DNA detected, respectively). *Echinococcus canadensis* was detected by the specific qPCR assay with a mean Cq of 22.7 (95% CI 12.9–32.5), ranging from 14.7 to 28.2 cycles (2.6 µg to 0.2 ng of DNA detected, respectively). From the qPCR inhibitor test, one CE sample showed the presence of PCR inhibitors, but was positive for the qPCR Eg cox3 (Cq = 16). For the other samples the mean Alea Cq was 34.7 cycles (95% CI 32.9–33.8).

From among the 47 FFPE samples, 45 were amplified (Table 1). *Echinococcus multilocularis* was detected by the specific qPCR assay with a mean Cq of 31.8 (95% CI 30.1–33.5), ranging from 22.9 to 39.2 cycles (25 ng to 0.2 pg of DNA detected, respectively). *Echinococcus granulosus* s.s. was detected by the specific qPCR assay with a mean Cq of 35.4 (95% CI 33.1–37.7), ranging from 27.1 to 39.9 cycles (3 ng and 0.4 pg of DNA). *Echinococcus ortleppi* was detected by the specific qPCR assay with a mean Cq of 25.8 and 34.7 cycles, (59.4 and 0.03 ng of DNA detected respectively). Two AE samples from 2011 to 2013 were not amplified and showed a Cq of approximately 41 cycles in the previous study with the rrn qPCR applied to 5 µL of extracted DNA, the other PCR techniques being negative. One CE sample presented in the previous study was not amplified and showed only a positive result for the multiplex PCR developed by Trachsel et al. [37] in the previous study. The mean Alea Cq was 34.7 (95% CI 34.4–35.0).

A Welch *t*-test was performed on the parasite qPCR Cq values obtained between fresh and FFPE samples, highlighting a significant difference (*p* < 0.001). FFPE samples with molecular confirmation first (MB) were sampled between 2012 and 2021 and FFPE samples with a pathology exam first (PATH) were sampled between 1997 and 2016. A Welch *t*-test was performed on the parasite qPCR Cq values obtained between the two groups and showed a significant difference (*p* = 0.027), with Cq values lower in samples with molecular confirmation first, being isolated more recently.

## Discussion

*Echinococcus* species typing is a key point in the diagnosis of echinococcosis in humans to differentiate alveolar from
cystic echinococcosis infections, which is essential for patient management and treatment. In this study, we developed an MLX qPCR assay able to detect the four species mainly involved in echinococcosis in Europe, and we showed that the high sensitivity of the technique enables testing with very small amounts of DNA. The MLX qPCR assay is also particularly helpful for identifying degraded DNA in paraffin-embedded samples.

In the present study, specificity was 100% for all targets, without cross-reaction between primer-pair couples used for the various parasite species. However, even though the number of *E. multilocularis* and *E. granulosus* s.s. specimens was adequate to test the specificity and sensitivity of the MLX qPCR technique, a more extensive sampling effort has to be made to conclude about performance of this assay for the detection of *E. ortleppi* and *E. canadensis*. In fact, only four fresh samples from *E. canadensis* and two FFPE from *E. ortleppi* were analysed in this study.

The MLX qPCR assays developed here show high sensitivity and allow for the detection of *Echinococcus* spp. that infect humans in Europe using a small volume of DNA extract. Due to the design of the primers and probes, targeting a small fragment of mitochondrial DNA, the assay is particularly efficient for diagnosis from FPPE, where DNA is fragmented. However, the qPCR failed for three FFPE samples. Further assays using higher DNA volumes were not attempted because of the lack of available left-DNA sample. Moreover, these samples, for which the DNA was extracted >5 years after paraffin inclusion could have become highly degraded during storage, as previously described [14, 19], and could hinder echinococcosis diagnosis, especially in cases of diagnostic wandering. An association with other PCR techniques may be necessary in such particular cases with degraded DNA, such as the multiplex PCR assay described by Trachsel et al. [37] and the Em-rm qPCR assay used with success on old samples [19].

A control for the presence of PCR inhibitors was integrated into the multiplex qPCR assays, allowing the validation of negative results. However, with high DNA concentrations, the Alea qPCR amplification can be negative, as observed in 1/56 routine samples. This could be due to consumption of the Taq polymerase during the first cycles of the qPCR for the parasite DNA amplification or the presence of inhibitors, without an impact on target parasite qPCR.

The MDL tests were performed on pure parasite DNA or with a minimum of contamination by the host DNA host. Importantly, contamination with human DNA occurs, especially in cases of AE lesions, because of the significant infiltration of the parasite into the intermediate host tissue. The DNA concentration values measured before the qPCR, e.g., by spectrophotometry, are not always informative. The sensitivity of the *Echinococcus* spp. PCR is below the picogram range of DNA for seven positive PCRs, providing the limit of parasite quantification in the tested samples. Beyond this limit, the parasite can be detected but not quantified. Moreover, the MDL can permit us to take a critical view on late amplifications, and if applicable, repeat the analyses to avoid false-positive results. We chose to apply 45 cycles to the MLX qPCR in order to permit late amplifications around 40 cycles to be completed. From the MDL tests, e.g. for *E. granulosus* s.s., the seven PCR tests remained positive

on the DNA concentration of $1 \times 10^{-4}$ ng/µL after 42 cycles of PCR. As for other infectious diseases, the positive threshold can be up to 40 cycles (example 43 cycles for *Aspergillus* spp. PCR) [41]. From quantitative results, we found significant differences based on the type of tissue conservation, i.e., fresh versus FFPE tissues, with lower Cq values obtained by qPCR for fresh than FFPE tissues, as previously observed for endpoint PCR [12]. We also observed a significant difference between recent and older FFPE samples. The storage time of FFPE samples is critical for PCR results, as previously observed, with a large decrease in PCR performance after four to five years of storage [14, 19]. Moreover, the sensitivity of the technique on FFPE samples may be linked to the macroscopic selection of the parasitic zone followed by the extraction of DNA from this zone, as in the estimation of the percentage of tumour cells presenting mutations in pathology examinations [25]. Concerning the DNA panel tested, the samples can be considered to be representative of specimens received for routine analysis in terms of the quality and concentration of parasite DNA and the presence of host DNA. Diagnosis can be made on FFPE samples included in paraffin several months or years before the analysis (data from the National Reference Centre for Echinococcoses).

The high sensitivity of the presented assays could permit the detection of parasites in other matrices, such as blood, cerebrospinal fluid, or even urine. Cell-free circulating DNA could be targeted, especially as the techniques for the diagnosis of both alveolar and cystic echinococcosis still need to be improved [2, 10, 18, 24, 36, 44] and replaced with newer approaches based on non-invasive, sensitive assays, such as those permitted by real-time qPCR.

The detection of early infections using molecular tools could allow for more efficient patient care and access to radical surgery, thus reducing the time of chemical treatment, reducing complications due to diagnostic wandering, and increasing patient quality of life. Moreover, in certain cases, imagery and serology as first-line diagnosis may not be conclusive, as observed for immunosuppressed patients [6], with co-morbidities making the diagnosis all the more difficult. Moreover, in cases of echinococcosis due to species other than *E. multilocularis* and *E. granulosus* s.s., for which the commercial immunodiagnostic kits are specifically designed, the diagnosis is even more difficult. Among the *E. granulosus* s.l. complex, *E. granulosus* s.s. associated with a dog-sheep life cycle is the most important cause of cystic echinococcosis in the world [1, 5]. However, infection by other species, such as *E. ortleppi* described in France [3, 13] and *E. canadensis* has been described (11% of CE due to *E. canadensis* G6–G7 [1]). A rapid screening test for multiple targets would be an efficient solution in cases of diagnostic wandering or the misidentification of the parasite species.

Here, we targeted cysticercosis and toxocariasis to help in the context of wandering diagnosis, as such molecular diagnosis is occasionally requested for some patients with atypical lesions (e.g. liver abscess either described in echinococcosis or toxocariasis) for immunosuppressed patients. However, no positive clinical samples were available in the present study, and only the theoretical limits of detection and the specificity could be assessed. Nevertheless, the qPCR assays targeting these species are now available for laboratories; the development of the
cystic echinococcosis infections, which is essential for patient management and treatment. In this study, we developed an MLX qPCR assay able to detect the four species mainly involved in echinococcosis in Europe, and we showed that the high sensitivity of the technique enables testing with very small amounts of DNA. The MLX qPCR assay is also particularly helpful for identifying degraded DNA in paraffin-embedded samples.

In the present study, specificity was 100% for all targets, without cross-reaction between primer-pair couples used for the various parasite species. However, even though the number of *E. multilocularis* and *E. granulosus* s.s. specimens was adequate to test the specificity and sensitivity of the MLX qPCR technique, a more extensive sampling effort has to be made to conclude about performance of this assay for the detection of *E. ortleppi* and *E. canadensis*. In fact, only four fresh samples from *E. canadensis* and two FFPE from *E. ortleppi* were analysed in this study.

The MLX qPCR assays developed here show high sensitivity and allow for the detection of *Echinococcus* spp. that infect humans in Europe using a small volume of DNA extract. Due to the design of the primers and probes, targeting a small fragment of mitochondrial DNA, the assay is particularly efficient for diagnosis from FPPE, where DNA is fragmented. However, the qPCR failed for three FFPE samples. Further assays using higher DNA volumes were not attempted because of the lack of available left-DNA sample. Moreover, these samples, for which the DNA was extracted >5 years after paraffin inclusion could have become highly degraded during storage, as previously described [14, 19], and could hinder echinococcosis diagnosis, especially in cases of diagnostic wandering. An association with other PCR techniques may be necessary in such particular cases with degraded DNA, such as the multiplex PCR assay described by Trachsel et al. [37] and the Em-rm qPCR assay used with success on old samples [19].

A control for the presence of PCR inhibitors was integrated into the multiplex qPCR assays, allowing the validation of negative results. However, with high DNA concentrations, the Alea qPCR amplification can be negative, as observed in 1/56 routine samples. This could be due to consumption of the Taq polymerase during the first cycles of the qPCR for the parasite DNA amplification or the presence of inhibitors, without an impact on target parasite qPCR.

The MDL tests were performed on pure parasite DNA or with a minimum of contamination by the host DNA host. Importantly, contamination with human DNA occurs, especially in cases of AE lesions, because of the significant infiltration of the parasite into the intermediate host tissue. The DNA concentration values measured before the qPCR, e.g., by spectrophotometry, are not always informative. The sensitivity of the *Echinococcus* spp. PCR is below the picogram range of DNA for seven positive PCRs, providing the limit of parasite quantification in the tested samples. Beyond this limit, the parasite can be detected but not quantified. Moreover, the MDL can permit us to take a critical view on late amplifications, and if applicable, repeat the analyses to avoid false-positive results. We chose to apply 45 cycles to the MLX qPCR in order to permit late amplifications around 40 cycles to be completed. From the MDL tests, e.g. for *E. granulosus* s.s., the seven PCR tests remained positive

on the DNA concentration of $1 \times 10^{-4}$ ng/µL after 42 cycles of PCR. As for other infectious diseases, the positive threshold can be up to 40 cycles (example 43 cycles for *Aspergillus* spp. PCR) [41]. From quantitative results, we found significant differences based on the type of tissue conservation, i.e., fresh versus FFPE tissues, with lower Cq values obtained by qPCR for fresh than FFPE tissues, as previously observed for endpoint PCR [12]. We also observed a significant difference between recent and older FFPE samples. The storage time of FFPE samples is critical for PCR results, as previously observed, with a large decrease in PCR performance after four to five years of storage [14, 19]. Moreover, the sensitivity of the technique on FFPE samples may be linked to the macroscopic selection of the parasitic zone followed by the extraction of DNA from this zone, as in the estimation of the percentage of tumour cells presenting mutations in pathology examinations [25]. Concerning the DNA panel tested, the samples can be considered to be representative of specimens received for routine analysis in terms of the quality and concentration of parasite DNA and the presence of host DNA. Diagnosis can be made on FFPE samples included in paraffin several months or years before the analysis (data from the National Reference Centre for Echinococcoses).

The high sensitivity of the presented assays could permit the detection of parasites in other matrices, such as blood, cerebrospinal fluid, or even urine. Cell-free circulating DNA could be targeted, especially as the techniques for the diagnosis of both alveolar and cystic echinococcosis still need to be improved [2, 10, 18, 24, 36, 44] and replaced with newer approaches based on non-invasive, sensitive assays, such as those permitted by real-time qPCR.

The detection of early infections using molecular tools could allow for more efficient patient care and access to radical surgery, thus reducing the time of chemical treatment, reducing complications due to diagnostic wandering, and increasing patient quality of life. Moreover, in certain cases, imagery and serology as first-line diagnosis may not be conclusive, as observed for immunosuppressed patients [6], with co-morbidities making the diagnosis all the more difficult. Moreover, in cases of echinococcosis due to species other than *E. multilocularis* and *E. granulosus* s.s., for which the commercial immunodiagnostic kits are specifically designed, the diagnosis is even more difficult. Among the *E. granulosus* s.l. complex, *E. granulosus* s.s. associated with a dog-sheep life cycle is the most important cause of cystic echinococcosis in the world [1, 5]. However, infection by other species, such as *E. ortleppi* described in France [3, 13] and *E. canadensis* has been described (11% of CE due to *E. canadensis* G6–G7 [1]). A rapid screening test for multiple targets would be an efficient solution in cases of diagnostic wandering or the misidentification of the parasite species.

Here, we targeted cysticercosis and toxocariasis to help in the context of wandering diagnosis, as such molecular diagnosis is occasionally requested for some patients with atypical lesions (e.g. liver abscess either described in echinococcosis or toxocariasis) for immunosuppressed patients. However, no positive clinical samples were available in the present study, and only the theoretical limits of detection and the specificity could be assessed. Nevertheless, the qPCR assays targeting these species are now available for laboratories; the development of the
protocol was made for DNA isolated from parasites (adult stage or isolated cyst, that were not or only slightly contaminated by host DNA), and additional clinical samples are needed for complete validation and evaluation in the clinical setting.

## Conclusion

The present work proposes a sensitive multi-target diagnosis on four *Echinococcus* species occurring in Europe, with PCR inhibitor control, and a substantial gain in sensitivity for FFPE specimens. The present molecular assays can be applied as an accurate screening technique in the context of clinical evaluation and imaging studies for a suspected echinococcosis infection, as well as for atypical forms and in differential diagnoses to rule out other parasites.

## Conflict of interest

None of the authors have any conflict of interest to declare.

*Acknowledgements.* We would also like to thank the Tumoroushèque Régionale de Franche-Comté (BB-0033-00024), the Pasteur Institute Paris for providing the *Taenia solium* specimens, the ELIZ Malzéville office and the CERFE Boult-aux-Bois laboratory for providing the *Toxocara* spp. specimens. This work was funded by the National Reference Centre for Echinococcoses.

## References

1. Alvarez Rojas CA, Romig T, Lightowlers MW. 2014. *Echinococcus granulosus* sensu lato genotypes infecting humans–review of current knowledge. International Journal for Parasitology 44, 9–18.

2. Baraquin A, Hervouet E, Richou C, Flori P, Peixoto P, Azizi A, Delabrousse E, Blagosklonov O, Umhang G, Bresson-Hadni S, Valot B, Grenouillet F, EchinoVista study group. 2018. Circulating cell-free DNA in patients with alveolar echinococcosis. Molecular and Biochemical Parasitology 222, 14–20.

3. Basmaciyan L, Burlet B, Ramla S, Blot M, Mahy S, Aubriot-Lorton M-H, Valot S, Grelat M, Sautour M, Grenouillet F, Knapp J, Millon L, Piroth L, Martin L, Dalle F. 2018. First case of human primary vertebral cystic echinococcosis due to *Echinococcus ortleppi*. Journal of Clinical Medicine 7, 443.

4. Can H, İnceboz T, Caner A, Atalay Şahar E, Karakavuk M, Döşkaya M, Çelebi F, Değirmenci Döşkaya A, Gülçe İz S, Gürüz Y, Korkmaz M. 2016. Detection of *Echinococcus granulosus* and *Echinococcus multilocularis* in cyst samples using a novel single tube multiplex real-time polymerase chain reaction. Mikrobiyoloji Bulteni 50, 266–277.

5. Casulli A, Massolo A, Saarma U, Umhang G, Santolamazza F, Santoro A. 2022. Species and genotypes belonging to *Echinococcus granulosus* sensu lato complex causing human cystic echinococcosis in Europe (2000–2021): a systematic review. Parasites & Vectors 15, 109.

6. Chauchet A, Grenouillet F, Knapp J, Richou C, Delabrousse E, Dentan C, Millon L, Di Martino V, Contreras R, Deconinck E, Blagosklonov O, Vuitton DA, Bresson-Hadni S, Network FrancEchino. 2014. Increased incidence and characteristics of alveolar echinococcosis in patients with immunosuppression-associated conditions. Clinical Infectious Diseases 59, 1095–1104.

7. Deibel A, Meyer Zu Schwabedissen C, Husmann L, Grimm F, Deplazes P, Reiner CS, Müllhaupt B. 2022. Characteristics and clinical course of alveolar echinococcosis in patients with immunosuppression-associated conditions: A retrospective cohort study. Pathogens 11, 441.

8. Despommier D. 2003. Toxocariasis: clinical aspects, epidemiology, medical ecology, and molecular aspects. Clinical Microbiology Reviews 16, 265–272.

9. Dinkel A, Kern S, Brinker A, Oehme R, Vaniscotte A, Giraudoux P, Mackenstedt U, Romig T. 2011. A real-time multiplex-nested PCR system for coprological diagnosis of *Echinococcus multilocularis* and host species. Parasitology Research 109, 493–498.

10. Fan H, Gai W, Zhang L, Ma Y, Wang H, Chen X, Dong J, Zhang Y, Bao H, Zhou Y, Ren L, Cairang Y, Hou L, Ren B, Wang Z, Wang Z, Song C. 2021. Parasite circulating cell-free DNA in the blood of alveolar echinococcosis patients as a diagnostic and treatment-status indicator. Clinical Infectious Diseases 73, e246–e251.

11. FAO/WHO. 2015. Multicriteria-based ranking for risk management of food-borne parasites. Rome, Italy: FAO, World Health Organization.

12. Funabashi KS, Barcelos D, Visoná I, e Silva MS, e Sousa MLAPO, de Franco MF, Iwamura ESM. 2012. DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: The effect of formalin fixation and paraffin embedding. Pathology – Research and Practice 208, 584–591.

13. Grenouillet F, Umhang G, Arbez-Gindre F, Mantion G, Delabrousse E, Millon L, Boué F. 2014. *Echinococcus ortleppi* infections in humans and cattle, France. Emerging Infectious Diseases 20, 2100–2102.

14. Grimm J, Krickl J, Beck A, Nell J, Bergmann M, Tappe D, Grüner B, Barth TF, Brehm K. 2021. Establishing and evaluation of a polymerase chain reaction for the detection of *Echinococcus multilocularis* in human tissue. PLOS Neglected Tropical Diseases 15, e0009155.

15. Guegan H, Fillaux J, Charpentier E, Robert-Gangneux F, Chauvin P, Guemas E, Boissier J, Valentin A, Cassaing S, Gangneux J-P, Berry A, Iriart X. 2019. Real-time PCR for diagnosis of imported schistosomiasis. PLoS Neglected Tropical Diseases 13, e0007711.

16. Ha KH, Song JE, Kim BS, Lee CH. 2016. Clinical characteristics and progression of liver abscess caused by toxocara. World Journal of Hepatology 8, 757–761.

17. Hospodsky D, Yamamoto N, Peccia J. 2010. Accuracy, precision, and method detection limits of quantitative PCR for airborne bacteria and fungi. Applied and Environmental Microbiology 76, 7004–7012.

18. Ji J, Li B, Li J, Danzeng W, Li J, Zhao Y, Qiangba G, Zhang Q, Renzhen N, Basang Z, Jia C, Gongsang Q, Ma J, Wang Y, Chen F, Zhou H, Huasang Yin J, Xie J, Pei N, Cai H, Jiang H, Yang H, Wang J, Asan Han X, Li J, Chen W, Yang D. 2020. Comprehensive characterization of plasma cell-free *Echinococcus* spp. DNA in echinococcosis patients using ultra-high-throughput sequencing. PLoS Neglected Tropical Diseases 14, e0008148.

19. Knapp J, Lallemand S, Monnien F, Felix S, Valmary-Degano S, Courquet S, Demonmerot F, Heyd B, Turco C, Doussot A, Bourgeois L, Bresson-Hadni S, Richou C, Millon L. 2022. Molecular diagnosis of alveolar echinococcosis in patients based on frozen and formalin-fixed paraffin-embedded tissue samples. Parasite 29, 4.

20. Knapp J, Millon L, Mouzon L, Umhang G, Raoul F, Ali ZS, Combes B, Comte S, Gbaguidi-Haore H, Grenouillet F, Giraudoux P. 2014. Real time PCR to detect the environmental faecal contamination by *Echinococcus multilocularis* from red fox stools. Veterinary Parasitology 201, 40–47.
protocol was made for DNA isolated from parasites (adult stage or isolated cyst, that were not or only slightly contaminated by host DNA), and additional clinical samples are needed for complete validation and evaluation in the clinical setting.

## Conclusion

The present work proposes a sensitive multi-target diagnosis on four *Echinococcus* species occurring in Europe, with PCR inhibitor control, and a substantial gain in sensitivity for FFPE specimens. The present molecular assays can be applied as an accurate screening technique in the context of clinical evaluation and imaging studies for a suspected echinococcosis infection, as well as for atypical forms and in differential diagnoses to rule out other parasites.

## Conflict of interest

None of the authors have any conflict of interest to declare.

*Acknowledgements.* We would also like to thank the Tumoroushèque Régionale de Franche-Comté (BB-0033-00024), the Pasteur Institute Paris for providing the *Taenia solium* specimens, the ELIZ Malzéville office and the CERFE Boult-aux-Bois laboratory for providing the *Toxocara* spp. specimens. This work was funded by the National Reference Centre for Echinococcoses.

## References

1. Alvarez Rojas CA, Romig T, Lightowlers MW. 2014. *Echinococcus granulosus* sensu lato genotypes infecting humans–review of current knowledge. International Journal for Parasitology 44, 9–18.

2. Baraquin A, Hervouet E, Richou C, Flori P, Peixoto P, Azizi A, Delabrousse E, Blagosklonov O, Umhang G, Bresson-Hadni S, Valot B, Grenouillet F, EchinoVista study group. 2018. Circulating cell-free DNA in patients with alveolar echinococcosis. Molecular and Biochemical Parasitology 222, 14–20.

3. Basmaciyan L, Burlet B, Ramla S, Blot M, Mahy S, Aubriot-Lorton M-H, Valot S, Grelat M, Sautour M, Grenouillet F, Knapp J, Millon L, Piroth L, Martin L, Dalle F. 2018. First case of human primary vertebral cystic echinococcosis due to *Echinococcus ortleppi*. Journal of Clinical Medicine 7, 443.

4. Can H, İnceboz T, Caner A, Atalay Şahar E, Karakavuk M, Döşkaya M, Çelebi F, Değirmenci Döşkaya A, Gülçe İz S, Gürüz Y, Korkmaz M. 2016. Detection of *Echinococcus granulosus* and *Echinococcus multilocularis* in cyst samples using a novel single tube multiplex real-time polymerase chain reaction. Mikrobiyoloji Bulteni 50, 266–277.

5. Casulli A, Massolo A, Saarma U, Umhang G, Santolamazza F, Santoro A. 2022. Species and genotypes belonging to *Echinococcus granulosus* sensu lato complex causing human cystic echinococcosis in Europe (2000–2021): a systematic review. Parasites & Vectors 15, 109.

6. Chauchet A, Grenouillet F, Knapp J, Richou C, Delabrousse E, Dentan C, Millon L, Di Martino V, Contreras R, Deconinck E, Blagosklonov O, Vuitton DA, Bresson-Hadni S, Network FrancEchino. 2014. Increased incidence and characteristics of alveolar echinococcosis in patients with immunosuppression-associated conditions. Clinical Infectious Diseases 59, 1095–1104.

7. Deibel A, Meyer Zu Schwabedissen C, Husmann L, Grimm F, Deplazes P, Reiner CS, Müllhaupt B. 2022. Characteristics and clinical course of alveolar echinococcosis in patients with immunosuppression-associated conditions: A retrospective cohort study. Pathogens 11, 441.

8. Despommier D. 2003. Toxocariasis: clinical aspects, epidemiology, medical ecology, and molecular aspects. Clinical Microbiology Reviews 16, 265–272.

9. Dinkel A, Kern S, Brinker A, Oehme R, Vaniscotte A, Giraudoux P, Mackenstedt U, Romig T. 2011. A real-time multiplex-nested PCR system for coprological diagnosis of *Echinococcus multilocularis* and host species. Parasitology Research 109, 493–498.

10. Fan H, Gai W, Zhang L, Ma Y, Wang H, Chen X, Dong J, Zhang Y, Bao H, Zhou Y, Ren L, Cairang Y, Hou L, Ren B, Wang Z, Wang Z, Song C. 2021. Parasite circulating cell-free DNA in the blood of alveolar echinococcosis patients as a diagnostic and treatment-status indicator. Clinical Infectious Diseases 73, e246–e251.

11. FAO/WHO. 2015. Multicriteria-based ranking for risk management of food-borne parasites. Rome, Italy: FAO, World Health Organization.

12. Funabashi KS, Barcelos D, Visoná I, e Silva MS, e Sousa MLAPO, de Franco MF, Iwamura ESM. 2012. DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: The effect of formalin fixation and paraffin embedding. Pathology – Research and Practice 208, 584–591.

13. Grenouillet F, Umhang G, Arbez-Gindre F, Mantion G, Delabrousse E, Millon L, Boué F. 2014. *Echinococcus ortleppi* infections in humans and cattle, France. Emerging Infectious Diseases 20, 2100–2102.

14. Grimm J, Krickl J, Beck A, Nell J, Bergmann M, Tappe D, Grüner B, Barth TF, Brehm K. 2021. Establishing and evaluation of a polymerase chain reaction for the detection of *Echinococcus multilocularis* in human tissue. PLOS Neglected Tropical Diseases 15, e0009155.

15. Guegan H, Fillaux J, Charpentier E, Robert-Gangneux F, Chauvin P, Guemas E, Boissier J, Valentin A, Cassaing S, Gangneux J-P, Berry A, Iriart X. 2019. Real-time PCR for diagnosis of imported schistosomiasis. PLoS Neglected Tropical Diseases 13, e0007711.

16. Ha KH, Song JE, Kim BS, Lee CH. 2016. Clinical characteristics and progression of liver abscess caused by toxocara. World Journal of Hepatology 8, 757–761.

17. Hospodsky D, Yamamoto N, Peccia J. 2010. Accuracy, precision, and method detection limits of quantitative PCR for airborne bacteria and fungi. Applied and Environmental Microbiology 76, 7004–7012.

18. Ji J, Li B, Li J, Danzeng W, Li J, Zhao Y, Qiangba G, Zhang Q, Renzhen N, Basang Z, Jia C, Gongsang Q, Ma J, Wang Y, Chen F, Zhou H, Huasang Yin J, Xie J, Pei N, Cai H, Jiang H, Yang H, Wang J, Asan Han X, Li J, Chen W, Yang D. 2020. Comprehensive characterization of plasma cell-free *Echinococcus* spp. DNA in echinococcosis patients using ultra-high-throughput sequencing. PLoS Neglected Tropical Diseases 14, e0008148.

19. Knapp J, Lallemand S, Monnien F, Felix S, Valmary-Degano S, Courquet S, Demonmerot F, Heyd B, Turco C, Doussot A, Bourgeois L, Bresson-Hadni S, Richou C, Millon L. 2022. Molecular diagnosis of alveolar echinococcosis in patients based on frozen and formalin-fixed paraffin-embedded tissue samples. Parasite 29, 4.

20. Knapp J, Millon L, Mouzon L, Umhang G, Raoul F, Ali ZS, Combes B, Comte S, Gbaguidi-Haore H, Grenouillet F, Giraudoux P. 2014. Real time PCR to detect the environmental faecal contamination by *Echinococcus multilocularis* from red fox stools. Veterinary Parasitology 201, 40–47.
21. Knapp J, Umhang G, Poulle M-L, Millon L. 2016. Development of a real-time PCR for a sensitive one-step coprodiagnosis allowing both the identification of carnivore feces and the detection of *Toxocara* spp. and *Echinococcus multilocularis*. Applied and Environmental Microbiology 82, 2950–2958.

22. Le TH, Pearson MS, Blair D, Dai N, Zhang LH, McManus DP. 2002. Complete mitochondrial genomes confirm the distinctiveness of the horse-dog and sheep-dog strains of *Echinococcus granulosus*. Parasitology 124, 97–112.

23. Li M-W, Lin R-Q, Song H-Q, Wu X-Y, Zhu X-Q. 2008. The complete mitochondrial genomes for three *Toxocara* species of human and animal health significance. BMC Genomics 9, 224.

24. Li R, Bao H, Liu C, Zhao L, Kang Y, Ge R-L, Fan H, Tang F. 2022. One-tube nested MGB Probe Real-time PCR assay for detection of *Echinococcus multilocularis* infection in plasma cell free DNA. Acta Tropica 232, 106518.

25. Luquain A, Arbez-Gindre F, Bedgedjian I, Felix S, Harimenshi J-M, Mihai I-M, Monnien F, Singeorzan C, Valmary-Degano S. 2016. Habilitation des pathologistes pour l'évaluation du pourcentage de cellules tumorales lors du contrôle morphologique avant extraction d'acides nucléiques. Annales de Pathologie 36, 268–274.

26. Nakao M, McManus DP, Schantz PM, Craig PS, Ito A. 2007. A molecular phylogeny of the genus *Echinococcus* inferred from complete mitochondrial genomes. Parasitology 134, 713–722.

27. Nakao M, Sako Y, Ito A. 2003. The mitochondrial genome of the tapeworm *Taenia solium*: a finding of the abbreviated stop codon U. Journal of Parasitology 89, 633–635.

28. Nakao M, Yokoyama N, Sako Y, Fukunaga M, Ito A. 2002. The complete mitochondrial DNA sequence of the cestode *Echinococcus multilocularis* (Cyclophyllidea: Taeniidae). Mitochondrion 1, 497–509.

29. PAHO/WHO. 2021. Guideline for preventive chemotherapy for the control of *Taenia solium* taeniasis. Pan American Health Organization.

30. Santa MA, Pastran SA, Klein C, Duignan P, Ruckstuhl K, Romig T, Massolo A. 2018. Detecting co-infections of *Echinococcus multilocularis* and *Echinococcus canadensis* in coyotes and red foxes in Alberta, Canada using real-time PCR. International Journal for Parasitology: Parasites and Wildlife 7, 111–115.

31. Silgado A, Armas M, Sánchez-Montalvá A, Goterris L, Ubals M, Temprana-Salvador J, Aparicio G, Chicharro C, Serre-Delcor N, Ferrer B, Molina I, García-Patos V, Pumarola T, Sulleiro E. 2021. Changes in the microbiological diagnosis and epidemiology of cutaneous leishmaniasis in real-time PCR era: a six-year experience in a referral center in Barcelona. PLoS Neglected Tropical Diseases 15, e0009884.

32. Temesgen TT, Robertson LJ, Tysnes KR. 2019. A novel multiplex real-time PCR for the detection of *Echinococcus multilocularis*, *Toxoplasma gondii*, and *Cyclospora cayetanensis* on berries. Food Research International 125, 108636.

33. Thompson R, McManus D. 2002. Aetiology: parasites and life-cycles, in WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern, Eckert J, Gemmell MA, Meslin F-X, Pawłowski ZS, Editors. World Organisation for Animal Health: Paris, France. p. 1–15.

34. Torgerson PR, Devleesschauwer B, Praet N, Speybroeck N, Willingham AL, Kasuga F, Rokni MB, Zhou X-N, Fèvre EM, Sripa B, Gargouri N, Fürst T, Budke CM, Carabin H, Kirk MD, Angulo FJ, Havelaar A, de Silva N. 2015. World Health Organization estimates of the global and regional disease burden of 11 foodborne parasitic diseases, 2010: A data synthesis. PLoS Medicine 12, e1001920.

35. Torgerson PR, Keller K, Magnotta M, Ragland N. 2010. The global burden of alveolar echinococcosis. PLoS Neglected Tropical Diseases 4, e722.

36. Toribio L, Santivanez S, Scott AL, Enriquez R, Sedano C, Soto-Becerra P, Garcia HH, Shiff CJ. Cysticercosis Working Group in Peru. 2020. Diagnostic urinary cfDNA detected in human cystic echinococcosis. Molecular and Biochemical Parasitology 239, 111314.

37. Trachsel D, Deplazes P, Mathis A. 2007. Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA. Parasitology, 134, 911–920.

38. Umhang G, Richomme C, Bastid V, Boucher J-M, Peytavin de Garam C, Itié-Hafez S, Danan C, Boué F. 2020. National survey and molecular diagnosis of *Echinococcus granulosus* sensu lato in livestock in France, 2012. Parasitology 147, 667–672.

39. Umhang G, Richomme C, Hormaz V, Boucher J-M, Boué F. 2014. Pigs and wild boar in Corsica harbor *Echinococcus canadensis* G6/7 at levels of concern for public health and local economy. Acta Tropica 133, 64–68.

40. Vuitton DA, McManus DP, Rogan MT, Romig T, Gottstein B, Naidich A, Tuxun T, Wen H, Menezes da Silva A, World Association of Echinococcosis. 2020. International consensus on terminology to be used in the field of echinococcoses. Parasite 27, 41.

41. White PL, Alanio A, Brown L, Cruciani M, Hagen F, Gorton R, Lackner M, Millon L, Morton CO, Rautemaa-Richardson R, Barnes RA, Donnelly JP, Loffler J, Initiative Fungal PCR. 2022. An overview of using fungal DNA for the diagnosis of invasive mycoses. Expert Review of Molecular Diagnostics 22, 169–184.

42. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. 2012. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134.

43. Zhang X, Jian Y, Guo Z, Duo H, Wei Y. 2022. Development of a triplex real-time PCR assay to detect *Echinococcus* species in canid fecal samples. Journal of Parasitology 108, 79–87.

44. Zhao Y, Shen S, Jin X, Wang W, Li J, Chen W. 2021. Cell-free DNA as a diagnostic tool for human echinococcosis. Trends in Parasitology 37, 943–946.

**Cite this article as:** Knapp J, Lallemand S, Monnien F, Felix S, Courquet S, Umhang G & Millon L. 2023. Real-time multiplex PCR for human echinococcosis and differential diagnosis. *Parasite* **30**, 3.
21. Knapp J, Umhang G, Poulle M-L, Millon L. 2016. Development of a real-time PCR for a sensitive one-step coprodiagnosis allowing both the identification of carnivore feces and the detection of *Toxocara* spp. and *Echinococcus multilocularis*. Applied and Environmental Microbiology 82, 2950–2958.

22. Le TH, Pearson MS, Blair D, Dai N, Zhang LH, McManus DP. 2002. Complete mitochondrial genomes confirm the distinctiveness of the horse-dog and sheep-dog strains of *Echinococcus granulosus*. Parasitology 124, 97–112.

23. Li M-W, Lin R-Q, Song H-Q, Wu X-Y, Zhu X-Q. 2008. The complete mitochondrial genomes for three *Toxocara* species of human and animal health significance. BMC Genomics 9, 224.

24. Li R, Bao H, Liu C, Zhao L, Kang Y, Ge R-L, Fan H, Tang F. 2022. One-tube nested MGB Probe Real-time PCR assay for detection of *Echinococcus multilocularis* infection in plasma cell free DNA. Acta Tropica 232, 106518.

25. Luquain A, Arbez-Gindre F, Bedgedjian I, Felix S, Harimenshi J-M, Mihai I-M, Monnien F, Singeorzan C, Valmary-Degano S. 2016. Habilitation des pathologistes pour l'évaluation du pourcentage de cellules tumorales lors du contrôle morphologique avant extraction d'acides nucléiques. Annales de Pathologie 36, 268–274.

26. Nakao M, McManus DP, Schantz PM, Craig PS, Ito A. 2007. A molecular phylogeny of the genus *Echinococcus* inferred from complete mitochondrial genomes. Parasitology 134, 713–722.

27. Nakao M, Sako Y, Ito A. 2003. The mitochondrial genome of the tapeworm *Taenia solium*: a finding of the abbreviated stop codon U. Journal of Parasitology 89, 633–635.

28. Nakao M, Yokoyama N, Sako Y, Fukunaga M, Ito A. 2002. The complete mitochondrial DNA sequence of the cestode *Echinococcus multilocularis* (Cyclophyllidea: Taeniidae). Mitochondrion 1, 497–509.

29. PAHO/WHO. 2021. Guideline for preventive chemotherapy for the control of *Taenia solium* taeniasis. Pan American Health Organization.

30. Santa MA, Pastran SA, Klein C, Duignan P, Ruckstuhl K, Romig T, Massolo A. 2018. Detecting co-infections of *Echinococcus multilocularis* and *Echinococcus canadensis* in coyotes and red foxes in Alberta, Canada using real-time PCR. International Journal for Parasitology: Parasites and Wildlife 7, 111–115.

31. Silgado A, Armas M, Sánchez-Montalvá A, Goterris L, Ubals M, Temprana-Salvador J, Aparicio G, Chicharro C, Serre-Delcor N, Ferrer B, Molina I, García-Patos V, Pumarola T, Sulleiro E. 2021. Changes in the microbiological diagnosis and epidemiology of cutaneous leishmaniasis in real-time PCR era: a six-year experience in a referral center in Barcelona. PLoS Neglected Tropical Diseases 15, e0009884.

32. Temesgen TT, Robertson LJ, Tysnes KR. 2019. A novel multiplex real-time PCR for the detection of *Echinococcus multilocularis*, *Toxoplasma gondii*, and *Cyclospora cayetanensis* on berries. Food Research International 125, 108636.

33. Thompson R, McManus D. 2002. Aetiology: parasites and life-cycles, in WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern, Eckert J, Gemmell MA, Meslin F-X, Pawłowski ZS, Editors. World Organisation for Animal Health: Paris, France. p. 1–15.

34. Torgerson PR, Devleesschauwer B, Praet N, Speybroeck N, Willingham AL, Kasuga F, Rokni MB, Zhou X-N, Fèvre EM, Sripa B, Gargouri N, Fürst T, Budke CM, Carabin H, Kirk MD, Angulo FJ, Havelaar A, de Silva N. 2015. World Health Organization estimates of the global and regional disease burden of 11 foodborne parasitic diseases, 2010: A data synthesis. PLoS Medicine 12, e1001920.

35. Torgerson PR, Keller K, Magnotta M, Ragland N. 2010. The global burden of alveolar echinococcosis. PLoS Neglected Tropical Diseases 4, e722.

36. Toribio L, Santivanez S, Scott AL, Enriquez R, Sedano C, Soto-Becerra P, Garcia HH, Shiff CJ, Cysticercosis Working Group in Peru. 2020. Diagnostic urinary cfDNA detected in human cystic echinococcosis. Molecular and Biochemical Parasitology 239, 111314.

37. Trachsel D, Deplazes P, Mathis A. 2007. Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA. Parasitology, 134, 911–920.

38. Umhang G, Richomme C, Bastid V, Boucher J-M, Peytavin de Garam C, Itié-Hafez S, Danan C, Boué F. 2020. National survey and molecular diagnosis of *Echinococcus granulosus* sensu lato in livestock in France, 2012. Parasitology 147, 667–672.

39. Umhang G, Richomme C, Hormaz V, Boucher J-M, Boué F. 2014. Pigs and wild boar in Corsica harbor *Echinococcus canadensis* G6/7 at levels of concern for public health and local economy. Acta Tropica 133, 64–68.

40. Vuitton DA, McManus DP, Rogan MT, Romig T, Gottstein B, Naidich A, Tuxun T, Wen H, Menezes da Silva A, World Association of Echinococcosis. 2020. International consensus on terminology to be used in the field of echinococcoses. Parasite 27, 41.

41. White PL, Alanio A, Brown L, Cruciani M, Hagen F, Gorton R, Lackner M, Millon L, Morton CO, Rautemaa-Richardson R, Barnes RA, Donnelly JP, Loffler J, Initiative Fungal PCR. 2022. An overview of using fungal DNA for the diagnosis of invasive mycoses. Expert Review of Molecular Diagnostics 22, 169–184.

42. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. 2012. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134.

43. Zhang X, Jian Y, Guo Z, Duo H, Wei Y. 2022. Development of a triplex real-time PCR assay to detect *Echinococcus* species in canid fecal samples. Journal of Parasitology 108, 79–87.

44. Zhao Y, Shen S, Jin X, Wang W, Li J, Chen W. 2021. Cell-free DNA as a diagnostic tool for human echinococcosis. Trends in Parasitology 37, 943–946.

**Cite this article as:** Knapp J, Lallemand S, Monnien F, Felix S, Courquet S, Umhang G & Millon L. 2023. Real-time multiplex PCR for human echinococcosis and differential diagnosis. *Parasite* **30**, 3.
<figure><img src="image_3.png" /></figure>

## PARASITE

An international open-access, peer-reviewed, online journal publishing high quality papers on all aspects of human and animal parasitology

Reviews, articles and short notes may be submitted. Fields include, but are not limited to: general, medical and veterinary parasitology; morphology, including ultrastructure; parasite systematics, including entomology, acarology, helminthology and protistology, and molecular analyses; molecular biology and biochemistry; immunology of parasitic diseases; host-parasite relationships; ecology and life history of parasites; epidemiology; therapeutics; new diagnostic tools.

All papers in *Parasite* are published in English. Manuscripts should have a broad interest and must not have been published or submitted elsewhere. No limit is imposed on the length of manuscripts.

**Parasite** (open-access) continues **Parasite** (print and online editions, 1994-2012) and **Annales de Parasitologie Humaine et Comparée** (1923-1993) and is the official journal of the Société Française de Parasitologie.

Editor-in-Chief:
Jean-Lou Justine, Paris

Submit your manuscript at
http://parasite.edmgr.com/
<figure><img src="image_4.png" /></figure>

PARASITE

An international open-access, peer-reviewed, online journal publishing high quality papers on all aspects of human and animal parasitology

Reviews, articles and short notes may be submitted. Fields include, but are not limited to: general, medical and veterinary parasitology; morphology, including ultrastructure; parasite systematics, including entomology, acarology, helminthology and protistology, and molecular analyses; molecular biology and biochemistry; immunology of parasitic diseases; host-parasite relationships; ecology and life history of parasites; epidemiology; therapeutics; new diagnostic tools.

All papers in *Parasite* are published in English. Manuscripts should have a broad interest and must not have been published or submitted elsewhere. No limit is imposed on the length of manuscripts.

**Parasite** (open-access) continues **Parasite** (print and online editions, 1994-2012) and **Annales de Parasitologie Humaine et Comparée** (1923-1993) and is the official journal of the Société Française de Parasitologie.

Editor-in-Chief:
Jean-Lou Justine, Paris

Submit your manuscript at
http://parasite.edmgr.com/