Detection of Intestinal Parasites With Automated Sedimax2®. A New Step in the Parasitological Diagnosis in High Throughput Laboratories.

Despite the low prevalence of parasitic infections in Europe, the diagnosis of intestinal parasites is dicult and laborious for microbiology laboratories. Currently, the antigens detection kits and the molecular biology have allowed an easier diagnosis. But these techniques have also limitations due to the fact that they do not detect all possible parasites presents in the samples. The objective of the study was to evaluate the accuracy and the usefulness of SediMAX2® (77 Elektronika, Budapest, Hungary) automated microscopy in the detection of parasitic structures from feces. A total of 197 formol-xed stool samples were processed in parallel with wet mount examination and by SediMAX2®. Sensitivities, specicities and predictive values were analyzed, reaching sensitivity of 89.51% and specicity of 98.15%. Predictive values were also calculated with a very good positive predictive value (99.22%). SediMAX2® is a good tool for a reliable diagnosis of intestinal parasitic infections. The easiness of use, processing and the exibility in the images analyse allows its incorporation in the day to day laboratory work as an extra step for the parasitologists workload.


Introduction
Parasitic diagnose still relies on microscopic examination although the limitations of these methods. Improving the parasitic diagnose is mandatory and combined with clinical symptoms, clinical and travel history, and geographic location of patient may help, especially in non endemic countries but there is still a lack. Despite the low prevalence of parasitic infections in Europe, the diagnosis of intestinal parasites is di cult and laborious for microbiology laboratories. In recent years, there has been a tremendous effort to focus the research on the development of newer diagnostic methods focusing on serological, molecular, and proteomic approaches (Ricciardi and Ndao, 2015) but these techniques have also limitations due to the fact that they do not detect all possible parasites, they are expensive and no available for all microbiology laboratories. Molecular tools have emerged to be the solution in a lot of infectious diseases (Yang and Rothman, 2004) and several targets have been developed but, more of them are homemade and not standardized.
Especially remarkable is the development of the loop-mediated isothermal ampli cation (LAMP) (Notomi et al., 2015) that has attracted much attention in the eld of parasitology. Several in house methods have been evaluated for different parasitic infectious (Fernández-Soto P et al., 2014; Mugambi et al., 2015), but is in the malaria diagnose in which this technology has represented the trigger for the development of new and available diagnostic tools showing best results than other point-of-care tests (De Koninck et al., 2017). In this case, there is the only commercial LAMP tool available in the market. The future of the malaria diagnosis has also changed with the incorporation of a new microscopic way to perform its detection. Parasight is an enhanced computer vision device for the diagnosis of malaria (Eshel et al., 2017) and it is able to novel malaria test system that is able to provide highly sensitive malaria evaluations faster and more accurate than current malaria tests. This is a big step in the evolution of the classical microscopic diagnose and leads to apply the technology to other pasitological areas.
In 2015, a group of Italian researchers published an interesting article which evaluate the accuracy of an autoanalyzer that it is used for the diagnosis of urinary tract infection, the sediMAX 1 (77 Elektronika, Budapest, Hungary) (Intra et al., 2016), and recently they have also improved the detection of protozoos with the new version of the SediMAX2® (77 Elektronika, Budapest, Hungary) (Intra et al., 2017).
Digitalized microscopy is already used in pathology departments with the advent of Whole-Slide Imaging.
SediMAX2® could be the rst step in the parasitological virtual microscopy.
We aimed to evaluate the accuracy and the usefulness of the SediMAX2® for the diagnosis of intestinal parasitic infections from formalin-xed stools and the usefulness in a high throughput laboratory.

Study design
This was a prospective cross-sectional observational study designed for the clinical evaluation of SediMAX2® compared with microscopy for the routine diagnosis of gastrointestinal parasitic infections at the Microbiology Department of the Germans Trias i Pujol University Hospital (HUGTiP, Badalona, Spain). The study was approved by the Ethics Committee at our institution. The need for informed consent was waived.

Study population and clinical samples
During the period January 2016-to December 2017 a total of 197 fecal samples from patients with suspected intestinal parasitic infection xed with sodium acetate-acetic acid-formalin (SAF) (Universal System 50 ml con SAF, Durviz, Valencia, Spain) and stored in the Microbiology Department of the HUGTiP were studied. Stools received from Monday to Friday during the morning schedule and processed at our hospital's clinical microbiology laboratory were included in this study. These samples were from patients with suspected parasitic infections in two different populations: (i) patients at the emergency department or hospitalized at HUGTiP and (ii) patients at primary care centers.

Stool samples processing
The samples were rst processed by microscopic ova and parasite examination. Firstly xed stools were diluted with 3 milliliters of ethyl acetate and the ltered and centrifuged 500 g during 5 minutes.
Then, the samples were analyzed using the automatic microscopy sediment analyzer, SediMAX2®. This autoanalyzer homogenizes and transfers 20 microliters the samples into special disposable cuvettes, which are centrifuged for a few seconds. Afterwards, whole-eld high de nition images are obtained. All images are stored on the computer, to be reviewed for a second independent reader. For the process in SediMAX2®, once the xed feces were concentrated, sediment was subsequently diluted with saline solution (1:20) for later analysis in SediMAX2®2 described by Intra et al (8). Subsequently, high de nition images of the different photographed elds will be obtained and evaluated. For each sample, a triple analysis will be carried out with SediMAX2®2, for which a total of 60 images will be reviewed.

Statistical analysis
The correlation of the results was evaluated using the Kappa coe cient (κ; CI 95%). Sensitivity (Se), speci city (Sp), positive and negative predictive values (PPV and NPV) for SediMAX2® were also calculated using openepi software, www.openepi.com (Emory University. Atlanta. USA).

Ethic statement.
Ethical approval from the Ethics Committee Research of Germans Trias i Pujol University Hospital Ethics Committee was obtained (PI-17-232) and the need for informed consent was waived.  In all samples 60 images were processed and reviewed but in 101 of the 143 positive samples (Fig. 1), the detection of the parasite was performed only with 20 images, reducing the time consuming problems of the parasitic diagnosis. In 18 cases 40 images were needed and in 23; 60 images were reviewed for a correct diagnosis. Additionally, a squared 15x15 micrometers was installed to allow the measure of the structures for a correct parasitological evaluation. Despite these results the evaluation of all the images performed is mandatory for a good diagnosis.

Discussion
Intestinal parasitic infections are a reality, and it is of great importance to nd the correct way to diagnose them to avoid further disease transmission and chronic illnesses. Although for many years, parasitic Although growth in international travel and migration from endemic areas, in our settings G. lamblia and E.vermicularis are still the most prevalent pathogens. In high throughput laboratories, tools for detection of these parasites are mandatory. The results obtained for the G. lamblia diagnosis suggest that SediMAX2® could be a good tool and it can be implemented for the detection of this protozoon (SE, SP, PPV and NPV for G. lamblia; 89,29%; 98,15%; 98,68%; 85,48%, respectively). Out of the 84 samples positives for G. lamblia, in 9 cases SediMAX2® was not able to detect them. In the other hand, it is important to remember that a negative result does not rule out parasitic infection because several parasites (particularly G. lamblia) have an intermittent shedding (CLSI, 2005) and the probability of parasite detection increases more than 95% when 3 stools are tested (Marti and Koella, 1993), so a serial parastilogical studies are still needed to con rm a high suspicion of G. lamblia infection in case of a previous negative result. For the rest of protozoa SediMAX2® was able to detect the protozoa with a pathogenic role, but only 4 E.hystolitica/dispar were included in the study.
In case of worm infections, all eggs were properly identi ed with the exception of E. vermicularis in all three cases, 2 A. lumbricoides and 1 case of S. stercoralis larvae. In the rest 15 worm infections from 13 patients, all eggs were detected. The discrepancies in the worm infections must be explained by the fact that in case of E. vermicularis, stool wet mount examination is not the recommended tool for the diagnosis and, even the presence in the microscopic mount, a tape slide-test must be sent to the laboratory to a correct diagnosis. For S. stercoralis, it is known that the visualization of rhabditiform larvae in stools in not always possible and complemented techniques (parasitological or serology-based test) is needed. Diagnosis of S.stercoralis, is often delayed due the presence of subclinical or poorlysymptomatic cases and the usually low parasite load and irregular larvae output. This fact makes that this worm is also known as there is but not seen (Montes et al., (2010).
The most remarkable fact in this automatic microscopic system is the reducing in hand and microscope time. With the possibility of a big storage of images, this system could reduce the time in microscope with a high positive predictive value.
Parasitic diagnosis is heavy workload and relies exclusively on the experience of the trained technicians. Thus, it is di cult to maintain enough people with expertise in diagnostic medical parasitology. A recognized image system based in the same principle that urines must be developed by the biomedical engineering to provide new tools in case of intestinal parasitic infections. Additionally, the improvements in the sample process to avoid detritus that can di cult the interpretation of the images would help to better parasitological diagnose.
This study is a rst step to implement engineering with medical practice to help medical microbiologist diagnose their patients. There is still an opportunity for improvement, especially with high throughput laboratories and when diagnosis is almost exclusively manual. A two-step algorithm including antigen detection and digital microscopic could be useful to help parasitologists in their day-to-day workload.
This study was partially funded by Menarini, S.A., distributor of SediMAX2® in Spain. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Declarations
Ethics approval and consent to participate The study was approved by the Ethics Committee at our institution (PI-17-232). The need for informed consent was waived.