한국인 환자에서 장관 감염성 원충의 검출을 위한 BD MAX Enteric Parasite Panel과 Seegene Allplex Gastrointestinal Parasite Assay의 비교 평가
Detection of Intestinal Protozoa in Korean Patients Using BD MAX Enteric Parasite Panel and Seegene Allplex Gastrointestinal Parasite Assay
1연세대학교 의과대학 진단검사의학교실
2연세대학과 의과대학 진단검사의학교실 세균내성연구소
4가천대 길병원 진단검사의학과
5연세대학교 대학원 의학과
1Department of Laboratory Medicine, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
2Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Seoul, Korea
3Armed Force Yangju Hospital, Gyeonggi-do, Korea
4Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea
5Department of Medicine, The Graduate School, Yonsei University, Seoul, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Lab Med Online 2020; 10(3): 221-226
Published July 1, 2020
Copyright © The Korean Society for Laboratory Medicine.
방법: 저자들은 295개의 대변 검체들을 EPP와 AGPA를 사용하여 검사하였고 이 중 양성 결과를 확인하기 위해 일반 PCR 및 nested PCR을 이용하여 재검하였다. EPP 또는 AGPA의 결과 및 일반 PCR, Nested PCR에서 동일한 결과를 진양성으로 간주하였다.
결과: 총 295개의 대변 검체에서 17개의 B. hominis 및 2개의 E. histolytica가 검출되었다. EPP는 검출 가능한 원충류 종류로 인한 한계로 비록 2개의 원충만 검출하였으나 이는 100% (2/2) 진양성이었다. AGPA는 총 24개의 검체에서 원충류를 검출하였으나 이 중 진양성 및 위양성은 각각 79.2% (19/24), 20.8% (5/24)였다.
결론: 원충류 감염, 특히 B. hominis의 감염은 예상보다 많을 수 있다. 따라서 AGPA는 원충류 감염의 스크리닝 목적으로 사용될 수 있을 것으로 판단된다.
Methods: We investigated 295 fecal samples using EPP and AGPA. Then we confirmed the positive results with the conventional and nested PCR. Consistent detection by conventional PCR, nested PCR, and one of the multiplex panels was considered “true positive.”
Results: Out of 295 samples, 17 were true positives for B. hominis and 2 were true positives for E. histolytica. EPP detected parasites in only two samples owing to its design; however, its true positive detection rate was 100% (2/2). AGPA detected parasites in 24 samples with 79.2% (19/24) true positives.
Conclusions: The incidence of protozoan, especially B. hominis, infection may be more prevalent than expected. AGPA could be an effective tool for screening protozoan infections.
Intestinal parasite infection is one of the main causes of gastrointestinal diseases ; however, parasitic infections in affected patients have been underreported compared to bacterial or viral infections . Intestinal protozoan parasites cause various gastrointestinal symptoms, ranging from asymptomatic to life-threatening watery or hemorrhagic diarrhea [3, 4]. Most laboratories use the microscopic ova and parasite examination for stool parasite testing. Usually direct smear microscopy, concentration techniques , or special stains such as Modified Fields’ stain, Giemsa stain, or iron hematoxylin stain are needed to detect protozoans ; however, these techniques are labor-intensive and require well-trained and highly skilled technicians for optimal interpretation. Furthermore, protozoan parasites are difficult to identify especially when they are present in low numbers; therefore, microscopic examination is not effective for accurate detection of the parasites due to low diagnostic sensitivity and specificity [3, 7-10]. Hence, molecular techniques involving polymerase chain reaction (PCR) have been developed for detecting protozoa to satisfy the need for unbiased and rapid analytical methods with high sensitivity and specificity [11, 12].
In this study, we compared EPP and AGPA to determine whether any of these assays could become a useful tool for detecting intestinal protozoan infections from fecal samples in Korean laboratory settings.
MATERIALS AND METHODS
1. Sample collection
A total of 295 fecal samples, including both loose and formed stool, from patients with symptoms of gastroenteritis or patients visiting for regular health check-up were collected from September 2017 to May 2018. Most of the 264 fecal samples from patients with gastroenteritis were not examined for intestinal parasites but were tested for bacterial or viral infections. The samples were stored at -80°C until used for DNA extraction. This study was approved by the Institutional Review Board of the Severance Hospital (IRB no. 4-2016-0946).
2. DNA extraction
Genomic DNA from 295 fecal samples was extracted using the QIAamp DNA mini kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. The DNA samples were stored at -20°C until tested.
3. Parasites detection using multiplex real-time PCR
The fecal samples were processed using fully automated real-time PCR systems; EPP designed to detect
4. Confirming positive results of EPP and AGPA through conventional and nested PCRs
The conventional and nested PCRs were designed to confirm the positive results of EPP and AGPA, and the primers used are shown in Table 1. We originally designed the first primer for
As shown in Table 2, EPP detected two
The 24 positive samples were re-tested using laboratory-designed conventional and nested PCR methods (Table 2). Overall, 19 out of 295 samples were positive, including 17
In this study, we compared the performance of two commercial multiplex panels in detecting intestinal protozoa using clinical stool samples (N=295) and confirmed the results with the conventional and nested PCRs. Many studies from outside of Asia have already approved the detection ability of EPP to be highly sensitive and specific [10, 13, 14]. In this study, EPP effectively detected
Despite the high sensitivity of EPP, AGPA could detect parasites from more fecal samples in this study since it is designed to detect
In this study, three
Therefore, despite EPP’s detection ability, it may not be the right choice for detecting intestinal parasites in Korean clinical setting; however, AGPA could possibly be used for screening purposes since it can help the laboratory to detect
Meanwhile, owing to the lack of prevalent parasites in Korea for both assays, complementary microscopic examination would be needed to increase the detection rate of parasites. Ironically, however, low prevalence of parasites makes it more difficult to create new detection methods. It is the reason why AGPA could not receive approval from the South Korea Ministry of Food and Drug Safety (MFDS), and the development of new multiplex PCR to detect prevalent parasites such as
We appreciate Dr. Eun Jeong Won and her colleagues from Chonnam University and faculties from Kyungsang University for supporting this study.
- Fletcher SM, Stark D, Harkness J, Ellis J. Enteric protozoa in the developed world: a public health perspective. Clin Microbiol Rev 2012;25:420-49.
- Won EJ, Kim SH, Kee SJ, Shin JH, Suh SP, Chai JY, et al. Multiplex real-time PCR assay targeting eight parasites customized to the Korean population: Potential use for detection in diarrheal stool samples from gastroenteritis patients. PLoS One 2016;11:e0166957.
- Autier B, Belaz S, Razakandrainibe R, Gangneux JP, Robert-Gangneux F. Comparison of three commercial multiplex PCR assays for the diagnosis of intestinal protozoa. Parasite 2018;25:48.
- Ocaña-Losada C, Cuenca-Gómez JA, Cabezas-Fernández MT, Vázquez-Villegas J, Soriano-Pérez MJ, Cabeza-Barrera I, et al. Clinical and epidemiological characteristics of intestinal parasite infection by
Blastocystis hominis. Rev Clin Esp 2018;218:115-20.
- Mergani MH, Mohammed MA, Khan N, Bano M, Khan AH. Detection of intestinal protozoa by using different methods. Dent Med Res 2014;2:28-32.
- Ragavan AD, Govind SK. Modified fields' stain: ideal to differentiate
Dientamoeba fragilisand Blastocystissp. Parasitol Res 2015;114:1163-6.
- McHardy IH, Wu M, Shimizu-Cohen R, Couturier MR, Humphries RM. Detection of intestinal protozoa in the clinical laboratory. J Clin Microbiol 2014;52:712-20.
- Van den Bossche D, Cnops L, Verschueren J, Van Esbroeck M. Comparison of four rapid diagnostic tests, ELISA, microscopy and PCR for the detection of
Giardia lamblia, Cryptosporidiumspp. and Entamoeba histolyticain feces. J Microbiol Methods 2015;110:78-84.
- Laude A, Valot S, Desoubeaux G, Argy N, Nourrisson C, Pomares C, et al. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identi cation of
Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominisand Entamoeba histolyticafrom stool samples? Evaluation of a new commercial multiplex PCR assay and literature review. Clin Microbiol Infect 2016;22:190.e1-8.
- Madison-Antenucci S, Relich RF, Doyle L, Espina N, Fuller D, Karchmer T, et al. Multicenter evaluation of BD Max Enteric Parasite Real-Time PCR Assay for detection of
Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica. J Clin Microbiol 2016;54:2681-8.
- Verweij JJ. Application of PCR-based methods for diagnosis of intestinal parasitic infections in the clinical laboratory. Parasitology 2014;141:1863-72.
- Adeyemo FE, Singh G, Reddy P, Stenström TA. Methods for the detection of
Cryptosporidium and Giardia: From microscopy to nucleic acid based tools in clinical and environmental regimes. Acta Trop 2018;184:15-28.
- Parčina M, Reiter-Owona I, Mockenhaupt FP, Vojvoda V, Gahutu JB, Hoerauf A, et al. Highly sensitive and specific detection of
Giardia duodenalis, Entamoeba histolytica, and Cryptosporidiumspp. in human stool samples by the BD MAX™ Enteric Parasite Panel. Parasitol Res 2018;117:447-51.
- Perry MD, Corden SA, Lewis White P. Evaluation of the BD MAX Enteric Parasite Panel for the detection of
Cryptosporidium parvum/hominis, Giardia duodenalisand Entamoeba histolytica. J Med Microbiol 2017;66:1118-23.
- Yoo J, Park J, Lee HK, Yu JK, Lee GD, Park KG, et al. Comparative evaluation of Seegene Allplex Gastrointestinal, Luminex xTAG Gastrointestinal Pathogen Panel, and BD MAX Enteric Assays for detection of gastrointestinal pathogens in clinical stool specimens. Arch Pathol Lab Med 2019;143:999-1005.
- Tan KS. New insights on classification, identification, and clinical relevance of
Blastocystisspp. Clin Microbiol Rev 2008;21:639-65.
- Roberts T, Stark D, Harkness J, Ellis J. Subtype distribution of
Blastocystisisolates from a variety of animals from New South Wales, Australia. Vet Parasitol 2013;196:85-9.
- Clark CG. Cryptic genetic variation in parasitic protozoa. J Med Microbiol 2000;49:489-91.
- Stenzel DJ, Boreham PF.
Blastocystis hominisrevisited. Clin Microbiol Rev 1996;9:563-84.
- Rajah Salim H, Suresh Kumar G, Vellayan S, Mak JW, Khairul Anuar A, Init I, et al.
Blastocystisin animal handlers. Parasitol Res 1999;85:1032-3.
- Kurt Ö, Doğruman Al F, Tanyüksel M. Eradication of
Blastocystisin humans: Really necessary for all? Parasitol Int 2016;65:797-801.
- Craven LJ, Nair Parvathy S, Tat-Ko J, Burton JP, Silverman MS. Extended screening costs associated with selecting donors for Fecal Microbiota Transplantation for treatment of metabolic syndrome-associated diseases. Open Forum Infect Dis 2017;4:ofx243.
- Zhang X, Qiao J, Wu X, Da R, Zhao L, Wei Z. In vitro culture of
Blastocystis hominisin three liquid media and its usefulness in the diagnosis of blastocystosis. Int J Infect Dis 2012;16:e23-8.