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ProCite
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BibTeX
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-
1.
Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, Smout M, Pairojkul C, Bhudhisawasdi V, Tesana S, Thinkamrop B, Bethony JM, Loukas A, Brindley PJ, 2007. Liver fluke induces cholangiocarcinoma. PLoS Med 4: e201.
-
2.
Belizario VY Jr, de Leon WU, Bersabe MJ, Purnomo, Baird JK, Bangs MJ, 2004. A focus of human infection by Haplorchis taichui (Trematoda: Heterophyidae) in the southern Philippines. J Parasitol 90: 1165–1169.
-
3.
Chai JY, Sohn WM, Jung BK, Yong TS, Eom KS, Min DY, Insisiengmay B, Insisiengmay S, Phommasack B, Rim HJ, 2015. Intestinal helminths recovered from humans in Xieng Khouang Province, Lao PDR with a particular note on Haplorchis pumilio infection. Korean J Parasitol 53: 439–445.
-
4.
Sayasone S, Vonghajack Y, Vanmany M, Rasphone O, Tesana S, Utzinger J, Akkhavong K, Odermatt P, 2009. Diversity of human intestinal helminthiasis in Lao PDR. Trans R Soc Trop Med Hyg 103: 247–254.
-
5.
Chai JY, Han ET, Guk SM, Shin EH, Sohn WM, Yong TS, Eom KS, Lee KH, Jeong HG, Ryang YS, Hoang EH, Phommasack B, Insisiengmay B, Lee SH, Rim HJ, 2007. High prevalence of liver and intestinal fluke infections among residents of Savannakhet Province in Laos. Korean J Parasitol 45: 213–218.
-
6.
Chai JY, Han ET, Shin EH, Sohn WM, Yong TS, Eom KS, Min DY, Um JY, Park MS, Hoang EH, Phommasack B, Insisiengmay B, Lee SH, Rim HJ, 2009. High prevalence of Haplorchis taichui, Phaneropsolus molenkampi, and other helminth infections among people in Khammouane province, Lao PDR. Korean J Parasitol 47: 243–247.
-
7.
Chai JY, Yong TS, Eom KS, Min DY, Shin EH, Banouvong V, Insisiengmay B, Insisiengmay S, Phommasack B, Rim HJ, 2010. Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR. Korean J Parasitol 48: 339–342.
-
8.
Sato M, Thaenkham U, Dekumyoy P, Waikagul J, 2009. Discrimination of O. viverrini, C. sinensis, H. pumilio and H. taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions. Acta Trop 109: 81–83.
-
9.
Duenngai K, Sithithaworn P, Rudrappa UK, Iddya K, Laha T, Stensvold CR, Strandgaard H, Johansen MV, 2008. Improvement of PCR for detection of Opisthorchis viverrini DNA in human stool samples. J Clin Microbiol 46: 366–368.
-
10.
Stensvold CR, Saijuntha W, Sithithaworn P, Wongratanacheewin S, Strandgaard H, Ornbjerg N, Johansen MV, 2006. Evaluation of PCR based coprodiagnosis of human opisthorchiasis. Acta Trop 97: 26–30.
-
11.
Wongratanacheewin S, Pumidonming W, Sermswan RW, Pipitgool V, Maleewong W, 2002. Detection of Opisthorchis viverrini in human stool specimens by PCR. J Clin Microbiol 40: 3879–3880.
-
12.
Muller B, Schmidt J, Mehlhorn H, 2007. PCR diagnosis of infections with different species of Opisthorchiidae using a rapid clean-up procedure for stool samples and specific primers. Parasitol Res 100: 905–909.
-
13.
Thaenkham U, Blair D, Nawa Y, Waikagul J, 2012. Families Opisthorchiidae and Heterophyidae: are they distinct? Parasitol Int 61: 90–93.
-
14.
Thaenkham U, Visetsuk K, Dung do T, Waikagul J, 2007. Discrimination of Opisthorchis viverrini from Haplorchis taichui using COI sequence marker. Acta Trop 103: 26–32.
-
15.
Lovis L, Mak TK, Phongluxa K, Soukhathammavong P, Sayasone S, Akkhavong K, Odermatt P, Keiser J, Felger I, 2009. PCR Diagnosis of Opisthorchis viverrini and Haplorchis taichui infections in a Lao Community in an area of endemicity and comparison of diagnostic methods for parasitological field surveys. J Clin Microbiol 47: 1517–1523.
-
16.
Arimatsu Y, Kaewkes S, Laha T, Hong SJ, Sripa B, 2012. Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP). Parasitol Int 61: 178–182.
-
17.
Kaewkong W, Intapan PM, Sanpool O, Janwan P, Thanchomnang T, Laummaunwai P, Lulitanond V, Doanh PN, Maleewong W, 2013. Molecular differentiation of Opisthorchis viverrini and Clonorchis sinensis eggs by multiplex real-time PCR with high resolution melting analysis. Korean J Parasitol 51: 689–694.
-
18.
Sanpool O, Intapan PM, Thanchomnang T, Janwan P, Lulitanond V, Doanh PN, Van Hien H, Dung do T, Maleewong W, Nawa Y, 2012. Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis. Parasitol Res 111: 89–96.
-
19.
Le TH, Van De N, Blair D, Sithithaworn P, McManus DP, 2006. Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination. Exp Parasitol 112: 109–114.
-
20.
Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP, 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22: 130–131, 134–138.
-
21.
Ririe KM, Rasmussen RP, Wittwer CT, 1997. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245: 154–160.
-
22.
Nicolas L, Milon G, Prina E, 2002. Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 51: 295–299.
-
23.
Pietila J, He Q, Oksi J, Viljanen MK, 2000. Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene. J Clin Microbiol 38: 2756–2759.
-
24.
Papin JF, Vahrson W, Dittmer DP, 2004. SYBR green-based real-time quantitative PCR assay for detection of West Nile Virus circumvents false-negative results due to strain variability. J Clin Microbiol 42: 1511–1518.
-
25.
Eom KS, Yong TS, Sohn WM, Chai JY, Min DY, Rim HJ, Jeon HK, Banouvong V, Insisiengmay B, Phommasack B, 2014. Prevalence of helminthic infections among inhabitants of Lao PDR. Korean J Parasitol 52: 51–56.
-
26.
Saijuntha W, Sithithaworn P, Wongkham S, Laha T, Chilton NB, Petney TN, Barton M, Andrews RH, 2008. Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes. Parasitology 135: 1479–1486.
-
27.
Ando K, Sithithaworn P, Nuchjungreed C, Tesana S, Srisawangwong T, Limviroj W, Chinzei Y, 2001. Nucleotide sequence of mitochondrial CO I and ribosomal ITS II genes of Opisthorchis viverrini in northeast Thailand. Southeast Asian J Trop Med Public Health 32 (Suppl 2): 17–22.
-
28.
Wilde J, Eiden J, Yolken R, 1990. Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J Clin Microbiol 28: 1300–1307.
-
29.
Abu Al-Soud W, Radstrom P, 2000. Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J Clin Microbiol 38: 4463–4470.
-
30.
Shepard GC, Lawson HL, Hawkins GA, Owen J, 2011. BsaXI/RFLP analysis of initial or selectively reamplified PCR product is unreliable in detecting the V617F mutation in JAK2. Int J Lab Hematol 33: 267–271.
-
31.
Cai XQ, Yu HQ, Li R, Yue QY, Liu GH, Bai JS, Deng Y, Qiu DY, Zhu XQ, 2014. Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis. ScientificWorldJournal 2014: 893981.
-
32.
Umesha KR, Kumar S, Parvathi A, Duenngai K, Sithithaworn P, Karunasagar I, 2008. Opisthorchis viverrini: detection by polymerase chain reaction (PCR) in human stool samples. Exp Parasitol 120: 353–356.
-
33.
Giboda M, Ditrich O, Scholz T, Viengsay T, Bouaphanh S, 1991. Human Opisthorchis and Haplorchis infections in Laos. Trans R Soc Trop Med Hyg 85: 538–540.
-
34.
Chai JY, Yong TS, Eom KS, Min DY, Jeon HK, Kim TY, Jung BK, Sisabath L, Insisiengmay B, Phommasack B, Rim HJ, 2013. Hyperendemicity of Haplorchis taichui infection among Riparian people in Saravane and Champasak province, Lao PDR. Korean J Parasitol 51: 305–311.
-
35.
Sripa B, Bethony JM, Sithithaworn P, Kaewkes S, Mairiang E, Loukas A, Mulvenna J, Laha T, Hotez PJ, Brindley PJ, 2011. Opisthorchiasis and Opisthorchis-associated cholangiocarcinoma in Thailand and Laos. Acta Trop 120 (Suppl 1): S158–S168.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 56 | 56 | 14 |
| Full Text Views | 364 | 111 | 1 |
| PDF Downloads | 167 | 42 | 0 |
Development of a PCR Assay for Diagnosing Trematode (Opisthorchis and Haplorchis) Infections in Human Stools
Pheophet Lamaningao
Pheophet Lamaningao
Department of Public Health, Kansai Medical University, Osaka, Japan.
Regenerative Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan.
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Regenerative Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan.
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Seiji Kanda
Seiji Kanda
Department of Public Health, Kansai Medical University, Osaka, Japan.
Regenerative Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan.
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Regenerative Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan.
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Sakhone Laimanivong
Sakhone Laimanivong
Center of Malariology, Parasitology, and Entomology, Ministry of Health, Vientiane, Lao People's Democratic Republic.
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Takaki Shimono
Takaki Shimono
Department of Public Health, Kansai Medical University, Osaka, Japan.
Regenerative Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan.
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Regenerative Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan.
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Andrew Waleluma Darcy
Andrew Waleluma Darcy
Department of Public Health, Kansai Medical University, Osaka, Japan.
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Amphay Phyaluanglath
Amphay Phyaluanglath
Clinical Laboratory, Mahosot Hospital, Ministry of Health, Vientiane, Lao People's Democratic Republic.
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Nobuyuki Mishima
Nobuyuki Mishima
Department of Public Health, Kansai Medical University, Osaka, Japan.
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Toshimasa Nishiyama
Toshimasa Nishiyama
Department of Public Health, Kansai Medical University, Osaka, Japan.
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We developed a combined conventional polymerase chain reaction (PCR) and real-time PCR (qPCR)-based assay for detecting and discriminating between Opisthorchis viverrini and Haplorchis taichui parasite infections. The first PCR amplifies the mitochondrial cytochrome c oxidase subunit I (COI) genes of parasites, and differential diagnosis is achieved by performing qPCR with specific primers and SYBR Green I. The detection limit of the assay was found to be 2.0 × 102 plasmid copies in a test in which a stool sample was spiked with a single egg, which is equivalent to 5 eggs per gram (EPG). The testing of 34 clinical stool samples that had been demonstrated to contain “Opisthorchis-like” eggs by microscopy showed that the novel assay exhibited a sensitivity of 100% for “Opisthorchis-like” parasitic infections, and 71% and 91% of these samples were found to be infected with O. viverrini and H. taichui, respectively. A further four parasitic infections were diagnosed in the 16 negative samples, and the microscopic findings of these samples were confirmed to be false negatives by sequencing analysis. The assay also displayed high specificity during the testing of 10 samples containing other common parasites. The fact that our qPCR SYBR Green I–based assay detected submicroscopic traces of parasitic DNA and was able to differentiate between parasites that produce eggs with similar morphologies indicates that it has a good potential for development of diagnostic application to use in areas where multiple parasites coexist.
Author Notes
Authors' addresses: Pheophet Lamaningao, Seiji Kanda, and Takaki Shimono, Department of Public Health, Kansai Medical University, Osaka, Japan, E-mails: lamaninp@hirakata.kmu.ac.jp, kandas@hirakata.kmu.ac.jp, and shimonot@hirakata.kmu.ac.jp. Sakhone Laimanivong, Center of Malariology, Parasitology, and Entomology, Ministry of Health, Vientiane, Lao PDR, E-mail: sakhone07@gmail.com. Andrew Waleluma Darcy, Nobuyuki Mishima, and Toshimasa Nishiyama, Department of Public Health, Kansai Medical University, Osaka, Japan, E-mails: darcyan@hirakata.kmu.ac.jp, mishiman@hirakata.kmu.ac.jp, and tnishi@hirakata.kmu.ac.jp. Amphay Phyaluanglath, Clinical Laboratory, Mahosot Hospital, Ministry of Health, Vientiane, Lao PDR, E-mail: amphay-phyaluanglath@hotmail.com.
ProCite
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Reference Manager
BibTeX
Zotero
EndNote
-
1.
Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, Smout M, Pairojkul C, Bhudhisawasdi V, Tesana S, Thinkamrop B, Bethony JM, Loukas A, Brindley PJ, 2007. Liver fluke induces cholangiocarcinoma. PLoS Med 4: e201.
-
2.
Belizario VY Jr, de Leon WU, Bersabe MJ, Purnomo, Baird JK, Bangs MJ, 2004. A focus of human infection by Haplorchis taichui (Trematoda: Heterophyidae) in the southern Philippines. J Parasitol 90: 1165–1169.
-
3.
Chai JY, Sohn WM, Jung BK, Yong TS, Eom KS, Min DY, Insisiengmay B, Insisiengmay S, Phommasack B, Rim HJ, 2015. Intestinal helminths recovered from humans in Xieng Khouang Province, Lao PDR with a particular note on Haplorchis pumilio infection. Korean J Parasitol 53: 439–445.
-
4.
Sayasone S, Vonghajack Y, Vanmany M, Rasphone O, Tesana S, Utzinger J, Akkhavong K, Odermatt P, 2009. Diversity of human intestinal helminthiasis in Lao PDR. Trans R Soc Trop Med Hyg 103: 247–254.
-
5.
Chai JY, Han ET, Guk SM, Shin EH, Sohn WM, Yong TS, Eom KS, Lee KH, Jeong HG, Ryang YS, Hoang EH, Phommasack B, Insisiengmay B, Lee SH, Rim HJ, 2007. High prevalence of liver and intestinal fluke infections among residents of Savannakhet Province in Laos. Korean J Parasitol 45: 213–218.
-
6.
Chai JY, Han ET, Shin EH, Sohn WM, Yong TS, Eom KS, Min DY, Um JY, Park MS, Hoang EH, Phommasack B, Insisiengmay B, Lee SH, Rim HJ, 2009. High prevalence of Haplorchis taichui, Phaneropsolus molenkampi, and other helminth infections among people in Khammouane province, Lao PDR. Korean J Parasitol 47: 243–247.
-
7.
Chai JY, Yong TS, Eom KS, Min DY, Shin EH, Banouvong V, Insisiengmay B, Insisiengmay S, Phommasack B, Rim HJ, 2010. Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR. Korean J Parasitol 48: 339–342.
-
8.
Sato M, Thaenkham U, Dekumyoy P, Waikagul J, 2009. Discrimination of O. viverrini, C. sinensis, H. pumilio and H. taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions. Acta Trop 109: 81–83.
-
9.
Duenngai K, Sithithaworn P, Rudrappa UK, Iddya K, Laha T, Stensvold CR, Strandgaard H, Johansen MV, 2008. Improvement of PCR for detection of Opisthorchis viverrini DNA in human stool samples. J Clin Microbiol 46: 366–368.
-
10.
Stensvold CR, Saijuntha W, Sithithaworn P, Wongratanacheewin S, Strandgaard H, Ornbjerg N, Johansen MV, 2006. Evaluation of PCR based coprodiagnosis of human opisthorchiasis. Acta Trop 97: 26–30.
-
11.
Wongratanacheewin S, Pumidonming W, Sermswan RW, Pipitgool V, Maleewong W, 2002. Detection of Opisthorchis viverrini in human stool specimens by PCR. J Clin Microbiol 40: 3879–3880.
-
12.
Muller B, Schmidt J, Mehlhorn H, 2007. PCR diagnosis of infections with different species of Opisthorchiidae using a rapid clean-up procedure for stool samples and specific primers. Parasitol Res 100: 905–909.
-
13.
Thaenkham U, Blair D, Nawa Y, Waikagul J, 2012. Families Opisthorchiidae and Heterophyidae: are they distinct? Parasitol Int 61: 90–93.
-
14.
Thaenkham U, Visetsuk K, Dung do T, Waikagul J, 2007. Discrimination of Opisthorchis viverrini from Haplorchis taichui using COI sequence marker. Acta Trop 103: 26–32.
-
15.
Lovis L, Mak TK, Phongluxa K, Soukhathammavong P, Sayasone S, Akkhavong K, Odermatt P, Keiser J, Felger I, 2009. PCR Diagnosis of Opisthorchis viverrini and Haplorchis taichui infections in a Lao Community in an area of endemicity and comparison of diagnostic methods for parasitological field surveys. J Clin Microbiol 47: 1517–1523.
-
16.
Arimatsu Y, Kaewkes S, Laha T, Hong SJ, Sripa B, 2012. Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP). Parasitol Int 61: 178–182.
-
17.
Kaewkong W, Intapan PM, Sanpool O, Janwan P, Thanchomnang T, Laummaunwai P, Lulitanond V, Doanh PN, Maleewong W, 2013. Molecular differentiation of Opisthorchis viverrini and Clonorchis sinensis eggs by multiplex real-time PCR with high resolution melting analysis. Korean J Parasitol 51: 689–694.
-
18.
Sanpool O, Intapan PM, Thanchomnang T, Janwan P, Lulitanond V, Doanh PN, Van Hien H, Dung do T, Maleewong W, Nawa Y, 2012. Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis. Parasitol Res 111: 89–96.
-
19.
Le TH, Van De N, Blair D, Sithithaworn P, McManus DP, 2006. Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination. Exp Parasitol 112: 109–114.
-
20.
Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP, 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22: 130–131, 134–138.
-
21.
Ririe KM, Rasmussen RP, Wittwer CT, 1997. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245: 154–160.
-
22.
Nicolas L, Milon G, Prina E, 2002. Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 51: 295–299.
-
23.
Pietila J, He Q, Oksi J, Viljanen MK, 2000. Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene. J Clin Microbiol 38: 2756–2759.
-
24.
Papin JF, Vahrson W, Dittmer DP, 2004. SYBR green-based real-time quantitative PCR assay for detection of West Nile Virus circumvents false-negative results due to strain variability. J Clin Microbiol 42: 1511–1518.
-
25.
Eom KS, Yong TS, Sohn WM, Chai JY, Min DY, Rim HJ, Jeon HK, Banouvong V, Insisiengmay B, Phommasack B, 2014. Prevalence of helminthic infections among inhabitants of Lao PDR. Korean J Parasitol 52: 51–56.
-
26.
Saijuntha W, Sithithaworn P, Wongkham S, Laha T, Chilton NB, Petney TN, Barton M, Andrews RH, 2008. Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes. Parasitology 135: 1479–1486.
-
27.
Ando K, Sithithaworn P, Nuchjungreed C, Tesana S, Srisawangwong T, Limviroj W, Chinzei Y, 2001. Nucleotide sequence of mitochondrial CO I and ribosomal ITS II genes of Opisthorchis viverrini in northeast Thailand. Southeast Asian J Trop Med Public Health 32 (Suppl 2): 17–22.
-
28.
Wilde J, Eiden J, Yolken R, 1990. Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J Clin Microbiol 28: 1300–1307.
-
29.
Abu Al-Soud W, Radstrom P, 2000. Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J Clin Microbiol 38: 4463–4470.
-
30.
Shepard GC, Lawson HL, Hawkins GA, Owen J, 2011. BsaXI/RFLP analysis of initial or selectively reamplified PCR product is unreliable in detecting the V617F mutation in JAK2. Int J Lab Hematol 33: 267–271.
-
31.
Cai XQ, Yu HQ, Li R, Yue QY, Liu GH, Bai JS, Deng Y, Qiu DY, Zhu XQ, 2014. Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis. ScientificWorldJournal 2014: 893981.
-
32.
Umesha KR, Kumar S, Parvathi A, Duenngai K, Sithithaworn P, Karunasagar I, 2008. Opisthorchis viverrini: detection by polymerase chain reaction (PCR) in human stool samples. Exp Parasitol 120: 353–356.
-
33.
Giboda M, Ditrich O, Scholz T, Viengsay T, Bouaphanh S, 1991. Human Opisthorchis and Haplorchis infections in Laos. Trans R Soc Trop Med Hyg 85: 538–540.
-
34.
Chai JY, Yong TS, Eom KS, Min DY, Jeon HK, Kim TY, Jung BK, Sisabath L, Insisiengmay B, Phommasack B, Rim HJ, 2013. Hyperendemicity of Haplorchis taichui infection among Riparian people in Saravane and Champasak province, Lao PDR. Korean J Parasitol 51: 305–311.
-
35.
Sripa B, Bethony JM, Sithithaworn P, Kaewkes S, Mairiang E, Loukas A, Mulvenna J, Laha T, Hotez PJ, Brindley PJ, 2011. Opisthorchiasis and Opisthorchis-associated cholangiocarcinoma in Thailand and Laos. Acta Trop 120 (Suppl 1): S158–S168.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 56 | 56 | 14 |
| Full Text Views | 364 | 111 | 1 |
| PDF Downloads | 167 | 42 | 0 |