c4f03aafa0b5123a3a5709962d8e4532 pmc_9927355.pdf 414081ee71b1a291129bd5d47278e460d8c65240 pmc_9927355.pdf 0810d000cc1ef12a0410cac5665dc380b4552207b2f1d3f96bcefd0a3c929dd9 pmc_9927355.pdf Title: Usefulness of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry in the Characterization of Leishmania Strains Causing Tegumentary Leishmaniasis in Bolivia versus hsp70 Gene Sequencing Subject: Research Article Keywords: Leishmania; characterization; MALDI-TOF MS; hsp70 gene sequencing; tegumentary leishmaniasis; Bolivia Author: Mary Cruz Torrico;Anna Fernández-Arévalo;Cristina Ballart;Marco Solano;Ernesto Rojas;Alba Abras;Fabiola Gonzales;Albert Arnau;Silvia Tebar;Teresa Llovet;Daniel Lozano;Eva Ariza-Vioque;Joaquim Gascón;Albert Picado;Faustino Torrico;Carmen Muñoz;Montserrat Gállego Creator: PTC Arbortext Advanced Print Publisher 11.1.4012/W Producer: Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version) CreationDate: Mon Jan 30 04:50:07 2023 CET ModDate: Wed Feb 1 16:06:12 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 9 Encrypted: no Page size: 584.957 x 782.986 pts Page rot: 0 File size: 611514 bytes Optimized: no PDF version: 1.4 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- LODLNC+AdvOTce3d9a73 Type 1C WinAnsi yes yes no 82 0 LODLND+AdvOT56309c18.I Type 1C WinAnsi yes yes no 83 0 LODLPD+AdvOT07517017 Type 1C WinAnsi yes yes no 366 0 LODLPE+AdvOT07517017+20 Type 1C WinAnsi yes yes no 367 0 LODLPF+AdvOT3b30f6db.B Type 1C WinAnsi yes yes no 33 0 LODLPG+AdvOT46dcae81 Type 1C WinAnsi yes yes no 34 0 LODLPH+AdvOT46dcae81+fb Type 1C Custom yes yes yes 35 0 LODMAH+AdvOT65f8a23b.I Type 1C WinAnsi yes yes no 36 0 LODMAI+AdvOT07517017+fb Type 1C Custom yes yes yes 368 0 LODMCJ+AdvOT46dcae81+20 Type 1C WinAnsi yes yes no 37 0 LODMCK+AdvOTd0125be5.BI Type 1C WinAnsi yes yes no 38 0 LODMPF+AdvP80675 Type 1C WinAnsi yes yes no 145 0 LODMPG+AdvP4C4E74 Type 1C Custom yes yes no 157 0 LODMPH+AdvP7DA6 Type 1C WinAnsi yes yes no 148 0 LODMPI+AdvP4C4E51 Type 1C WinAnsi yes yes no 151 0 LODMPJ+AdvOT8817665d Type 1C WinAnsi yes yes no 154 0 LODNBK+AdvOT46dcae81+01 Type 1C WinAnsi yes yes no 201 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-01-11 02:29:36 CET RepresentationInformation: pmc_9927355.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-23 02:12:04 CET Size: 611514 Format: PDF Version: 1.4 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 410 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: true NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: RESULTS Destination: flink1 Children: Item: Title: Characterization via hsp70 gene sequencing. Destination: slink1 Item: Title: MALDI-TOF MS. Destination: slink2 Item: Title: DISCUSSION Destination: flink2 Item: Title: MATERIALS AND METHODS Destination: flink3 Children: Item: Title: Leishmania isolates. Destination: slink3 Item: Title: hsp70 gene sequencing. Destination: slink4 Item: Title: MALDI-TOF MS. Destination: slink5 Item: Title: Ethical aspects. Destination: slink6 Item: Title: Data availability. Destination: slink7 Item: Title: ACKNOWLEDGMENTS Destination: flink4 Item: Title: REFERENCES Destination: flink5 Info: Title: Usefulness of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry in the Characterization of Leishmania Strains Causing Tegumentary Leishmaniasis in Bolivia versus hsp70 Gene Sequencing Author: Mary Cruz Torrico;Anna Fernández-Arévalo;Cristina Ballart;Marco Solano;Ernesto Rojas;Alba Abras;Fabiola Gonzales;Albert Arnau;Silvia Tebar;Teresa Llovet;Daniel Lozano;Eva Ariza-Vioque;Joaquim Gascón;Albert Picado;Faustino Torrico;Carmen Muñoz;Montserrat Gállego Subject: Research Article Keywords: Leishmania; characterization; MALDI-TOF MS; hsp70 gene sequencing; tegumentary leishmaniasis; Bolivia Creator: PTC Arbortext Advanced Print Publisher 11.1.4012/W Producer: Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version) CreationDate: Mon Jan 30 05:20:07 CET 2023 ModDate: Wed Feb 01 16:36:12 CET 2023 ID: 0x3fc544079936b518299e1c066b802ae3, 0x72dc7c26557220577563f6b542b2dcf1 Filters: FilterPipeline: FlateDecode Images: Image: NisoImageMetadata: FormatName: image/png ImageWidth: 67 ImageHeight: 67 BitsPerSample: 8 BitsPerSampleUnit: integer Filter: FlateDecode Fonts: Type1: Font: BaseFont: LODLPF+AdvOT3b30f6db.B FontSubset: true FirstChar: 45 LastChar: 122 FontDescriptor: FontName: LODLPF+AdvOT3b30f6db.B Flags: Nonsymbolic FontBBox: -39, -250, 875, 897 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODLPG+AdvOT46dcae81 FontSubset: true FirstChar: 37 LastChar: 253 FontDescriptor: FontName: LODLPG+AdvOT46dcae81 Flags: Nonsymbolic FontBBox: -46, -250, 838, 879 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODLPH+AdvOT46dcae81+fb FontSubset: true FirstChar: 1 LastChar: 3 FontDescriptor: FontName: LODLPH+AdvOT46dcae81+fb Flags: Symbolic FontBBox: 0, 0, 742, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: LODMAH+AdvOT65f8a23b.I FontSubset: true FirstChar: 40 LastChar: 122 FontDescriptor: FontName: LODMAH+AdvOT65f8a23b.I Flags: Nonsymbolic FontBBox: -112, -250, 894, 873 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODMCJ+AdvOT46dcae81+20 FontSubset: true FirstChar: 146 LastChar: 150 FontDescriptor: FontName: LODMCJ+AdvOT46dcae81+20 Flags: Nonsymbolic FontBBox: -9, -205, 1126, 868 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODMCK+AdvOTd0125be5.BI FontSubset: true FirstChar: 48 LastChar: 115 FontDescriptor: FontName: LODMCK+AdvOTd0125be5.BI Flags: Nonsymbolic FontBBox: -95, -250, 930, 889 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODNBK+AdvOT46dcae81+01 FontSubset: true FirstChar: 138 LastChar: 154 FontDescriptor: FontName: LODNBK+AdvOT46dcae81+01 Flags: Nonsymbolic FontBBox: -46, -227, 857, 873 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODLPD+AdvOT07517017 FontSubset: true FirstChar: 40 LastChar: 243 FontDescriptor: FontName: LODLPD+AdvOT07517017 Flags: Nonsymbolic FontBBox: -49, -250, 816, 867 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODLPE+AdvOT07517017+20 FontSubset: true FirstChar: 146 LastChar: 146 FontDescriptor: FontName: LODLPE+AdvOT07517017+20 Flags: Nonsymbolic FontBBox: -6, -209, 1046, 868 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODMAI+AdvOT07517017+fb FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: LODMAI+AdvOT07517017+fb Flags: Symbolic FontBBox: 0, 0, 683, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: LODMPF+AdvP80675 FontSubset: true FirstChar: 44 LastChar: 50 FontDescriptor: FontName: LODMPF+AdvP80675 Flags: Nonsymbolic FontBBox: -20, -229, 1010, 812 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODLNC+AdvOTce3d9a73 FontSubset: true FirstChar: 40 LastChar: 243 FontDescriptor: FontName: LODLNC+AdvOTce3d9a73 Flags: Nonsymbolic FontBBox: -42, -250, 858, 889 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODLND+AdvOT56309c18.I FontSubset: true FirstChar: 48 LastChar: 115 FontDescriptor: FontName: LODLND+AdvOT56309c18.I Flags: Nonsymbolic FontBBox: -103, -250, 914, 882 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODMPH+AdvP7DA6 FontSubset: true FirstChar: 97 LastChar: 109 FontDescriptor: FontName: LODMPH+AdvP7DA6 Flags: Nonsymbolic FontBBox: -20, -208, 1000, 770 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODMPI+AdvP4C4E51 FontSubset: true FirstChar: 62 LastChar: 62 FontDescriptor: FontName: LODMPI+AdvP4C4E51 Flags: Nonsymbolic FontBBox: -31, -239, 1041, 750 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODMPJ+AdvOT8817665d FontSubset: true FirstChar: 35 LastChar: 35 FontDescriptor: FontName: LODMPJ+AdvOT8817665d Flags: Nonsymbolic FontBBox: -30, -210, 1000, 779 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: LODMPG+AdvP4C4E74 FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: LODMPG+AdvP4C4E74 Flags: Symbolic FontBBox: -20, -958, 1114, 770 FontFile3: true EncodingDictionary: Differences: true XMP: Usefulness of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry in the Characterization of Leishmania Strains Causing Tegumentary Leishmaniasis in Bolivia versus hsp70 Gene Sequencing 10.1128/spectrum.03477-22 Mary Cruz Torrico Anna Fernández-Arévalo Cristina Ballart Marco Solano Ernesto Rojas Alba Abras Fabiola Gonzales Albert Arnau Silvia Tebar Teresa Llovet Daniel Lozano Eva Ariza-Vioque Joaquim Gascón Albert Picado Faustino Torrico Carmen Muñoz Montserrat Gállego Research Article Research Article Leishmania characterization MALDI-TOF MS hsp70 gene sequencing tegumentary leishmaniasis Bolivia American Society for Microbiology Torrico et al. Copyright © 2023 Torrico et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. 20230112 2023 ABSTRACT Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a proteomic technique with proven efficiency in the identification of microorganisms, such as bacteria, fungi, and parasites. The present study aimed to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia using hsp70 gene sequencing as a reference technique. 55 Leishmania strains that were isolated from patients with tegumentary leishmaniasis were analyzed. MALDI-TOF MS identified two species of the L. braziliensis complex (L. braziliensis, n = 26; L. braziliensis outlier, n = 18), one species of the L. guyanensis complex (L. guyanensis, n = 1), one species of the L. lainsoni complex (L. lainsoni, n = 2), and two species of the L. mexicana complex (L. amazonensis, n = 5; and L. garnhami, n = 3). All of the strains were correctly identified at the subgenus, genus, and complex level, but 10 of them (18%) were misidentified as other species within the same complex by the hsp70 gene sequencing, with 7 of these corresponding to possible hybrids. Thus, one L. braziliensis corresponded to L. peruviana, two L. braziliensis corresponded to L. braziliensis/L. peruviana possible hybrids, two L. amazonensis corresponded to L. mexicana, and three L. garnhami and two L. amazonensis corresponded to L. mexicana/L. amazonensis possible hybrids. Accordingly, MALDI-TOF MS could be used as an alternative to molecular techniques for the identification of Leishmania spp., as it is low cost, simple to apply, and able to quickly produce results. In Bolivia, its application would allow for the improvement of the management of patient follow-ups, the updating of the epidemiological data of the Leishmania species, and a contribution to the control of tegumentary leishmaniasis. IMPORTANCE The objective of the study was to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia, in comparison with the sequencing of the hsp70 gene. In our study, all of the isolates could be identified, and no misidentifications were observed at the complex level. Although the equipment implies a high initial investment in our context, MALDI-TOF MS can be used in different areas of microbiology and significantly reduces the cost of testing. Once the parasite culture is obtained, the technique quickly yields information by accessing a free database that is available online. This would allow for the improvement of the management of patients and follow-ups, the updating of the epidemiological data of the species, and a contribution to the control of tegumentary leishmaniasis in Bolivia. Likewise, it can be used to determine a specific treatment to be given, according to the causal species of Leishmania, when there are protocols in this regard in the area. 20221002 20221123 2165-0497 Pages: Page: Label: 1 Annotations: Annotation: Subtype: Link Rect: 50, 545, 119, 554 Annotation: Subtype: Link Rect: 436, 545, 479, 554 Annotation: Subtype: Link Rect: 417, 133, 513, 140 Annotation: Subtype: Link Rect: 400, 124, 456, 131 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Annotation: Subtype: Link Rect: 508, 683, 544, 719 Page: Label: 2 Annotations: Annotation: Subtype: Link Rect: 188, 486, 193, 495 Destination: 161 Annotation: Subtype: Link Rect: 152, 450, 156, 459 Destination: 161 Annotation: Subtype: Link Rect: 149, 426, 153, 435 Destination: 161 Annotation: Subtype: Link Rect: 206, 414, 211, 423 Destination: 161 Annotation: Subtype: Link Rect: 194, 378, 199, 387 Destination: 161 Annotation: Subtype: Link Rect: 203, 378, 208, 387 Destination: 161 Annotation: Subtype: Link Rect: 155, 366, 160, 375 Destination: 161 Annotation: Subtype: Link Rect: 164, 366, 168, 375 Destination: 161 Annotation: Subtype: Link Rect: 341, 366, 345, 375 Destination: 161 Annotation: Subtype: Link Rect: 301, 330, 305, 339 Destination: 161 Annotation: Subtype: Link Rect: 310, 330, 319, 339 Destination: 161 Annotation: Subtype: Link Rect: 268, 270, 277, 279 Destination: 161 Annotation: Subtype: Link Rect: 281, 270, 290, 279 Destination: 161 Annotation: Subtype: Link Rect: 145, 246, 154, 255 Destination: 161 Annotation: Subtype: Link Rect: 159, 246, 168, 255 Destination: 161 Annotation: Subtype: Link Rect: 172, 246, 181, 255 Destination: 161 Annotation: Subtype: Link Rect: 145, 234, 154, 243 Destination: 161 Annotation: Subtype: Link Rect: 107, 198, 116, 207 Destination: 202 Annotation: Subtype: Link Rect: 120, 198, 129, 207 Destination: 202 Annotation: Subtype: Link Rect: 357, 186, 366, 195 Destination: 202 Annotation: Subtype: Link Rect: 366, 186, 370, 195 Destination: 202 Annotation: Subtype: Link Rect: 42, 174, 50, 183 Destination: 202 Annotation: Subtype: Link Rect: 475, 734, 544, 741 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Page: Label: 3 Annotations: Annotation: Subtype: Link Rect: 190, 336, 210, 345 Destination: 41 Annotation: Subtype: Link Rect: 210, 152, 237, 161 Destination: 55 Annotation: Subtype: Link Rect: 268, 43, 277, 52 Destination: 202 Annotation: Subtype: Link Rect: 281, 43, 290, 52 Destination: 202 Annotation: Subtype: Link Rect: 475, 734, 544, 741 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Page: Label: 4 Annotations: Annotation: Subtype: Link Rect: 164, 476, 172, 485 Destination: 202 Annotation: Subtype: Link Rect: 176, 476, 185, 485 Destination: 202 Annotation: Subtype: Link Rect: 284, 452, 293, 461 Destination: 202 Annotation: Subtype: Link Rect: 297, 452, 306, 461 Destination: 202 Annotation: Subtype: Link Rect: 291, 440, 300, 449 Destination: 202 Annotation: Subtype: Link Rect: 71, 416, 80, 425 Destination: 202 Annotation: Subtype: Link Rect: 84, 416, 93, 425 Destination: 202 Annotation: Subtype: Link Rect: 326, 392, 335, 401 Destination: 202 Annotation: Subtype: Link Rect: 212, 344, 221, 353 Destination: 202 Annotation: Subtype: Link Rect: 90, 308, 99, 317 Destination: 202 Annotation: Subtype: Link Rect: 104, 308, 113, 317 Destination: 202 Annotation: Subtype: Link Rect: 259, 284, 353, 293 Annotation: Subtype: Link Rect: 359, 247, 369, 256 Destination: 161 Annotation: Subtype: Link Rect: 42, 235, 51, 244 Destination: 202 Annotation: Subtype: Link Rect: 55, 235, 64, 244 Destination: 202 Annotation: Subtype: Link Rect: 68, 235, 77, 244 Destination: 202 Annotation: Subtype: Link Rect: 341, 187, 351, 196 Destination: 202 Annotation: Subtype: Link Rect: 323, 175, 332, 184 Destination: 202 Annotation: Subtype: Link Rect: 286, 151, 295, 160 Destination: 202 Annotation: Subtype: Link Rect: 76, 91, 85, 100 Destination: 202 Annotation: Subtype: Link Rect: 139, 79, 148, 88 Destination: 202 Annotation: Subtype: Link Rect: 359, 43, 368, 52 Destination: 202 Annotation: Subtype: Link Rect: 475, 734, 544, 741 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Page: Label: 5 Annotations: Annotation: Subtype: Link Rect: 319, 702, 328, 711 Destination: 202 Annotation: Subtype: Link Rect: 233, 677, 242, 686 Destination: 202 Annotation: Subtype: Link Rect: 44, 653, 53, 662 Destination: 202 Annotation: Subtype: Link Rect: 74, 567, 83, 576 Destination: 161 Annotation: Subtype: Link Rect: 87, 567, 96, 576 Destination: 202 Annotation: Subtype: Link Rect: 100, 567, 110, 576 Destination: 202 Annotation: Subtype: Link Rect: 257, 531, 266, 540 Destination: 202 Annotation: Subtype: Link Rect: 228, 409, 233, 418 Destination: 161 Annotation: Subtype: Link Rect: 237, 409, 247, 418 Destination: 202 Annotation: Subtype: Link Rect: 154, 384, 163, 393 Destination: 161 Annotation: Subtype: Link Rect: 167, 384, 177, 393 Destination: 202 Annotation: Subtype: Link Rect: 226, 348, 236, 357 Destination: 202 Annotation: Subtype: Link Rect: 78, 299, 87, 308 Destination: 202 Annotation: Subtype: Link Rect: 91, 299, 100, 308 Destination: 202 Annotation: Subtype: Link Rect: 283, 287, 292, 296 Destination: 202 Annotation: Subtype: Link Rect: 242, 262, 251, 271 Destination: 202 Annotation: Subtype: Link Rect: 176, 226, 185, 235 Destination: 202 Annotation: Subtype: Link Rect: 306, 201, 315, 210 Destination: 202 Annotation: Subtype: Link Rect: 359, 164, 368, 173 Destination: 202 Annotation: Subtype: Link Rect: 77, 140, 86, 149 Destination: 202 Annotation: Subtype: Link Rect: 314, 128, 323, 137 Destination: 202 Annotation: Subtype: Link Rect: 336, 79, 341, 88 Destination: 161 Annotation: Subtype: Link Rect: 475, 734, 544, 741 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Page: Label: 6 Annotations: Annotation: Subtype: Link Rect: 115, 580, 124, 589 Destination: 202 Annotation: Subtype: Link Rect: 295, 568, 305, 577 Destination: 202 Annotation: Subtype: Link Rect: 309, 568, 318, 577 Destination: 202 Annotation: Subtype: Link Rect: 323, 568, 332, 577 Destination: 202 Annotation: Subtype: Link Rect: 350, 458, 354, 467 Destination: 161 Annotation: Subtype: Link Rect: 359, 458, 369, 467 Destination: 202 Annotation: Subtype: Link Rect: 42, 446, 51, 455 Destination: 202 Annotation: Subtype: Link Rect: 189, 421, 198, 430 Destination: 202 Annotation: Subtype: Link Rect: 148, 397, 153, 406 Destination: 161 Annotation: Subtype: Link Rect: 87, 372, 96, 381 Destination: 202 Annotation: Subtype: Link Rect: 166, 360, 175, 369 Destination: 202 Annotation: Subtype: Link Rect: 309, 360, 318, 369 Destination: 202 Annotation: Subtype: Link Rect: 233, 348, 242, 357 Destination: 202 Annotation: Subtype: Link Rect: 265, 299, 274, 308 Destination: 202 Annotation: Subtype: Link Rect: 231, 275, 239, 284 Destination: 202 Annotation: Subtype: Link Rect: 132, 177, 141, 186 Destination: 161 Annotation: Subtype: Link Rect: 337, 104, 341, 113 Destination: 161 Annotation: Subtype: Link Rect: 183, 67, 191, 76 Destination: 202 Annotation: Subtype: Link Rect: 475, 734, 544, 741 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Page: Label: 7 Annotations: Annotation: Subtype: Link Rect: 159, 457, 166, 465 Destination: 202 Annotation: Subtype: Link Rect: 85, 390, 89, 397 Destination: 161 Annotation: Subtype: Link Rect: 79, 342, 87, 349 Destination: 202 Annotation: Subtype: Link Rect: 117, 197, 125, 205 Destination: 202 Annotation: Subtype: Link Rect: 175, 178, 253, 186 Annotation: Subtype: Link Rect: 475, 734, 544, 741 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Page: Label: 8 Annotations: Annotation: Subtype: Link Rect: 71, 599, 107, 606 Annotation: Subtype: Link Rect: 111, 599, 146, 606 Annotation: Subtype: Link Rect: 162, 599, 194, 606 Annotation: Subtype: Link Rect: 198, 599, 230, 606 Annotation: Subtype: Link Rect: 234, 599, 325, 606 Annotation: Subtype: Link Rect: 69, 359, 283, 366 Annotation: Subtype: Link Rect: 54, 350, 65, 357 Annotation: Subtype: Link Rect: 167, 331, 283, 339 Annotation: Subtype: Link Rect: 54, 322, 73, 329 Annotation: Subtype: Link Rect: 84, 294, 284, 302 Annotation: Subtype: Link Rect: 54, 285, 282, 292 Annotation: Subtype: Link Rect: 54, 275, 174, 283 Annotation: Subtype: Link Rect: 108, 247, 283, 255 Annotation: Subtype: Link Rect: 54, 238, 233, 246 Annotation: Subtype: Link Rect: 253, 210, 283, 218 Annotation: Subtype: Link Rect: 54, 201, 151, 208 Annotation: Subtype: Link Rect: 260, 117, 283, 125 Annotation: Subtype: Link Rect: 54, 108, 180, 115 Annotation: Subtype: Link Rect: 113, 61, 266, 69 Annotation: Subtype: Link Rect: 314, 369, 451, 376 Annotation: Subtype: Link Rect: 511, 351, 544, 358 Annotation: Subtype: Link Rect: 314, 341, 544, 349 Annotation: Subtype: Link Rect: 314, 332, 402, 340 Annotation: Subtype: Link Rect: 342, 296, 476, 304 Annotation: Subtype: Link Rect: 353, 260, 500, 268 Annotation: Subtype: Link Rect: 361, 224, 496, 231 Annotation: Subtype: Link Rect: 435, 188, 543, 195 Annotation: Subtype: Link Rect: 314, 179, 378, 186 Annotation: Subtype: Link Rect: 488, 152, 543, 159 Annotation: Subtype: Link Rect: 314, 143, 377, 150 Annotation: Subtype: Link Rect: 441, 115, 543, 123 Annotation: Subtype: Link Rect: 314, 106, 353, 114 Annotation: Subtype: Link Rect: 412, 79, 543, 87 Annotation: Subtype: Link Rect: 314, 70, 335, 78 Annotation: Subtype: Link Rect: 392, 43, 522, 51 Annotation: Subtype: Link Rect: 475, 734, 544, 741 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Page: Label: 9 Annotations: Annotation: Subtype: Link Rect: 261, 667, 283, 675 Annotation: Subtype: Link Rect: 54, 658, 166, 666 Annotation: Subtype: Link Rect: 208, 622, 283, 630 Annotation: Subtype: Link Rect: 54, 613, 108, 621 Annotation: Subtype: Link Rect: 132, 506, 251, 513 Annotation: Subtype: Link Rect: 147, 488, 262, 495 Annotation: Subtype: Link Rect: 173, 470, 283, 477 Annotation: Subtype: Link Rect: 54, 461, 236, 468 Annotation: Subtype: Link Rect: 134, 434, 268, 441 Annotation: Subtype: Link Rect: 228, 407, 283, 414 Annotation: Subtype: Link Rect: 54, 398, 164, 405 Annotation: Subtype: Link Rect: 260, 371, 283, 378 Annotation: Subtype: Link Rect: 54, 362, 196, 369 Annotation: Subtype: Link Rect: 54, 335, 167, 342 Annotation: Subtype: Link Rect: 116, 308, 255, 315 Annotation: Subtype: Link Rect: 54, 272, 174, 279 Annotation: Subtype: Link Rect: 54, 209, 216, 217 Annotation: Subtype: Link Rect: 443, 694, 541, 701 Annotation: Subtype: Link Rect: 383, 657, 519, 664 Annotation: Subtype: Link Rect: 361, 591, 472, 599 Annotation: Subtype: Link Rect: 397, 554, 533, 561 Annotation: Subtype: Link Rect: 374, 526, 527, 534 Annotation: Subtype: Link Rect: 350, 489, 508, 496 Annotation: Subtype: Link Rect: 512, 461, 543, 469 Annotation: Subtype: Link Rect: 314, 452, 421, 459 Annotation: Subtype: Link Rect: 468, 424, 543, 431 Annotation: Subtype: Link Rect: 314, 414, 372, 422 Annotation: Subtype: Link Rect: 396, 387, 543, 394 Annotation: Subtype: Link Rect: 314, 377, 364, 385 Annotation: Subtype: Link Rect: 367, 377, 493, 385 Annotation: Subtype: Link Rect: 466, 349, 543, 357 Annotation: Subtype: Link Rect: 314, 340, 379, 347 Annotation: Subtype: Link Rect: 314, 293, 449, 301 Annotation: Subtype: Link Rect: 370, 256, 511, 264 Annotation: Subtype: Link Rect: 441, 228, 543, 236 Annotation: Subtype: Link Rect: 314, 219, 370, 226 Annotation: Subtype: Link Rect: 444, 200, 543, 208 Annotation: Subtype: Link Rect: 314, 191, 336, 199 Annotation: Subtype: Link Rect: 475, 734, 544, 741 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Checksum: 24fe5a24 Type: CRC32 Checksum: c4f03aafa0b5123a3a5709962d8e4532 Type: MD5 Checksum: 414081ee71b1a291129bd5d47278e460d8c65240 Type: SHA-1 Checksum: 0810d000cc1ef12a0410cac5665dc380b4552207b2f1d3f96bcefd0a3c929dd9 Type: SHA-256