Letter to the Editor
Multiplex PCR assay for genotyping of Mycobacterium tuberculosis in Lima, Peru
PCR Múltiple para el Genotipaje de Mycobacterium tuberculosis en Lima, Perú
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Twenty-four hours later they were fixed and nuclear DNA was stained with 4′,6,-diaminodino-2-phenylindole (DAPI) (Invitrogen™). DAPI and GFP expression were monitored under a fluorescent microscope; the obtained images were merged into the same picture using the ImageJ program. (A) Observed expression of GFP in all the cytoplasms of infected cells with the presence of multinucleated cells (arrow), and cytoplasmic inclusion bodies (triangle). (B) Advanced infections showed evidence of apoptosis with cell shrinkage, condensed and pyknotic nuclei and apoptotic bodies.</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Carolina A. Silvestro, Ana C. Bratanich" "autores" => array:2 [ 0 => array:2 [ "nombre" => "Carolina A." "apellidos" => "Silvestro" ] 1 => array:2 [ "nombre" => "Ana C." "apellidos" => "Bratanich" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S032575411730055X?idApp=UINPBA00004N" "url" => "/03257541/0000004900000003/v1_201708180123/S032575411730055X/v1_201708180123/en/main.assets" ] "itemAnterior" => array:19 [ "pii" => "S0325754117300159" "issn" => "03257541" "doi" => "10.1016/j.ram.2017.02.001" "estado" => "S300" "fechaPublicacion" => "2017-07-01" "aid" => "168" "copyright" => "Asociación Argentina de Microbiología" "documento" => "simple-article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "cor" "cita" => "Rev Argent Microbiol. 2017;49:297-8" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 872 "formatos" => array:3 [ "EPUB" => 46 "HTML" => 552 "PDF" => 274 ] ] "en" => array:10 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Cartas al Editor</span>" "titulo" => "Plasmid-mediated colistin resistance in <span class="elsevierStyleItalic">Escherichia coli</span> recovered from healthy poultry" "tienePdf" => "en" "tieneTextoCompleto" => "en" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "297" "paginaFinal" => "298" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Resistencia a colistina mediada por plásmido en <span class="elsevierStyleItalic">Escherichia coli</span> recuperadas de aves de corral sanas" ] ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Johana E. Dominguez, Roque A. Figueroa Espinosa, Leandro M. Redondo, Daniela Cejas, Gabriel O. Gutkind, Pablo A. Chacana, José A. Di Conza, Mariano E. Fernández-Miyakawa" "autores" => array:8 [ 0 => array:2 [ "nombre" => "Johana E." "apellidos" => "Dominguez" ] 1 => array:2 [ "nombre" => "Roque A." "apellidos" => "Figueroa Espinosa" ] 2 => array:2 [ "nombre" => "Leandro M." "apellidos" => "Redondo" ] 3 => array:2 [ "nombre" => "Daniela" "apellidos" => "Cejas" ] 4 => array:2 [ "nombre" => "Gabriel O." "apellidos" => "Gutkind" ] 5 => array:2 [ "nombre" => "Pablo A." "apellidos" => "Chacana" ] 6 => array:2 [ "nombre" => "José A." "apellidos" => "Di Conza" ] 7 => array:2 [ "nombre" => "Mariano E." "apellidos" => "Fernández-Miyakawa" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754117300159?idApp=UINPBA00004N" "url" => "/03257541/0000004900000003/v1_201708180123/S0325754117300159/v1_201708180123/en/main.assets" ] "en" => array:16 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Letter to the Editor</span>" "titulo" => "Multiplex PCR assay for genotyping of <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> in Lima, Peru" "tieneTextoCompleto" => true "saludo" => "Dear Editor," "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "298" "paginaFinal" => "300" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Carlos A. Tello, Nelly Borja, Ronnie G. Gavilan, Ruth Garcia-de-la-Guarda" "autores" => array:4 [ 0 => array:4 [ "nombre" => "Carlos A." "apellidos" => "Tello" "email" => array:1 [ 0 => "carolusmagnum@gmail.com" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] 1 => array:3 [ "nombre" => "Nelly" "apellidos" => "Borja" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 2 => array:3 [ "nombre" => "Ronnie G." "apellidos" => "Gavilan" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 3 => array:3 [ "nombre" => "Ruth" "apellidos" => "Garcia-de-la-Guarda" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">c</span>" "identificador" => "aff0015" ] ] ] ] "afiliaciones" => array:3 [ 0 => array:3 [ "entidad" => "Hospital Guillermo Almenara Irigoyen, Lima, Peru" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Instituto Nacional de Salud, Lima, Peru" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Universidad Nacional Mayor de San Marcos, Lima, Peru" "etiqueta" => "c" "identificador" => "aff0015" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "PCR Múltiple para el Genotipaje de <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> en Lima, Perú" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1712 "Ancho" => 3418 "Tamanyo" => 563968 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">The dendogram of fragment to Multiplex PCR of 50 strains of <span class="elsevierStyleItalic">M. tuberculosis</span> was constructed using the similarity coefficient of Dice and UPGMA, using a tolerance of 1.2% and optimization of 0.17%. The <span class="elsevierStyleItalic">clusters</span> CI, CII, CIII, CIV, CV, CVI, CVII y CVIII are indicate in the dendogram. The banding patterns obtained by PCR-based methodology are in the right of the dendogram. The attached table contains demographic and drug resistant date of the cases.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0005" class="elsevierStylePara elsevierViewall">In South American countries it is necessary to have molecular methods as epidemiological tools as well as low costs and good level of discrimination capacity for polymorphisms that may enable molecular laboratories to perform the tasks for other regional laboratories having minimum standards. The methodology proposed in this article addresses this need.</p><p id="par0010" class="elsevierStylePara elsevierViewall">We used the Proportion Method<a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">2</span></a> for susceptibility testing of <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> isolates against isoniazid (INH), rifampicin (RMP), ethambutol (EMB), para-aminosalicylic acid (PAS), thioacetazone (T), kanamycin (KM) and streptomycin (SM). Cultures were grown on Lowenstein Jensen medium at 37<span class="elsevierStyleHsp" style=""></span>°C for 21 days, and slides were processed with the Ziehl–Neelsen stain. DNA from samples was extracted by using the phenol chloroform method<a class="elsevierStyleCrossRef" href="#bib0030"><span class="elsevierStyleSup">6</span></a>. In a simple PCR for <span class="elsevierStyleItalic">M. tuberculosis</span> strain differentiation, primer Mtb2 (5′-CGGCGGCAACGGCGGCA) was used with primers IS1 (5′-CGGACTCACCGGGGCGGTTCA) and IS2 (5′-CGGACATGCCGGGGCGGTTCA) that anneal at the inverted repeats flanking IS<span class="elsevierStyleItalic">6110</span><a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a>. PCR was done in a mixture containing 25<span class="elsevierStyleHsp" style=""></span>pmol of each primer, 1<span class="elsevierStyleHsp" style=""></span>U of Platinum <span class="elsevierStyleItalic">Taq</span> DNA polymerase (Invitrogen), 0.2<span class="elsevierStyleHsp" style=""></span>mM of each deoxyribonucleotide triphosphate, 10<span class="elsevierStyleHsp" style=""></span>mM Tris–HCl (pH 8.4), 1.65<span class="elsevierStyleHsp" style=""></span>mM MgCl<span class="elsevierStyleInf">2</span>, 50<span class="elsevierStyleHsp" style=""></span>mM KCl, and 0.1% Triton X-100 and overlaid with mineral oil. Cycling conditions were as follows: denaturation at 94<span class="elsevierStyleHsp" style=""></span>°C for 5<span class="elsevierStyleHsp" style=""></span>min, followed by amplification for 35 cycles of 94<span class="elsevierStyleHsp" style=""></span>°C for 1<span class="elsevierStyleHsp" style=""></span>min, 62<span class="elsevierStyleHsp" style=""></span>°C for 1<span class="elsevierStyleHsp" style=""></span>min, and 72<span class="elsevierStyleHsp" style=""></span>°C for 1<span class="elsevierStyleHsp" style=""></span>min, followed by a final extension at 72<span class="elsevierStyleHsp" style=""></span>°C for 10<span class="elsevierStyleHsp" style=""></span>min. A total of 20<span class="elsevierStyleHsp" style=""></span>μl of amplified DNA was subjected to electrophoresis in a 2% agarose gel, detected by ethidium bromide staining, and visualized under UV light. For the genetic polymorphism study, we used the Bionumerics program version 5.0 (Applied-Maths).</p><p id="par0015" class="elsevierStylePara elsevierViewall">The drug resistance study showed that out of 49 strains belonging to TB patients in the Guillermo Almenara Irigoyen National Hospital, 18 strains (36.74%) were drug-sensitive, 10 (20.4%) were drug-resistant TB (DR) and 21 (42.8%) were multidrug-resistant TB (MDR), 3 of which were TB/HIV cases. Forty-two (42) different banding patterns were observed, which were classified into 10 clusters (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). We suspect that cases MT009 and MT029 belong to heteroresistant strains, i.e. mixed wild-type and mutant strains because the banding pattern seems to overlap<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a>.</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0020" class="elsevierStylePara elsevierViewall">The transmission study showed Cluster II. Two male patients with MDR-TB, one of whom (MT014) had undergone previous MDR-TB treatment, were both hospitalized; Cluster IV. Two male patients with TB/HIV co-infection, both residing in the same district; Cluster VI. Two male patients with TB/HIV co-infection, both living in the same district and receiving their treatment in the same hospital, one of them (MT048) with DR TB and the other (MT054) with a sensitive case; Cluster VIII. Three male patients aged 44, 34 and 28 years, respectively, two of whom were brothers (MT033 and MT041) and the other a neighbor (MT032), all of them sensitive cases. The other clusters did not have an epidemiological link. A statistical risk study was performed<a class="elsevierStyleCrossRefs" href="#bib0020"><span class="elsevierStyleSup">4,13</span></a> and the result was that the patients with HIV infections had the highest contagion risk in our population (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.174; OR<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>3.150; CI<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.568–17.477). The repetition rate was good (Cronbach's alpha<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.82). This genotyping method could be an alternative for other PCR-based typing procedures, such as spoligotyping and MIRU-VNTR typing as cited in other studies<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a> and could help in the study of transmission relationship with heteroresistance, HIV-TB patients and outbreaks. Our TB survey system has many complications<a class="elsevierStyleCrossRefs" href="#bib0015"><span class="elsevierStyleSup">3,9,11</span></a> and the lack of surveillance in DOTS<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a> results in patients having a great diversity of genotypes and drug-resistant profiles<a class="elsevierStyleCrossRefs" href="#bib0005"><span class="elsevierStyleSup">1,5</span></a>, as well as heteroresistance of wild type to resistant, resistant to resistant, and wild type to MDR strains<a class="elsevierStyleCrossRefs" href="#bib0005"><span class="elsevierStyleSup">1,4,8,10,12,13</span></a>. Our country needs a strategy based on epidemiology with molecular tools that will assist us in the analysis of the genetic diversity existing in Peru.</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0005">Ethical disclosures</span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Protection of human and animal subjects</span><p id="par0025" class="elsevierStylePara elsevierViewall">The authors declare that no experiments were performed on humans or animals for this study.</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Confidentiality of data</span><p id="par0030" class="elsevierStylePara elsevierViewall">The authors declare that they have followed the protocols of their work center on the publication of patient data.</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Right to privacy and informed consent</span><p id="par0035" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article.</p></span></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Funding</span><p id="par0040" class="elsevierStylePara elsevierViewall">Universidad Nacional Mayor de San Marcos: CON-CON 2006 – Con asignación a la Investigación y con incentivo al investigador.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:4 [ 0 => array:3 [ "identificador" => "sec0005" "titulo" => "Ethical disclosures" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0010" "titulo" => "Protection of human and animal subjects" ] 1 => array:2 [ "identificador" => "sec0015" "titulo" => "Confidentiality of data" ] 2 => array:2 [ "identificador" => "sec0020" "titulo" => "Right to privacy and informed consent" ] ] ] 1 => array:2 [ "identificador" => "sec0025" "titulo" => "Funding" ] 2 => array:2 [ "identificador" => "xack295020" "titulo" => "Acknowledgments" ] 3 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "multimedia" => array:1 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1712 "Ancho" => 3418 "Tamanyo" => 563968 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">The dendogram of fragment to Multiplex PCR of 50 strains of <span class="elsevierStyleItalic">M. tuberculosis</span> was constructed using the similarity coefficient of Dice and UPGMA, using a tolerance of 1.2% and optimization of 0.17%. The <span class="elsevierStyleItalic">clusters</span> CI, CII, CIII, CIV, CV, CVI, CVII y CVIII are indicate in the dendogram. The banding patterns obtained by PCR-based methodology are in the right of the dendogram. The attached table contains demographic and drug resistant date of the cases.</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:13 [ 0 => array:3 [ "identificador" => "bib0005" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Perfiles genéticos (RFLP-IS6110) y resistencia a drogas en aislamientos de <span class="elsevierStyleItalic">M. tuberculosis</span> de pacientes internados en un hospital referencial del Callao, Perú" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "C. Baldeviano" 1 => "N. Quispe" 2 => "C. Bonilla" 3 => "D. Gastiaburu" 4 => "J. 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Van Embden" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1086/314930" "Revista" => array:6 [ "tituloSerie" => "J Infect Dis" "fecha" => "1999" "volumen" => "180" "paginaInicial" => "726" "paginaFinal" => "736" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/10438361" "web" => "Medline" ] ] ] ] ] ] ] ] ] ] ] ] "agradecimientos" => array:1 [ 0 => array:4 [ "identificador" => "xack295020" "titulo" => "Acknowledgments" "texto" => "<p id="par0045" class="elsevierStylePara elsevierViewall">The authors acknowledge to Dr. Pablo Ramirez Roca and Dra. Debora Alvarado Iparraguirre for the use of the Molecular Microbiology Laboratory (Universidad Nacional Mayor de San Marcos, Peru), Dr. Ernesto Montoro and Dr. Raul Diaz of Instituto Pedro Kouri (Cuba) for the advice and review of this investigation.</p>" "vista" => "all" ] ] ] "idiomaDefecto" => "en" "url" => "/03257541/0000004900000003/v1_201708180123/S032575411730041X/v1_201708180123/en/main.assets" "Apartado" => array:4 [ "identificador" => "37864" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Carta al Editor" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/03257541/0000004900000003/v1_201708180123/S032575411730041X/v1_201708180123/en/main.pdf?idApp=UINPBA00004N&text.app=https://www.elsevier.es/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S032575411730041X?idApp=UINPBA00004N" ]
| Year/Month | Html | Total | |
|---|---|---|---|
| 2024 November | 1 | 0 | 1 |
| 2024 October | 24 | 9 | 33 |
| 2024 September | 38 | 4 | 42 |
| 2024 August | 46 | 5 | 51 |
| 2024 July | 42 | 11 | 53 |
| 2024 June | 25 | 7 | 32 |
| 2024 May | 20 | 1 | 21 |
| 2024 April | 20 | 3 | 23 |
| 2024 March | 28 | 8 | 36 |
| 2024 February | 31 | 9 | 40 |
| 2024 January | 24 | 4 | 28 |
| 2023 December | 11 | 8 | 19 |
| 2023 November | 42 | 15 | 57 |
| 2023 October | 30 | 10 | 40 |
| 2023 September | 22 | 0 | 22 |
| 2023 August | 24 | 8 | 32 |
| 2023 July | 21 | 6 | 27 |
| 2023 June | 30 | 6 | 36 |
| 2023 May | 41 | 5 | 46 |
| 2023 April | 36 | 2 | 38 |
| 2023 March | 27 | 5 | 32 |
| 2023 February | 22 | 2 | 24 |
| 2023 January | 16 | 11 | 27 |
| 2022 December | 13 | 3 | 16 |
| 2022 November | 14 | 7 | 21 |
| 2022 October | 19 | 10 | 29 |
| 2022 September | 24 | 8 | 32 |
| 2022 August | 10 | 7 | 17 |
| 2022 July | 13 | 10 | 23 |
| 2022 June | 9 | 7 | 16 |
| 2022 May | 13 | 8 | 21 |
| 2022 April | 35 | 10 | 45 |
| 2022 March | 28 | 12 | 40 |
| 2022 February | 22 | 3 | 25 |
| 2022 January | 42 | 8 | 50 |
| 2021 December | 33 | 11 | 44 |
| 2021 November | 35 | 14 | 49 |
| 2021 October | 23 | 12 | 35 |
| 2021 September | 11 | 8 | 19 |
| 2021 August | 38 | 7 | 45 |
| 2021 July | 20 | 6 | 26 |
| 2021 June | 23 | 13 | 36 |
| 2021 May | 17 | 8 | 25 |
| 2021 April | 32 | 21 | 53 |
| 2021 March | 19 | 13 | 32 |
| 2021 February | 12 | 11 | 23 |
| 2021 January | 14 | 16 | 30 |
| 2020 December | 12 | 3 | 15 |
| 2020 November | 10 | 7 | 17 |
| 2020 October | 7 | 3 | 10 |
| 2020 September | 14 | 9 | 23 |
| 2020 August | 18 | 16 | 34 |
| 2020 July | 16 | 13 | 29 |
| 2020 June | 17 | 10 | 27 |
| 2020 May | 19 | 8 | 27 |
| 2020 April | 10 | 8 | 18 |
| 2020 March | 6 | 4 | 10 |
| 2020 February | 13 | 13 | 26 |
| 2020 January | 15 | 12 | 27 |
| 2019 December | 12 | 10 | 22 |
| 2019 November | 14 | 6 | 20 |
| 2019 October | 14 | 3 | 17 |
| 2019 September | 20 | 1 | 21 |
| 2019 August | 15 | 2 | 17 |
| 2019 July | 13 | 9 | 22 |
| 2019 June | 47 | 8 | 55 |
| 2019 May | 96 | 9 | 105 |
| 2019 April | 36 | 15 | 51 |
| 2019 March | 15 | 3 | 18 |
| 2019 February | 18 | 2 | 20 |
| 2019 January | 11 | 4 | 15 |
| 2018 December | 23 | 6 | 29 |
| 2018 November | 27 | 10 | 37 |
| 2018 October | 25 | 16 | 41 |
| 2018 September | 20 | 1 | 21 |
| 2018 August | 17 | 3 | 20 |
| 2018 July | 12 | 4 | 16 |
| 2018 June | 17 | 4 | 21 |
| 2018 May | 23 | 2 | 25 |
| 2018 April | 16 | 3 | 19 |
| 2018 March | 13 | 2 | 15 |
| 2018 February | 14 | 11 | 25 |
| 2018 January | 22 | 7 | 29 |
| 2017 December | 17 | 3 | 20 |
| 2017 November | 31 | 1 | 32 |
| 2017 October | 83 | 5 | 88 |
| 2017 September | 27 | 5 | 32 |
| 2017 August | 9 | 10 | 19 |
| 2017 July | 3 | 10 | 13 |
| 2017 June | 1 | 37 | 38 |
| 2017 May | 0 | 2 | 2 |
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