Effect of Demographics and Time to Sample Processing on the qPCR Detection of Pathogenic <i>Leptospira</i> spp. from Human Samples in the National Reference Laboratory for Leptospirosis, Brazil
Leptospirosis diagnosis by MAT requires antibody levels that are typically present only after the first week of symptoms, many days after infection. To improve testing capacity and to develop a fast and reliable solution for the diagnosis of this disease in the first few days after clinical manifest...
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MDPI AG
2023-02-01
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| Series: | Tropical Medicine and Infectious Disease |
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| Online Access: | https://www.mdpi.com/2414-6366/8/3/151 |
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| author | Romulo Leão Silva Neris Mariana Cristina da Silva Mariana da Silva Batista Keila de Cássia Ferreira de Almeida Silva Ilana Teruszkin Balassiano Kátia Eliane Santos Avelar |
| author_facet | Romulo Leão Silva Neris Mariana Cristina da Silva Mariana da Silva Batista Keila de Cássia Ferreira de Almeida Silva Ilana Teruszkin Balassiano Kátia Eliane Santos Avelar |
| author_sort | Romulo Leão Silva Neris |
| collection | DOAJ |
| description | Leptospirosis diagnosis by MAT requires antibody levels that are typically present only after the first week of symptoms, many days after infection. To improve testing capacity and to develop a fast and reliable solution for the diagnosis of this disease in the first few days after clinical manifestations, the National Reference Laboratory for Leptospirosis/WHO Collaborating Center in Brazil implemented a duplex molecular method by qPCR for human samples for the detection of the gene <i>lip</i>L32, conserved in pathogenic <i>Leptospira</i> spp. In this paper, we describe the overall performance of this protocol in the first 3 months as a standard routine. Detection of pathogenic <i>Leptospira</i> spp. DNA was similar between blood, plasma, and tissue samples, with a limit of detection as low as one cell per sample, and among 391 samples from suspected cases, 174 (44.6%) were positive. The average <i>RNASEP1</i> control gene detection cycle thresholds (Ct) were 28.4 and 29.8 for positive and negative samples, respectively. The median sample collection interval from the beginning of symptoms was 3 days for positive and 4 days for negative samples, respectively. Neither age, sex, nor the time intervals between sample collection and DNA extraction significantly influenced the results. Surprisingly, positivity was related to the time between DNA extraction and the qPCR reaction. These data support the use of this routine as a diagnostic approach to strengthen the molecular detection of leptospirosis and to develop new strategies. |
| first_indexed | 2024-03-11T05:48:11Z |
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| language | English |
| last_indexed | 2024-03-11T05:48:11Z |
| publishDate | 2023-02-01 |
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| record_format | Article |
| series | Tropical Medicine and Infectious Disease |
| spelling | doaj.art-680ed8f095824efc9347de9f5dcead132023-11-17T14:15:08ZengMDPI AGTropical Medicine and Infectious Disease2414-63662023-02-018315110.3390/tropicalmed8030151Effect of Demographics and Time to Sample Processing on the qPCR Detection of Pathogenic <i>Leptospira</i> spp. from Human Samples in the National Reference Laboratory for Leptospirosis, BrazilRomulo Leão Silva Neris0Mariana Cristina da Silva1Mariana da Silva Batista2Keila de Cássia Ferreira de Almeida Silva3Ilana Teruszkin Balassiano4Kátia Eliane Santos Avelar5Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial da Saúde para Leptospirose, Instituto Oswaldo Cruz, Rio de Janeiro 3304557, BrazilLaboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial da Saúde para Leptospirose, Instituto Oswaldo Cruz, Rio de Janeiro 3304557, BrazilLaboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial da Saúde para Leptospirose, Instituto Oswaldo Cruz, Rio de Janeiro 3304557, BrazilLaboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial da Saúde para Leptospirose, Instituto Oswaldo Cruz, Rio de Janeiro 3304557, BrazilLaboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial da Saúde para Leptospirose, Instituto Oswaldo Cruz, Rio de Janeiro 3304557, BrazilLaboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial da Saúde para Leptospirose, Instituto Oswaldo Cruz, Rio de Janeiro 3304557, BrazilLeptospirosis diagnosis by MAT requires antibody levels that are typically present only after the first week of symptoms, many days after infection. To improve testing capacity and to develop a fast and reliable solution for the diagnosis of this disease in the first few days after clinical manifestations, the National Reference Laboratory for Leptospirosis/WHO Collaborating Center in Brazil implemented a duplex molecular method by qPCR for human samples for the detection of the gene <i>lip</i>L32, conserved in pathogenic <i>Leptospira</i> spp. In this paper, we describe the overall performance of this protocol in the first 3 months as a standard routine. Detection of pathogenic <i>Leptospira</i> spp. DNA was similar between blood, plasma, and tissue samples, with a limit of detection as low as one cell per sample, and among 391 samples from suspected cases, 174 (44.6%) were positive. The average <i>RNASEP1</i> control gene detection cycle thresholds (Ct) were 28.4 and 29.8 for positive and negative samples, respectively. The median sample collection interval from the beginning of symptoms was 3 days for positive and 4 days for negative samples, respectively. Neither age, sex, nor the time intervals between sample collection and DNA extraction significantly influenced the results. Surprisingly, positivity was related to the time between DNA extraction and the qPCR reaction. These data support the use of this routine as a diagnostic approach to strengthen the molecular detection of leptospirosis and to develop new strategies.https://www.mdpi.com/2414-6366/8/3/151leptospirosishuman samplesqPCRdiagnosticsDNA extraction |
| spellingShingle | Romulo Leão Silva Neris Mariana Cristina da Silva Mariana da Silva Batista Keila de Cássia Ferreira de Almeida Silva Ilana Teruszkin Balassiano Kátia Eliane Santos Avelar Effect of Demographics and Time to Sample Processing on the qPCR Detection of Pathogenic <i>Leptospira</i> spp. from Human Samples in the National Reference Laboratory for Leptospirosis, Brazil Tropical Medicine and Infectious Disease leptospirosis human samples qPCR diagnostics DNA extraction |
| title | Effect of Demographics and Time to Sample Processing on the qPCR Detection of Pathogenic <i>Leptospira</i> spp. from Human Samples in the National Reference Laboratory for Leptospirosis, Brazil |
| title_full | Effect of Demographics and Time to Sample Processing on the qPCR Detection of Pathogenic <i>Leptospira</i> spp. from Human Samples in the National Reference Laboratory for Leptospirosis, Brazil |
| title_fullStr | Effect of Demographics and Time to Sample Processing on the qPCR Detection of Pathogenic <i>Leptospira</i> spp. from Human Samples in the National Reference Laboratory for Leptospirosis, Brazil |
| title_full_unstemmed | Effect of Demographics and Time to Sample Processing on the qPCR Detection of Pathogenic <i>Leptospira</i> spp. from Human Samples in the National Reference Laboratory for Leptospirosis, Brazil |
| title_short | Effect of Demographics and Time to Sample Processing on the qPCR Detection of Pathogenic <i>Leptospira</i> spp. from Human Samples in the National Reference Laboratory for Leptospirosis, Brazil |
| title_sort | effect of demographics and time to sample processing on the qpcr detection of pathogenic i leptospira i spp from human samples in the national reference laboratory for leptospirosis brazil |
| topic | leptospirosis human samples qPCR diagnostics DNA extraction |
| url | https://www.mdpi.com/2414-6366/8/3/151 |
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