Favacho Joana da Felicidade Ribeiro, Leite Keren Kariene, Jacomasso Thiago, Farias Aline Burda, Franco Filho Luciano Chaves, Gomes Samara Tatielle Monteiro, Dos Reis Herald Souza, Mota Gardene Dourado, Schluga Pedro Henrique de Caires, Tassi Walleyd Sami, Rampazzo Rita de Cássia Pontello, West Sheila Kay, Gaydos Charlotte Ann, da Cunha Antonio José Ledo Alves, Costa Alexandre Dias Tavares
Evandro Chagas Institute, Secretariat of Health and Environment Surveillance, Ministry of Health (IEC/SVSA/MS), Ananindeua 67030-000, PA, Brazil.
Institute of Molecular Biology of Paraná (IBMP), Curitiba 81350-010, PR, Brazil.
Diagnostics (Basel). 2024 Apr 25;14(9):892. doi: 10.3390/diagnostics14090892.
Trachoma is the world-leading infectious cause of preventable blindness and is caused by the bacteria . In developing countries, diagnosis is usually based on clinical evaluation. Serological-based tests are cheaper than molecular-based ones, but the latter are more sensitive and specific. The present study developed a new duplex qPCR which concomitantly detects the cryptic plasmid and the human 18S rRNA gene, with an LOD95% for DNA of 13.04 genome equivalents per reaction. The new qPCR was tested using 50 samples from an endemic area and 12 from a non-endemic area that were previously characterized using direct immunofluorescence assay (DFA) and clinical evaluation. Among the 50 endemic samples, 3 were found to be positive by clinical evaluation (6%), 18 were found to be positive by DFA (36%), and 48 were found to be positive by qPCR (96%). Next, the new duplex qPCR was validated using 50 samples previously characterized by qPCR. Validation was carried out on a benchtop instrument (ABI7500) or on a portable point-of-care instrument (Q3-Plus), showing 95% specificity and 100% sensitivity. The ubiquitous presence of DNA in samples from the endemic region confirms that constant monitoring is of paramount importance for the effective measurement of the elimination of trachoma. The newly developed duplex qPCR presented in this study, along with its validation in a portable qPCR system, constitutes important tools toward achieving this goal.
沙眼是全球可预防失明的主要感染性病因，由细菌引起。在发展中国家，诊断通常基于临床评估。血清学检测比分子检测便宜，但后者更敏感、更具特异性。本研究开发了一种新的双重定量聚合酶链反应（qPCR），可同时检测隐蔽质粒和人类18S rRNA基因，每个反应中DNA的95%最低检测限为13.04个基因组当量。使用来自流行地区的50个样本和来自非流行地区的12个样本对新的qPCR进行了测试，这些样本之前已通过直接免疫荧光测定（DFA）和临床评估进行了特征分析。在50个流行样本中，临床评估发现3个呈阳性（6%），DFA发现18个呈阳性（36%），qPCR发现48个呈阳性（96%）。接下来，使用之前通过qPCR进行特征分析的50个样本对新的双重qPCR进行了验证。验证在台式仪器（ABI7500）或便携式即时检测仪器（Q3-Plus）上进行，显示出95%的特异性和100%的敏感性。流行地区样本中普遍存在该DNA证实，持续监测对于有效衡量沙眼的消除至关重要。本研究中提出的新开发的双重qPCR及其在便携式qPCR系统中的验证，构成了实现这一目标的重要工具。
