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      Comparison of molecular methods for Bartonella henselae detection in blood donors

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          Abstract

          The Bartonella genus consists of neglected pathogens associated with potentially transfusional-transmitted and fatal human diseases. We aimed to evaluate Bartonella sp. prevalence in 500 blood donors and compare the results with the data already published about these samples. We used molecular diagnostic methods to detect Bartonella sp.-DNA from blood and liquid culture samples: (A) conventional PCR for two gene regions, the ITS targeting the genus Bartonella and the specific gltA Bartonella henselae; (B) nested PCR for the ftsZ gene and (C) qualitative real-time PCR for the gltA gene, both B. henselae specific. We obtained 30/500 (6%) DNA detections from the blood samples; 77/500 (15.4%) DNA detections from liquid culture samples and five (1%) samples had DNA detection from both. In total, we detected B. henselae DNA from 102/500 (20.4%) donors. The samples used in this study had already been submitted for Bartonella sp.-DNA detection using only a conventional PCR in liquid culture. Sixteen samples (3.2%) were positive previously, and from these 16 samples, 13 were negative in the new investigation. We concluded that the use of liquid culture combined with different molecular tests increases the possibility of detecting Bartonella sp.-DNA, but the tests do not avoid false-negative results. More than a fifth of blood donors had at least one PCR that detected Bartonella sp.-DNA among the eight molecular reactions performed now (four reactions in whole blood and four in liquid culture). Seven percent had B. henselae-DNA detection for two or more distinct regions. Considering the results obtained previously, the DNA of Bartonella spp. was detected or the agent isolated in 23% of analyzed blood donors. The results establish that the low bacteremia and the fastidious characteristics of the bacterium are challenges to laboratory diagnosis and can make it difficult to confirm the infection in patients with bartonelloses.

          Author summary

          Bartonella are bacteria that can infect humans and cause fatal diseases. They can cause chronic infection and can potentially be transmitted by transfusion since they infect red blood cells. They are difficult to cultivate in a laboratory, and they are present in low numbers in blood. There is no laboratory test that is sufficiently sensitive to detect them. To compare several laboratory diagnostic tests, we searched for these bacterial DNAs in blood and in microbiological liquid cultures of 500 blood donors. We used diverse molecular techniques and then compared the results with the previously published project. We concluded that the use of liquid culture combined with different molecular tests increases the possibility of detecting Bartonella sp.-DNA, but the tests do not avoid false-negative results. We found Bartonella henselae DNA in the blood of at least one in five donors. Hemovigilance programs are unlikely to contribute substantially to the identification of chronic posttransfusion infections since they are designed to identify well-defined acute outcomes, so it is urgent to review B. henselae transfusional risk of transmission and Bartonella sp. infection diagnosis in donors and in patients.

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          STARD 2015 guidelines for reporting diagnostic accuracy studies: explanation and elaboration

          Diagnostic accuracy studies are, like other clinical studies, at risk of bias due to shortcomings in design and conduct, and the results of a diagnostic accuracy study may not apply to other patient groups and settings. Readers of study reports need to be informed about study design and conduct, in sufficient detail to judge the trustworthiness and applicability of the study findings. The STARD statement (Standards for Reporting of Diagnostic Accuracy Studies) was developed to improve the completeness and transparency of reports of diagnostic accuracy studies. STARD contains a list of essential items that can be used as a checklist, by authors, reviewers and other readers, to ensure that a report of a diagnostic accuracy study contains the necessary information. STARD was recently updated. All updated STARD materials, including the checklist, are available at http://www.equator-network.org/reporting-guidelines/stard. Here, we present the STARD 2015 explanation and elaboration document. Through commented examples of appropriate reporting, we clarify the rationale for each of the 30 items on the STARD 2015 checklist, and describe what is expected from authors in developing sufficiently informative study reports.
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            This review provides the basic principle and rational for ROC analysis of rating and continuous diagnostic test results versus a gold standard. Derived indexes of accuracy, in particular area under the curve (AUC) has a meaningful interpretation for disease classification from healthy subjects. The methods of estimate of AUC and its testing in single diagnostic test and also comparative studies, the advantage of ROC curve to determine the optimal cut off values and the issues of bias and confounding have been discussed.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: MethodologyRole: Writing – original draft
                Role: Formal analysisRole: MethodologyRole: Writing – original draft
                Role: Formal analysisRole: MethodologyRole: Writing – original draft
                Role: ConceptualizationRole: Writing – original draft
                Role: ConceptualizationRole: Formal analysisRole: MethodologyRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                PLOS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                1 June 2023
                June 2023
                : 17
                : 6
                : e0011336
                Affiliations
                [1 ] Applied Research in Dermatology and Bartonella Infection Laboratory, University of Campinas—UNICAMP; Campinas, Sao Paulo, Brazil
                [2 ] Hematology and Hemotherapy Center, University of Campinas–UNICAMP, Campinas, Sao Paulo, Brazil
                [3 ] College of Veterinary Medicine, Western University of Health Sciences, Pomona, California, United States of America
                [4 ] Division of Dermatology, Department of Medicine, UNICAMP, Campinas, Sao Paulo, Brazil
                Laos-Oxford-Mahosot Hospital Wellcome Trust Research Unit, LAO PEOPLE’S DEMOCRATIC REPUBLIC
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0001-7901-2351
                Article
                PNTD-D-22-01039
                10.1371/journal.pntd.0011336
                10234562
                37262044
                be3f31b1-9f5a-4cf9-a212-32b56b31ac4a
                © 2023 Drummond et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 12 August 2022
                : 25 April 2023
                Page count
                Figures: 4, Tables: 2, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 159717/2013-2
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 301900 / 2015-9
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001807, Fundação de Amparo à Pesquisa do Estado de São Paulo;
                Award ID: 2013/14211-3
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 4570/2018
                Award Recipient :
                The authors received funding from Doctoral scholarship by The Brazilian National Council for Scientific and Technological Development (CNPq) 159717/2013-2 (to MRD), The Brazilian National Council for Scientific and Technological Development (CNPq) productivity grant 301900 / 2015- 9 (to PENFV); The São Paulo Research Foundation (FAPESP) Regular Research Assistance 2013/14211-3 (to PENFV) and Master's Scholarship by The Brazilian National Council for Scientific and Technological Development (CNPq) 4570/2018 (to LSDS). https://www.gov.br/cnpq/pt-br, https://fapesp.br/en. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Organisms
                Bacteria
                Bartonella
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Bartonella
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Bartonella
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Biology and Life Sciences
                Physiology
                Body Fluids
                Blood
                Medicine and Health Sciences
                Health Care
                Blood Donors
                Research and analysis methods
                Extraction techniques
                DNA extraction
                Medicine and Health Sciences
                Medical Conditions
                Infectious Diseases
                Bacterial Diseases
                Bacteremia
                Medicine and Health Sciences
                Diagnostic Medicine
                Clinical Laboratory Sciences
                Transfusion Medicine
                Blood Transfusion
                Medicine and Health Sciences
                Hematology
                Transfusion Medicine
                Blood Transfusion
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Nested Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Nested Polymerase Chain Reaction
                Custom metadata
                All relevant data are within the manuscript and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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