Pierre H. H. Schneeberger

Experiences and Lessons from a Multicountry NIDIAG Study on Persistent Digestive Disorders in the Tropics.

Persistent digestive disorders can be defined as any diarrhea (i.e., three or more loose stools per day) lasting for at least two weeks and/or abdominal pain that persists for two weeks or longer [1–3]. These disorders cause considerable morbidity and human suffering, and hence, are reasons why people might seek primary health care. However, in resource-constrained settings of the tropics and subtropics, accurate point-of-care diagnostics are often lacking and treatment is empiric, particularly in remote rural areas with no laboratory infrastructure. As a result, the relative contribution of selected pathogens to the syndrome of persistent digestive disorders is poorly understood, and evidence-based guidelines for patient management in different social-ecological settings are scarce [4–6]. In order to improve the clinical management of patients with disorders caused by neglected tropical diseases (NTDs), the European Commission (EC) funded a five-year study—the Neglected Infectious diseases DIAGnosis (NIDIAG) research consortium. The overarching goal of the NIDIAG consortium is to develop and validate patient-centered diagnosis–treatment guidelines for use at the primary health care level in low- and middle-income countries (http://www.nidiag.org) [3,7–9]. Emphasis is placed on three syndromes: (i) persistent digestive disorders described here; (ii) persistent fever; and (iii) neurological disorders, the latter two of which are detailed in companion pieces published in the same issue of PLOS Neglected Tropical Diseases. With regard to the study on persistent digestive disorders, the main aims are (i) to identify the most important NTDs and other infectious agents that give rise to this clinical syndrome, including their relative frequency; (ii) to assess and compare the accuracy of different diagnostic methods; and (iii) to determine clinical responses to commonly employed empiric treatment options for persistent digestive disorders [9]. To this end, a case–control study has been implemented in four countries: Cote d’Ivoire and Mali in West Africa and Indonesia and Nepal in Asia. An integral part of the NIDIAG consortium is to ensure that good clinical practice (GCP) and good clinical laboratory practice (GCLP) are adhered to while conducting the studies [10,11]. A quality assurance system, which included the development and implementation of a set of standard operating procedures (SOPs), along with on-the-spot staff training and internal and external quality control activities, has been developed at the project level and introduced at each study site. The development of, and adherence to, SOPs within harmonized study protocols were considered crucial steps for maximizing the integrity of laboratory and clinical data across study settings. They also provided the basis on which quality control activities could be performed. For Which Procedures Have SOPs Been Developed? For the study on persistent digestive disorders, 33 specific SOPs have been developed (Supporting Information). As summarized in Table 1, detailed steps on clinical and laboratory procedures, data management, and quality assurance were described. With regard to clinical investigations, SOPs on history taking and clinical examination, assessing inclusion and exclusion criteria, patient recruitment, and study flow were developed (S1-S6). Detailed instructions on how to perform a set of laboratory diagnostic techniques for the detection of helminth and intestinal protozoa infections were included in the laboratory SOPs. Different conventional stool microscopy techniques were combined with more recent rapid antigen detection tests to encompass a broad spectrum of potentially implicated pathogens with high diagnostic accuracy (S7-S20). An overview of the employed diagnostic methods is provided in Table 2. Pertaining to data management, SOPs on completion of case report forms (CRFs) and on various activities (such as data entry, data cleaning, querying, database locking, and backing up data) were also included. To ensure quality control, SOPs on internal quality control activities, external monitoring, and laboratory supervision visits were jointly developed for the three syndromes (S21-S33). Table 1 Set of standard operating procedures (SOPs) used in the NIDIAG study on persistent digestive disorders. Table 2 Laboratory diagnostic techniques used and internally compared in the NIDIAG study on persistent digestive disorders. Of note, all SOPs were developed in English (for use in Nepal) and subsequently translated into French (for use in Cote d’Ivoire and Mali) and Bahasa Indonesia (for use in Indonesia). This comprehensive set of closely interconnected SOPs—which provides guidance on all essential procedures from the first presentation of an individual at a health care center until the final processing of all patient and laboratory data—is displayed in Fig 1. Fig 1 Principal elements of the NIDIAG digestive study and the respective standard operating procedures (SOPs) used. How Was the Development of SOPs Coordinated, and Which Quality Control Measures Were Adopted? The development and harmonization of the various SOPs was coordinated by the quality assurance group of the NIDIAG consortium and the trial management group (TMG) of the digestive syndrome study and followed a standard template and consortium-wide guidelines stipulated in the SOP entitled “SOP on SOP” (S24). This allowed different authors with varied background and writing styles to convey key messages and pass on their expert knowledge in a systematic, standardized manner for the benefit of the end user of all the SOPs. In addition, it provided clear instructions on how the SOPs should be numbered, reviewed, and approved to allow for strict version control. The authors of the SOPs were chosen from within the NIDIAG consortium, and allocation of topics was based on expertise and track record in the clinical, laboratory, data management, and quality assurance components of the study. Experts in the field, at the bench, and at the bedside carefully reviewed and revised the draft SOPs. Before the start of recruitment, local clinical and laboratory teams were trained on the set of SOPs through two hands-on workshops lasting three days each that were conducted on site by relevant experts of the NIDIAG consortium. During these workshops, feedback from the local partners was incorporated to refine the already developed SOPs, and additional SOPs were jointly developed to meet specific demands of local clinical, epidemiologic, and laboratory conditions. For example, in Indonesia, where Kinyoun staining was not available, an SOP pertaining to a slightly modified acid-fast staining technique was developed for the local team instead. Finally, once an SOP was finalized, a member of the TMG would approve it. A quality assurance member of the NIDIAG consortium was tasked to compile and keep updated the final set of SOPs and ensure that the latest versions were available on the NIDIAG intranet for distribution among the different country partners.

A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

&NA; Molecular diagnostics of plant pathogens are crucial to prevent disease spread and to enhance food quality and security. A comparative genomics approach using genomes of different Xanthomonas species and pathovars was applied to identify highly specific targets in the genome of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, listed under quarantine regulations in Europe. A reliable and sensitive loop‐mediated isothermal amplification (LAMP) assay was designed using a unique marker, providing a highly specific and rapid detection technique, convenient for on‐site detection. Specificity of the designed assay was tested on 37 strains from a culture collection of X. fragariae, 82 strains of other Xanthomonas species and pathovars and 11 strains of other bacterial genera isolated from strawberry leaves. A detection limit of 102 fg was achieved, approximating to 20 genome copies per reaction. When performing analyses with crude plant material, a consistent lower detection efficiency of 102 CFU mL−1 was achieved. The LAMP assay designed in this study was adapted to work on crude plant material without any prior extensive extraction steps or incubation period; moreover, it does not require advanced analytical knowledge or a fully equipped laboratory. Results were produced within 7–20 min, depending on the pathogen concentration, thus providing a high‐throughput and user‐friendly method for detection and screening of plant material in support of quarantine regulations.

Development and evaluation of a bioinformatics approach for designing molecular assays for viral detection

Background Viruses belonging to the Flaviviridae and Bunyaviridae families show considerable genetic diversity. However, this diversity is not necessarily taken into account when developing diagnostic assays, which are often based on the pairwise alignment of a limited number of sequences. Our objective was to develop and evaluate a bioinformatics workflow addressing two recurrent issues of molecular assay design: (i) the high intraspecies genetic diversity in viruses and (ii) the potential for cross-reactivity with close relatives. Methodology The workflow developed herein was based on two consecutive BLASTn steps; the first was utilized to select highly conserved regions among the viral taxon of interest, and the second was employed to assess the degree of similarity of these highly-conserved regions to close relatives. Subsequently, the workflow was tested on a set of eight viral species, including various strains from the Flaviviridae and Bunyaviridae families. Principal findings The genetic diversity ranges from as low as 0.45% variable sites over the complete genome of the Japanese encephalitis virus to more than 16% of variable sites on segment L of the Crimean-Congo hemorrhagic fever virus. Our proposed bioinformatics workflow allowed the selection—based on computing scores—of the best target for a diagnostic molecular assay for the eight viral species investigated. Conclusions/Significance Our bioinformatics workflow allowed rapid selection of highly conserved and specific genomic fragments among the investigated viruses, while considering up to several hundred complete genomic sequences. The pertinence of this workflow will increase in parallel to the number of sequences made publicly available. We hypothesize that our workflow might be utilized to select diagnostic molecular markers for higher organisms with more complex genomes, provided the sequences are made available.