Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples

Prior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evaluated four SeeGene Allplex real-time PCR assays amplifying a total of 28 bacteria, microsporidal and parasitic nucleic acid sequence targets in human stool samples in a multicentric approach. In the assessments with strongly positive samples, sensitivity values ranging between 13% and 100% were recorded for bacteria, between 0% and 100% for protozoa and between 7% and 100% for helminths and microsporidia; for the weakly positive samples, the recorded sensitivity values for bacteria ranged from 0% to 100%; for protozoa, from 0% to 40%; and for helminths and microsporidia, from 0% to 53%. For bacteria, the recorded specificity was in the range between 87% and 100%, while a specificity of 100% was recorded for all assessed PCRs targeting parasites and microsporidia. The intra- and inter-assay variations were generally low. Specifically for some helminth species, the sensitivity could be drastically increased by applying manual nucleic acid extraction instead of the manufacturer-recommended automatic procedure, while such effects were less obvious for the bacteria and protozoa. In summary, the testing with the chosen positive control samples showed varying degrees of discordance between the evaluated Allplex assays and the applied in-house reference assays associated with higher cycle threshold values in the Allplex assays, suggesting that samples with very low pathogen densities might be missed. As the targeted species can occur as harmless colonizers in the gut of individuals in high-endemicity settings as well, future studies should aim at assessing the clinical relevance of the latter hint.Peer Reviewe

Evaluation of a duplex real-time PCR in human serum for simultaneous detection and differentiation of Schistosoma mansoni and Schistosoma haematobium infections - cross-sectional study

Background: We evaluated a one-tube multiplex real-time PCR targeting DNA of Schistosoma haematobium complex and S. mansoni complex in serum samples obtained at different German diagnostic centers. Methods: Simplex real-time PCR protocols for the detection of the multi-copy DNA-repeats Dra1 of S. haematobium complex and Sm1-7 of S. mansoni complex in serum were combined to a new one-tube multiplex format. The new PCR was subjected to full validation including evaluation in a diagnostic real-life setting with travelers and migrants. PCR results were compared with those of stool and urine microscopy, serology, and circulating cathodic antigen (CCA) rapid diagnostic tests in urine. Sensitivity and specificity of the diagnostic approaches were analyzed using latent class analysis (LCA). Results: LCA assessment indicated sensitivity and specificity of 94.9% and 98.4%, respectively, for serum PCR if serology was included in the calculation, and 100% and 95.6%, respectively, if serology was not included as a parameter not necessarily associated with active infection. Agreement between the compared diagnostic procedures at genus level was fair (kappa 0.273) if serology was included and moderate (kappa 0.420) if serology was not included. Discussion: The PCR assay proved to be highly reliable for the diagnosis of schistosomiasis in travelers and migrants

Optimization of Case Definitions for Sensitivity as a Preventive Strategy—A Modelling Exemplified with Rapid Diagnostic Test-Based Prevention of Sexual HIV Transmission

In clinical studies, case definitions are usually designed to optimally match the desired clinical state, because lacking specificity is associated with a risk of bias regarding the study outcome. In preventive medicine, however, high sensitivity is sometimes considered as more critical in order not to overlook infectious individuals, because the latter may be associated with ongoing spread of a transmittable disease. Accordingly, this work was focused on a theoretical model on how the sensitivity of case definitions can be optimized by adding clinical symptoms to diagnostic results for preventive purposes, if the associated reduction in specificity is considered as acceptable. The model was exemplified with an analysis on whether and in how far exposure risk can be reduced by the inclusion of observable symptoms during seroconversion syndrome in case of rapid diagnostic test-based prevention of sexual HIV transmission. The approach provided a high level of safety (negative predictive values close to 1) for the price of a considerably number of false positives (positive predictive values < 0.01 for some subpopulations). When applying such a sensitivity-optimized screening as a “diagnostics as prevention” strategy, the advantages of excellent negative predictive values need to be cautiously balanced against potential undesirable consequences of low positive predictive values

Testing as Prevention of Resistance in Bacteria Causing Sexually Transmitted Infections—A Population-Based Model for Germany

Prescribed antibiotic treatments which do not match the therapeutic requirements of potentially co-existing undetected sexually transmitted infections (STIs) can facilitate the selection of antibiotic-drug-resistant clones. To reduce this risk, this modelling assessed the potential applicability of reliable rapid molecular test assays targeting bacterial STI prior to the prescription of antibiotic drugs. The modelling was based on the prevalence of three bacterial STIs in German heterosexual and men-having-sex-with-men (MSM) populations, as well as on reported test characteristics of respective assays. In the case of the application of rapid molecular STI assays for screening, the numbers needed to test in order to correctly identify any of the included bacterial STIs ranged from 103 to 104 for the heterosexual population and from 5 to 14 for the MSM population. The number needed to harm—defined as getting a false negative result for any of the STIs and a false positive signal for another one, potentially leading to an even more inappropriate adaptation of antibiotic therapy than without any STI screening—was at least 208,995 for the heterosexuals and 16,977 for the MSM. Therefore, the screening approach may indeed be suitable to avoid unnecessary selective pressure on bacterial causes of sexually transmitted infections

New Developments in PCR-Based Diagnostics for Bacterial Pathogens Causing Gastrointestinal Infections—A Narrative Mini-Review on Challenges in the Tropics

The application of modern PCR approaches for the diagnosis of bacterial gastrointestinal pathogens is on the rise due to their rapidly available results combined with high sensitivity. While multiple studies describe the ongoing implementation of this technique for routine diagnostic purposes in laboratories in Western industrialized countries, reports on successful and also sustainable respective approaches in resource-poor tropical settings are still scarce. In order to shed light on potential reasons for this marked discrepancy, this narrative review summarizes identified challenges for the application of diagnostic PCR targeting bacterial gastrointestinal pathogens from stool samples in the tropics. The identified and discussed issues comprise the lack of generally accepted definitions for (1) minimum standards regarding sample acquisition, storage and transport time for diagnostic PCR analyses in the tropics, (2) nucleic acid extraction standards allowing an optimum detection of all types of pathogens which may be responsible for gastroenteritis in the tropics, (3) validation standards to ensure comparable quality of applied diagnostic assays, and (4) cut-offs for a reliable discrimination of infection and mere colonization in areas where semi-immunity due to repeated exposition associated with poor hygiene conditions has to be expected. Further implementation research is needed to solve those issues

Comparison of Self-Reported Sexual Activity Among Heterosexuals with Sexual Spread of Poorly Transmittable Agents: A Minimalistic Approach to Estimating Sexual Activity Based on HIV Incidence

The aim of this study was to assess whether epidemics of sexually transmitted infections caused by poorly transmittable agents corresponded to self-reported sexual activity in a distinct population. To exemplify this, a model was used to investigate whether HIV infection incidences corresponded to the extent of sexual activity as assessed by a questionnaire-based study. The model suggested between 97 and 486 sexual contacts per German individual during a sexually active lifetime based on the annual HIV incidence of 680 among the heterosexual population reported by the German National Health Authority. This is in line with the estimated 296 sexual contacts during one’s lifetime, which was indicated by questionnaire respondents. The model confirms the correspondence of self-reported sexual activity with HIV incidence as reported by the German National Health Authority. Accordingly, HIV incidence- and prevalence-based modeling of sexual activity in a population provides crude estimations in situations where a range of uncertainty is acceptable. The model’s veracity is limited by a number of assumptions necessitated by the paucity of data. Nevertheless, the model may be suitable in settings where severe reporting bias has to be expected for legal or socio-cultural reasons

Metagenomic Sequencing for the Diagnosis of Plasmodium spp. with Different Levels of Parasitemia in EDTA Blood of Malaria Patients—A Proof-of-Principle Assessment

Molecular diagnostic approaches are increasingly included in the diagnostic workup and even in the primary diagnosis of malaria in non-endemic settings, where it is difficult to maintain skillful microscopic malaria detection due to the rarity of the disease. Pathogen-specific nucleic acid amplification, however, bears the risk of overlooking other pathogens associated with febrile illness in returnees from the tropics. Here, we assessed the discriminatory potential of metagenomic sequencing for the identification of different Plasmodium species with various parasitemia in EDTA blood of malaria patients. Overall, the proportion of Plasmodium spp.-specific sequence reads in the assessed samples showed a robust positive correlation with parasitemia (Spearman r = 0.7307, p = 0.0001) and a robust negative correlation with cycle threshold (Ct) values of genus-specific real-time PCR (Spearman r = −0.8626, p ≤ 0.0001). Depending on the applied bioinformatic algorithm, discrimination on species level was successful in 50% (11/22) to 63.6% (14/22) instances. Limiting factors for the discrimination on species level were very low parasitemia, species-depending lacking availability of reliable reference genomes, and mixed infections with high variance of the proportion of the infecting species. In summary, metagenomic sequencing as performed in this study is suitable for the detection of malaria in human blood samples, but the diagnostic detection limit for a reliable discrimination on species level remains higher than for competing diagnostic approaches like microscopy and PCR