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.

As qualified microscopy of enteric parasitoses as defined by high diagnostic accuracy is difficult to maintain in non-endemic areas due to scarce opportunities for practicing with positive sample materials, molecular diagnostic options provide less investigator-dependent alternatives. Here, we compared three molecular targets for the real-time PCR-based detection of Cryptosporidium spp. From a population of 1000 individuals comprising both Ghanaian HIV (human immunodeficiency virus) patients and military returnees after deployment in the tropics, stool samples were assessed for Cryptosporidium spp. by real-time PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene, the Cryptosporidium oocyst wall (COWP) gene, and the DnaJ-like protein gene (DnaJ), respectively. In declining order, sensitivity of 100% for the SSU rRNA gene PCR, 90.0% for the COWP PCR and 88.8% for the DnaJ PCR, respectively, as well as specificity of 99.6% for the COWP PCR and 96.9% for both the SSU rRNA gene PCR and the DnaJ PCR, respectively, were recorded. Substantial agreement (kappa value 0.663) between the three assays was observed. Further, an accuracy-adjusted Cryptosporidium spp. prevalence of 6.0% was calculated for the study population. In conclusion, none of the assessed real-time PCR assays were associated with perfect test accuracy. However, a combination of highly sensitive SSU rRNA gene PCR for screening purposes and more specific COWP PCR for confirmatory testing should allow reliable diagnosis of Cryptosporidium spp. in stool samples even in low prevalence settings.

Diagnostic real-time PCR for the detection of Cyclospora cayetanensis in human stool samples has been applied for two decades. However, recent comparative assessments between in-house and commercial assays suggested room for improvement regarding the agreement of positive signals of the applied real-time PCRs. In order to assess the effect of the choice of the target sequence, 3 inhouse real time PCR assays targeting the 18S rRNA gene (n = 2, one of them later referred to as SSU rRNA gene assay to avoid confusion) and the hsp70 gene of C. cayetanensis were compared in a head-to-head comparison with 905 samples with high pretest probability for C. cayetanensis infections from Ghanaian HIV patients in a test comparison without a reference standard. Only slight agreement kappa of 0.095 was observed. In the assays targeting the SSU rRNA gene, the 18S rRNA gene, and hsp70, positive signals were recorded in 63, 45, and 0 instances, respectively, with latent class analysis-based estimation of sensitivity of 32.2%, 23.3%, 0% as well as of specificity of 99.7%, 99.9% and 100%, respectively. High cycle threshold values with an average of about 35 indicated low quantities of target DNA in the samples with similar Ct values in concordantly and discordantly positive samples. In conclusion, the study suggested target-gene-specific differences in the diagnostic accuracy of real-time PCR-based diagnosis of C. cayetanensis as well as an ongoing need for further standardization of this diagnostic approach.

Background: COVID-19 can show a variable course, from asymptomatic infections to acute respiratory failure and death. For efficient allocation of resources, patients should be stratified according to their risk for a severe course as early as possible.Methods: 135 hospitalized patients with COVID-19 pneumonia at four German hospitals were prospectively included in this observational study. A standardized clinical laboratory profile was taken at hospital admission and a panel of serum markers with possible roles in the COVID-associated cytokine storm were also determined. 112 patients could be evaluated. The primary endpoint of ventilator requirement or death within 30 days of symptom onset was met by 13 patients.Results: Serum elevations of interleukin-6 (IL-6), procalcitonin (PCT), and C-reactive protein (CRP) at hospital admission were each highly significantly (p < 0.001) associated with ventilator requirement/death within 30 days of symptom onset. With a sensitivity of 92% and a specificity of 65–67%, IL-6 ≥ 52.8 pg/ml, PCT ≥ 0.11 ng/ml, and CRP ≥ 71.1 mg/L were predictive of a severe course of COVID-19. Positive likelihood ratios were between 2.6–2.8 and negative likelihood ratios were between 0.11–0.13 for these three markers.Conclusion: Negative likelihood ratios indicate that IL-6, PCT, and CRP at hospital admission can be used for identifying patients at low risk for severe COVID-19 progression.

The study was conducted to identify cluster patterns of enteric microorganisms with potential etiological relevance for infectious gastroenteritis in stool samples of individuals from Ghana, which is a known high-endemicity setting for infectious gastroenteritis. These patterns were compared to previous observations with specimens from Colombian indigenous people in order to assess potentially stable clustering for temporally and spatially distinct populations from high-endemicity regions. By doing so, the study aimed to identify stable clusters as markers of microbial interaction with potential importance for etiological relevance assignment in cases of multiple enteric pathogen detections. Stool samples from 1569 Ghanaian individuals (875 from HIV patients, 30 from HIV-negative control adult patients, and 644 from children < 2 years of age) were assessed for enteric microorganisms by applying real-time PCR. As a result, nucleic acids of bacterial microorganisms were most frequently detected, followed by protozoa, microsporidia, and helminths. Interestingly, the cluster assessment confirmed interaction patterns known from the previous analysis with Colombian indigenous people, demonstrating a high likelihood of Blastocystis hominis for clustering with other microorganisms and a prominent, potentially mediating role of Dientamoeba fragilis for microbial interactions within the clusters. In conclusion, the assessment confirmed conserved clustering of enteric microorganisms with potential etiological relevance for human infectious gastroenteritis over geographically distinct high-endemicity settings. Furthermore, the composition of abundant microorganisms is more important than regional factors for the determination of the interplay of enteric microorganisms in the human gut. Thereby, some microbial pathogens and commensals seem more susceptible to a changing microbial composition in the human gut than others.

This study was performed to comparably assess two commercial real-time PCR assays for the identification of Trypanosoma cruzi DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for infections with either T. cruzi or apathogenic Trypanosoma rangeli were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL, ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with specificity for T. cruzi and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS, order no. 611013, referred to as the RealStar assay in the following) targeting a kinetoplast sequence of both T. cruzi and T. rangeli without further discrimination. To discriminate between T. cruzi- and T. rangeli-specific real-time PCR amplicons, Sanger sequencing results were available for a minority of cases with discordant real-time PCR results, while the amplicons of the remaining discordant samples were subjected to nanopore sequencing. The study assessment indicated a proportion of 18.1% (n = 94) T. cruzi-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite T. rangeli. The observed diagnostic accuracy as expressed by sensitivity and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and 96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced specificity resulted from cross-reaction with T. rangeli in all instances (3 cross-reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar assay). DNA from the six discrete typing units (DTUs) of T. cruzi was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of T. cruzi from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic T. rangeli according to the RealStar assay may be a disadvantage in areas of co-circulation with T. cruzi, while the test performance of the two compared assays will be quite similar in geographic settings where T. rangeli infections are unlikely.