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.

A comparison of two PCR protocols for the differentiation of Plasmodium ovale species and implications for clinical management in travellers returning to Germany: a 10-year cross-sectional study.

BACKGROUND: To assess the occurrence of Plasmodium ovale wallikeri and Plasmodium ovale curtisi species in travellers returning to Germany, two real-time PCR protocols for the detection and differentiation of the two P. ovale species were compared. Results of parasite differentiation were correlated with patient data. METHODS: Residual nucleic acid extractions from EDTA blood samples of patients with P. ovale spp. malaria, collected between 2010 and 2019 at the National Reference Centre for Tropical Pathogens in Germany, were subjected to further parasite discrimination in a retrospective assessment. All samples had been analysed by microscopy and by P. ovale spp.-specific real-time PCR without discrimination on species level. Two different real-time PCR protocols for species discrimination of P. o. curtisi and P. o. wallikeri were carried out. Results were correlated with patient data on gender, age, travel destination, thrombocyte count, and duration of parasite latency. RESULTS: Samples from 77 P. ovale spp. malaria patients were assessed, with a male:female ratio of about 2:1 and a median age of 30 years. Parasitaemia was low, ranging from few visible parasites up to 1% infected erythrocytes. Discriminative real-time PCRs revealed 41 cases of P. o. curtisi and 36 cases of P. o. wallikeri infections. Concordance of results by the two PCR approaches was 100%. Assessment of travel destinations confirmed co-existence of P. o. curtisi and P. o. wallikeri over a wide range of countries in sub-Saharan Africa. Latency periods for the two P. ovale species were similar, with median values of 56.0 days for P. o. curtisi and 58.0 days for P. o. wallikeri; likewise, there was no statistically significant difference in thrombocyte count with median values of 138.5/µL for patients with P. o. curtisi and 152.0/µL for P. o. wallikeri-infected patients. CONCLUSIONS: Two different real-time PCR protocols were found to be suitable for the discrimination of P. o. curtisi and P. o. wallikeri with only minor differences in sensitivity. Due to the overall low parasitaemia and the lack of differences in severity-related aspects like parasite latency periods or thrombocyte counts, this study supports the use of P. ovale spp. PCR without discrimination on species level to confirm the diagnosis and to inform clinical management of malaria in these patients.

Modelling of doxycycline-based prevention of bacterial sexually transmitted infections in comparison to condom-based and test-based prevention.

Background: Doxycycline-based prevention of bacterial sexually transmitted infections (STIs) has been assessed in various studies and has been recommended by the European AIDS Clinical Society to be proposed to persons with repeated STIs on a case-by-case basis. However, while good preventive effects could be shown for Chlamydia trachomatis and Treponema pallidum in Europe, no reliable prevention against doxycycline resistance-affected bacterial causes of STIs like Neisseria gonorrhoeae and Mycoplasma genitalium was confirmed. Methods: In a modelling-approach, we assessed potential beneficial effects even against the latter microorganisms in case of optimized adherence with doxycycline prevention. These effects were modelled for Germany in comparison to traditional prevention schemes like condom-based STI-prevention and testing-as-prevention. Results: With estimated risk reduction in the ranges of 86% for N. gonorrhoeae and of 82% for Mycoplasma genitalium, expectable preventive efficacy similar to alternative preventive approaches could be calculated in case of optimized adherence with doxycycline prevention. In case of repeated risk exposure, the preventive potential of condom-based prevention was decreased compared to both optimized doxycycline prevention and testing-as-prevention. Conclusions: As suggested by the applied modelling, the preventive effect of optimized doxycycline prevention against bacterial STIs is in a similar range, like other common prevention strategies.

Clustering of Gastrointestinal Microorganisms in Human Stool Samples from Ghana.

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.

Information Technology Systems for Infection Control in German University Hospitals-Results of a Structured Survey a Year into the Severe Acute Respiratory Syndrome Coronavirus 2 Pandemic.

BACKGROUND: Digitalization is playing a major role in mastering the current coronavirus 2019 (COVID-19) pandemic. However, several outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in German hospitals last year have shown that many of the surveillance and warning mechanisms related to infection control (IC) in hospitals need to be updated. OBJECTIVES: The main objective of the following work was to assess the state of information technology (IT) systems supporting IC and surveillance in German university hospitals in March 2021, almost a year into the SARS-CoV-2 pandemic. METHODS: As part of the National Research Network for Applied Surveillance and Testing project within the Network University Medicine, a cross-sectional survey was conducted to assess the situation of IC IT systems in 36 university hospitals in Germany. RESULTS: Among the most prominent findings were the lack of standardization of IC IT systems and the predominant use of commercial IC IT systems, while the vast majority of hospitals reported inadequacies in the features their IC IT systems provide for their daily work. However, as the pandemic has shown that there is a need for systems that can help improve health care, several German university hospitals have already started this upgrade independently. CONCLUSIONS: The deep challenges faced by the German health care sector regarding the integration and interoperability of IT systems designed for IC and surveillance are unlikely to be solved through punctual interventions and require collaboration between educational, medical, and administrative institutions.

Comparison of the Diagnostic Accuracy of Three Real-Time PCR Assays for the Detection of Arcobacter butzleri in Human Stool Samples Targeting Different Genes in a Test Comparison without a Reference Standard.

Potential etiological relevance for gastroenteric disorders including diarrhea has been assigned to Arcobacter butzleri. However, standard routine diagnostic algorithms for stool samples of patients with diarrhea are rarely adapted to the detection of this pathogen and so, A. butzleri is likely to go undetected unless it is specifically addressed, e.g., by applying pathogen-specific molecular diagnostic approaches. In the study presented here, we compared three real-time PCR assays targeting the genes hsp60, rpoB/C (both hybridization probe assays) and gyrA (fluorescence resonance energy transfer assay) of A. butzleri in a test comparison without a reference standard using a stool sample collection with a high pretest probability from the Ghanaian endemicity setting. Latent class analysis was applied with the PCR results obtained with a collection of 1495 stool samples showing no signs of PCR inhibition to assess the real-time PCR assays' diagnostic accuracy. Calculated sensitivity and specificity were 93.0% and 96.9% for the hsp60-PCR, 100% and 98.2% for the rpoB/C-PCR, as well as 12.7% and 99.8% for the gyrA-PCR, respectively. The calculated A. butzleri prevalence within the assessed Ghanaian population was 14.7%. As indicated by test results obtained with high-titer spiked samples, cross-reactions of the hsp60-assay and rpoB/C-assay with phylogenetically related species such as A. cryaerophilus can occur but are less likely with phylogenetically more distant species like, e.g., A. lanthieri. In conclusion, the rpoB/C-assay showed the most promising performance characteristics as the only assay with sensitivity >95%, albeit associated with a broad 95%-confidence interval. In addition, this assay showed still-acceptable specificity of >98% in spite of the known cross-reactivity with phylogenetically closely related species such as A. cryaerophilus. If higher certainty is desired, the gyrA-assay with specificity close to 100% can be applied for confirmation testing with samples showing positive rpoB/C-PCR results. However, in case of a negative result in the gyrA-assay, this cannot reliably exclude the detection of A. butzleri in the rpoB/C-assay due to the gyrA-assay's very low sensitivity.

Limited Reliability of the Molecular Detection of Plasmodium spp. from Incubated Blood Culture Samples for Forensic Purposes.

The suitability of incubated blood culture material for forensic molecular malaria diagnosis was assessed for non-endemic settings for cases in which the differential diagnosis malaria was initially overlooked. For the proof-of-principle assessment, residual blood culture materials from febrile patients from tropical Ghana were investigated by real-time PCR and compared with available historic microscopic results. In 2114 samples, for which microscopical results and real-time PCR results were available, microscopical results comprised 711 P. falciparum detections, 7 P. malariae detections, 1 microscopically not-further-discriminable Plasmodium spp. detection as well as 13 detections of mixed infections comprising 12 cases of P. falciparum/P. malariae co-infections and 1 case of a P. falciparum/P. ovale complex co-infection, while real-PCR indicated 558 P. falciparum detections, 95 P. malariae detections, 10 P. ovale complex detections, 1 P. vivax detection and 4 detected P. falciparum/P. malariae co-infections. Concordance of routine microscopy and real-time PCR was imperfect. Using routine microscopy as reference was associated with a seemingly low agreement of positive real-time PCR results of 90.9%. However, if positive samples, either by routine microscopy or real-time PCR or both, were applied as a combined reference, the agreement of positive results obtained with real-time PCR was increased from 74.0% to 77.9%, while the agreement of positive results obtained with routine microscopy was decreased from 100% to 85.3%. The predictive value of routine microscopy for negative results in the reference was slightly better with 90.9% compared to real-time PCR with 86.9%; the concordance between routine microscopy and real-time PCR was imperfect. In conclusion, even suboptimal sample materials such as incubated blood culture materials can be applied for forensic malaria diagnosis, if more suitable sample materials are not available, but the molecular detection rate of positive results in routine microscopy is much lower than previously reported for non-incubated blood.

Molecular Epidemiology of Escherichia coli with Resistance against Third-Generation Cephalosporines Isolated from Deployed German Soldiers-A Retrospective Assessment after Deployments to the African Sahel Region and Other Sites between 2007 and 2016.

Colonization and infection with bacteria with acquired antibiotic resistance are among the risks for soldiers on international deployments. Enterobacterales with resistance against third-generation cephalosporines are amongst the most frequently imported microorganisms. To contribute to the scarcely available epidemiological knowledge on deployment-associated resistance migration, we assessed the molecular epidemiology of third-generation cephalosporine-resistant Escherichia coli isolated between 2007 and 2016 from German soldiers after deployments, with a particular focus on the African Sahel region. A total of 51 third-generation cephalosporine-resistant E. coli isolated from 51 military returnees from deployment collected during the assessment period between 2007 and 2016 were subjected to short-read next-generation sequencing analysis. Returnees from the Sahel region (Djibouti, Mali, South Sudan, Sudan, Sudan, and Uganda) comprised a proportion of 52.9% (27/51). Repeatedly isolated sequence types according to the Warwick University scheme from returnees from the Sahel region were ST38, ST131, and ST648, confirming previous epidemiological assessments from various sub-Saharan African regions. Locally prevalent resistance genes in isolates from returnees from the Sahel region associated with third-generation resistance were blaCTX-M-15, blaCTX-M-27, blaCTX-M-1, blaTEM-169, blaCTX-M-14, blaCTX-M-99-like, blaCTX-M-125, blaSHV-12, and blaDHA-1, while virulence genes were east1, sat, and tsh in declining order of frequency of occurrence each. In line with phenotypically observed high resistance rates for aminoglycosides and trimethoprim/sulfamethoxazole, multiple associated resistance genes were observed. A similar, slightly more diverse situation was recorded for the other deployment sites. In summary, this assessment provides first next-generation sequencing-based epidemiological data on third-generation cephalosporine-resistant E. coli imported by deployed German soldiers with a particular focus on deployments to the Sahel region, thus serving as a small sentinel. The detected sequence types are well in line with the results from previous epidemiological assessments in sub-Saharan Africa.

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.

Comparative Evaluation of Real-Time Screening PCR Assays for Giardia duodenalis and of Assays Discriminating the Assemblages A and B.

Due to superior sensitivity compared to traditional microscopy, real-time PCR has been well established for the diagnosis of Giardia duodenalis in human stool samples. In this study, screening real-time PCRs for different target genes of G. duodenalis, i.e., the 18S rRNA gene, the gdh (glutamate dehydrogenase) gene and the bg (beta-giardin) gene, were comparatively assessed next to various real-time PCR assays for the discrimination of the assemblages A and B of G. duodenalis targeting the bg gene with and without locked nucleic acid-containing probes as well as the tpi (triose phosphate isomerase) gene. The screening PCRs were assessed by including 872 non-preselected samples with a high pre-test probability for G. duodenalis in the statistical analysis, while 53 G. duodenalis-positive samples as indicated by at least two screening PCRs were finally included in the assessment of the assemblage-specific PCRs. For the screening PCRs, sensitivity estimated with latent class analysis (LCA) ranged from 17.5% to 100%, specificity from 92.3% to 100% with an accuracy-adjusted prevalence of 7.2% for G. duodenalis within the non-preselected sample collection. In detail, sensitivity and specificity were 100% and 100% for the 18S rRNA gene-specific assay, 17.5% and 92.3% for the gdh gene-specific assay, and 31.7% and 100% for the bg gene-specific assay, respectively. Agreement kappa was slight with only 15.5%. For the assemblage-specific PCRs, estimated sensitivity ranged from 82.1% to 100%, specificity from 84.0% to 100% with nearly perfect agreement kappa of 90.1% for assemblage A and yet substantial agreement of 74.8% for assemblage B. In detail for assemblage A, sensitivity and specificity were 100% and 100% for the bg gene-specific assay without locked nucleic acids (LNA) as well as 100% and 97.8% for both the bg gene-specific assay with LNA and the tri gene-specific assay, respectively. For assemblage B, sensitivity and specificity were 100% and 100% for the bg gene-specific assay without LNA, 96.4% and 84.0% for the bg gene-specific assay with LNA, and 82.1% and 100% for the tri gene-specific assay, respectively. Within the assessed sample collection, the observed proportion comprised 15.1% G. duodenalis assemblage A, 52.8% G. duodenalis assemblage B and 32.1% non-resolved assemblages. Only little differences were observed regarding the cycle threshold (Ct) values when comparing the assays. In conclusion, best diagnostic accuracy was shown for an 18S rRNA gene-specific screening assay for G. duodenalis and for a differentiation assay discriminating the G. duodenalis assemblages A and B by targeting the bg gene with probes not containing locked nucleic acids. By adding additional highly specific competitor assays for confirmation testing, diagnostic specificity can be further increased on the cost of sensitivity if optimized specificity is desired.

Comparison of Three Real-Time PCR Assays for the Detection of Cyclospora cayetanensis in Stool Samples Targeting the 18S rRNA Gene and the hsp70 Gene.

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.

Intestinal Helminth Infections in Ghanaian Children from the Ashanti Region between 2007 and 2008-A Retrospective Cross-Sectional Real-Time PCR-Based Assessment.

In spite of ongoing eradication programs, helminth infections are still a medical issue in Ghana. For follow-up assessments on the decline of regional helminth infections, historic baseline prevalence values obtained with standardized diagnostic procedures can be helpful. In this retrospective cross-sectional study, real-time PCR targeting the nematodes Ancylostoma spp. (ITS2), Ascaris lumbricoides (ITS1), Enterobius vermicularis (ITS1), Necator americanus (ITS2), Strongyloides stercoralis (18S rRNA) and Trichuris trichiura (18S rRNA), the trematodes Schistosoma spp. (ITS2) as well as the cestodes Hymenolepis nana (ITS1), Taenia saginata (ITS1) and Taenia solium (ITS1) was applied with 2046 DNA eluates from stool samples of Ghanaian children from the Ashanti region collected between 2007 and 2008 in order to retrospectively define prevalence values. The overall prevalence was low with 3.8% (n = 77) and only 0.1% (n = 2) double infections with helminths were recorded. The three most frequently detected enteric helminth species comprised 2% S. stercoralis (n = 41), 0.8% H. nana (n = 16), and 0.7% N. americanus (n = 14), while only sporadic infection events were recorded for other helminth species comprising 0.1% E. vermicularis (n = 2), 0.1% Schistosoma spp. (n = 2), 0.1% T. saginata (n = 1) and 0.1% T. trichiura (n = 1). A. lumbricoides, Ancylostoma spp. and T. solium were not detected at all. In conclusion, the retrospective assessment suggests a low prevalence of enteric helminth infections in Ghanaian children from the Ashanti Region within the assessment period between 2007 and 2008.

Epidemiology of Plasmids in Escherichia coli and Klebsiella pneumoniae with Acquired Extended Spectrum Beta-Lactamase Genes Isolated from Chronic Wounds in Ghana.

Little information is available on the local epidemiology of mobile genetic elements such as plasmids harboring acquired beta-lactamase genes in Western African Ghana. In the present study, we screened for plasmids in three Escherichia coli and four Klebsiella pneumoniae isolates expressing extended spectrum beta-lactamases (ESBL) mediated by the blaCTX-M-15 gene from chronically infected wounds of Ghanaian patients. Bacterial isolates were subjected to combined short-read and long-read sequencing to obtain the sequences of their respective plasmids. In the blaCTX-M-15-gene-carrying plasmids of the four ESBL-positive K. pneumoniae isolates, IncFIB/IncFII (n = 3) and FIA (n = 1) sequences were detected, while in the blaCTX-M-15-gene-carrying plasmids of the three ESBL-positive E. coli isolates, IncFIA/IncFIB (n = 2) and IncFIB (n = 1) sequences were found. The three IncFIB/IncFII sequence-containing plasmids were almost identical to a K. pneumoniae plasmid reported from France. They belonged to the clonal lineages ST17, ST36 and ST39 of K. pneumoniae, suggesting transversal spread of this obviously evolutionary successful plasmid in Ghana. Other resistance gene-encoding plasmids observed in the assessed Enterobacterales harbored IncFIA/IncR and IncFII sequences. International spread was confirmed by the high genetic similarity to resistance-mediating plasmids published from Asia, Australia, Europe and Northern America, including a blaCTX-M-15-gene-carrying plasmid isolated from a wild bird in Germany. In conclusion, the study contributed to the scarcely available information on the epidemiology of third-generation cephalosporine resistance-mediating plasmids in Ghana. Furthermore, the global spread of resistance-mediating plasmids provided hints on the evolutionary success of individual resistance-harboring plasmids by transversal spread among K. pneumoniae lineages in Ghana.

Correction: Tanida et al. Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples. Pathogens 2021, 10, 656.

In the original publication [...].

Antimicrobial resistance of the enteric protozoon Giardia duodenalis - A narrative review.

INTRODUCTION: As therapy-refractory giardiasis is an emerging health issue, this review aimed at summarizing mechanisms of reduced antimicrobial susceptibility in Giardia duodenalis and strategies to overcome this problem. METHODS: A narrative review on antimicrobial resistance in G. duodenalis was based upon a selective literature research. RESULTS: Failed therapeutic success has been observed for all standard therapies of giardiasis comprising nitroimidazoles like metronidazole or tinidazole as first line substances but also benznidazoles like albendazole and mebendazole, the nitrofuran furazolidone, the thiazolide nitazoxanide, and the aminoglycoside paromomycin. Multicausality of the resistance phenotypes has been described, with differentiated gene expression due to epigenetic and post-translational modifications playing a considerable bigger role than mutational base exchanges in the parasite DNA. Standardized resistance testing algorithms are not available and clinical evidence for salvage therapies is scarce in spite of research efforts targeting new giardicidal drugs. CONCLUSION: In case of therapeutic failure of first line nitroimidazoles, salvage strategies including various options for combination therapy exist in spite of limited evidence and lacking routine diagnostic-compatible assays for antimicrobial susceptibility testing in G. duodenalis. Sufficiently powered clinical and diagnostic studies are needed to overcome both the lacking evidence regarding salvage therapy and the diagnostic neglect of antimicrobial resistance.

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.

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.

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Isolates from Northern Africa and the Middle East.

At the Bundeswehr Hospitals of Hamburg and Westerstede, patients repatriated from subtropical war and crisis zones of Northern Africa and the Middle East were medically treated, including microbiological assessment. Within a six-year interval, 16 Acinetobacter spp. strains, including 14 Acinetobacter baumannii (Ab) isolates with resistance against carbapenems and origins in Afghanistan (n = 4), Iraq (n = 2), Libya (n = 2), and Syria (n = 8) were collected. While clonal relationships of Libyan and Syrian strains had been assessed by superficial next generation sequencing (NGS) and "DiversiLab" repetitive elements sequence-based (rep-)PCR so far, this study provides core genome-based sequence typing and thus more detailed epidemiological information. In detail, sequencing allowed a definitive species identification and comparison with international outbreak-associated Ab strains by core genome multi locus sequence typing (cgMLST) and the identification of MLST lineages, as well as the identification of known resistance genes. The sequence analysis allowed for the confirmation of outbreak-associated clonal clusters among the Syrian and Afghan Ab isolates, indicating likely transmission events. The identified acquired carbapenem resistance genes comprised blaOXA-23, blaOXA-58, blaNDM-1, and blaGES-11, next to other intrinsic and acquired, partly mobile resistance-associated genes. Eleven out of 14 Ab isolates clustered with the previously described international clonal lineages IC1 (4 Afghan strains), IC2 (6 Syrian strains), and IC7 (1 Syrian strain). Identified Pasteur sequence types of the 14 Ab strains comprised ST2 (Syrian), ST25 (Libyan), ST32 (Iraqi), ST81 (Afghan), ST85 (Libyan), and ST1112 (Syrian), respectively. In conclusion, the study revealed a broad spectrum of resistance genes in Ab isolated from war-injured patients from Northern Africa and the Middle East, thereby broadening the scarcely available data on locally abundant clonal lineages and resistance mechanisms.