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Filtering face piece (FFP) masks according to EN 149 Respiratory protective devices - Filtering half masks to protect against particles - Requirements, testing, marking are essential components of personal protective equipment against biological agents from an occupational health and hospital hygiene perspective. Therefore, shortages due to increased demand or supply bottlenecks can lead to staff threats due to the risk of infection. To determine whether FFP masks could be made reusable in a hospital setting, a thermal reprocessing concept (steam at 105 °C with a holding phase of 10 min) was evaluated in a bed reprocessing chamber. The results indicate that it is logistically possible to establish a reprocessing concept. Of 267 reprocessed masks, 48 were rejected by inspection because of defect strapping, trapped hair, misfolding, and missing lot number or deformation, and 22 masks were rejected by bacteriological examination because of contamination > 10 CFU of total bacteria per 25 cm 2 or the presence of Staphylococcus aureus . Two selected mask models maintained the expected mask performance equivalent to the FFP2 standard after reprocessing. Thermal reprocessing resulted in a virucidal effect. The results show that reprocessing of FFP masks in hospitals is possible. However, the success of reprocessing depends on the type of mask used. This study identified a suitable mask type for which the reported method is bactericidal and virucidal without impairing mask performance. The reported method required the use of a stationary hospital bed reprocessing chamber (sanitizing washer), so it cannot be used everywhere. Other methods and procedures should be tested to be independent of a bed reprocessing chamber and therefore may be more mobile and flexible.

Objective: To molecularly characterize several extensively drug-resistant isolates from a single hospital admission screening of a war-injured patient from Ukraine., Methods: Admission screening included swabs from skin, wounds, catheters, nasopharyngeum and rectum. Bacterial identification, antimicrobial susceptibility testing and rapid multiplex PCR assays targeting resistance genes were performed during routine diagnostics. Isolates positive by PCR had their genomes sequenced using short- and long read-platforms (MiSeq and MinION) to confirm species, identify resistance genes and plasmids and investigate clonality with core-genome MLST., Results: Seven Gram-negative pathogens (Acinetobacter baumannii (n = 2; ST78, ST2), Klebsiella pneumoniae (n = 2; ST395), Pseudomonas aeruginosa (n = 1; ST1047), Escherichia coli (n = 1; ST46), Enterobacter cloacae complex (n = 1; ST231)) were molecularly confirmed non-identical. Antimicrobial susceptibility testing showed resistance to carbapenems (7/7 isolates) and last-resort treatment options such as ceftazidime-avibactam (6/7 isolates) and cefiderocol (4/7 isolates). All isolates were colistin susceptible. Sequencing identified the E. cloacae complex as Enterobacter hormaechei subsp. xiangfangensis. Six acquired carbapenemase genes (bla IMP-1 , bla NDM-1 , bla OXA-48 , bla NDM-5 , bla OXA-23 and bla OXA-72 ) were detected. Both A. baumannii isolates differed in sequence type, carbapenemases and cefiderocol susceptibility. Both K. pneumoniae isolates shared sequence type and some resistance genes on an IncR plasmid but were different in core-genome MLST and carbapenemases (OXA-48 or NDM-1). One vancomycin-resistant Enterococcus faecium was also detected (VanA)., Conclusions: War-injured patients from Ukraine may carry different clones of multidrug-resistant pathogens with limited treatment options and diverse resistance genes at risk for dissemination. Infection control measures should include early molecular characterization of isolates for detection of routes of transmission., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction . Fungal infections are relevant health risks for individuals with acquired immunodeficiency in the resource-limited tropics, but available surveillance data are scarce. For Candida auris and Cryptococcus spp., the evolution from environmental reservoirs to human pathogens causing life-threatening diseases is currently discussed as a public health concern in the context of climate change and limited treatment options. Gap statement . Uncovering the gastrointestinal tract as an epidemiological niche of fungi emerging from the environment into individuals for whom fungal infections are not diagnosed. Aim . To contribute to data on the local epidemiology of C. auris and Cryptococcus spp. in Western African Ghana by analysing gastrointestinal samples of Ghanaian individuals. Methodology . Four real-time PCR assays targeting C. auris and five real-time PCR assays targeting Cryptococcus spp. were applied with stool samples of 875 non-age-stratified Ghanaian HIV patients and 30 Ghanaian control individuals without known HIV infection. Also, 664 samples from Ghanaian children under 2 years of age were investigated. The true abundance of the target micro-organism was considered as unlikely in the case of one or fewer positive signals, likely in the case of two to three positive signals and highly likely in the case of four or more positive signals per sample in the real-time PCR assays. Results . The combined application of sensitive, target-specific real-time PCR assays indicates that neither C. auris , Cryptococcus neoformans complex nor Cryptococcus gattii complex were part of the gut microbiota of Ghanaian individuals with or without HIV infection. Conclusion . Despite the significant disease burden from these pathogens in immunosuppressed Ghanaian individuals, detection from gastrointestinal samples was unlikely, which should be taken into account when discussing screening strategies for these fungi of public health concern. In contrast, the detection of these fungi from such samples should not routinely be considered as commensal colonization flora.

Background: Colombian indigenous Wiwas are exposed to a variety of partly complex medical conditions with a predominance of infectious diseases. The study provided here aims at verifying of falsifying previous suspicions on therapeutic shortcomings and neglect of disease categories., Material and Methods: Local diagnoses within various subpopulations of indigenous Wiwas obtained by a study physician and local health brigades and health points between 2017 and 2018 were coded following the ICD 10 classification from 2019. Proportions of diagnoses per ICD-10 sub-chapter were evaluated to find diseases and to rank the occurrence of diagnoses in the population of indigenous people. Thereafter, the available medication provided by the indigenous health care provider Dusakawi for the treatment of the indigenous patients was analyzed in regard of its sufficiency to cover the recorded diseases., Results: The majority of the diseases found in the communities cannot at all (32%) or only partially (56%) be treated according to available guidelines. Only few (12%), predominantly infectious diseases, were covered completely by the provided medication. Notably, there are some ICD chapters with diseases that do only rarely appear at all in the gained datasets, e.g., complications during birth, mental disorders or cancer., Conclusions: An expansion and revision of the medical supply for the indigenous population of the Sierra Nevada de Santa Marta is needed. An emergency kit for medical brigades and health points should be provided and in place. Awareness for neglected diseases needs to be created., (© 2024. The Author(s).)

Multiple microbial detections in stool samples of indigenous individuals suffering from chronic gastroenteric disorder of a likely infectious origin, characterized by recurring diarrhea of variable intensity, in the rural north-east of Colombia are common findings, making the assignment of etiological relevance to individual pathogens challenging. In a population of 773 indigenous people from either the tribe Wiwa or Kogui, collider bias analysis was conducted comprising 32 assessed microorganisms including 10 bacteria ( Aeromonas spp., Campylobacter spp., enteroaggregative Escherichia coli (EAEC), enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), Salmonella spp., Shiga toxin-producing Escherichia coli (STEC), Shigella spp./enteroinvasive Escherichia coli (EIEC), Tropheryma whipplei and Yersinia spp.), 11 protozoa ( Blastocystis spp., Cryptosporidium spp., Cyclospora spp., Dientamoeba fragilis , Entamoeba coli , Entamoeba bangladeshi / dispar / histolytica / moshkovskii complex, Entamoeba histolytica , Endolimax nana , Giardia duodenalis , Iodamoeba buetschlii and Pentatrichomonas hominis ), 8 helminths ( Ascaris spp., Enterobius vermicularis , Hymenolepis spp., Necator americanus , Schistosoma spp., Strongyloides spp., Taenia spp. and Trichuris spp.), microsporidia ( Encephalocytozoon spp.) and fungal elements (microscopically observed conidia and pseudoconidia). The main results indicated that negative associations potentially pointing towards collider bias were infrequent events (n = 14), while positive associations indicating increased likelihood of co-occurrence of microorganisms quantitatively dominated (n = 88). Microorganisms showing the most frequent negative associations were EPEC (n = 6) and Blastocystis spp. (n = 3), while positive associations were most common for Trichuris spp. (n = 16), Dientamoeba fragilis (n = 15), Shigella spp./EIEC (n = 12), Ascaris spp. (n = 11) and Blastocystis spp. (n = 10). Of note, positive associations quantitively dominated for Blastocystis spp. In conclusion, collider bias assessment did not allow clear-cut assignment of etiological relevance for detected enteric microorganisms within the assessed Colombian indigenous population. Instead, the results suggested complex microbial interactions with potential summative effects. Future studies applying alternative biostatistical approaches should be considered to further delineate respective interactions.

Background : For indigenous people in Colombia, high infection rates with Chagas disease (CD) are known. Methods : In 2018 and 2020, nine villages were screened for CD. CD-positive patients could enter a drug observed treatment. While, in 2018, Benznidazole (BNZ) was provided as the first-line drug by the government, nifurtimox (NFX) was administered in 2020. Results : Of 121 individuals treated with BNZ, 79 (65%) suffered from at least one adverse event (AE). Of 115 treated with NFX, at least one AE occurred in 96 (84%) patients. In 69% of BNZ cases, the side effects did not last longer than one day; this applied to 31% of NFX cases. Excluding extreme outlier values, average duration of AEs differed highly significantly: BNZ ( M = 0.7, SD = 1.4) and NFX ( M = 1.7, SD = 1.5, p < 0.001). Using an intensity scale, AEs were highly significantly more severe for NFX ( M = 2.1, SD = 0.58) compared to BZN ( M = 1.1, SD = 0.38), p < 0.001. When analyzing the duration in relation to the intensity, the burden of AEs caused by NFX was significantly more pronounced. Dropouts ( n = 2) due to AEs were in the NFX-group only. Conclusions : Side effects caused by BNZ were significantly fewer, as well as milder, shorter in duration, and more easily treatable, compared to NFX.

Indigenous people live in remote areas of Colombia. Multiple infections with bacteria, protozoa and/or helminths are common, as well as colonization in various forms. This study focused on the question of whether and to what extent various pathogens interact with each other. Therefore, a mathematical approach was retrospectively applied to PCR-based data of 244 stool samples, collected in two datasets. A stable cluster solution of the pathogens assessed was determined, and a unique configuration between Blastocystis hominis / Campylobacter spp./ Giardia lamblia forming cluster 1 and Dientaemoeba fragilis was verified. A pathogen density-dependent interplay appeared between the B. hominis / Campylobacter spp./ G. lamblia cluster, D. fragilis and Ascaris lumbricoides . The applied mathematical approach demonstrated that co-infections with parasites of questionable pathological relevance such as B. hominis and D. fragilis can be of diagnostic relevance due to their ability to promote or repress other pathogens. With the increasing availability of highly sensitive multiplexed molecular diagnostic approaches even in resource-limited settings, where multiple colonization of infection events with enteric pathogens in parallel are common, the importance of interpreting whole pathogen patterns rather than just individual pathogen detection may become more and more relevant.

Medical complications during pregnancy have been frequently reported from Western Africa with a particular importance of infectious complications. Placental tissue can either become the target of infectious agents itself, such as, e.g., in the case of urogenital schistosomiasis, or be subjected to contamination with colonizing or infection-associated microorganisms of the cervix or the vagina during vaginal delivery. In the retrospective cross-sectional assessment presented here, the quantitative dimension of infection or colonization with selected resistant or pathogenic bacteria and parasites was regionally assessed. To do so, 274 collected placental tissues from Ivory Coastal and Ghanaian women were subjected to selective growth of resistant bacteria, as well as to molecular screening for beta-lactamase genes, Schistosoma spp. and selected bacterial causative agents of sexually transmitted infections (STI). Panton-Valentine-negative methicillin-resistant Staphylococcus aureus (MRSA) was grown from 1.8% of the tissue samples, comprising the spa types t008 and t688, as well as the newly detected ones, t12101 (n = 2) and t12102. While the culture-based recovery of resistant Enterobacterales and nonfermentative rod-shaped Gram-negative bacteria failed, molecular assessments confirmed beta-lactamase genes in 31.0% of the samples with multiple detections of up to four resistance genes per sample and bla CTX-M , bla IMP , bla GES , bla VIM , bla OXA-58 -like, bla NDM , bla OXA-23 -like, bla OXA-48 -like and bla KPC occurring in descending order of frequency. The beta-lactamase genes bla OXA-40/24 -like, bla NMC&#95;A/IMI , bla BIC , bla SME , bla GIM and bla DIM were not detected. DNA of the urogenital schistosomiasis-associated Schistosoma haematobium complex was recorded in 18.6% of the samples, but only a single positive signal for S. mansoni with a high cycle-threshold value in real-time PCR was found. Of note, higher rates of schistosomiasis were observed in Ghana (54.9% vs. 10.3% in Ivory Coast) and Cesarean section was much more frequent in schistosomiasis patients (61.9% vs. 14.8% in women without Schistosoma spp. DNA in the placenta). Nucleic acid sequences of nonlymphogranuloma-venereum-associated Chlamydia trachomatis and of Neisseria gonorrhoeae were recorded in 1.1% and 1.9% of the samples, respectively, while molecular attempts to diagnose Treponema pallidum and Mycoplasma genitalium did not lead to positive results. Molecular detection of Schistosoma spp. or STI-associated pathogens was only exceptionally associated with multiple resistance gene detections in the same sample, suggesting epidemiological distinctness. In conclusion, the assessment confirmed considerable prevalence of urogenital schistosomiasis and resistant bacterial colonization, as well as a regionally expected abundance of STI-associated pathogens. Continuous screening offers seem advisable to minimize the risks for the pregnant women and their newborns.

Phlebotomine sand flies of the genus Sergentomyia are considered to be of minor importance as vectors of Leishmania parasites pathogenic to humans, but are known to transmit lizard parasites of the subgenus Sauroleishmania, including L. (S.) adleri. However, knowledge on the geographic distribution of Sauroleishmania spp. and the infection rates in the vectors is very limited. Therefore, our study aimed (1) to further elucidate the distribution and prevalence of Sauroleishmania spp. in their respective vectors and (2) to assess the potential risk for occasional transmission of Leishmania parasites to international military personnel deployed in camps in Mali and Niger. A total of 1,482 wild-caught sand flies (Sergentomyia spp. and closely related Grassomyia spp.) were screened by real-time PCR for the presence of Leishmania DNA. Thirty-two sand fly pools were tested positive, with six from Mali and 26 from Niger. The DNA of four representative isolates was sequenced. The resulting sequences revealed a homology to L. adleri, which leads to the first report of this species from Mali and Niger to the best of our knowledge. The results suggest that Sergentomyia (Sintonius) clydei might be the natural sand fly vector, while Grassomyia spp. appear to be refractory. No Leishmania sp. pathogenic to humans was detected in these sand flies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

Extended spectrum beta-lactamases (ESBL) are frequently found in Enterobacterales isolates from Western Africa. However, information on the molecular epidemiology of regional ESBL-positive Enterobacterales strains is scarce. In order to provide epidemiological information, ESBL-positive Escherichia coli isolates from stool samples of European soldiers with diarrhea deployed to a field camp in Mali were subjected to whole-genome sequencing (Illumina MiSeq and Oxford Nanopore MinION) and antimicrobial susceptibility testing. With two exemptions, sequence-based analysis suggested an absence of transmission events between soldiers as indicated by a high genetic diversity of isolates and sequence types, confirming previous rep-PCR results. Third-generation cephalosporin resistance was associated with the presence of bla CTX-M-15 genes with ( n  = 14) and without ( n  = 5) co-occurring bla TEM-1b genes. Between 0 and 6 virulence and resistance plasmids per isolate were recorded. The detected resistance plasmids could be categorized into five types, which, in turn, share different sequence-identical segments, representing particular antimicrobial resistance gene-associated mobile genetic elements (MGEs). Phenotypic resistance rates within the 19 assessed isolates that showed distinguishable colony morphologies were 94.7% (18/19) against ampicillin-sulbactam and trimethoprim/sulfamethoxazole, 68.4% (13/19) against moxifloxacin, 31.6% (6/19) against ciprofloxacin, 42.1% (8/19) against gentamicin, 31.6% (6/19) against tobramycin, and 21.1% (4/19) against piperacillin-tazobactam and fosfomycin. Virulence-associated genes mediating infectious gastroenteritis were rarely detected. The gene aggR , which is characteristic for enteroaggregative E. coli , was only detected in one single isolate. In summary, we found a variety of different strains and clonal lineages of ESBL-carrying E. coli. Transmission either between soldiers or from common contaminated sources was demonstrated in two cases and played only a minor role in this military field camp, while there were indications that resistance gene bearing MGEs had been exchanged between antimicrobial resistance gene-(ARG-)carrying plasmids., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hoffmann, Riediger, Tersteegen, Marquardt, Kahlfuß, Kaasch, Hagen, Frickmann and Zautner.)

Objectives: Military deployments to the tropics are associated with specific infection risks. To add to the available epidemiological information, infectious disease risks in German military personnel returning from predominantly tropical deployments were assessed., Methods: Since 2006, German soldiers returning from predominantly tropical deployments have been offered the opportunity of returnee screenings at the Department of Tropical Medicine and Infectious Diseases of the Bundeswehr Hospital Hamburg. Case files and diagnostic results recorded between 2006 and 2018 were retrospectively assessed to identify deployment-associated infectious disease risks., Results: Along with high enteric colonisation rates with apathogenic protozoa and resistant Enterobacteriaceae , direct or indirect proof of infections among the 764 assessed cases comprised Plasmodium spp (n=37), Giardia duodenalis (n=21), Schistosoma spp (n=14), Yersinia enterocolitica (n=5), Strongyloides stercoralis (n=3), Campylobacter jejuni (n=1), Leishmania spp (n=1) and Salmonella enterica (n=1), as well as latent infections with Mycobacterium tuberculosis complex (n=8). The infections were mainly imported from the African region and Eastern Mediterranean region and high proportions of cases lacked typical symptoms. Reported side effect rates of antimalarial chemoprophylaxis for mefloquine (n=121), atovaquone/proguanil (n=49) and doxycycline (n=6) were 36.3%, 19.3% and 11.8%, respectively, while non-compliance rates were 12.9%, 13.0% and 5.9%, respectively., Conclusions: Considerable rates of infections with sometimes atypical or absent symptoms confirm a need for returnee screenings after tropical deployments. High reported side effect rates for mefloquine support its replacement by atovaquone/proguanil or doxycycline for antimalarial chemoprophylaxis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

An Indigenous agropastoralist population called the Wiwa from the Sierra Nevada de Santa Marta, in North-East Colombia, shows high rates of gastrointestinal infections. Chronic gut inflammatory processes and dysbiosis could be a reason, suggesting an influence or predisposing potential of the gut microbiome composition. The latter was analyzed by 16S rRNA gene amplicon next generation sequencing from stool samples. Results of the Wiwa population microbiomes were associated with available epidemiological and morphometric data and compared to control samples from a local urban population. Indeed, locational-, age-, and gender-specific differences in the Firmicutes/Bacteriodetes ratio, core microbiome, and overall genera-level microbiome composition were shown. Alpha- and ß-diversity separated the urban site from the Indigenous locations. Urban microbiomes were dominated by Bacteriodetes , whereas Indigenous samples revealed a four times higher abundance of Proteobacteria . Even differences among the two Indigenous villages were noted. PICRUSt analysis identified several enriched location-specific bacterial pathways. Moreover, on a general comparative scale and with a high predictive accuracy, we found Sutterella associated with the abundance of enterohemorrhagic Escherichia coli (EHEC), Faecalibacteria associated with enteropathogenic Escherichia coli (EPEC) and helminth species Hymenolepsis nana and Enterobius vermicularis . Parabacteroides , Prevotella , and Butyrivibrio are enriched in cases of salmonellosis, EPEC, and helminth infections. Presence of Dialister was associated with gastrointestinal symptoms, whereas Clostridia were exclusively found in children under the age of 5 years. Odoribacter and Parabacteroides were exclusively identified in the microbiomes of the urban population of Valledupar. In summary, dysbiotic alterations in the gut microbiome in the Indigenous population with frequent episodes of self-reported gastrointestinal infections were confirmed with epidemiological and pathogen-specific associations. Our data provide strong hints of microbiome alterations associated with the clinical conditions of the Indigenous population.

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 bla CTX-M-15 , bla CTX-M-27 , bla CTX-M-1 , bla TEM-169 , bla CTX-M-14 , bla CTX-M-99 -like, bla CTX-M-125 , bla SHV-12 , and bla DHA-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.

Background: Salmonella enterica is among the major burdens for public health at global level. Typing of salmonellae below the species level is fundamental for different purposes, but traditional methods are expensive, technically demanding, and time-consuming, and therefore limited to reference centers. Fourier transform infrared (FTIR) spectroscopy is an alternative method for bacterial typing, successfully applied for classification at different infra-species levels., Aim: This study aimed to address the challenge of subtyping Salmonella enterica at O-serogroup level by using FTIR spectroscopy. We applied machine learning to develop a novel approach for S. enterica typing, using the FTIR-based IR Biotyper® system (IRBT; Bruker Daltonics GmbH & Co. KG, Germany). We investigated a multicentric collection of isolates, and we compared the novel approach with classical serotyping-based and molecular methods., Methods: A total of 958 well characterized Salmonella isolates (25 serogroups, 138 serovars), collected in 11 different centers (in Europe and Japan), from clinical, environmental and food samples were included in this study and analyzed by IRBT. Infrared absorption spectra were acquired from water-ethanol bacterial suspensions, from culture isolates grown on seven different agar media. In the first part of the study, the discriminatory potential of the IRBT system was evaluated by comparison with reference typing method/s. In the second part of the study, the artificial intelligence capabilities of the IRBT software were applied to develop a classifier for Salmonella isolates at serogroup level. Different machine learning algorithms were investigated (artificial neural networks and support vector machine). A subset of 88 pre-characterized isolates (corresponding to 25 serogroups and 53 serovars) were included in the training set. The remaining 870 samples were used as validation set. The classifiers were evaluated in terms of accuracy, error rate and failed classification rate., Results: The classifier that provided the highest accuracy in the cross-validation was selected to be tested with four external testing sets. Considering all the testing sites, accuracy ranged from 97.0% to 99.2% for non-selective media, and from 94.7% to 96.4% for selective media., Conclusions: The IRBT system proved to be a very promising, user-friendly, and cost-effective tool for Salmonella typing at serogroup level. The application of machine learning algorithms proved to enable a novel approach for typing, which relies on automated analysis and result interpretation, and it is therefore free of potential human biases. The system demonstrated a high robustness and adaptability to routine workflows, without the need of highly trained personnel, and proving to be suitable to be applied with isolates grown on different agar media, both selective and unselective. Further tests with currently circulating clinical, food and environmental isolates would be necessary before implementing it as a potentially stand-alone standard method for routine use., Competing Interests: Declaration of Competing Interest O.J.L, F.G., S.L., M.A., M.P., S.Z., M.B., J.S., H.F., D.D., Y.F., Z.N., J.S., L.O., A.C.V., U.S.J., H.M.H., A.L., S.A., S.P., L.S., A.W., S.R., R.M.H., J.M. and A.B.P. declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. M.C., N.M. and M.K. are Bruker's employees., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Both Schistosoma spp. (species) and Leishmania spp. are prevalent in Ghana in West Africa. However, little is known about their local occurrence in immunocompromised individuals. In the study presented here, the real-time PCR-(polymerase chain reaction-)based screening for repetitive DNA (deoxyribonucleotide acid) sequences from the genomes of Leishmania (L.) spp. and Schistosoma (S.) spp. was performed in the serum of HIV-(human immunodeficiency virus-)infected Ghanaian patients. In 1083 assessed serum samples from HIV-positive and HIV-negative Ghanian patients, Leishmania spp.-specific DNA was not detected, while the diagnostic accuracy-adjusted prevalence estimation suggested a 3.6% prevalence of the S. mansoni complex and a 0.5% prevalence of the S. haematobium complex. Associations of schistosomiasis with younger age, as well as with the male sex, could be shown but not with an HIV status. Weakly significant signals for the associations of schistosomiasis with an increased viral load, reduced CD4+ (CD = cluster of differentiation) T cell count, and a reduced CD4+/CD8+ ratio could be observed but was inconsistently lost in the case of the stratification on the species complex level. So, it is concluded that factors other than HIV status are more likely to have influenced the occurrence of Schistosoma spp. infections in the assessed Ghanaian patients. Potential associations between HIV infection-associated factors, such as the viral load and the immune status of the patients, for which weak signals were observed in this hypothesis-forming retrospective assessment, should be confirmed by prospective, sufficiently powered investigations.

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.

Rickettsiae may cause febrile infections in humans in tropical and subtropical regions. From Madagascar, no molecular data on the role of rickettsioses in febrile patients are available. Blood samples from patients presenting with fever in the area of the capital Antananarivo were screened for the presence of rickettsial DNA. EDTA (ethylenediaminetetraacetic acid) blood from 1020 patients presenting with pyrexia > 38.5 °C was analyzed by gltA -specific qPCR. Positive samples were confirmed by ompB -specific qPCR. From confirmed samples, the gltA amplicons were sequenced and subjected to phylogenetic analysis. From five gltA -reactive samples, two were confirmed by ompB -specific qPCR. The gltA sequence in the sample taken from a 38-year-old female showed 100% homology with R. typhi . The other sample taken from a 1.5-year-old infant was 100% homologous to R. felis . Tick-borne rickettsiae were not identified. The overall rate of febrile patients with molecular evidence for a rickettsial infection from the Madagascan study site was 0.2% (2/1020 patients). Flea-borne rickettsiosis is a rare but neglected cause of infection in Madagascar. Accurate diagnosis may prompt adequate antimicrobial treatment.

The study was performed to provide an overview of the molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in Afghanistan isolated by the German military medical service during the Afghanistan conflict. A total of 18 isolates were collected between 2012 and 2018 at the microbiological laboratory of the field hospital in Camp Marmal near Mazar-e Sharif, Afghanistan, from Afghan patients. The isolates were subjected to phenotypic and genotypic differentiation and antimicrobial susceptibility testing as well as to a core genome multi-locus sequence typing (cgMLST) approach based on whole-genome next-generation sequence (wgNGS) data. Next to several sporadic isolates, four transmission clusters comprising strains from the international clonal lineages IC1, IC2, and IC9 were identified. Acquired carbapenem resistance was due to bla OXA-23 in 17/18 isolates, while genes mediating resistance against sulfonamides, macrolides, tetracyclines, and aminoglycosides were frequently identified as well. In conclusion, the assessment confirmed both the frequent occurrence of A. baumannii associated with outbreak events and a variety of different clones in Afghanistan. The fact that acquired carbapenem resistance was almost exclusively associated with bla OXA-23 may facilitate molecular resistance screening based on rapid molecular assays targeting this resistance determinant.

Cystoisospora (C.) belli is a coccidian parasite associated with acute or chronic gastroenteritis in immunocompromised patients. Dissatisfactory sensitivity of microscopy as the diagnostic standard approach has been described. Here, we comparatively evaluated two real-time PCRs targeting ribosomal RNA gene sequences of C. belli in stool in a test comparison without a reference standard applying latent class analysis. Therefore, 1000 stool samples from Ghanaian HIV (human immunodeficiency virus) patients (n = 905) as well as military returnees from the tropics (n = 95) were assessed by both assays in parallel. After the exclusion of 33 samples showing PCR inhibition, 29 and 33 positive results were recorded with the 5.8S rRNA gene/ITS-2 sequence PCR and the ITS-2 sequence PCR, respectively, resulting in an accuracy-adjusted prevalence of 3.2%. Nearly perfect agreement between both assays was indicated by Fleiss' kappa of 0.933 with sensitivity and specificity of 92.8% and 100% as well as 100% and 99.8% for the 5.8S rRNA gene/ITS-2 sequence PCR and the ITS-2 sequence PCR, respectively. Both assays proved to be suitable for the diagnosis of C. belli in human stool samples with slightly better sensitivity of the ITS-2 sequence assay, while the 5.8S rRNA gene/ITS-2 sequence PCR may be considered for confirmatory testing.

To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three L eishmania -specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-( gpi -)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi -PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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.

Typhoidal and para-typhoidal Salmonella are major causes of bacteraemia in resource-limited countries. Diagnostic alternatives to laborious and resource-demanding serotyping are essential. Fourier transform infrared spectroscopy (FTIRS) is a rapidly developing and simple bacterial typing technology. In this study, we assessed the discriminatory power of the FTIRS-based IR Biotyper (Bruker Daltonik GmbH, Bremen, Germany), for the rapid and reliable identification of biochemically confirmed typhoid and paratyphoid fever-associated Salmonella isolates. In total, 359 isolates, comprising 30 S . Typhi, 23 S . Paratyphi A, 23 S . Paratyphi B, and 7 S . Paratyphi C, respectively and other phylogenetically closely related Salmonella serovars belonging to the serogroups O:2, O:4, O:7 and O:9 were tested. The strains were derived from clinical, environmental and food samples collected at different European sites. Applying artificial neural networks, specific automated classifiers were built to discriminate typhoidal serovars from non-typhoidal serovars within each of the four serogroups. The accuracy of the classifiers was 99.9%, 87.0%, 99.5% and 99.0% for Salmonella Typhi, Salmonella Paratyphi A, B and Salmonella Paratyphi C, respectively. The IR Biotyper is a promising tool for fast and reliable detection of typhoidal Salmonella . Hence, IR biotyping may serve as a suitable alternative to conventional approaches for surveillance and diagnostic purposes.

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 bla OXA-23 , bla OXA-58 , bla NDM-1 , and bla GES-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.

Following the rapid spread of a new type of coronavirus (SARS-CoV-2), nearly all countries have introduced temporary restrictions affecting daily life, with "social distancing" as a key intervention for slowing the spread of the virus. Despite the pandemic, the development or actualization of medical guidelines, especially in the rapidly changing field of oncology, needs to be continued to provide up-to-date evidence- and consensus-based recommendations for shared decision making and maintaining the treatment quality for patients. In this viewpoint, we describe the potential strengths and limitations of online conferences for medical guideline development. This viewpoint will assist guideline developers in evaluating whether online conferences are an appropriate tool for their guideline conference and audience., (©Theresa Steeb, Markus Follmann, Ralf Matthias Hagen, Carola Berking, Markus Vincent Heppt. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 29.12.2020.)

Introduction: The study was performed to assess the infection risk of German police officers on predominantly tropical deployments, mostly United Nations missions, with gastrointestinal pathogens., Methods: Police officers were offered PCR-based screening for gastrointestinal pathogens before and after deployment. The screening panel comprised enteroinvasive bacteria (Salmonella spp., Shigella spp./enteroinvasive Escherichia coli, Campylobacter jejuni, and Yersinia spp.), enteropathogenic protozoa (Entamoeba histolytica, Giardia duodenalis, Cryptosporidium spp., and Cyclospora cayetanensis), as well as enteric helminths (Ancyclostoma spp., Ascaris lumbricoides, Enterobius vermicularis, Hymenolepis nana, Necator americanus, African Schistosoma spp., Strongyloides stercoralis, Taenia saginata, Taenia solium, and Trichuris trichiura)., Results: G. duodenalis (n = 3), C. jejuni (n = 2), Salmonella spp. (n = 1), Shigella spp./enteroinvasive E. coli (n = 3), and S. stercoralis (n = 3) were detect in 12 out of 133 (9.0%) police officers. The majority had shown gastrointestinal symptoms on deployment and all were asymptomatic at the time of medical assessment. The major infection sites were Sub-Saharan Africa followed by Northern Africa and the Middle East., Conclusions: Deployment of police officers to tropical deployment sites on United Nations missions is associated with a considerable acquisition risk of gastrointestinal pathogens in a quantitatively relevant minority. Post-deployment screening is advisable to facilitate therapeutic and hygiene-related consequences.

Diarrhoea is a frequent symptom associated with travelling to tropical regions, but the cause is often not found. Epidemiology was assessed including up-to-date real-time PCR approaches.We analysed datasets of 528 patients who presented at the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany, between 2006 and 2010 for screening purposes or because of diarrhoea. Stool samples were obtained and investigated by microscopy, bacterial culture, two PCR assays targeting Entamoeba histolytica, Entamoeba dispar, Giardia duodenalis, and Cryptosporidium parvum, or Salmonella spp., Shigella/EIEC spp., Campylobacter jejuni, and Yersinia spp.Among patients with gastrointestinal symptoms, 51% tested positive for bacteria or parasites, of which 66% had a known enteropathogenic potential. In patients without diarrhoea, 53% (n = 80) were positive, and 33% of these cases harboured agents of pathogenic potential. Association with clinical symptoms was primarily found for bacterial infections. Blastocystis hominis, however, was more frequent in asymptomatic than in symptomatic travellers.In conclusion, the study stresses the etiological relevance of bacterial gastroenteritis in travellers returning from the tropics, the need for molecular approaches to increase diagnostic sensitivity and demonstrates that asymptomatic carriage of enteropathogens after prolonged stays in the tropics is similarly frequent compared with symptomatic infections in travellers.

Recently, a total of 32 carbapenem- and fluoroquinolone-resistant Acinetobacter baumannii (Ab) isolates was isolated from war-injured patients who were treated at German Bundeswehr Hospitals, and preliminarily typed by "DiversiLab" repetitive elements sequence-based (rep-) PCR. Core genome-based sequence typing was also used to provide more detailed epidemiological information. From the clusters observed by rep-PCR, selected Ab strains were subjected to Next Generation Sequencing (NGS) in order to compare them with international outbreak-associated Ab strains and to identify MLST (multi-locus sequence type) lineages, as well as to identify known resistance genes. Accordingly, NGS indicated higher diversity than rep-PCR, but also confirmed likely transmission events. The identified acquired carbapenem-resistant genes comprised bla OXA-23 , bla OXA-72 and bla GES-12 , as well as various other intrinsic and acquired resistance-associated genetic elements. All isolates clustered with the previously identified international clonal lineages IC1, IC2, IC6 and IC7, with corresponding Pasteur sequence types ST1, ST2, ST78 and ST25, respectively. In conclusion, the assessment confirmed a broad spectrum of resistance-associated genes in Ab isolated from war-injured patients from the Eastern Ukraine, and provided the first insights into locally abundant clonal lineages.

Background: Intestinal infections remain a major public health burden in developing countries. Due to social, ecological, environmental, and cultural conditions, Indigenous peoples in Colombia are at particularly high risk., Materials: 137 stool samples were analyzed by microscopy and real-time-Polymerase Chain Reaction (RT-PCR), targeting protozoan parasites ( Giardia intestinalis, Entamoeba histolytica, Cryptosporidium spp., and Cyclospora cayetanensis ), bacteria ( Campylobacter jejuni, Salmonella spp., Shigella ssp. / enteroinvasive E. coli ( EIEC) , Yersinia spp., enterohemorrhagic E. coli (EHEC) , enteropathogenic E. coli ( EPEC) , enterotoxin-producing E. coli ( ETEC) , enteroaggregative E. coli ( EAEC) , and Tropheryma whipplei), and helminths ( Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Ancylostoma spp., Trichuris. trichiura, Taenia spp., Hymenolepis nana , Enterobius vermicularis , and Schistosoma spp.). Microscopy found additional cases of helminth infections., Results: At least one pathogen was detected in 93% of the samples. The overall results revealed protozoa in 79%, helminths in 69%, and bacteria in 41%. G. intestinalis (48%) , Necator/hookworm (27%), and EAEC (68%) were the most common in each group. Noteworthy, T. whipplei was positive in 7% and T. trichirua in 23% of the samples. A significant association of one infection promoting the other was determined for G. intestinalis and C. jejuni, helminth infections, and EIEC., Conclusions: The results illustrate the high burden of gastrointestinal pathogens among Indigenous peoples compared to other developing countries. Countermeasures are urgently required.

The SARS-CoV-2 infection can be seen as a single disease, but it also affects patients with relevant comorbidities who may have an increased risk of a severe course of infection. In this report, we present a 77-year-old patient with a heart transplant receiving relevant immunosuppressive therapy who tested positive for SARS-CoV-2 after several days of dyspnea, dry cough, and light general symptoms. Computed tomography confirmed interstitial pneumonia. The patient received antiviral therapy with hydroxychloroquine and showed no further deterioration of the clinical state. After 12 days of hospitalization, the patient was released; he was SARS-CoV-2 negative and completely asymptomatic., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Two commercial PCR assays were assessed in a retrospective study to determine their reliability as tools for the differentiation of Plasmodium species in human blood., Methods: A total of 1022 blood samples from 817 patients with suspected or confirmed malaria submitted to the German National Reference Centre for Tropical Pathogens were subjected to malaria microscopy using thick and thin blood films as well as to a genus-specific malaria real-time PCR. Parasite-positive samples were analysed by RealStar Malaria S&T PCR Kit 1.0 (altona Diagnostics) and FTD Malaria Differentiation (Fast Track Diagnostics) multiplex real-time PCR assays targeting species-specific Plasmodium DNA., Results: Out of the 1022 blood samples, 247 (24.2%) tested positive for Plasmodium spp. The two multiplex assays showed rather similar performance characteristics and provided concordant species information in 98.9% of samples positive by malaria microscopy and in 95.1% (RealStar) and 96.8% (FTD) of samples positive by genus-specific PCR. Compared to FTD, RealStar revealed slightly reduced sensitivity for submicroscopic, low-level P. falciparum infections, while FTD was unable to detect P. knowlesi., Conclusions: The two commercial malaria PCR assays assessed are suitable for discriminating Plasmodium species in clinical samples, and can provide additional information in cases of microscopically uncertain findings., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In-house loop-mediated isothermal amplification (LAMP) procedures for the detection of paratyphoid fever-associated bacteria on serovar level were evaluated. Therefore, LAMP primers for Salmonella genus, for two LAMP schemes for S. Paratyphi A, for S. Paratyphi B and for S. Paratyphi C were tested with DNA from culture isolates from strain collections and spiked blood cultures against published PCR protocols targeting the same micro-organisms. Sensitivity and specificity for DNA from culture isolates verified by LAMP ranged from 80·0 to 100·0% and 96·1 to 100·0% vs 65 to 100% and 98·7 to 100% for the PCR approaches. For the spiked blood culture materials, sensitivity and specificity for LAMP ranged from 87·5 to 100·0% and 96·7 to 100·0% vs from 60 to 100% and 98·2 to 100% for PCR. In conclusion, LAMP for paratyphoid fever shows comparable performance characteristics as PCR. Due to its easy application, the procedure is well suited for surveillance purposes in resource-limited settings. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of easy-to-apply, point-of-care-testing-like loop-mediated isothermal amplification (LAMP) for the diagnosis of paratyphoid fever is evaluated. This approach can contribute to low-threshold availability of surveillance options for resource limited settings. Easy-to-teach and easy-to-apply LAMP schemes with similar performance characteristics as PCR are provided. The described test evaluation is of particular use for surveillance and public health experts., (© 2019 The Society for Applied Microbiology.)

Background: The potential of next-generation sequencing (NGS) for hypothesis-free pathogen diagnosis from (poly-)microbially contaminated, formalin-fixed, paraffin embedded tissue samples from patients with invasive fungal infections and amebiasis was investigated. Samples from patients with chromoblastomycosis (n = 3), coccidioidomycosis (n = 2), histoplasmosis (n = 4), histoplasmosis or cryptococcosis with poor histological discriminability (n = 1), mucormycosis (n = 2), mycetoma (n = 3), rhinosporidiosis (n = 2), and invasive Entamoeba histolytica infections (n = 6) were analyzed by NGS (each one Illumina v3 run per sample). To discriminate contamination from putative infections in NGS analysis, mean and standard deviation of the number of specific sequence fragments (paired reads) were determined and compared in all samples examined for the pathogens in question., Results: For matches between NGS results and histological diagnoses, a percentage of species-specific reads greater than the 4th standard deviation above the mean value of all 23 assessed sample materials was required. Potentially etiologically relevant pathogens could be identified by NGS in 5 out of 17 samples of patients with invasive mycoses and in 1 out of 6 samples of patients with amebiasis., Conclusions: The use of NGS for hypothesis-free pathogen diagnosis from contamination-prone formalin-fixed, paraffin-embedded tissue requires further standardization.

Background: The objective of this study was to assess an in-house loop-mediated isothermal amplification (LAMP) platform for malaria parasite detection and identification on species level., Methods: LAMP primers specific for the human Plasmodium spp., namely, P. falciparum, P. vivax, P. ovale, P. malariae , and P. knowlesi , as well as genus-specific primers, were tested against a composite gold standard comprising microscopy from thick and thin blood films, commercial genus-specific Meridian illumigene Malaria LAMP, in-house real-time polymerase chain reaction (PCR), and commercial fast-track diagnostics (FTD) Malaria differentiation PCR., Results: Of the 523 blood samples analyzed, the composite gold standard indicated 243 Plasmodium-species-DNA-containing samples (46.5%). Sensitivity and specificity of the analyzed genus- and species-specific LAMP primers were 71.0%-100.0% and 90.8%-100.0%, respectively. The influence of parasitemia was best documented for P. falciparum -specific LAMP with sensitivity values of 35.5% (22/62) for microscopically negative samples containing P. falciparum DNA, 50% (19/38) for parasitemia ≤50/μL, 84% (21/25) for parasitemia ≤500/μL, and 100% (92/92) for parasitemia >500/μL., Conclusions: In our hands, performance characteristics of species-specific in-house LAMP for malaria lack reliability required for diagnostic laboratories. The use of the easy-to-apply technique for surveillance purposes may be considered.

Two new DNA FISH-probes for Campylobacter fetus were designed, in silico checked for cross-reactions and successfully evaluated in a multi-centric approach with 41 Campylobacter fetus isolates including isolates of all three know subspecies: Campylobacter fetus ssp. fetus, Campylobacter fetus ssp. venerealis, and Campylobacter fetus ssp. testudinum and 40 strains of five non-target Campylobacter species., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: We assessed a commercial loop-mediated amplification (LAMP) platform for its reliability as a screening tool for malaria parasite detection., Methods: A total of 1000 blood samples from patients with suspected or confirmed malaria submitted to the German National Reference Center for Tropical Pathogens were subjected to LAMP using the Meridian illumigene Malaria platform. Results were compared with microscopy from thick and thin blood films in all cases. In case of discordant results between LAMP and microscopy (n = 60), confirmation testing was performed with real-time PCR. Persistence of circulating parasite DNA was analyzed by serial assessments of blood samples following malaria treatment., Results: Out of 1000 blood samples analyzed, 238 were positive for malaria parasites according to microscopy (n = 181/1000) or PCR (additional n = 57/60). LAMP demonstrated sensitivity of 98.7% (235/238), specificity of 99.6% (759/762), positive predictive value (PPV) of 98.7% (235/238) and negative predictive value (NPV) of 99.6% (759/762), respectively. For first slides of patients with malaria and for follow-up slides, sensitivity values were 99.1% (106/107) and 98.5% (129/131), respectively., Conclusions: The performance of the Meridian illumigene Malaria platform is suitable for initial screening of patients suspected of clinical malaria., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The European Union Training Mission Mali (EUTM MLI) is a multinational military training deployment to the Western African tropical nation of Mali. Based on routinely collected disease and non-battle injury surveillance data, this study quantifies the true impact of infectious diseases for this tropical mission and potential seasonal variations in infectious disease threats., Methods: Categorized health events during the EUTM MLI mission and associated lost working days were reported using the EpiNATO-2 report. Infection-related health events were descriptively analyzed for a 4-year period from the 12th week in 2013 to the 13th week in 2017. Aggregated EpiNATO-2 data collected from all missions other than EUTM MLI were used as a comparator., Results: Among the infectious diseases reported by EUTM MLI, non-severe upper respiratory infections and gastrointestinal diseases dominated quantitatively, accounting for 1.65 and 1.42 consultations per 100 person-weeks, respectively. The number of recorded infectious disease-associated lost working days during the whole study interval was 723. Seasonal changes in disease frequency were detectable. More gastrointestinal infections were seen in the rainy season, and more respiratory infections occurred in the dry season; these were associated with peaks of more than 2.5 consultations per 100 person-weeks for both categories., Conclusions: Despite initial concerns focused on tropical infectious diseases during this mission in tropical Mali, upper respiratory tract and gastrointestinal infections predominate. The relatively low number of reported lost working days may indicate that these infections are at the milder end of the spectrum of infectious diseases despite a likely reporting bias.

In western and eastern Africa, rickettsioses are one cause of fever in humans. Little is known regarding the presence of Rickettsia sp. in northern Cameroon. The present work was conducted in order to identify potential tick-borne spotted fever group Rickettsia in the Adamawa region of northern Cameroon, which may contribute filling some of the knowledge gaps of these pathogens. Ticks were collected from cattle in the municipal slaughterhouse of Ngaoundere in the Adamawa region of northern Cameroon. After morphological identification of tick species, extracted DNA was analyzed by PCR targeting the rickettsial ompB gene and the intergenic spacers dksA-xerC, mppA-purC and rpmE-tRNA fMet . Of the 316 adult ticks collected, 149 (47.1%) were Amblyomma variegatum, 92 (29%) Rhipicephalus spp. and 75 (23.7%) Hyalomma spp. Through the use of conventional PCR assays for the rickettsial ompB gene, rickettsial DNA was detected in 104 (32.9%) samples (85 Amblyomma sp., 14 Hyalomma spp. and 5 Rhipicephalus spp.). The ompB gene and the three intergenic were sequenced for 10 ticks in order to determine the rickettsial species. Rickettsia africae was detected in Amblyomma variegatum, Rickettsia aeschlimannii in Hyalomma rufipes and Hyalomma truncatum, Rickettsia sibirica in H. truncatum, Rickettsia massiliae in Rhipicephalus lunulatus and Candidatus Rickettsia barbariae in R. lunulatus. To the best of the author's knowledge, this report represents the first molecular evidence of rickettsial infection in ticks in the Adamawa region of northern Cameroon, which suggests a possible exposure of the human population in this region., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Introduction: We assessed the molecular epidemiology of multidrug-resistant bacteria colonizing or infecting war-injured patients from Libya and Syria who were treated at the Bundeswehr hospitals Hamburg and Westerstede, Germany., Methods: Enterobacteriaceae and Gram-negative rod-shaped nonfermentative bacteria with resistance against third-generation methoxyimino cephalosporins or carbapenems as well as methicillin-resistant Staphylococcus aureus (MRSA) from war-injured patients from Libya and Syria were assessed by molecular typing, i.e., spa typing for MRSA strains and rep-PCR and next-generation sequencing (NGS) for Gram-negative isolates., Results: A total of 66 isolates were assessed - comprising 44 Enterobacteriaceae, 16 nonfermentative rod-shaped bacteria, and 6 MRSA from 22 patients - and 8 strains from an assessment of the patient environment comprising 5 Enterobacteriaceae and 3 nonfermentative rod-shaped bacteria. Although 24 out of 66 patient strains were isolated more than 3 days after hospital admission, molecular typing suggested only 7 likely transmission events in the hospitals. Identified clonal clusters primarily suggested transmission events in the country of origin or during the medical evacuation flights., Conclusions: Nosocomial transmissions in hospital can be efficiently prevented by hygiene precautions in spite of heavy colonization. Transmission prior to hospital admission like on evacuation flights or in crises zones needs further assessment.

Background: Non-typhoidal Salmonella (NTS) cause the majority of bloodstream infections in Ghana, however the mode of transmission and source of invasive NTS in Africa are poorly understood. This study compares NTS from water sources and invasive bloodstream infections in rural Ghana., Methods: Blood from hospitalised, febrile children and samples from drinking water sources were analysed for Salmonella spp. Strains were serotyped to trace possible epidemiological links between human and water-derived isolates.. Antibiotic susceptibility testing was performed, RESULTS: In 2720 blood culture samples, 165 (6%) NTS were isolated. S. Typhimurium (70%) was the most common serovar followed by S. Enteritidis (8%) and S. Dublin (8%). Multidrug resistance (MDR) was found in 95 (58%) NTS isolates, including five S. Enteritidis. One S. Typhimurium showed reduced fluroquinolone susceptibility. In 511 water samples, 19 (4%) tested positive for S. enterica with two isolates being resistant to ampicillin and one isolate being resistant to cotrimoxazole. Serovars from water samples were not encountered in any of the clinical specimens., Conclusion: Water analyses demonstrated that common drinking water sources were contaminated with S. enterica posing a potential risk for transmission. However, a link between S. enterica from water sources and patients could not be established, questioning the ability of water-derived serovars to cause invasive bloodstream infections.

The occurrence of tick-borne relapsing fever and leptospirosis in humans in Madagascar remains unclear despite the presence of their potential vectors and reservoir hosts. We screened 255 Amblyomma variegatum ticks and 148 Rhipicephalus microplus ticks from Zebu cattle in Madagascar for Borrelia-specific DNA. Borrelia spp. DNA was detected in 21 Amblyomma variegatum ticks and 2 Rhipicephalus microplus ticks. One Borrelia found in one Rhipicephalus microplus showed close relationship to Borrelia theileri based on genetic distance and phylogenetic analyses on 16S rRNA and flaB sequences. The borreliae from Amblyomma variegatum could not be identified due to very low quantities of present DNA reflected by high cycle threshold values in real-time-PCR. It is uncertain whether these low numbers of Borrelia spp. are sufficient for transmission of infection from ticks to humans. In order to determine whether spirochaete infections are relevant in humans, blood samples of 1009 patients from the highlands of Madagascar with fever of unknown origin were screened for Borrelia spp. - and in addition for Leptospira spp. - by real-time PCR. No target DNA was detected, indicating a limited relevance of these pathogens for humans in the highlands of Madagascar., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Direct growth on blood and screening agar for methicillin-resistant Staphylococcus aureus (MRSA) at a tropical surveillance site was compared with broth enrichment and subsequent growth on selective MRSA agar after international sample transport. In Madagascar, 1548 swabs from an MRSA surveillance study were assessed for growth on Columbia blood agar enriched with 5% sheep blood and MRSA screening agar at the surveillance site with subsequent cold storage of the samples and shipment to Germany. In Germany, 1541 shipped samples were analyzed by non-selective broth enrichment with subsequent culture on MRSA selective agar. A total of 28 MRSA isolates were detected. Of these, 20 strains were isolated from direct culture on blood and MRSA screening agars at the surveillance site, 24 MRSA strains were isolated using the broth enrichment method in Germany, and 16 MRSA strains were identified by both approaches. In spite of the observed die-off of individual strains due to long-term storage and transport, broth enrichment with subsequent screening on MRSA selective agar after international sample shipment led to comparable sensitivity of MRSA detection like streaking on blood and MRSA agar at the tropical surveillance site.

Brucellosis, Q fever and melioidosis are zoonoses, which can lead to pyrexia. These diseases are often under-ascertained and underreported because of their unspecific clinical signs and symptoms, insufficient awareness by physicians and public health officers and limited diagnostic capabilities, especially in low-resource countries. Therefore, the presence of Brucella spp., Coxiella burnetii and Burkholderia pseudomallei was investigated in Malagasy patients exhibiting febrile illness. In addition, we analyzed zebu cattle and their ticks as potential reservoirs for Brucella and C. burnetii, respectively. Specific quantitative real-time PCR assays (qPCRs) were performed on 1020 blood samples drawn from febrile patients. In total, 15 samples (1.5%) were Brucella-positive, mainly originating from patients without travel history, while DNA from C. burnetii and Bu. pseudomallei was not detected. Anti-C. burnetii antibodies were found in four out of 201 zebu serum samples (2%), whereas anti-Brucella antibodies could not be detected. Brucella DNA was detected in a single zebu sample. Three out of 330 ticks analyzed (1%) were positively tested for C. burnetii DNA but with high Ct values in the qPCR assay. Our data suggest that zebus as well as Amblyomma and Boophilus ticks have to be considered as a natural reservoir or vector for C. burnetii, but the risk of cattle-to-human transmission is low. Since bovine brucellosis does not seem to contribute to human infections in Madagascar, other transmission routes have to be assumed., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Modern molecular diagnostic approaches in the diagnostic microbiological laboratory like real-time quantitative polymerase chain reaction (qPCR) have led to a considerable increase of diagnostic sensitivity. They usually outperform the diagnostic sensitivity of culture-based approaches. Culture-based diagnostics were found to be insufficiently sensitive for the assessment of the composition of biofilms in chronic wounds and poorly standardized for screenings for enteric colonization with multi-drug resistant bacteria. However, the increased sensitivity of qPCR causes interpretative challenges regarding the attribution of etiological relevance to individual pathogen species in case of multiple detections of facultative pathogenic microorganisms in primarily non-sterile sample materials. This is particularly the case in high-endemicity settings, where continuous exposition to respective microorganisms leads to immunological adaptation and semi-resistance while considerable disease would result in case of exposition of a non-adapted population. While biofilms in chronic wounds show higher pathogenic potential in case of multi-species composition, detection of multiple pathogens in respiratory samples is much more difficult to interpret and asymptomatic enteric colonization with facultative pathogenic microorganisms is frequently observed in high endemicity settings. For respiratory samples and stool samples, cycle-threshold-value-based semi-quantitative interpretation of qPCR results has been suggested. Etiological relevance is assumed if cycle-threshold values are low, suggesting high pathogen loads. Although the procedure is challenged by lacking standardization and methodical issues, first evaluations have led to promising results. Future studies should aim at generally acceptable quantitative cut-off values to allow discrimination of asymptomatic colonization from clinically relevant infection.

The effectiveness of a disinfectant-based decolonization strategy for multidrug-resistant bacteria like extended spectrum β-lactamase (ESBL)-positive Gram-negative bacteria with or without additional fluoroquinolon and carbapenem resistance as well as vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus was assessed. Between 2011 and 2015, 25 patients from Libya, Syria, and the Ukraine with war traumata were treated at the Bundeswehr hospital Hamburg. The patients were heavily colonized and infected with multidrug-resistant bacteria, altogether comprising 371 distinct combinations of pathogens and isolation sites. Local disinfection was assessed for effectiveness regarding successful decolonization of multidrug-resistant bacteria. Altogether, 170 cases of successful decolonization were observed, comprising 95 (55.8%) such events at sampling sites that were accessible to disinfecting procedures. The remaining 75 (44.2%) decolonization events had to be considered as spontaneous. In contrast, 95 out of 172 (55.2%) colonized isolation sites that were accessible to disinfection procedures were successfully decolonized. Patient compliance with the enforced hygiene procedures was associated with decolonization success. Systemic antibiotic therapy did not relevantly affect isolation time. Disinfecting washing moderately supports local decolonization of multidrug-resistant pathogens in comparison with spontaneous decolonization rates if the patients' compliance with the applied hygiene procedures is ensured.

Early identification of microbial pathogens is essential for rational and conservative antibiotic use especially in the case of known regional resistance patterns. Here, we describe fluorescence in situ hybridization (FISH) as one of the rapid methods for easy identification of microbial pathogens, and its advantages and disadvantages for the diagnosis of pathogens in human infections in the laboratory diagnostic routine. Binding of short fluorescence-labeled DNA or nucleic acid-mimicking PNA probes to ribosomes of infectious agents with consecutive analysis by fluorescence microscopy allows identification of bacterial and eukaryotic pathogens at genus or species level. FISH analysis leads to immediate differentiation of infectious agents without delay due to the need for microbial culture. As a microscopic technique, FISH has the unique potential to provide information about spatial resolution, morphology and identification of key pathogens in mixed species samples. On-going automation and commercialization of the FISH procedure has led to significant shortening of the time-to-result and increased test reliability. FISH is a useful tool for the rapid initial identification of microbial pathogens, even from primary materials. Among the rapidly developing alternative techniques, FISH serves as a bridging technology between microscopy, microbial culture, biochemical identification and molecular diagnostic procedures.

Background: The German Military Medical Service contributed to the medical screening of unaccompanied minor refugees (UMRs) coming to Germany in 2014 and 2015. In this study, a broad range of diagnostic procedures was applied to identify microorganisms with clinical or public health significance. Previously, those tests had only been used to screen soldiers returning from tropical deployments. This instance is the first time the approach has been studied in a humanitarian context., Methods: The offered screenings included blood cell counts, hepatitis B serology and microscopy of the stool to look for protozoa and worm eggs as well as PCR from stool samples targeting pathogenic bacteria, protozoa and helminths. If individuals refused certain assessments, their decision to do so was accepted. A total of 219 apparently healthy male UMRs coming from Afghanistan, Egypt, Somalia, Eritrea, Syria, Ghana, Guinea, Iran, Algeria, Iraq, Benin, Gambia, Libya, Morocco, Pakistan, and Palestine were assessed. All UMRs who were examined at the study department were included in the assessment., Results: We detected decreasing frequencies of pathogens that included diarrhoea-associated bacteria [C ampylobacter (C.) jejuni , enteropathogenic Escherichia (E.) coli (EPEC), enterotoxic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC)/ Shigella spp.), Giardia (G.) duodenalis , helminths (comprising Schistosoma spp., Hymenolepis (H.) nana , Strongyloides (S.) stercoralis ] as well as hepatitis B virus. Pathogenic microorganisms dominated the samples by far. While G. duodenalis was detected in 11.4% of the assessed UMRs, the incidence of newly identified cases in the German population was 4.5 cases per 100,000 inhabitants., Conclusions: We conclude that the applied in-house PCR screening systems, which have proven to be useful for screening military returnees from tropical deployments, can also be used for health assessment of immigrants from the respective sites. Apparently healthy UMRs may be enterically colonized with a broad variety of pathogenic and apathogenic microorganisms. Increased colonization rates, as shown for G. duodenalis, can pose a hygiene problem in centralized homes for asylum seekers.