Collider Bias Assessment in Colombian Indigenous Wiwa and Kogui Populations with Chronic Gastroenteric Disorder of Likely Infectious Etiology Suggests Complex Microbial Interactions Rather Than Clear Assignments of Etiological Relevance.

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.

Chagas Disease: Comparison of Therapy with Nifurtimox and Benznidazole in Indigenous Communities in Colombia.

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.

Gastrointestinal Pathogens in Multi-Infected Individuals: A Cluster Analysis of Interaction.

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.

Screening for Resistant Bacteria, Antimicrobial Resistance Genes, Sexually Transmitted Infections and Schistosoma spp. in Tissue Samples from Predominantly Vaginally Delivered Placentae in Ivory Coast and Ghana.

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 blaCTX-M, blaIMP, blaGES, blaVIM, blaOXA-58-like, blaNDM, blaOXA-23-like, blaOXA-48-like and blaKPC occurring in descending order of frequency. The beta-lactamase genes blaOXA-40/24-like, blaNMC_A/IMI, blaBIC, blaSME, blaGIM and blaDIM 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.

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.

Molecular epidemiology of enterically colonizing Escherichia coli with resistance against third-generation cephalosporins isolated from stool samples of European soldiers with concomitant diarrhea on deployment in Western African Mali.

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 blaCTX-M-15 genes with (n = 14) and without (n = 5) co-occurring blaTEM-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.

The Gut Microbiome of an Indigenous Agropastoralist Population in a Remote Area of Colombia with High Rates of Gastrointestinal Infections and Dysbiosis.

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.

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.

Machine learning-based typing of Salmonella enterica O-serogroups by the Fourier-Transform Infrared (FTIR) Spectroscopy-based IR Biotyper system.

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.

Colitis caused by Entamoeba histolytica identified by real-time-PCR and fluorescence in situ hybridization from formalin-fixed, paraffin-embedded tissue.

Intestinal amoebiasis in a 35-year-old German patient with a 3 weeks travel history in Indonesia was initially misidentified as non-steroidal anti-inflammatory-drug associated colitis in colonoscopy and histopathological analysis. Furthermore, initial stool examination by microscopy and Entamoeba faecal antigen ELISA did not reveal any protozoan infection. When cessation of non-steroidal anti-inflammatory drug (NSAID) use and mesalazine treatment did not lead to clinical improvement, the patient presented to a specialist for tropical diseases. An intensive reinvestigation including a workup of formalin-fixed, paraffin-embedded colonic biopsies by molecular analysis with real-time PCR and fluorescence in situ hybridization (FISH) proofed the diagnosis of Entamoeba histolytica colitis. Molecular methods including real-time PCR and FISH for the diagnosis of amoebiasis from histopathological samples are rarely used for the diagnosis of E. histolytica infections. Bloody diarrhoea vanished after the onset of metronidazole treatment. In conclusion, the here-presented case demonstrates how modern molecular diagnostics may help to diagnose E. histolytica-associated colitis, even from difficult specimens like paraffin-embedded, formalin-fixed tissue.

Screening for Schistosoma spp. and Leishmania spp. DNA in Serum of Ghanaian Patients with Acquired Immunodeficiency.

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.

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 Carbapenem-Resistant Acinetobacter baumannii Strains Isolated at the German Military Field Laboratory in Mazar-e Sharif, Afghanistan.

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 blaOXA-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 blaOXA-23 may facilitate molecular resistance screening based on rapid molecular assays targeting this resistance determinant.

Molecular Evidence for Flea-Borne Rickettsiosis in Febrile Patients from Madagascar.

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.

Comparison of Two Real-Time PCR Assays Targeting Ribosomal Sequences for the Identification of Cystoisospora belli in Human Stool Samples.

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.

Comparison of Three In-House Real PCR Assays Targeting Kinetoplast DNA, the Small Subunit Ribosomal RNA Gene and the Glucose-6-Phosphate Isomerase Gene for the Detection of Leishmania spp. in Human Serum.

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 Leishmania-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.

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.

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.

Classification of Salmonella enterica of the (Para-)Typhoid Fever Group by Fourier-Transform Infrared (FTIR) Spectroscopy.

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.

Implications of the COVID-19 Pandemic for the Development and Update of Clinical Practice Guidelines: Viewpoint.

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.