16S rRNA Gene Sequence-Based Identification of Bacteria in Automatically Incubated Blood Culture Materials from Tropical Sub-Saharan Africa.

BACKGROUND: The quality of microbiological diagnostic procedures depends on pre-analytic conditions. We compared the results of 16S rRNA gene PCR and sequencing from automatically incubated blood culture materials from tropical Ghana with the results of cultural growth after automated incubation. METHODS: Real-time 16S rRNA gene PCR and subsequent sequencing were applied to 1500 retained blood culture samples of Ghanaian patients admitted to a hospital with an unknown febrile illness after enrichment by automated culture. RESULTS: Out of all 1500 samples, 191 were culture-positive and 98 isolates were considered etiologically relevant. Out of the 191 culture-positive samples, 16S rRNA gene PCR and sequencing led to concordant results in 65 cases at species level and an additional 62 cases at genus level. PCR was positive in further 360 out of 1309 culture-negative samples, sequencing results of which suggested etiologically relevant pathogen detections in 62 instances, detections of uncertain relevance in 50 instances, and DNA contamination due to sample preparation in 248 instances. In two instances, PCR failed to detect contaminants from the skin flora that were culturally detectable. Pre-analytical errors caused many Enterobacteriaceae to be missed by culture. CONCLUSIONS: Potentially correctable pre-analytical conditions and not the fastidious nature of the bacteria caused most of the discrepancies. Although 16S rRNA gene PCR and sequencing in addition to culture led to an increase in detections of presumably etiologically relevant blood culture pathogens, the application of this procedure to samples from the tropics was hampered by a high contamination rate. Careful interpretation of diagnostic results is required

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 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

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 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

Comparison of Three Real-Time PCR Assays Targeting the SSU rRNA Gene, the COWP Gene and the DnaJ-Like Protein Gene for the Diagnosis of Cryptosporidium spp. in Stool Samples

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

Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples

Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen's kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied

Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples (vol 10, 656, 2021)

In the original publication [...

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

Summarized results of cultural growth and 16S rRNA gene PCR.

<p>Summarized results of cultural growth and 16S rRNA gene PCR.</p

Results of 917 bp 16S rRNA gene sequencing and consecutive 357 bp 16S rRNA gene sequencing in case of negative 917 bp 16S rRNA gene PCR results of culture-positive samples.

<p>^aComprising <i>Escherichia coli</i> (17), <i>Escherichia hermannii</i> (1), <i>Enterobacter cloacae</i> (1), <i>Klebsiella pneumoniae</i> (5), <i>Salmonella enterica</i> (11), <i>Salmonella</i> spp. (8), <i>Serratia</i> sp. (1) according to biochemical identification.</p><p>^bIncluding Gram-negative, not further identified coliform bacteria (3), Gram-negative, not further identified non-fermentative rod-shaped bacteria (1), <i>Sphingomonas paucimobilis</i> (2), <i>Streptococcus</i> Lancefield Group G (2), viridians group streptococci (1).</p><p>^cIncluding <i>Micrococcus</i> sp. (6), and <i>Rhizobium radiobacter</i> (1).</p><p>^d<i>Bacillus</i> sp. identified by PCR and sequencing.</p><p>^eBiochemically confirmed <i>Escherichia coli</i> not differentiable between <i>Escherichia coli</i> and <i>Shigella</i> sp. (4) based on 16S rRNA gene sequencing or identified as <i>Enterobacter</i> sp. (1), biochemical result <i>Serratia</i> sp. vs. sequencing result <i>Enterobacter</i> sp. (1), biochemical result <i>Klebsiella pneumoniae</i> vs. sequencing result <i>Enterobacter</i> sp. (1), biochemical result <i>Salmonella</i> sp. vs. sequencing result <i>Diaphorobacter</i> sp. (1).</p><p>^fBiochemically identification <i>Pseudomonas luteola</i> (2) vs. sequence-based identification as <i>Acinetobacter baumannii</i> (1) and <i>Acinetobacter</i> sp. (1), respectively.</p><p>^gCultural suspicion of Gram-negative, coliform bacteria (2) vs. sequence results <i>Bacillus</i> sp. (1) and <i>Microbacterium</i> sp. (1), cultural suspicion of Gram-negative, non-fermentative rod-shaped bacteria (1) vs. sequence result <i>Nesterenkonia</i> sp., biochemically identified <i>Sphingomonas paucimobilis</i> (2) vs. sequence results <i>Paenibacillus xylanilyticus</i> (1) and <i>Microbacterium oxydans</i> (1).</p><p>^hCultural suspicion of coagulase-negative staphylococci (13) vs. sequencing results <i>Bacterium</i> spp. (2), <i>Corynebacterium</i> sp. (1), <i>Diaphorobacter</i> spp. (3), <i>Haematobacter massiliensis</i> (1), <i>Kocuria</i> sp. (1), <i>Micrococcus luteus</i> (1), <i>Micrococcus</i> sp. (1), <i>Pseudomonas</i> sp. (1), <i>Sphingomonas</i> sp. (1), and <i>Staphylococcus aureus</i> (1).</p><p>ⁱCultural suspicion of <i>Corynebacterium</i> spp. (5) vs. sequencing results <i>Arthrobacter</i> spp. (2), <i>Diaphorobacter</i> spp. (2), <i>Massilia aurea</i> (1), and <i>Microbacterium</i> sp. (1).</p><p>^jCultural suspicion of <i>Micrococcus</i> spp. (3) vs. sequence results <i>Diaphorobacter</i> sp. (1), <i>Kocuria marina</i> (1), and <i>Staphylococcus</i> sp. (1).</p><p>Results of 917 bp 16S rRNA gene sequencing and consecutive 357 bp 16S rRNA gene sequencing in case of negative 917 bp 16S rRNA gene PCR results of culture-positive samples.</p