Viral and Bacterial Zoonotic Agents in Dromedary Camels from Southern Tunisia: A Seroprevalence Study.

The rapid spread of SARS-CoV-2 clearly demonstrated the potential of zoonotic diseases to cause severe harm to public health. Having limited access to medical care combined with severe underreporting and a lack of active surveillance, Africa carries a high burden of neglected zoonotic diseases. Therefore, the epidemiological monitoring of pathogen circulation is essential. Recently, we found extensive Middle East respiratory syndrome coronavirus (MERS-CoV) prevalence in free-roaming dromedary camels from southern Tunisia. In this study, we aimed to investigate the seroprevalence, and thus the risk posed to public health, of two additional viral and two bacterial pathogens in Tunisian dromedaries: Rift Valley fever virus (RVFV), foot-and-mouth disease virus (FMDV), Coxiella burnetii and Brucella spp. via ELISA. With 73.6% seropositivity, most animals had previously been exposed to the causative agent of Q fever, C. burnetii. Additionally, 7.4% and 1.0% of the dromedaries had antibodies against Brucella and RVFV, respectively, while no evidence was found for the occurrence of FMDV. Our studies revealed considerable immunological evidence of various pathogens within Tunisian dromedary camels. Since these animals have intense contact with humans, they pose a high risk of transmitting serious zoonotic diseases during active infection. The identification of appropriate countermeasures is therefore highly desirable.

In-Depth Analysis of Bacillus anthracis 16S rRNA Genes and Transcripts Reveals Intra- and Intergenomic Diversity and Facilitates Anthrax Detection.

Analysis of 16S rRNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ, in vitro, and in silico assays to assess the unknown 16S state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing, and bioinformatics, we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B. anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis SRA data sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons. IMPORTANCE For severe infectious diseases, precise pathogen detection is crucial for antibiotic therapy and patient survival. Identification of Bacillus anthracis, the causative agent of the zoonosis anthrax, can be challenging when querying specific nucleotide sequences such as in small subunit rRNA (16S rRNA) genes, which are commonly used for typing of bacteria. This study analyzed on a broad genomic scale a cryptic and hitherto underappreciated allelic variant of the bacterium's 16S rRNA genes and their transcripts using a set of in situ, in vitro, and in silico assays and found significant intra- and intergenomic heterogeneity in the distribution of the allele and overall rRNA operon copy numbers. This allelic variation was uniquely species specific, which enabled sensitive pathogen detection on both DNA and transcript levels. The methodology used here is likely also applicable to other pathogens that are otherwise difficult to discriminate from their less harmful relatives.

Development and Validation of Ribosomal RNA-Targeted Reverse Transcription Real-Time PCR Assays for the Sensitive and Rapid Diagnostics of High Consequence Pathogens.

Real-time PCR (rtPCR) has become an essential tool in clinical microbiology and has been used for the acute diagnostics of many pathogens. Key performance indicators of rtPCR assays are their specificity as well as their analytical and clinical sensitivity. One way to maximize the sensitivity of such diagnostic rtPCRs is the use of genomic targets, which are present in several copies in the target cells. Here, we use the naturally pre-amplified ribosomal RNA as target for specific and highly sensitive reverse-transcription rtPCR detection of two high consequence pathogens, Yersinia pestis and Francisella tularensis. We determined their analytical sensitivity and illustrate that the newly designed assays are superior compared with other previous published rtPCR assays. Furthermore, we used spiked clinical sample matrices to evaluate their clinical applicability. Finally, we demonstrate that these assays can be applied on heat-inactivated samples without the need of time-consuming nucleic acid extraction.

Prevalence of Middle East Respiratory Syndrome Coronavirus in Dromedary Camels, Tunisia.

Free-roaming camels, especially those crossing national borders, pose a high risk for spreading Middle East respiratory syndrome coronavirus (MERS-CoV). To prevent outbreaks, active surveillance is necessary. We found that a high percentage of dromedaries in Tunisia are MERS-CoV seropositive (80.4%) or actively infected (19.8%), indicating extensive MERS-CoV circulation in Northern Africa.

Rapid detection of SARS-CoV-2 by pulse-controlled amplification (PCA).

In the current pandemic of SARS-CoV-2, rapid identification of infected individuals is crucial for management and control of the outbreak. However, transport of samples, sample processing and RT-qPCR analysis in laboratories are time-consuming. Here we present a prototype of a novel nucleic acid-based test format - pulse controlled amplification - that allows detection of SARS-CoV-2 directly from up to eight swab samples simultaneously without the need for RNA extraction within 25 min with a sensitivity of 100 % for samples with a viral load of ≥ 1.6 × 10e3 copies/µl This new principle might pave the way to rapid and sensitive point of care testing.

Pulse-Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions.

BACKGROUND: Molecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity. METHODS/PRINCIPLE FINDINGS: Here we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis. CONCLUSIONS/SIGNIFICANCE: PCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests.

Whole genome sequencing and phylogenetic classification of Tunisian SARS-CoV-2 strains from patients of the Military Hospital in Tunis.

In the present work, two complete genome sequences of SARS-CoV-2 were obtained from nasal swab samples of Tunisian SARS-CoV-2 PCR-positive patients using nanopore sequencing. The virus genomes of two of the patients examined, a Tunisian soldier returning from a mission in Morocco and a member of another Tunisian family, showed significant differences in analyses of the total genome and single nucleotide polymorphisms (SNPs). Phylogenetic relationships with known SARS-CoV-2 genomes in the African region, some European and Middle Eastern countries and initial epidemiological conclusions indicate that the introduction of SARS-CoV-2 into Tunisia from two independent sources was travel-related.

NOTIFy (non-toxic lyophilized field)-FISH for the identification of biological agents by Fluorescence in situ Hybridization.

The rapid and reliable diagnostics of highly pathogenic bacteria under restricted field conditions poses one of the major challenges to medical biodefense, especially since false positive or false negative reports might have far-reaching consequences. Fluorescence in situ hybridization (FISH) has the potential to represent a powerful microscopy-based addition to the existing molecular-based diagnostic toolbox. In this study, we developed a set of FISH-probes for the fast, matrix independent and simultaneous detection of thirteen highly pathogenic bacteria in different environmental and clinical sample matrices. Furthermore, we substituted formamide, a routinely used chemical that is toxic and volatile, by non-toxic urea. This will facilitate the application of FISH under resource limited field laboratory conditions. We demonstrate that hybridizations performed with urea show the same specificity and comparable signal intensities for the FISH-probes used in this study. To further simplify the use of FISH in the field, we lyophilized the reagents needed for FISH. The signal intensities obtained with these lyophilized reagents are comparable to freshly prepared reagents even after storage for a month at room temperature. Finally, we show that by the use of non-toxic lyophilized field (NOTIFy)-FISH, specific detection of microorganisms with simple and easily transportable equipment is possible in the field.

Detection of Coxiella burnetii in heart valve sections by fluorescence in situ hybridization.

PURPOSE: Infective endocarditis is a severe and potentially fatal disease. Nearly a third of all cases remain culture-negative, making a targeted and effective antibiotic therapy of patients challenging. In the past years, fluorescence in situ hybridization (FISH) has proven its value for the diagnosis of infective endocarditis, particularly when it is caused by fastidious bacteria. To increase the number of infective endocarditis causing agents, which can be identified by FISH, we designed and optimized a FISH-probe for the specific detection of Coxiella burnetii in heart valve tissue. METHODOLOGY: Even with specific probes the detection and identification of bacteria can be complicated by the high autofluorescence due to calcification of the analysed tissue. To overcome this problem, we developed a protocol to detect C. burnetii by hybridizing, stripping and reprobing the identical section with different species-specific probes repeatedly.Results/Key findings. The newly designed specific FISH probe and the developed protocol exemplarily allowed us to unequivocally identify C. burnetii in tissue sections of a patient with infective endocarditis. CONCLUSION: This method provides an add-on to existing protocols for the unambiguous diagnosis of bacteria directly within tissues or other difficult tissue samples in cases with small sample size and limited sections.

Deep Sequencing of RNA from Blood and Oral Swab Samples Reveals the Presence of Nucleic Acid from a Number of Pathogens in Patients with Acute Ebola Virus Disease and Is Consistent with Bacterial Translocation across the Gut.

In this study, samples from the 2013-2016 West African Ebola virus outbreak from patients in Guinea with Ebola virus disease (EVD) were analyzed to discover and classify what other pathogens were present. Throat swabs were taken from deceased EVD patients, and peripheral blood samples were analyzed that had been taken from patients when they presented at the treatment center with acute illness. High-throughput RNA sequencing (RNA-seq) and bioinformatics were used to identify the potential microorganisms. This approach confirmed Ebola virus (EBOV) in all samples from patients diagnosed as acute positive for the virus by quantitative reverse transcription-PCR in deployed field laboratories. Nucleic acid mapping to Plasmodium was also used on the patient samples, confirming results obtained with an antigen-based rapid diagnostic test (RDT) conducted in the field laboratories. The data suggested that a high Plasmodium load, as determined by sequence read depth, was associated with mortality and influenced the host response, whereas a lower parasite load did not appear to affect outcome. The identifications of selected bacteria from throat swabs via RNA-seq were confirmed by culture. The data indicated that the potential pathogens identified in the blood samples were associated with translocation from the gut, suggesting the presence of bacteremia, which transcriptome data suggested may induce or aggravate the acute-phase response observed during EVD. Transcripts mapping to different viruses were also identified, including those indicative of lytic infections. The development of high-resolution analysis of samples from patients with EVD will help inform care pathways and the most appropriate general antimicrobial therapy to be used in a resource-poor setting. IMPORTANCE Our results highlight the identification of an array of pathogens in the blood of patients with Ebola virus disease (EVD). This has not been done before, and the data have important implications for the treatment of patients with EVD, particularly considering antibiotic stewardship. We show that EVD patients who were also infected with Plasmodium, particularly at higher loads, had more adverse outcomes than patients with lower levels of Plasmodium. However, the presence of Plasmodium did not influence the innate immune response, and it is likely that the presence of EBOV dominated this response. Several viruses other than EBOV were identified, and bacteria associated with sepsis were also identified. These findings were indicative of bacterial translocation across the gut during the acute phase of EVD.

MinION as part of a biomedical rapidly deployable laboratory.

Fast turnaround times are of utmost importance for biomedical reconnaissance, particularly regarding dangerous pathogens. Recent advances in sequencing technology and its devices allow sequencing within a short time frame outside stationary laboratories close to the epicenter of the outbreak. In our study, we evaluated the portable sequencing device MinION as part of a rapidly deployable laboratory specialized in identification of highly pathogenic agents. We tested the device in the course of a NATO live agent exercise in a deployable field laboratory in hot climate conditions. The samples were obtained from bio-terroristic scenarios that formed part of the exercise and contained unknown bacterial agents. To simulate conditions of a resource-limited remote deployment site, we operated the sequencer without internet access. Using a metagenomic approach, we were able to identify the causative agent in the analyzed samples. Furthermore, depending on the obtained data, we were able to perform molecular typing down to strain level. In our study we challenged the device and discuss advances as well as remaining limitations for sequencing biological samples outside of stationary laboratories. Nevertheless, massive parallel sequencing as a non-selective methodology yields important information and is able to support outbreak investigation - even in the field.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015.

BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

Unique human immune signature of Ebola virus disease in Guinea.

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4(+) and CD8(+) T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Mobile diagnostics in outbreak response, not only for Ebola: a blueprint for a modular and robust field laboratory.

We established a modular, rapidly deployable laboratory system that provides diagnostic support in resource-limited, remote areas. Developed as a quick response asset to unusual outbreaks of infectious diseases worldwide, several of these laboratories have been used as part of the World Health Organization response to the Ebola virus outbreaks by teams of the 'European Mobile Lab' project in West Africa since March 2014. Within three days from deployment, the first European mobile laboratory became operational at the Ebola Treatment Unit (ETU) in Guéckédou, southern Guinea. Deployment in close proximity to the ETU decreased the turnaround time to an average of 4 h instead of several days in many cases. Between March 2014 and May 2015, more than 5,800 samples were tested in this field laboratory. Further EMLab units were deployed to Nigeria, Liberia and Sierra Leone in the following months of the Ebola outbreak. The technical concept of the EMLab units served as a blueprint for other mobile Ebola laboratories which have been set up in Mali, Côte d'Ivoire, Sierra Leone and other countries in West Africa. Here, we describe design, capabilities and utility of this deployable laboratory system for use in response to disease outbreaks, epidemiological surveillance and patient management.

Field Evaluation of Capillary Blood Samples as a Collection Specimen for the Rapid Diagnosis of Ebola Virus Infection During an Outbreak Emergency.

BACKGROUND: Reliable reverse transcription polymerase chain reaction (RT-PCR)-based diagnosis of Ebola virus infection currently requires a blood sample obtained by intravenous puncture. During the current Ebola outbreak in Guinea, we evaluated the usability of capillary blood samples collected from fingersticks of patients suspected of having Ebola virus disease (EVD) for field diagnostics during an outbreak emergency. METHODS: A total of 120 venous and capillary blood samples were collected from 53 patients admitted to the Ebola Treatment Centre in Guéckédou, Guinea, between July and August 2014. All sample specimens were analyzed by RT-PCR using the RealStar Filovirus Screen RT-PCR Kit 1.0 from altona Diagnostics (Germany). We compared samples obtained by venipuncture and those obtained by capillary blood sampling absorbed onto swab devices. RESULTS: The resulting sensitivity and specificity of tests performed with capillary blood samples were 86.8% (95% confidence interval [CI], 71.9%-95.6%; 33/38 patients) and 100% (95% CI, 84.6%-100%; 22/22 patients), respectively. CONCLUSIONS: Our data suggest that capillary blood samples could serve as an alternative to venous blood samples for the diagnosis of EVD in resource-limited settings during a crisis. This can be of particular advantage in cases when venipuncture is difficult to perform-for example, with newborns and infants or when adult patients reject venipuncture for cultural or religious reasons.

Evidence for a decrease in transmission of Ebola virus--Lofa County, Liberia, June 8-November 1, 2014.

Lofa County has one of the highest cumulative incidences of Ebola virus disease (Ebola) in Liberia. Recent situation reports from the Liberian Ministry of Health and Social Welfare (MoHSW) have indicated a decrease in new cases of Ebola in Lofa County. In October 2014, the Liberian MoHSW requested the assistance of CDC to further characterize recent trends in Ebola in Lofa County. Data collected during June 8-November 1, 2014 from three sources were analyzed: 1) aggregate data for newly reported cases, 2) case-based data for persons admitted to the dedicated Ebola treatment unit (ETU) for the county, and 3) test results for community decedents evaluated for Ebola. Trends from all three sources suggest that transmission of Ebola virus decreased as early as August 17, 2014, following rapid scale-up of response activities in Lofa County after a resurgence of Ebola in early June 2014. The comprehensive response strategy developed with participation from the local population in Lofa County might serve as a model to implement in other affected areas to accelerate control of Ebola.

Response to comments on "Invasive harlequin ladybird carries biological weapons against native competitors".

Comments by de Jong et al., Solter et al., and Sloggett question the ecological relevance of the abundant microsporidia found in the invasive ladybird Harmonia axyridis. We contend that there is abundant evidence that native ladybirds feed on H. axyridis eggs and that interspecific microsporidial transfer is a common phenomenon, supporting the proposed role of these parasites as biological weapons.

Invasive harlequin ladybird carries biological weapons against native competitors.

Invasive species that proliferate after colonizing new habitats have a negative environmental and economic impact. The reason why some species become successful invaders, whereas others, even closely related species, remain noninvasive is often unclear. The harlequin ladybird Harmonia axyridis, introduced for biological pest control, has become an invader that is outcompeting indigenous ladybird species in many countries. Here, we show that Harmonia carries abundant spores of obligate parasitic microsporidia closely related to Nosema thompsoni. These microsporidia, while not harming the carrier Harmonia, are lethal pathogens for the native ladybird Coccinella septempunctata. We propose that intraguild predation, representing a major selective force among competing ladybird species, causes the infection and ultimate death of native ladybirds when they feed on microsporidia-contaminated Harmonia eggs or larvae.

A straightforward DOPE (double labeling of oligonucleotide probes)-FISH (fluorescence in situ hybridization) method for simultaneous multicolor detection of six microbial populations.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is an essential tool for the cultivation-independent identification of microbes within environmental and clinical samples. However, one of the major constraints of conventional FISH is the very limited number of different target organisms that can be detected simultaneously with standard epifluorescence or confocal laser scanning microscopy. Recently, this limitation has been overcome via an elegant approach termed combinatorial labeling and spectral imaging FISH (CLASI-FISH) (23). This technique, however, suffers compared to conventional FISH from an inherent loss in sensitivity and potential probe binding biases caused by the competition of two differentially labeled oligonucleotide probes for the same target site. Here we demonstrate that the application of multicolored, double-labeled oligonucleotide probes enables the simultaneous detection of up to six microbial target populations in a straightforward and robust manner with higher sensitivity and less bias. Thus, this newly developed technique should be an attractive option for all researchers interested in applying conventional FISH methods for the study of microbial communities.

UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili.

Archaea, like bacteria and eukaryotes, contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life. Species of the order Sulfolobales of hyperthermophilic crenarchaeota are equipped with a strongly UV-inducible type IV pilus system that promotes cellular aggregation. Here we demonstrate by fluorescence in situ hybridization that cellular aggregates are formed based on a species-specific recognition process and that UV-induced cellular aggregation mediates chromosomal marker exchange with high frequency. Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Knockout strains of Sulfolobus acidocaldarius incapable of pilus production could not self-aggregate, but were partners in mating experiments with wild-type strains indicating that one cellular partner can mediate the DNA transfer. Since pilus knockout strains showed decreased survival upon UV treatment, we conclude that the UV-inducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosome integrity in Sulfolobus. It might also contribute substantially to the frequent chromosomal DNA exchange and horizontal gene transfer in these archaea in their natural habitat.