Ongoing mpox outbreak in Kamituga, South Kivu province, associated with monkeypox virus of a novel Clade I sub-lineage, Democratic Republic of the Congo, 2024.

Since the beginning of 2023, the number of people with suspected monkeypox virus (MPXV) infection have sharply increased in the Democratic Republic of the Congo (DRC). We report near-to-complete MPXV genome sequences derived from six cases from the South Kivu province. Phylogenetic analyses reveal that the MPXV affecting the cases belongs to a novel Clade I sub-lineage. The outbreak strain genome lacks the target sequence of the probe and primers of a commonly used Clade I-specific real-time PCR.

Circulation, viral diversity and genomic rearrangement in mpox virus in the Netherlands during the 2022 outbreak and beyond.

Mpox is an emerging zoonotic disease which has now spread to over 113 countries as of August 2023, with over 89,500 confirmed human cases. The Netherlands had one of the highest incidence rates in Europe during the peak of the outbreak. In this study, we generated 158 near-complete mpox virus (MPXV) genomes (12.4% of nationwide cases) that were collected throughout the Netherlands from the start of the outbreak in May 2022 to August 2023 to track viral evolution and investigate outbreak dynamics. We detected 14 different viral lineages, suggesting multiple introductions followed by rapid initial spread within the country. The estimated evolutionary rate was relatively high compared to previously described in orthopoxvirus literature, with an estimated 11.58 mutations per year. Genomic rearrangement events occurred at a rate of 0.63% and featured a large deletion event. In addition, based on phylogenetics, we identified multiple potential transmission clusters which could be supported by direct source- and contact tracing data. This led to the identification of at least two main transmission locations at the beginning of the outbreak. We conclude that whole genome sequencing of MPXV is essential to enhance our understanding of outbreak dynamics and evolution of a relatively understudied and emerging zoonotic pathogen.

Mpox outbreak among men who have sex with men in Amsterdam and Rotterdam, the Netherlands: no evidence for undetected transmission prior to May 2022, a retrospective study.

Since May 2022, over 21,000 mpox cases have been reported from 29 EU/EEA countries, predominantly among men who have sex with men (MSM). The Netherlands was the fourth most affected country in Europe, with more than 1,200 cases and a crude notification rate of 70.7 per million population. The first national case was reported on 10 May, yet potential prior transmission remains unknown. Insight into prolonged undetected transmission can help to understand the current outbreak dynamics and aid future public health interventions. We performed a retrospective study and phylogenetic analysis to elucidate whether undetected transmission of human mpox virus (hMPXV) occurred before the first reported cases in Amsterdam and Rotterdam. In 401 anorectal and ulcer samples from visitors to centres for sexual health in Amsterdam or Rotterdam dating back to 14 February 2022, we identified two new cases, the earliest from 6 May. This coincides with the first cases reported in the United Kingdom, Spain and Portugal. We found no evidence of widespread hMPXV transmission in Dutch sexual networks of MSM before May 2022. Likely, the mpox outbreak expanded across Europe within a short period in the spring of 2022 through an international highly intertwined network of sexually active MSM.

Host DNA depletion can increase the sensitivity of Mycobacterium spp. detection through shotgun metagenomics in sputum.

Identification and phenotypic drug-susceptibility testing for mycobacteria are time-consuming and challenging but essential for managing mycobacterial infections. Next-generation sequencing (NGS) technologies can increase diagnostic speed and quality, but standardization is still lacking for many aspects (e.g., unbiased extraction, host depletion, bioinformatic analysis). Targeted PCR approaches directly on sample material are limited by the number of targets that can be included. Unbiased shotgun metagenomics on direct material is hampered by the massive amount of host DNA, which should be removed to improve the microbial detection sensitivity. For this reason, we developed a method for NGS-based diagnosis of mycobacteria directly from patient material. As a model, we used the non-tuberculous mycobacterium (NTM) Mycobacterium abscessus. We first compared the efficiency of three different DNA extraction kits for isolating DNA (quality and concentration). The two most efficient kits were then used in a follow-up study using artificial sputum. Finally, one extraction kit was selected and further evaluated for DNA isolation from a patients' sputum mixture spiked with M. abscessus at three concentrations (final concentrations 108, 107, 106 CFU/ml). The spiked sputum samples were processed with and without saponin treatment (ST) in combination with DNAse treatment prior to bacterial DNA extraction to evaluate the recovery of bacteria and depletion of host DNA by PCR and Illumina sequencing. While Ct values of the qPCR targeting mycobacterial ITS DNA remained rather stable, Ct values in the qPCR targeting the human ß-actin gene increased by five Ct values in ST samples. In subsequent Illumina sequencing, a decrease of 89% of reads mapped to the human genome was observed in ST samples. The percentage of reads mapped to M. abscessus (108 CFU/ml) increased by 89%, and the sequencing depth increased two times when undergoing ST. In conclusion, the sensitivity of M. abscessus detection in artificial sputum was increased using a saponin pre-treatment step. The saponin followed by the DNase I treatment approach could be efficiently applied to detect and characterize mycobacterial infections, including tuberculosis, directly from sputum.

Genomic characterization of coxsackievirus A22 from a regional university hospital in the Netherlands.

BACKGROUND: Enteroviruses are highly diverse with a wide spectrum of genotypes and clinical manifestations. Coxsackievirus A22 (CVA22) has been detected globally from sewage surveillance; however, currently there is limited information on its prevalence in patients, as well as available genomic data. OBJECTIVE: We aimed to provide genomic and relative frequency data on CVA22 from a regional hospital perspective between 2013-2020. STUDY DESIGN: Sanger sequencing was performed on all samples with a positive enterovirus RT-qPCR result (≤Ct 32). Viral targeted sequence capture (ViroCap) and next-generation sequencing (NGS) (Illumina NextSeq 500) was used to characterize and generate near-complete CVA22 genomes for enteroviruses without genotyping results from Sanger sequencing. Epidemiological and phylogenetic analysis was performed using maximum likelihood trees on MEGA-11. RESULTS: A total of twenty detections derived from fecal material from sixteen patients were observed between 2013- 2020. One transplant recipient had five different CVA22 infection episodes over five years, with phylogenetic analysis indicating possible reinfection with an additional prolonged infection (>3 weeks). Furthermore, we report the first two near-complete CVA22 sequences from Europe, which grouped with a strain previously isolated from Bangladesh in 1999. CONCLUSIONS: We show a highly diverse enterovirus genotype which causes infections annually, typically in autumn and winter, and is capable of recurrent infection in an immunocompromised patient. Furthermore, we highlight the use of NGS to complement conventional targeted Sanger sequencing.

Exploring a prolonged enterovirus C104 infection in a severely ill patient using nanopore sequencing.

Chronic enterovirus infections can cause significant morbidity, particularly in immunocompromised patients. This study describes a fatal case associated with a chronic untypeable enterovirus infection in an immunocompromised patient admitted to a Dutch university hospital over nine months. We aimed to identify the enterovirus genotype responsible for the infection and to determine potential evolutionary changes. Long-read sequencing was performed using viral targeted sequence capture on four respiratory and one faecal sample. Phylogenetic analysis was performed using a maximum likelihood method, along with a root-to-tip regression and time-scaled phylogenetic analysis to estimate evolutionary changes between sample dates. Intra-host variant detection, using a Fixed Ploidy algorithm, and selection pressure, using a Fixed Effect Likelihood and a Mixed Effects Model of Evolution, were also used to explore the patient samples. Near-complete genomes of enterovirus C104 (EV-C104) were recovered in all respiratory samples but not in the faecal sample. The recovered genomes clustered with a recently reported EV-C104 from Belgium in August 2018. Phylodynamic analysis including ten available EV-C104 genomes, along with the patient sequences, estimated the most recent common ancestor to occur in the middle of 2005 with an overall estimated evolution rate of 2.97 × 10-3 substitutions per year. Although positive selection pressure was identified in the EV-C104 reference sequences, the genomes recovered from the patient samples alone showed an overall negative selection pressure in multiple codon sites along the genome. A chronic infection resulting in respiratory failure from a relatively rare enterovirus was observed in a transplant recipient. We observed an increase in single-nucleotide variations between sample dates from a rapidly declining patient, suggesting mutations are weakly deleterious and have not been purged during selection. This is further supported by the persistence of EV-C104 in the patient, despite the clearance of other viral infections. Next-generation sequencing with viral enrichment could be used to detect and characterise challenging samples when conventional workflows are insufficient.

Application of shotgun metagenomics sequencing and targeted sequence capture to detect circulating porcine viruses in the Dutch-German border region.

Porcine viruses have been emerging in recent decades, threatening animal and human health, as well as economic stability for pig farmers worldwide. Next-generation sequencing (NGS) can detect and characterize known and unknown viruses but has limited sensitivity when an unbiased approach, such as shotgun metagenomics sequencing, is used. To increase the sensitivity of NGS for the detection of viruses, we applied and evaluated a broad viral targeted sequence capture (TSC) panel and compared it to an unbiased shotgun metagenomic approach. A cohort of 36 pooled porcine nasal swab and blood serum samples collected from both sides of the Dutch-German border region were evaluated. Overall, we detected 46 different viral species using TSC, compared to 40 viral species with a shotgun metagenomics approach. Furthermore, we performed phylogenetic analysis on recovered influenza A virus (FLUAV) genomes from Germany and revealed a close similarity to a zoonotic influenza strain previously detected in the Netherlands. Although TSC introduced coverage bias within the detected viruses, it improved sensitivity, genome sequence depth and contig length. In-depth characterization of the swine virome, coupled with developing new enrichment techniques, can play a crucial role in the surveillance of circulating porcine viruses and emerging zoonotic pathogens.

Off-season circulation and characterization of enterovirus D68 with respiratory and neurological presentation using whole-genome sequencing.

To explore an off-season enterovirus D68 (EV-D68) upsurge in the winter season of 2019/2020, we adapted a whole-genome sequencing approach for Nanopore Sequencing for 20 hospitalized patients with accompanying respiratory or neurological presentation. Applying phylodynamic and evolutionary analysis on Nextstrain and Datamonkey respectively, we report a highly diverse virus with an evolutionary rate of 3.05 × 10-3 substitutions per year (entire EV-D68 genome) and a positive episodic/diversifying selection with persistent yet undetected circulation likely driving evolution. While the predominant B3 subclade was identified in 19 patients, one A2 subclade was identified in an infant presenting with meningitis. Exploring single nucleotide variations using CLC Genomics Server showed high levels of non-synonymous mutations, particularly in the surface proteins, possibly highlighting growing problems with routine Sanger sequencing for typing enteroviruses. Surveillance and molecular approaches to enhance current knowledge of infectious pathogens capable of pandemic potential are paramount to early warning in health care facilities.

Future potential of metagenomics in microbiology laboratories.

Rapid and sensitive diagnostic strategies are necessary for patient care and public health. Most of the current conventional microbiological assays detect only a restricted panel of pathogens at a time or require a microbe to be successfully cultured from a sample. Clinical metagenomics next-generation sequencing (mNGS) has the potential to unbiasedly detect all pathogens in a sample, increasing the sensitivity for detection and enabling the discovery of unknown infectious agents. High expectations have been built around mNGS; however, this technique is far from widely available. This review highlights the advances and currently available options in terms of costs, turnaround time, sensitivity, specificity, validation, and reproducibility of mNGS as a diagnostic tool in clinical microbiology laboratories. The need for a novel diagnostic tool to increase the sensitivity of microbial diagnostics is clear. mNGS has the potential to revolutionize clinical microbiology. However, its role as a diagnostic tool has yet to be widely established, which is crucial for successfully implementing the technique. A clear definition of diagnostic algorithms that include mNGS is vital to show clinical utility. Similarly to real-time PCR, mNGS will one day become a vital tool in any testing algorithm.

First detection of porcine respirovirus 1 in Germany and the Netherlands.

Porcine respirovirus 1, also referred to as porcine parainfluenza virus 1 (PPIV-1), was first detected in deceased pigs from Hong Kong in 2013. It has since then been found in the USA, Chile and most recently in Hungary. Information on the pathogenicity and global spread is sparse. However, it has been speculated to play a role in the porcine respiratory disease complex. To investigate the porcine virome, we screened 53 pig samples from 26 farms within the Dutch-German border region using shotgun metagenomics sequencing (SMg). After detecting PPIV-1 in five farms through SMg, a real-time reverse transcriptase PCR (RT-qPCR) assay was designed, which not only confirmed the presence of the virus in 1 of the 5 farms but found an additional 6 positive farms. Phylogenetic analysis found the closest match to be the first detected PPIV-1 strain in Hong Kong. The Dutch-German region represents a significant area of pig farming within Europe and could provide important information on the characterization and circulation of porcine viruses, such as PPIV-1. With its recent detection in Hungary, these findings suggest widespread circulation of PPIV-1 in Central Europe, highlighting the need for further research on persistence, pathogenicity and transmission in Europe.

Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples.

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.

Author Correction: Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.

High throughput sequencing has been proposed as a one-stop solution for diagnostics and molecular typing directly from patient samples, allowing timely and appropriate implementation of measures for treatment, infection prevention and control. However, it is unclear how the variety of available methods impacts the end results. We applied shotgun metagenomics on diverse types of patient samples using three different methods to deplete human DNA prior to DNA extraction. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed using methods likely to be available in clinical microbiology laboratories using genomics. The results of microbial identification were compared to standard culture-based microbiological methods. On average, 75% of the reads corresponded to human DNA, being a major determinant in the analysis outcome. None of the kits was clearly superior suggesting that the initial ratio between host and microbial DNA or other sample characteristics were the major determinants of the proportion of microbial reads. Most pathogens identified by culture were also identified through metagenomics, but substantial differences were noted between the taxonomic classification tools. In two cases the high number of human reads resulted in insufficient sequencing depth of bacterial DNA for identification. In three samples, we could infer the probable multilocus sequence type of the most abundant species. The tools and databases used for taxonomic classification and antimicrobial resistance identification had a key impact on the results, recommending that efforts need to be aimed at standardization of the analysis methods if metagenomics is to be used routinely in clinical microbiology.

Genomic characterisation of clinical and environmental Pseudomonas putida group strains and determination of their role in the transfer of antimicrobial resistance genes to Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas putida is a Gram-negative, non-fermenting bacterium frequently encountered in various environmental niches. P. putida rarely causes disease in humans, though serious infections and outbreaks have been reported from time to time. Some have suggested that P. putida functions as an exchange platform for antibiotic resistance genes (ARG), and thus represents a serious concern in the spread of ARGs to more pathogenic organisms within a hospital. Though poorly understood, the frequency of ARG exchange between P. putida and the more virulent Pseudomonas aeruginosa and its clinical relevance are particularly important for designing efficient infection control strategies, such as deciding whether high-risk patients colonized with a multidrug resistant but typically low pathogenic P. putida strain should be contact isolated or not. RESULTS: In this study, 21,373 screening samples (stool, rectal and throat swab) were examined to determine the presence of P. putida in a high-risk group of haemato-oncology patients during a 28-month period. A total of 89 P. putida group strains were isolated from 85 patients, with 41 of 89 (46.1%) strains harbouring the metallo-beta-lactamase gene bla VIM. These 41 clinical isolates, plus 18 bla VIM positive environmental P. putida isolates, and 17 bla VIM positive P. aeruginosa isolates, were characterized by whole genome sequencing (WGS). We constructed a maximum-likelihood tree to separate the 59 bla VIM positive P. putida group strains into eight distinct phylogenetic clusters. Bla VIM-1 was present in 6 clusters while bla VIM-2 was detected in 4 clusters. Five P. putida group strains contained both, bla VIM-1 and bla VIM-2 genes. In contrast, all P. aeruginosa strains belonged to a single genetic cluster and contained the same ARGs. Apart from bla VIM-2 and sul genes, no other ARGs were shared between P. aeruginosa and P. putida. Furthermore, the bla VIM-2 gene in P. aeruginosa was predicted to be only chromosomally located. CONCLUSION: These data provide evidence that no exchange of comprehensive ARG harbouring mobile genetic elements had occurred between P. aeruginosa and P. putida group strains during the study period, thus eliminating the need to implement enhanced infection control measures for high-risk patients colonized with a bla VIM positiv P. putida group strains in our clinical setting.

Development of a quantitative high-performance thin-layer chromatographic method for sucralose in sewage effluent, surface water, and drinking water.

Sucralose, a persistent chlorinated substance used as sweetener, can already be found in waste water, and various countries focused on the release of sucralose into the aquatic environment. A quantitative high-performance thin-layer chromatography (HPTLC) method, which is orthogonal to existing methods, was developed to analyze sucralose in water. After sample preparation, separation of up to 17 samples was performed in parallel on a HPTLC plate silica gel 60 F(254) with a mixture of isopropyl acetate, methanol and water (15:3:1, v/v/v) within 15 min. Due to the weak native UV absorption of sucralose (≤200 nm), various post-chromatographic derivatization reactions were compared to selectively detect sucralose in effluent and surface water matrices. Thereby p-aminobenzoic acid reagent was discovered as a new derivatization reagent for sucralose. Compared to the latter and to ß-naphthol, derivatization with aniline diphenylamine o-phosphoric acid reagent was slightly preferred and densitometry was performed by absorbance measurement at 400 nm. The limit of quantification (LOQ) of sucralose in drinking and surface water was calculated to be 100 ng/L for a given recovery rate of 80% and the extraction of a 0.5 L water sample. The sucralose content determined in four water samples obtained during an interlaboratory trial in 2008 was in good agreement to the mean laboratory values of that trial. According to the t-test, which compares the results with the target value, the means obtained by HPTLC were not significantly different from the respective means of six laboratories, analyzed by HPLC-MS/MS or HPLC-TOF-MS with the use of mostly isotopically labeled standards. The good accuracy and high sample throughput capacity proved HPTLC as a well suited method regarding quantification of sucralose in various aqueous matrices.