Three cases of Cutibacterium avidum prosthetic valve infective endocarditis at a single heart center.

OBJECTIVE: To resolve an exceptional clustering of Cutibacterium avidum prosthetic valve infective endocarditis at a single heart center. METHODS: During a period of 21 months, three patients experienced C. avidum bacteraemia 24-128 days after aortic valve replacement. Operative procedures and electronic prescriptions of antimicrobials were surveyed, and bacterial isolates were genome sequenced. RESULTS: The prosthetic valves were inserted by separate surgical teams. In one case, echocardiographic confirmation of infective endocarditis was not achieved until four months after the first positive blood culture, but the causative agents were irrefutably documented in all cases by culture, or amplification of bacterial DNA, from removed prosthetic material. Whole genome sequencing clustered isolates to a distinctive subgroup of the species, but did not suggest inter-patient transmission of isolates. CONCLUSIONS: Despite vigorous sampling of blood and tissue, detection of C. avidum was not unconditional, neither by culture nor PCR. The causative agent is likely underreported and should be meticulously searched for in culture-negative prosthetic valve endocarditis.

The best of both worlds: a proposal for further integration of Candidatus names into the International Code of Nomenclature of Prokaryotes.

The naming of prokaryotes is governed by the International Code of Nomenclature of Prokaryotes (ICNP) and partially by the International Code of Nomenclature for Algae, Fungi and Plants (ICN). Such codes must be able to determine names of taxa in a universal and unambiguous manner, thus serving as a common language across different fields and activities. This unity is undermined when a new code of nomenclature emerges that overlaps in scope with an established, time-tested code and uses the same format of names but assigns different nomenclatural status values to the names. The resulting nomenclatural confusion is not beneficial to the wider scientific community. Such ambiguity is expected to result from the establishment of the 'Code of Nomenclature of Prokaryotes Described from DNA Sequence Data' ('SeqCode'), which is in general and specific conflict with the ICNP and the ICN. Shortcomings in the interpretation of the ICNP may have exacerbated the incompatibility between the codes. It is reiterated as to why proposals to accept sequences as nomenclatural types of species and subspecies with validly published names, now implemented in the SeqCode, have not been implemented by the International Committee on Systematics of Prokaryotes (ICSP), which oversees the ICNP. The absence of certain regulations from the ICNP for the naming of as yet uncultivated prokaryotes is an acceptable scientific argument, although it does not justify the establishment of a separate code. Moreover, the proposals rejected by the ICSP are unnecessary to adequately regulate the naming of uncultivated prokaryotes. To provide a better service to the wider scientific community, an alternative proposal to emend the ICNP is presented, which would result in Candidatus names being regulated analogously to validly published names. This proposal is fully consistent with previous ICSP decisions, preserves the essential unity of nomenclature and avoids the expected nomenclatural confusion.

Genome Characterisation of Invasive Haemophilus influenzae in Pregnancy: The Noticeable Placental Tissue Tropism Is Distributed across the Species Rather Than Linked with Capsulation or Particular Clones.

Pregnancy is associated with a 5-26 times increased risk of invasive Haemophilus influenzae infection and subsequent adverse pregnancy outcomes. Incidence rate and outcome are published in some regions, but the characterisation of bacterial isolates is limited. We performed comparative genomic analyses of isolates from 12 pregnancy-associated cases, cultured from maternal bacteraemia in pregnancy (nine), postpartum bacteraemia (one), neonatal bacteraemia (one), and placental tissue (one). In two bacteraemia cases, identical isolates were also cultured from cervical swabs. Eight cases occurred early in pregnancy (gestational week 7-26), and seven of them resulted in miscarriage or neonatal death. All bacterial genomes were devoid of capsule loci, and they were evenly distributed in the major phylogenetic group I of the species. The conspicuous tropism of H. influenzae for pregnancy and placental tissue is associated with the species rather than specific clonal subtypes.

Culture on Selective Media and Amplicon-Based Sequencing of 16S rRNA from Spontaneous Brain Abscess-the View from the Diagnostic Laboratory.

Forty-one stored samples from cases of spontaneous brain abscess were investigated to gain insight into the natural history, causative agents, and relevant laboratory diagnostics of a rare infection. Samples from a larger collection were selected based on retrospective analysis of patient records. All samples were subjected to amplicon sequencing of 16S rRNA gene fragments. Supplementary culture on selected media was performed as suggested by bioinformatics analysis. For three cases, no microorganism was disclosed, while Toxoplasma gondii, Aspergillus fumigatus, and various bacteria were the cause of 1, 2, and 35 cases, respectively. Bacterial infections were monomicrobial in 20 cases and polymicrobial in 15; the microorganisms of the latter cases were restricted to residents of cavum oris. Amplicon sequencing did not further enhance the importance of the Streptococcus anginosus group, which was involved in 17 cases, and the single primer set used may be suboptimal for amplification of Actinomyces and Nocardia. But, amplicon-based sequencing unquestionably expanded the number of polybacterial infections, with focus on the Fusobacterium nucleatum group, Parvimonas, and Porphyromonas. Culture on selective media confirmed the presence of F. nucleatum group bacteria, which attained a prominence in spontaneous brain abscess similar to the S. anginosus group. Metagenomics is a powerful tool to disclose the spectrum of agents in polymicrobial infections, but a reliable cutoff value for substantial detection is complex. Commercial media for isolation of F. nucleatum group bacteria from mixed infections are available, and these pathogens should be carefully characterized. Isolation of Parvimonas and Porphyromonas in polymicrobial infections has not been resolved. IMPORTANCE Polymicrobial brain abscess is a challenge to the clinical microbiology laboratory due to the aggregative nature of the dental and oral microbiota. Because polymicrobial infections may escape detection by conventional culture methods, directed therapy toward a single detected bacterium is problematic. Amplicon-based sequencing provides important clues to these infections, but only cultured microorganisms can be fully characterized, subjected to antimicrobial susceptibility testing, and formally named. By use of specific selective culture plates, we successfully isolated bacteria of the Fusobacterium nucleatum group, and these bacteria rose to the same prominence as the widely recognized pathogen, the Streptococcus anginosus group. Named and unnamed members of the Fusobacterium nucleatum group must be further investigated to gain insight into a rare but grave disease.

Induction of Broad ß-lactam Resistance in Achromobacter ruhlandii by Exposure to Ticarcillin Is Primarily Linked to Substitutions in Murein Peptide Ligase Mpl.

Achromobacter species are emerging pathogens in cystic fibrosis with inherent resistance to several classes of antimicrobial agents. We exposed strains with wild-type antimicrobial susceptibility to ticarcillin and generated mutants with broad ß-lactam resistance. Within the detection limit of the assay, the capability to develop mutational resistance was strain-specific and reproducible. Mutational resistance was observed for all three tested strains of Achromobacter ruhlandii, for one of seven strains of Achromobacter xylosoxidans, and for none of five strains of Achromobacter insuavis. All mutants were resistant to piperacillin-tazobactam, while minimal inhibitory concentration of several other ß-lactams increased 4-32-fold. Whole genome sequencing identified 1-4 non-synonymous mutations in known genes per mutant. All mutants encoded amino acid substitutions in cell wall recycling proteins, primarily Mpl, and the observed resistance is probably caused by hyperproduction of OXA-114-like ß-lactamases. Related, but not identical substitutions were detected in clinical strains expressing acquired antimicrobial resistance.

Multilocus Sequence Typing of Aggregatibacter actinomycetemcomitans Competently Depicts the Population Structure of the Species.

We developed a multilocus sequence typing scheme (MLST) for Aggregatibacter actinomycetemcomitans based on seven housekeeping genes, adk, atpG, frdB, mdh, pgi, recA, and zwf. A total of 188 strains of seven serotypes were separated into 57 sequence types. Whole-genome sequences were available for 140 strains, and in contrast to comparison of 16S rRNA genes, phylogenetic analysis of concatenated MLST gene fragments was in accordance with the population structure revealed by alignment of 785 core genes. MLST could not decisively identify the so-called JP2 clone associated with rapidly progressing periodontitis in adolescents, but noticeable clustering of JP2 genotype strains was revealed. The MLST scheme of A. actinomycetemcomitans can be assessed at www.pubmlst.org. IMPORTANCE Accurate diagnosis of infectious disease comprise identification, typing, and antimicrobial resistance of the infective agent. Bacteria are sometimes grouped within their species according to expression of specific toxins or particular antimicrobial resistance traits, but explicit typing for infection control and survey of pathogenesis necessitates genetic analysis such as multilocus sequence typing (MLST). Schemes for the most prevalent human pathogens have been available for more than 10 years, and time has come to extend the scrutiny to second-line infectious agents. One such pathogen is Aggregatibacter actinomycetemcomitans, which is commonly involved in periodontitis, and more rarely as the cause of infective endocarditis or spontaneous brain abscess. A MLST scheme for A. actinomycetemcomitans is now available at www.pubmlst.org. Whole-genome sequencing of a large number of isolates confirms that MLST competently depicts the population structure of the species.

Danish Whole-Genome-Sequenced Candida albicans and Candida glabrata Samples Fit into Globally Prevalent Clades.

Candida albicans and Candida glabrata are opportunistic fungal pathogens with increasing incidence worldwide and higher-than-expected prevalence in Denmark. We whole-genome sequenced yeast isolates collected from Danish Clinical Microbiology Laboratories to obtain an overview of the Candida population in the country. The majority of the 30 C. albicans isolates were found to belong to three globally prevalent clades, and, with one exception, the remaining isolates were also predicted to cluster with samples from other geographical locations. Similarly, most of the eight C. glabrata isolates were predicted to be prevalent subtypes. Antifungal susceptibility testing proved all C. albicans isolates to be susceptible to both azoles and echinocandins. Two C. glabrata isolates presented azole-resistant phenotypes, yet all were susceptible to echinocandins. There is no indication of causality between population structure and resistance phenotypes for either species.

Haemophilus influenzae one day in Denmark: prevalence, circulating clones, and dismal resistance to aminopenicillins.

Haemophilus influenzae is a common cause of mucosal infections that warrants accurate surveillance. We aimed to assess the prevalence of the species in clinical specimens, and characterise population structure and resistance to aminopenicillins by whole genome sequencing.We assessed the point prevalence by entering the database records of 1 day in Denmark and examined the genome sequences of nationwide, collected isolates from the same day. The prevalence of H. influenzae in clinical samples on the 10th of January 2018 was 1.78 per 100,000 person-days (all samples), and 2.47 per 1000 hospital bed-days (hospital samples). Of 2009 bacteria deemed clinically relevant and collected in a concerted action by the Danish departments of clinical microbiology, 62 (3.1%) were H. influenzae. All 62 isolates belonged to phylogenetic group I and were unencapsulated. Three strains from separate Danish regions had identical core genome sequences, but a small number of intergenic mutations testified to circulating clones, rather than individual cases of patient-to-patient transmission. The TEM-1 ß-lactamase gene was present in 24 strains, while 13 strains were genetically categorised as ampicillin-resistant due to substitutions in penicillin-binding protein 3; shared patterns of amino acid substitutions in unrelated strains indicated putative lateral transfer of chromosomal resistance. Circulating clones of H. influenzae are frequent, and host factors, rather than direct transmission of epidemic strains, may be the primary cause of infection. The bleak presence of ampicillin resistance revealed by sequencing of point prevalence strains underscores the necessity for close examination of testing methods.

Genome Sequence of Staphylococcus epidermidis AUH4567, a Clinical Isolate from an Infected Central Venous Catheter.

Staphylococcus epidermidis is a common cause of implant-associated infections, and this is related to its ability to form biofilms. Strain-to-strain variability in biofilm formation is likely caused by genetic differences. Here, we present a draft genome of S. epidermidis AUH4567, which was isolated from a central venous catheter infection.

Transmission and Antibiotic Resistance of Achromobacter in Cystic Fibrosis.

Achromobacter species are increasingly being detected in patients with cystic fibrosis (CF), and this emerging pathogen is associated with antibiotic resistance and more-severe disease outcomes. Nonetheless, little is known about the extent of transmission and antibiotic resistance development in Achromobacter infections. We sequenced the genomes of 101 Achromobacter clinical isolates (identified as Achromobacter xylosoxidans based on matrix-assister laser desorption ionization-time of flight [MALDI-TOF] or API N20 typing) collected from 51 patients with CF-the largest longitudinal data set to date. We performed phylogenetic analysis on the genomes and combined this with epidemiological and antibiotic resistance data to identify patient-to-patient transmission and the development of antibiotic resistance. We confirmed that the MALDI-TOF or API N20 method was not sufficient for Achromobacter species-level typing and that the population of Achromobacter isolates was composed of five different species, among which A. xylosoxidans accounted for 52% of infections. Most patients were infected by unique Achromobacter clone types; nonetheless, suspected patient-to-patient transmission cases identified by shared clone types were observed in 35% (n = 18) of patients. In 15 of 16 cases, the suspected transmissions were further supported by genome- or clinic visit-based epidemiological analysis. Finally, we found that resistance developed over time. We show that whole-genome sequencing (WGS) is essential for Achromobacter species typing and identification of patient-to-patient transmission, which was revealed for Achromobacter ruhlandii, A. xylosoxidans, and, for the first time, Achromobacter insuavis Furthermore, we show that the development of antibiotic resistance is associated with chronic Achromobacter infections. Our findings emphasize that transmission and antibiotic resistance should be considered in future treatment strategies.

Comparative in vitro activities of meropenem in combination with colistin, levofloxacin, or chloramphenicol against Achromobacter xylosoxidans strains isolated from patients with cystic fibrosis.

OBJECTIVES: Achromobacter xylosoxidans is an emerging pathogen in cystic fibrosis (CF). Relatively little is known about its clinical impact and optimal management. In the present study, the in vitro bactericidal activities of meropenem, either alone or in combination with colistin, levofloxacin, or chloramphenicol, were assessed using A. xylosoxidans strains isolated from CF patients. The synergistic interactions of these combinations were also investigated. METHODS: Minimal inhibitory concentrations (MICs) were determined by microbroth dilution. Bactericidal and synergistic effects of the tested antibiotic combinations were assessed by using the time-kill curve technique. RESULTS: Based on the time-kill curves, we found that meropenem-colistin combinations have bactericidal and synergistic activities for 24 h against A. xylosoxidans strains, both at 1 × MIC and 4 × MIC. Although synergistic interactions were seen with meropenem-levofloxacin combinations, no bactericidal interactions were observed. Additionally, the meropenem-chloramphenicol combinations were found to be neither bactericidal nor synergistic. No antagonism was observed with any combination tested. CONCLUSIONS: This study's findings could have important implications for empirical or combination antimicrobial therapy with tested antibiotics.

Prevalence of hypermutator isolates of Achromobacter spp. from cystic fibrosis patients.

Bacteria colonising the lungs of cystic fibrosis (CF) patients encounter high selective pressures. Hypermutation facilitates adaptation to fluctuating environments, and hypermutator strains are frequently isolated from CF patients. We investigated the prevalence of hypermutator isolates of Achromobacter spp. among patients affiliated with the CF Centre in Aarhus, Denmark. By exposure to rifampicin, the mutation frequency was determined for 90 isolates of Achromobacter spp. cultured from 42 CF patients; 20 infections were categorised as chronic, 22 as intermittent. The genetic mechanisms of hypermutation were examined by comparing DNA repair gene sequences from hypermutator and normomutator isolates. Achromobacter spp. cultured from 11 patients were categorised as hypermutators, and this phenotype was exclusively associated with chronic infections. Isolates of the Danish epidemic strain (DES) of Achromobacter ruhlandii cultured from patients from both Danish CF centres showed elevated mutation frequencies. The hypermutator state of Achromobacter spp. was most commonly associated with nonsynonymous mutations in the DNA mismatch repair gene mutS; a single clone had developed a substitution in the S-adenosyl-L-methionine-dependent methyltransferase putatively involved in DNA repair mechanisms, but not previously linked to the hypermutator phenotype. Hypermutation is prevalent among clinical isolates of Achromobacter spp. and could be a key determinant for the extraordinary adaptation and persistence of DES.

Comparative genomic analysis identifies X-factor (haemin)-independent Haemophilus haemolyticus: a formal re-classification of 'Haemophilus intermedius'.

The heterogeneous and highly recombinogenic genus Haemophilus comprises several species, some of which are pathogenic to humans. All share an absolute requirement for blood-derived factors during growth. Certain species, such as the pathogen Haemophilus influenzae and the commensal Haemophilus haemolyticus, are thought to require both haemin (X-factor) and nicotinamide adenine dinucleotide (NAD, V-factor), whereas others, such as the informally classified 'Haemophilus intermedius subsp. intermedius', and Haemophilus parainfluenzae, only require V-factor. These differing growth requirements are commonly used for species differentiation, although a number of studies are now revealing issues with this approach. Here, we perform large-scale phylogenomics of 240 Haemophilus spp. genomes, including five 'H. intermedius' genomes generated in the current study, to reveal that strains of the 'H. intermedius' group are in fact haemin-independent H. haemolyticus (hiHh). Closer examination of these hiHh strains revealed that they encode an intact haemin biosynthesis pathway, unlike haemin-dependent H. haemolyticus and H. influenzae, which lack most haemin biosynthesis genes. Our results suggest that the common ancestor of modern-day H. haemolyticus and H. influenzae lost key haemin biosynthesis loci, likely as a consequence of specialized adaptation to otorhinolaryngeal and respiratory niches during their divergence from H. parainfluenzae. Genetic similarity analysis demonstrated that the haemin biosynthesis loci acquired in the hiHh lineage were likely laterally transferred from a H. parainfluenzae ancestor, and that this event probably occurred only once in hiHh. This study further challenges the validity of phenotypic methods for differentiating among Haemophilus species, and highlights the need for whole-genome sequencing for accurate characterization of species within this taxonomically challenging genus.

Differential Cell Lysis Among Periodontal Strains of JP2 and Non-JP2 Genotype of Aggregatibacter actinomycetemcomitans Serotype B Is Not Reflected in Dissimilar Expression and Production of Leukotoxin.

Leukotoxic potential of Aggregatibacter actinomycetemcomitans strains has been studied by the use of several methods, and results differ depending on the methods used. The aim of the present study was to perform a comprehensive examination of the leukotoxic potential of a collection of A. actinomycetemcomitans strains by use of three quantitative methods, Western blotting, ELISA, and mRNA expression assay and compare these results with previous data obtained by a cell lysis assay. A higher leukotoxic potential among JP2 genotype strains compared to non-JP2 genotype strains of A. actinomycetemcomitans was found by Western blotting, ELISA and mRNA expression assay. Leukotoxicity as determined by cell lysis assay showed a variation among strains examined, not only depending on being part of JP2 genotype vs. non-JP2 genotype group of A. actinomycetemcomitans. The leukotoxicity of A. actinomycetemcomitans strains as determined by cell lysis assay did not correspond to the leukotoxic potential of A. actinomycetemcomitans strains as determined by three quantitative methods. A comparison of the results obtained by ELISA and mRNA expression assay showed a reasonable correlation between these two methods. It seems important to use more than one method to assess the LtxA-related virulence capacity of A. actinomycetemcomitans in order to obtain comprehensive understanding of the leukotoxic potential of A. actinomycetemcomitans strains.

Aggregatibacter Actinomycetemcomitans: Clinical Significance of a Pathobiont Subjected to Ample Changes in Classification and Nomenclature.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that is part of the oral microbiota. The aggregative nature of this pathogen or pathobiont is crucial to its involvement in human disease. It has been cultured from non-oral infections for more than a century, while its portrayal as an aetiological agent in periodontitis has emerged more recently. A. actinomycetemcomitans is one species among a plethora of microorganisms that constitute the oral microbiota. Although A. actinomycetemcomitans encodes several putative toxins, the complex interplay with other partners of the oral microbiota and the suppression of host response may be central for inflammation and infection in the oral cavity. The aim of this review is to provide a comprehensive update on the clinical significance, classification, and characterisation of A. actinomycetemcomitans, which has exclusive or predominant host specificity for humans.

Whole Genome Sequencing of Aggregatibacter actinomycetemcomitans Cultured from Blood Stream Infections Reveals Three Major Phylogenetic Groups Including a Novel Lineage Expressing Serotype a Membrane O Polysaccharide.

Twenty-nine strains of Aggregatibacter actinomycetemcomitans cultured from blood stream infections in Denmark were characterised. Serotyping was unremarkable, with almost equal proportions of the three major types plus a single serotype e strain. Whole genome sequencing positioned the serotype e strain outside the species boundary; moreover, one of the serotype a strains was unrelated to other strains of the major serotypes and to deposited sequences in the public databases. We identified five additional strains of this type in our collections. The particularity of the group was corroborated by phylogenetic analysis of concatenated core genes present in all strains of the species, and by uneven distribution of accessory genes only present in a subset of strains. Currently, the most accurate depiction of A. actinomycetemcomitans is a division into three lineages that differ in genomic content and competence for transformation. The clinical relevance of the different lineages is not known, and even strains excluded from the species sensu stricto can cause serious human infections. Serotyping is insufficient for characterisation, and serotypes a and e are not confined to specific lineages.

Comprehensive antimicrobial susceptibility testing of a large collection of clinical strains of Aggregatibacter actinomycetemcomitans does not identify resistance to amoxicillin.

AIM: The present study aims to determine the susceptibility of Aggregatibacter actinomycetemcomitans to amoxicillin by investigating a large collection of oral strains of diverse geographical origin. METHODS: Two hundred and fifty-seven A. actinomycetemcomitans strains were serotyped using a multiplex polymerase chain reaction, and minimal inhibitory concentration (MIC) values of amoxicillin were determined using the agar dilution method (range 0.25-8.0 mg/L). The plates were spot-wise inoculated with approximately 104 colony-forming units, incubated in 5% CO2 at 37 C°, and visually inspected after 24 and 48 hr. A MIC ≤ 2.00 mg/L was categorised as susceptible using EUCAST interpretative criteria for Haemophilus species. RESULTS: Amoxicillin MIC values varied from 0.25 mg/L to 2.00 mg/L, and all tested strains, including strains previously reported as resistant, were susceptible to amoxicillin. The MIC50 was 1.00 mg/L and the MIC90 was 2.00 mg/L. CONCLUSION: Meticulous investigation of strains including isolates previously reported as resistant could not confirm the emergence of resistance to ß-lactams in A. actinomycetemcomitans. Based on the present in vitro results, amoxicillin can be considered a key oral antimicrobial agent for treatment of A. actinomycetemcomitans.

Motility, Biofilm Formation and Antimicrobial Efflux of Sessile and Planktonic Cells of Achromobacter xylosoxidans.

Achromobacter xylosoxidans is an innately multidrug-resistant bacterium capable of forming biofilms in the respiratory tract of cystic fibrosis (CF) patients. During the transition from the planktonic stage to biofilm growth, bacteria undergo a transcriptionally regulated differentiation. An isolate of A. xylosoxidans cultured from the sputum of a CF patient was separated into sessile and planktonic stages in vitro, and the transcriptomes were compared. The selected genes of interest were subsequently inactivated, and flagellar motility was found to be decisive for biofilm formation in vitro. The spectrum of a new resistance-nodulation-cell division (RND)-type multidrug efflux pump (AxyEF-OprN) was characterized by inactivation of the membrane fusion protein. AxyEF-OprN is capable of extruding some fluoroquinolones (levofloxacin and ciprofloxacin), tetracyclines (doxycycline and tigecycline) and carpabenems (ertapenem and imipenem), which are classes of antimicrobials that are widely used for treatment of CF pulmonary infections.

A new DNA sensor system for specific and quantitative detection of mycobacteria.

In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely: (1) the ability to isolate mycobacterial TOP1A from crude samples and (2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU) per mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.