Diagnosis of Clostridium difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC) should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has now been advocated by international guidelines in order to optimize diagnostic accuracy. Despite these recommendations, testing methods between hospitals vary widely, which impacts CDI incidence rates. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

Multistep algorithmic testing in which a sensitive nucleic acid amplification test (NAAT) is followed by a specific toxin A and toxin B enzyme immunoassay (EIA) is among the most accurate methods for infection (CDI) diagnosis. The obvious shortcoming of this approach is that multiple tests must be performed to establish a CDI diagnosis, which may delay treatment. Therefore, we sought to determine whether a preliminary diagnosis could be made on the basis of the quantitative results of the first test in algorithmic testing, which provide a measure of organism burden. To do so, we retrospectively analyzed two large collections of samples ( = 2,669 and = 1,718) that were submitted to the laboratories of two Dutch hospitals for CDI testing. Both hospitals apply a two-step testing algorithm in which a NAAT is followed by a toxin A/B EIA. Of all samples, 208 and 113 samples, respectively, tested positive by NAAT. Among these NAAT-positive samples, significantly lower mean quantification cycle ( ) values were found for patients whose stool eventually tested positive for toxin, compared with patients who tested negative for toxin (mean values of 24.4 versus 30.4 and 26.8 versus 32.2; < 0.001 for both cohorts). Receiver operating characteristic curve analysis was performed to investigate the ability of values to predict toxin status and yielded areas under the curve of 0.826 and 0.854. Using the optimal cutoff values, prediction of the eventual toxin A/B EIA results was accurate for 78.9% and 80.5% of samples, respectively. In conclusion, values can serve as predictors of toxin status but, due to the suboptimal correlation between the two tests, additional toxin testing is still needed.

There is a need to develop diagnostic and analytical tools that allow noninvasive monitoring of bacterial growth and dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification, and they should allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer, 99mTc-UBI29–41-Cy5, which contains both a fluorescent and a radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh-muscle infection with 99mTc-UBI29–41-Cy5-labeled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence-imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the 99mTc-UBI29–41-Cy5-labeled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period: 52% of the total injected radioactivity per gram of tissue (%ID/g) at 4 h postinfection (pi) versus 44%ID/g at 28 h pi (15% decrease). After inoculation, a portion of the bacteria disseminated from the abscess, and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with 99mTc-UBI29–41-Cy5 allowed noninvasive bacterial-cell tracking during a 28 h period. Given the versatility of the presented bacterial-tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled-human-infection studies.

Professional antigen-presenting cells (APCs), like macrophages (Mϕs) and dendritic cells (DCs), are central players in the induction of natural and vaccine-induced immunity to malaria, yet very little is known about the interaction of SPZ with human APCs. Intradermal delivery of whole-sporozoite vaccines reduces their effectivity, possibly due to dermal immunoregulatory effects. Therefore, understanding these interactions could prove pivotal to malaria vaccination. We investigated human APC responses to recombinant circumsporozoite protein (recCSP), SPZ and anti-CSP opsonized SPZ both in monocyte derived MoDCs and MoMϕs. Both MoDCs and MoMϕs readily took up recCSP but did not change phenotype or function upon doing so. SPZ are preferentially phagocytosed by MoMϕs instead of DCs and phagocytosis greatly increased after opsonization. Subsequently MoMϕs show increased surface marker expression of activation markers as well as tolerogenic markers such as Programmed Death-Ligand 1 (PD-L1). Additionally they show reduced motility, produce interleukin 10 and suppressed interferon gamma (IFNγ) production by antigen specific CD8 + T cells. Importantly, we investigated phenotypic responses to SPZ in primary dermal APCs isolated from human skin explants, which respond similarly to their monocyte-derived counterparts. These findings are a first step in enhancing our understanding of pre-erythrocytic natural immunity and the pitfalls of intradermal vaccinationinduced immunity.

Many, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found in Methanosarcina species that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studied Methanosarcina species, Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, to the quinone pool size in the bacterium Escherichia coli. We found the quantity of MPh per cell increases as cultures transition from exponential growth to stationary phase, and absolute quantities of MPh were 3-fold higher in M. acetivorans than in M. barkeri. The concentration of MPh suggests the cell membrane of M. acetivorans, but not of M. barkeri, is electrically quantized as if it were a single conductive metal sheet and near optimal for rate of electron transport. Similarly, stationary (but not exponentially growing) E. coli cells also have electrically quantized membranes on the basis of quinone content. Consistent with our hypothesis, we demonstrated that the exogenous addition of phenazine increases the growth rate of M. barkeri three times that of M. acetivorans. Our work suggests electron flux through MPh is naturally higher in M. acetivorans than in M. barkeri and that hydrogen cycling is less efficient at conserving energy than scalar proton translocation using MPh.IMPORTANCE Can we grow more from less? The ability to optimize and manipulate metabolic efficiency in cells is the difference between commercially viable and nonviable renewable technologies. Much can be learned from methane-producing archaea (methanogens) which evolved a successful metabolic lifestyle under extreme thermodynamic constraints. Methanogens use highly efficient electron transport systems and supramolecular complexes to optimize electron and carbon flow to control biomass synthesis and the production of methane. Worldwide, methanogens are used to generate renewable methane for heat, electricity, and transportation. Our observations suggest Methanosarcina acetivorans, but not Methanosarcina barkeri, has electrically quantized membranes. Escherichia coli, a model facultative anaerobe, has optimal electron transport at the stationary phase but not during exponential growth. This study also suggests the metabolic efficiency of bacteria and archaea can be improved using exogenously supplied lipophilic electron carriers. The enhancement of methanogen electron transport through methanophenazine has the potential to increase renewable methane production at an industrial scale.