High variance in quantification of Mycobacterium tuberculosis at low bacterial loads and with differentially detectable mycobacteria.

We examined the correlation between three different methods of Mycobacterium tuberculosis quantification: time to positivity (TTP), log10 CFU, and an assay to detect differentially detectable M. tuberculosis (DD Mtb) from three different prospective studies. Participants with DD Mtb have significantly more variation in the CFU/TTP correlation than participants with no DD Mtb (P < 0.001). This may impact the design of early bactericidal activity studies that use TTP as the primary outcome.

Characterization of Differentially Detectable Mycobacterium tuberculosis in the Sputum of Subjects with Drug-Sensitive or Drug-Resistant Tuberculosis before and after Two Months of Therapy.

Standard methods for enumerating Mycobacterium tuberculosis in patient sputum can miss large populations of viable M. tuberculosis cells that are unable to grow either on solid medium or in liquid medium unless the medium has been extensively diluted. Because these bacteria can be detected in liquid medium after limiting dilution, they have been termed differentially culturable or differentially detectable M. tuberculosis (DD-Mtb). Treatment with isoniazid (H), rifampin (R), pyrazinamide (Z), and ethambutol (E) (HRZE) for 1 to 2 weeks has been shown to increase the representation of DD-Mtb in the sputum of drug-sensitive (DS) tuberculosis (TB) patients. However, little is known about DD-Mtb after longer periods of treatment with HRZE or in patients with drug-resistant (DR) TB who receive second-line therapies. Here, we measured the proportion of DD-Mtb cells in the sputum of 47 subjects, 29 with DS TB and 18 with DR TB, before initiation of treatment and at 2 weeks and 2 months thereafter. Prior to treatment, DD-Mtb cells represented the majority of M. tuberculosis cells in the sputum of 21% of subjects with DS TB, and this proportion rose to 65% after 2 weeks of treatment with first-line drugs. In subjects with DR TB, DD-Mtb cells were found in the sputum of 29% of subjects prior to treatment initiation, and this proportion remained steady at 31% after 2 weeks of treatment with second-line drugs. By 2 months, DD-Mtb cells were detected in the sputum of only 2/15 (13.3%) subjects with DS TB and in 0/15 of subjects with DR TB. One of the DS subjects whose sputum was positive for DD-Mtb at month 2 later experienced treatment failure.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations.

Mycobacterium tuberculosis (Mtb) encounters stresses during the pathogenesis and treatment of tuberculosis (TB) that can suppress replication of the bacteria and render them phenotypically tolerant to most available drugs. Where studied, the majority of Mtb in the sputum of most untreated subjects with active TB have been found to be nonreplicating by the criterion that they do not grow as colony-forming units (cfus) when plated on agar. However, these cells are viable because they grow when diluted in liquid media. A method for generating such "differentially detectable" (DD) Mtb in vitro would aid studies of the biology and drug susceptibility of this population, but lack of independent confirmation of reported methods has contributed to skepticism about their existence. Here, we identified confounding artifacts that, when avoided, allowed development of a reliable method of producing cultures of ≥90% DD Mtb in starved cells. We then characterized several drugs according to whether they contribute to the generation of DD Mtb or kill them. Of the agents tested, rifamycins led to DD Mtb generation, an effect lacking in a rifampin-resistant strain with a mutation in rpoB, which encodes the canonical rifampin target, the ß subunit of RNA polymerase. In contrast, thioridazine did not generate DD Mtb from starved cells but killed those generated by rifampin.

Implementation of Molecular Surveillance After a Cluster of Fatal Toxoplasmosis at 2 Neighboring Transplant Centers.

After a cluster of fatal toxoplasmosis among stem cell transplant recipients at 2 hospitals, surveillance with polymerase chain reaction (PCR) (blood) was instituted. Rate of reactivation among seropositive recipients was 2.2 and 16%. Parasitemia was successfully managed with preemptive treatment. For seropositive recipients unable to take prophylaxis, toxoplasma PCR surveillance should be routinely performed.

Factors associated with inconsistent condom use in adolescents with negative or unknown HIV status in Northwest Cameroon.

The purpose of this study is to evaluate the association between utilization of HIV testing and condom use amongst Cameroonian youths/adolescents who are not known to be HIV-infected. Worldwide, HIV is spreading most quickly amongst youths/adolescents. Between 44% and 82% of sexually active youths in Cameroon report inconsistent condom use. Data regarding utilization of HIV testing and condom use are lacking. A cross-sectional survey was administered to 431 youths ages 12-26 years in Cameroon from September 2011 to December 2011. Data on sociodemographics, sexual risk behaviors, self-reported HIV status, and condom use were collected. We compared rates of inconsistent condom use between those with known HIV negative status who utilized testing (HIV-N) and those with unknown status due to unutilized testing (HIV-U). Inconsistent condom use was defined as responding "never," "sometimes," or "usually," while consistent condom use was defined as responding "always" to questions regarding frequency of condom use. Generalized estimating equations were applied to assess the association between HIV testing and inconsistent condom use, adjusting for other confounders. Of 414 eligible respondents, 205 were HIV-U and 209 were HIV-N. HIV-U subjects were younger (mean age = 16.4 vs. 17.9, p < 0.001) and more likely to report living in an urban area (p = 0.002) than HIV-N subjects. Seventy-two percent (137/191) of sexually active youths reported inconsistent condom use. After adjusting for potential confounders, HIV-U status (odds ratio [OR] = 3.97, 95% confidence interval [CI] = 1.68-6.01) was associated with inconsistent condom use. Similarly, female gender (OR = 3.2, 95% CI = 1.29-7.89) was associated with inconsistent condom use, while older age at sexual debut was associated with a decreased risk for inconsistent condom use (OR = 0.67, 95% CI = 0.56-0.81). Cameroonian adolescents report high rates of inconsistent condom use which we found to be associated with self-reported unknown HIV status due to unutilized HIV testing. Successful HIV prevention programs among African youths/adolescents may benefit from expanded HIV testing programs.

Clinically encountered growth phenotypes of tuberculosis-causing bacilli and their in vitro study: A review.

The clinical manifestations of tuberculosis (TB) vary widely in severity, site of infection, and outcomes of treatment-leading to simultaneous efforts to individualize therapy safely and to search for shorter regimens that can be successfully used across the clinical spectrum. In these endeavors, clinicians and researchers alike employ mycobacterial culture in rich media. However, even within the same patient, individual bacilli among the population can exhibit substantial variability in their culturability. Bacilli in vitro also demonstrate substantial heterogeneity in replication rate and cultivation requirements, as well as susceptibility to killing by antimicrobials. Understanding parallels in clinical, ex vivo and in vitro growth phenotype diversity may be key to identifying those phenotypes responsible for treatment failure, relapse, and the reactivation of bacilli that progresses TB infection to disease. This review briefly summarizes the current role of mycobacterial culture in the care of patients with TB and the ex vivo evidence of variability in TB culturability. We then discuss current advances in in vitro models that study heterogenous subpopulations within a genetically identical bulk culture, with an emphasis on the effect of oxidative stress on bacillary cultivation requirements. The review highlights the complexity that heterogeneity in mycobacterial growth brings to the interpretation of culture in clinical settings and research. It also underscores the intricacies present in the interplay between growth phenotypes and antimicrobial susceptibility. Better understanding of population dynamics and growth requirements over time and space promises to aid both the attempts to individualize TB treatment and to find uniformly effective therapies.

Transcriptional Biomarkers of Differentially Detectable Mycobacterium tuberculosis in Patient Sputum.

Certain populations of Mycobacterium tuberculosis go undetected by standard diagnostics but can be enumerated using limiting dilution assays. These differentially detectable M. tuberculosis (DD M. tuberculosis) populations may have relevance for persistence due to their drug tolerance. It is unclear how well DD M. tuberculosis from patients is modeled by a recently developed in vitro model in which M. tuberculosis starved in phosphate-buffered saline is incubated with rifampin to produce DD M. tuberculosis (the PBS-RIF model). This study attempted to answer this question. We selected 14 genes that displayed differential expression in the PBS-RIF model and evaluated their expression in patient sputa containing various proportions of DD M. tuberculosis. The expression of 12/14 genes correlated with the relative abundance of DD M. tuberculosis in patient sputa. Culture filtrate (CF), which promotes recovery of DD M. tuberculosis from certain patient sputa, improved these correlations in most cases. The gene whose reduced expression relative to M. tuberculosis 16S rRNA showed the greatest association with the presence and relative abundance of DD M. tuberculosis in patient sputa, icl1, was recently shown to play a functional role in restraining DD M. tuberculosis formation in the PBS-RIF model. Expression of icl1, combined with two additional DD M. tuberculosis-related genes, showed strong performance for predicting the presence or absence of DD M. tuberculosis in patient sputa (receiver operating characteristic [ROC] area under the curve [AUC] = 0.88). Thus, the in vitro DD M. tuberculosis model developed by Saito et al. (K. Saito, T. Warrier, S. Somersan-Karakaya, L. Kaminski, et al., Proc Natl Acad Sci U S A 114:E4832-E4840, 2017, https://doi.org/10.1073/pnas.1705385114) bears a resemblance to DD M. tuberculosis found in tuberculosis (TB) patients, and DD M. tuberculosis transcriptional profiles may be useful for monitoring DD M. tuberculosis populations in patient sputum. IMPORTANCE Differentially detectable M. tuberculosis (DD M. tuberculosis), which is detectable by limiting dilution assays but not by CFU, is present and enriched for in TB patient sputum after initiation of first-line therapy. These cryptic cells may play a role in disease persistence due to their phenotypic tolerance to anti-TB drugs. A recently developed in vitro model of DD M. tuberculosis (the PBS-RIF model) has expanded our understanding of these cells, though how well it translates to DD M. tuberculosis in patients is currently unknown. To answer this question, we selected 14 genes that displayed differential expression in the PBS-RIF model and evaluated their expression in TB patient sputa. We found that 12/14 of these genes showed a similar expression profile in patient sputa that correlated with the relative abundance of DD M. tuberculosis. Further, the expression of three of these genes showed strong performance for predicting the presence or absence of DD M. tuberculosis in patient sputa. The use of DD M. tuberculosis transcriptional profiles may allow for easier monitoring of DD M. tuberculosis populations in patient sputum in comparison to limiting dilution assays.

Oxidative damage and delayed replication allow viable Mycobacterium tuberculosis to go undetected.

"Viable but nonculturable" states of bacteria pose challenges for environmental and clinical microbiology, but their biological mechanisms remain obscure. Mycobacterium tuberculosis (Mtb), the leading cause of death from infection until the coronavirus disease 2019 pandemic, affords a notable example of this phenotype. Mtb can enter into a "differentially detectable" (DD) state associated with phenotypic antimicrobial resistance. In this state, Mtb cells are viable but undetectable as colony-forming units. We found that Mtb cells enter the DD state when they undergo sublethal oxidative stress that damages their DNA, proteins, and lipids. In addition, their replication process is delayed, allowing time for repair. Mycobacterium bovis and its derivative, BCG, fail to enter the DD state under similar conditions. These findings have implications for tuberculosis latency, detection, relapse, treatment monitoring, and development of regimens that overcome phenotypic antimicrobial resistance.

Profiling of immune dysfunction in COVID-19 patients allows early prediction of disease progression.

With a rising incidence of COVID-19-associated morbidity and mortality worldwide, it is critical to elucidate the innate and adaptive immune responses that drive disease severity. We performed longitudinal immune profiling of peripheral blood mononuclear cells from 45 patients and healthy donors. We observed a dynamic immune landscape of innate and adaptive immune cells in disease progression and absolute changes of lymphocyte and myeloid cells in severe versus mild cases or healthy controls. Intubation and death were coupled with selected natural killer cell KIR receptor usage and IgM+ B cells and associated with profound CD4 and CD8 T-cell exhaustion. Pseudo-temporal reconstruction of the hierarchy of disease progression revealed dynamic time changes in the global population recapitulating individual patients and the development of an eight-marker classifier of disease severity. Estimating the effect of clinical progression on the immune response and early assessment of disease progression risks may allow implementation of tailored therapies.

Kinetic evidence for dihapto (eta(2)) pi-aryl participation in acid-catalyzed ring opening of diarylhomobenzoquinone epoxides.

The BF(3)-catalyzed ring-opening reaction of variously endo/exo m- and p-substituted diarylhomobenzoquinone epoxides proceeded through a transannular S(E)2-Ar cyclization of endo-aryl groups to give the tricyclic diketo-alcohols and cyclohexadienone spiro-linked tricyclic diketo-alcohols. Kinetics of these reactions has been investigated in CDCl(3) at 30 degrees C in order to elucidate the possible remote pi-aryl participation. The rates were significantly increased with increasing electron-donating ability of the endo-aryl substituents X (k(p-MeO)/k(p-CF3) = 8200) but only negligibly influenced by the distal exo-aryl substituents Y (k(p-MeO)/k(p-CF3) = 2.1). For the endo-X substituted series, an excellent linear free energy relationship, log k(rel)(endo) = -2.49sigma(ipso) - 1.62sigma(ortho) - 0.108 (R(2) = 0.98, n = 8), was attained using two modified site-dependent substituent parameters sigma(ipso) (using sigma(p)(+) for p-X and sigma(m) for m-X) and sigma(ortho) (using sigma(m) for p-X and sigma(p)(+) for m-X). This means that the dihapto(eta(2)) pi-coordination occurs in the pi-aryl participation, with the ipso pi-electron donation contributing 1.6 times more effectively than the ortho one. On the other hand, the distal exo-Y substituted series gave an acceptable Yukawa-Tsuno equation with small polar and resonance contributions; log k(rel)(exo) = -0.912(sigma(0) + 0.237Deltasigma(R)(+)) (R(2) = 0.96, n = 8). These kinetic substituent effects were compared with those of the acid-catalyzed pi-aryl assisted transannular S(E)2-Ar cyclization of the cyclobutene-fused diarylhomobenzoquinones. It was found that the geometrical characteristics of the vacant oxirane Walsh orbital and the cyclobutene antibonding orbital play a crucial role in the topological features of eta(2) pi-aryl participation.

Longitudinal immune profiling of mild and severe COVID-19 reveals innate and adaptive immune dysfunction and provides an early prediction tool for clinical progression.

With a rising incidence of COVID-19-associated morbidity and mortality worldwide, it is critical to elucidate the innate and adaptive immune responses that drive disease severity. We performed longitudinal immune profiling of peripheral blood mononuclear cells from 45 patients and healthy donors. We observed a dynamic immune landscape of innate and adaptive immune cells in disease progression and absolute changes of lymphocyte and myeloid cells in severe versus mild cases or healthy controls. Intubation and death were coupled with selected natural killer cell KIR receptor usage and IgM+ B cells and associated with profound CD4 and CD8 T cell exhaustion. Pseudo-temporal reconstruction of the hierarchy of disease progression revealed dynamic time changes in the global population recapitulating individual patients and the development of an eight-marker classifier of disease severity. Estimating the effect of clinical progression on the immune response and early assessment of disease progression risks may allow implementation of tailored therapies.

Dual-Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis.

The historical view of ß-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a ß-lactamase inhibitor. However, most antimycobacterial ß-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 ß-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3' that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a ß-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A ß-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent ß-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.

Differentially Detectable Mycobacterium tuberculosis Cells in Sputum from Treatment-Naive Subjects in Haiti and Their Proportionate Increase after Initiation of Treatment.

Recent reports indicate that the sputum of 80% or more of treatment-naive subjects with tuberculosis recruited in England or South Africa contained more viable Mycobacterium tuberculosis cells detected by limiting dilution (LD) in liquid culture than detected as CFU. Efforts to generate such differentially detectable (DD) M. tuberculosis populations in vitro have been difficult to reproduce, and the LD assay is prone to artifact. Here, we applied a stringent version of the LD assay to sputum from 33 treatment-naive, HIV-negative Haitian subjects with drug-sensitive tuberculosis (TB) and to a second sputum sample after two weeks of standard treatment with isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) for 13 of these subjects. Twenty-one percent had statistically defined levels of DD M. tuberculosis in their pretreatment sputum at an average proportional excess over CFU of 3-fold. Sixty-nine percent of those who received HRZE had statistically defined levels of DD M. tuberculosis in their sputum, and of these, the mean proportionate excess over CFU was 7.9-fold. Thus, DD M. tuberculosis is detectable in pretreatment sputum from a significant proportion of subjects in the Western Hemisphere, and certain drugs or drug regimens, while reducing CFU, may at the same time increase the proportional representation of DD M. tuberculosis among the surviving bacilli. Monitoring DD M. tuberculosis may improve our ability to predict the efficacy of efforts to shorten treatment.IMPORTANCE Measurement of the reduction in CFU in sputum of patients with TB up to 2 weeks after the initiation of treatment is the gateway test for a new TB treatment. Reports have suggested that CFU assays fail to detect the majority of viable M. tuberculosis cells in sputum samples from the majority of patients when the number of M. tuberculosis is estimated by limiting dilution (LD). In an effort to avoid potential methodologic confounders, we applied a modified version of the LD assay in a study of a geographically distinct population. We confirmed that differentially detectable (DD) M. tuberculosis is often found before treatment, albeit at lower proportionate levels than in earlier reports. Strikingly, the prevalence and proportionate representation of DD M. tuberculosis increased during standard treatment. Sublethal exposure to certain antibiotics may help generate DD M. tuberculosis cells or enrich their representation among the surviving bacteria, and this may contribute to the need for prolonged treatment with those agents in order to achieve durable cures.

Rifamycin congeners kanglemycins are active against rifampicin-resistant bacteria via a distinct mechanism.

Rifamycin antibiotics (Rifs) target bacterial RNA polymerases (RNAPs) and are widely used to treat infections including tuberculosis. The utility of these compounds is threatened by the increasing incidence of resistance (RifR). As resistance mechanisms found in clinical settings may also occur in natural environments, here we postulated that bacteria could have evolved to produce rifamycin congeners active against clinically relevant resistance phenotypes. We survey soil metagenomes and identify a tailoring enzyme-rich family of gene clusters encoding biosynthesis of rifamycin congeners (kanglemycins, Kangs) with potent in vivo and in vitro activity against the most common clinically relevant RifR mutations. Our structural and mechanistic analyses reveal the basis for Kang inhibition of RifR RNAP. Unlike Rifs, Kangs function through a mechanism that includes interfering with 5'-initiating substrate binding. Our results suggest that examining soil microbiomes for new analogues of clinically used antibiotics may uncover metabolites capable of circumventing clinically important resistance mechanisms.

Retrospective analysis of the prevalence of and factors associated with condom use among young HIV-infected women in Cameroon.

OBJECTIVES: Young women are more likely to be infected with HIV globally, in sub-Saharan Africa, and in Cameroon. Despite its clear clinical and public health benefits, condom use among HIV-infected women continues to be low. The objective of this study was to describe the prevalence of inconsistent condom use among HIV-infected women in Cameroon and the factors associated with it. METHODS: We conducted a cross-sectional study of HIV-infected young women aged 17-26 years from three semi-urban HIV clinics in the Northwest Region of Cameroon. This study was a subgroup analysis of a previously reported study on inconsistent condom use in HIV-infected and -uninfected youth. Inconsistent condom use was defined as reporting "sometimes" or "never" to questions regarding frequency of condom use. Logistic regression modeling was used to determine factors associated with inconsistent condom use. RESULTS: A total of 84 participants were recruited and submitted completed questionnaires for analysis. Median age was 24 years (interquartile range = 22-25) and the median age at HIV diagnosis was 21 years (interquartile range = 20-23). Fifty percent of the participants reported no prior schooling or only primary school education. Overall, 61/84 (73%) reported inconsistent condom use. After adjusting for potential confounders, education to the secondary school level was protective against inconsistent condom use (odds ratio = 0.19; confidence interval: 0.04-0.95), and having ≥2 pregnancies was associated with inconsistent condom use (odds ratio = 7.52; confidence interval: 1.67-34.00). CONCLUSION: There is a high prevalence of inconsistent condom use among young HIV-infected women in Cameroon, which appears to be associated with lower levels of educational attainment and higher parity. Further larger studies assessing the factors associated with poor condom use in this population are warranted and may inform public health policy in resource-limited settings with high HIV prevalence.