Different degree of cytokinemia and T-cell activation according to serum IL-6 levels in critical COVID-19.

Introduction: Tocilizumab, a humanized anti-interleukin-6 receptor (IL-6R) antibody, is recommended for the treatment of severe to critical coronavirus diseases 2019 (COVID-19). However, there were conflicting results on the efficacy of tocilizumab. Therefore, we hypothesized that the differences in tocilizumab efficacy may stem from the different immune responses of critical COVID-19 patients. In this study, we described two groups of immunologically distinct COVID-19 patients, based on their IL-6 response. Methods: We prospectively enrolled critical COVID-19 patients, requiring oxygen support with a high flow nasal cannula or a mechanical ventilator, and analyzed their serial samples. An enzyme-linked immunosorbent assay and flow cytometry were used to evaluate the cytokine kinetics and cellular immune responses, respectively. Results: A total of nine patients with critical COVID-19 were included. The high (n = 5) and low IL-6 (n = 4) groups were distinguished by their peak serum IL-6 levels, using 400 pg/mL as the cut-off value. Although the difference of flow cytometric data did not reach the level of statistical significance, the levels of pro-inflammatory cytokines and the frequencies of intermediate monocytes (CD14+CD16+), IFN-γ+ CD4+ or CD8+ T cells, and HLA-DR+PD-1+ CD4+ T cells were higher in the high IL-6 group than in the low IL-6 group. Conclusion: There were distinctive two groups of critical COVID-19 according to serum IL-6 levels having different degrees of cytokinemia and T-cell responses. Our results indicate that the use of immune modulators should be more tailored in patients with critical COVID-19.

Accurate Diagnosis of COVID-19 from Self-Collectable Biospecimens Using Synthetic Apolipoprotein H Peptide-Coated Nanoparticle Assay.

A high-throughput, accurate screening is crucial for the prevention and control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current methods, which involve sampling from the nasopharyngeal (NP) area by medical staffs, constitute a fundamental bottleneck in expanding the testing capacity. To meet the scales required for population-level surveillance, self-collectable specimens can be used; however, its low viral load has hindered its clinical adoption. Here, we describe a magnetic nanoparticle functionalized with synthetic apolipoprotein H (ApoH) peptides to capture, concentrate, and purify viruses. The ApoH assay demonstrates a viral enrichment efficiency of >90% for both SARS-CoV-2 and its variants, leading to an order of magnitude improvement in analytical sensitivity. For validation, we apply the assay to a total of 84 clinical specimens including nasal, oral, and mouth gargles obtained from COVID-19 patients. As a result, a 100% positivity rate is achieved from the patient-collected nasal and gargle samples, which exceeds that of the traditional NP swab method. The simple 12 min pre-enrichment assay enabling the use of self-collectable samples will be a practical solution to overcome the overwhelming diagnostic capacity. Furthermore, the methodology can easily be built on various clinical protocols, allowing its broad applicability to various disease diagnoses.

Tuberculosis drugs' distribution and emergence of resistance in patient's lung lesions: A mechanistic model and tool for regimen and dose optimization.

BACKGROUND: The sites of mycobacterial infection in the lungs of tuberculosis (TB) patients have complex structures and poor vascularization, which obstructs drug distribution to these hard-to-reach and hard-to-treat disease sites, further leading to suboptimal drug concentrations, resulting in compromised TB treatment response and resistance development. Quantifying lesion-specific drug uptake and pharmacokinetics (PKs) in TB patients is necessary to optimize treatment regimens at all infection sites, to identify patients at risk, to improve existing regimens, and to advance development of novel regimens. Using drug-level data in plasma and from 9 distinct pulmonary lesion types (vascular, avascular, and mixed) obtained from 15 hard-to-treat TB patients who failed TB treatments and therefore underwent lung resection surgery, we quantified the distribution and the penetration of 7 major TB drugs at these sites, and we provide novel tools for treatment optimization. METHODS AND FINDINGS: A total of 329 plasma- and 1,362 tissue-specific drug concentrations from 9 distinct lung lesion types were obtained according to optimal PK sampling schema from 15 patients (10 men, 5 women, aged 23 to 58) undergoing lung resection surgery (clinical study NCT00816426 performed in South Korea between 9 June 2010 and 24 June 2014). Seven major TB drugs (rifampin [RIF], isoniazid [INH], linezolid [LZD], moxifloxacin [MFX], clofazimine [CFZ], pyrazinamide [PZA], and kanamycin [KAN]) were quantified. We developed and evaluated a site-of-action mechanistic PK model using nonlinear mixed effects methodology. We quantified population- and patient-specific lesion/plasma ratios (RPLs), dynamics, and variability of drug uptake into each lesion for each drug. CFZ and MFX had higher drug exposures in lesions compared to plasma (median RPL 2.37, range across lesions 1.26-22.03); RIF, PZA, and LZD showed moderate yet suboptimal lesion penetration (median RPL 0.61, range 0.21-2.4), while INH and KAN showed poor tissue penetration (median RPL 0.4, range 0.03-0.73). Stochastic PK/pharmacodynamic (PD) simulations were carried out to evaluate current regimen combinations and dosing guidelines in distinct patient strata. Patients receiving standard doses of RIF and INH, who are of the lower range of exposure distribution, spent substantial periods (>12 h/d) below effective concentrations in hard-to-treat lesions, such as caseous lesions and cavities. Standard doses of INH (300 mg) and KAN (1,000 mg) did not reach therapeutic thresholds in most lesions for a majority of the population. Drugs and doses that did reach target exposure in most subjects include 400 mg MFX and 100 mg CFZ. Patients with cavitary lesions, irrespective of drug choice, have an increased likelihood of subtherapeutic concentrations, leading to a higher risk of resistance acquisition while on treatment. A limitation of this study was the small sample size of 15 patients, performed in a unique study population of TB patients who failed treatment and underwent lung resection surgery. These results still need further exploration and validation in larger and more diverse cohorts. CONCLUSIONS: Our results suggest that the ability to reach and maintain therapeutic concentrations is both lesion and drug specific, indicating that stratifying patients based on disease extent, lesion types, and individual drug-susceptibility profiles may eventually be useful for guiding the selection of patient-tailored drug regimens and may lead to improved TB treatment outcomes. We provide a web-based tool to further explore this model and results at http://saviclab.org/tb-lesion/.

MiR-9, miR-21, and miR-155 as potential biomarkers for HPV positive and negative cervical cancer.

BACKGROUND: Cervical cancer is the second leading cause of death among female patients with cancer in the world. High risk human papillomavirus has causal roles in cervical cancer initiation and progression by deregulating several cellular processes. However, HPV infection is not sufficient for cervical carcinoma development. Therefore, other genetic and epigenetic factors may be involved in this complex disease, and the identification of which may lead to better diagnosis and treatment. Our aim was to analyze the expression of microRNAs in cervical cancer cases positive or negative for HPV E6/E7 mRNA, and to assess their diagnostic usefulness and relevance. METHODS: The expression of three different microRNAs (miR-9, miR-21, and miR-155) in 52 formalin-fixed paraffin-embedded (FFPE) primary cervical cancer tissue samples and 50 FFPE normal cervical tissue samples were evaluated. RESULTS: MiR-9, miR-21, and miR-155 were significantly overexpressed in cervical cancer tissues compared to normal tissues (P < 0.001). MiR-21 and miR-155 expression combined with the HPV E6/E7 mRNA assay in HPV E6/E7 negative cervical cancer showed increased AUC of 0.7267 and 0.7000, respectively (P = 0.01, P = 0.04), demonstrating their potential as diagnostic tools. Moreover, miR-21 and miR-155 were predictors showing a 7 fold and 10.3 fold higher risk for HPV E6/E7 negative patients with cervical cancer (P = 0.024 and P = 0.017, respectively) while miR-155 was a predictor showing a 27.9 fold higher risk for HPV E6/E7 positive patients with cervical cancer (P < 0.0001). CONCLUSIONS: There is a strong demand for additional, alternative molecular biomarkers for diagnosis and management of precancer patients. MiR-21 and miR-155 may be helpful in the prediction of both HPV positive and HPV negative cases of cervical cancer.

Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

Complete genome sequence of Mycobacterium tuberculosis K from a Korean high school outbreak, belonging to the Beijing family.

Mycobacterium tuberculosis K, a member of the Beijing family, was first identified in 1999 as the most prevalent genotype in South Korea among clinical isolates of M. tuberculosis from high school outbreaks. M. tuberculosis K is an aerobic, non-motile, Gram-positive, and non-spore-forming rod-shaped bacillus. A transmission electron microscopy analysis displayed an abundance of lipid bodies in the cytosol. The genome of the M. tuberculosis K strain was sequenced using two independent sequencing methods (Sanger and Illumina). Here, we present the genomic features of the 4,385,518-bp-long complete genome sequence of M. tuberculosis K (one chromosome, no plasmid, and 65.59 % G + C content) and its annotation, which consists of 4194 genes (3447 genes with predicted functions), 48 RNA genes (3 rRNA and 45 tRNA) and 261 genes with peptide signals.

Co-immunization of plasmid DNA encoding IL-12 and IL-18 with Bacillus Calmette-Guérin vaccine against progressive tuberculosis.

PURPOSE: Bacillus Calmette-Guérin (BCG) vaccine has widely been used to immunize against tuberculosis, but its protective efficacy is variable in adult pulmonary tuberculosis, while it is not efficiently protective against progressive infection of virulent Mycobacterium tuberculosis strains. In this study, the protective effects of plasmid DNA vaccine constructs encoding IL-12 or IL-18 with the BCG vaccine were evaluated against progressive infection of M. tuberculosis, using mouse aerosol challenge model. MATERIALS AND METHODS: Plasmid DNA vaccine constructs encoding IL-12 or IL-18 were constructed and mice were immunized with the BCG vaccine or with IL-12 DNA or IL-18 DNA vaccine constructs together with the BCG vaccine. RESULTS: The BCG vaccine induced high level of interferon gamma (IFN-γ) but co-immunization of IL-12 or IL-18 DNA vaccine constructs with the BCG vaccine induced significantly higher level of IFN-γ than a single BCG vaccine. The BCG vaccine was highly protective at early stage of M. tuberculosis infection, but its protective efficacy was reduced at later stage of infection. The co-immunization of IL-12 DNA vaccine constructs with the BCG vaccine was slightly more protective at early stage of infection and was significantly more protective at later stage infection than a single BCG vaccine. CONCLUSION: Co-immunization of IL-12 DNA vaccine with the BCG vaccine induced more protective immunity and was more effective for protection against progressive infection of M. tuberculosis.

Improved rapid molecular diagnosis of multidrug-resistant tuberculosis using a new reverse hybridization assay, REBA MTB-MDR.

Rapid diagnosis of multidrug-resistant tuberculosis (MDR-TB) is essential for the prompt initiation of effective second-line therapy to improve treatment outcome and limit transmission of this obstinate disease. A variety of molecular methods that enable the rapid detection of mutations implicated in MDR-TB have been developed. The sensitivity of the methods is dependent, in principle, on the repertoire of mutations being detected, which is typically limited to mutations in the genes rpoB, katG and the promoter region of inhA. In this study, a new reverse hybridization assay, REBA MTB-MDR (M&D), that probes mutations in the oxyR-ahpC intergenic region, in addition to those in rpoB, katG and the inhA promoter region, was evaluated. A set of 240 Mycobacterium tuberculosis clinical isolates from patients receiving retreatment regimens was subjected to conventional phenotypic drug-susceptibility testing (DST) and the REBA MTB-MDR assay. The nucleotide sequences of the loci known to be involved in drug resistance were determined for comparison. In brief, the results showed that the REBA MTB-MDR assay efficiently recognized nucleotide changes in the oxyR-ahpC intergenic region as well as those in rpoB, katG and the inhA promoter region with higher sensitivity, resulting in an 81.0 % detection rate for isoniazid resistance. Inclusion of the oxyR-ahpC intergenic region in the REBA MTB-MDR assay improved the overall sensitivity of molecular DST for MDR-TB from 73.1 to 79.9 %.

Polymorphisms associated with resistance and cross-resistance to aminoglycosides and capreomycin in Mycobacterium tuberculosis isolates from South Korean Patients with drug-resistant tuberculosis.

The aminoglycosides streptomycin, amikacin, and kanamycin and the cyclic polypeptide capreomycin are all widely used in second-line therapy for patients who develop multidrug-resistant tuberculosis. We have characterized a set of 106 clinical isolates of Mycobacterium tuberculosis using phenotypic drug susceptibility testing (DST) to determine the extent of resistance to each agent and cross-resistance between agents. These results were compared with polymorphisms in the DNA sequences of ribosome-associated genes previously implicated in resistance and with the clinical outcomes of subjects from whom these isolates were obtained. Thirty-six (34%) of these isolates displayed resistance to one or more of these agents, and the majority of these (20 of 36) showed cross-resistance to one or more agents. Most (33 of 36) of the resistant isolates showed polymorphisms in the 16S ribosome components RpsL and rrs. Three resistant strains (3 of 36) were identified that had no known polymorphisms in ribosomal constituents. For kanamycin and streptomycin, molecular DST significantly outperformed phenotypic DST using the absolute concentration method for predicting 4-month sputum conversion (likelihood ratios of 4.0 and 2.0, respectively) and was equivalent to phenotypic DST using the National Committee for Clinical Laboratory Standards (NCCLS)-approved agar proportion method for estimating MIC (likelihood ratio, 4.0). These results offer insight into mechanisms of resistance and cross-resistance among these agents and suggest that the development of rapid molecular tests to distinguish polymorphisms would significantly enhance clinical utility of this important class of second-line antituberculosis drugs.

Novel polymorphic region of the rpoB gene containing Mycobacterium species-specific sequences and its use in identification of mycobacteria.

Sequence analysis of a specific region of the mycobacterium rpoB gene in 35 mycobacterial strains representing 26 different mycobacterial species of clinical importance showed that there exists a highly polymorphic region. Based on the sequences of the polymorphic region, the oligonucleotide probes of 14 mycobacterial species with relatively high clinical importance were designed and shown to be specific to their corresponding mycobacterial species by dot blot hybridization. The results showed that the probes designed in this study are highly specific to each mycobacterial species, which suggests that these sequences may be useful for the species identification of mycobacteria.