Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

https://journals.asm.org/doi/pdf/10.1128/CMR.00022-06

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

BACKGROUND: The efficacy of interleukin-6 receptor antagonists in critically ill patients with coronavirus disease 2019 (Covid-19) is unclear. METHODS: We evaluated tocilizumab and sarilumab in an ongoing international, multifactorial, adaptive platform trial. Adult patients with Covid-19, within 24 hours after starting organ support in the intensive care unit (ICU), were randomly assigned to receive tocilizumab (8 mg per kilogram of body weight), sarilumab (400 mg), or standard care (control). The primary outcome was respiratory and cardiovascular organ support-free days, on an ordinal scale combining in-hospital death (assigned a value of -1) and days free of organ support to day 21. The trial uses a Bayesian statistical model with predefined criteria for superiority, efficacy, equivalence, or futility. An odds ratio greater than 1 represented improved survival, more organ support-free days, or both. RESULTS: Both tocilizumab and sarilumab met the predefined criteria for efficacy. At that time, 353 patients had been assigned to tocilizumab, 48 to sarilumab, and 402 to control. The median number of organ support-free days was 10 (interquartile range, -1 to 16) in the tocilizumab group, 11 (interquartile range, 0 to 16) in the sarilumab group, and 0 (interquartile range, -1 to 15) in the control group. The median adjusted cumulative odds ratios were 1.64 (95% credible interval, 1.25 to 2.14) for tocilizumab and 1.76 (95% credible interval, 1.17 to 2.91) for sarilumab as compared with control, yielding posterior probabilities of superiority to control of more than 99.9% and of 99.5%, respectively. An analysis of 90-day survival showed improved survival in the pooled interleukin-6 receptor antagonist groups, yielding a hazard ratio for the comparison with the control group of 1.61 (95% credible interval, 1.25 to 2.08) and a posterior probability of superiority of more than 99.9%. All secondary analyses supported efficacy of these interleukin-6 receptor antagonists. CONCLUSIONS: In critically ill patients with Covid-19 receiving organ support in ICUs, treatment with the interleukin-6 receptor antagonists tocilizumab and sarilumab improved outcomes, including survival. (REMAP-CAP ClinicalTrials.gov number, NCT02735707.).

Interleukin-6 Receptor Antagonists in Critically Ill Patients with Covid-19.

Adaptive immunity to SARS-CoV-2 and COVID-19.

Iron-sulphur (Fe-S) clusters have long been recognized as essential and versatile cofactors of proteins involved in catalysis, electron transport and sensing of ambient conditions. Despite the relative simplicity of Fe-S clusters in terms of structure and composition, their synthesis and assembly into apoproteins is a highly complex and coordinated process in living cells. Different biogenesis machineries in both bacteria and eukaryotes have been discovered that assist Fe-S-protein maturation according to uniform biosynthetic principles. The importance of Fe-S proteins for life is documented by an increasing number of diseases linked to these components and their biogenesis.

Function and biogenesis of iron–sulphur proteins

Ferritins: a family of molecules for iron storage, antioxidation and more.

Iron and Neurodegeneration

causes an iron-deficient phenotype with mitochondrial iron overload RNA silencing of the mitochondrial ABCB7 transporter in HeLa cells

An HIV elite controller patient carrying the homozygous H63D variant in the homeostatic iron regulator gene: A case report.

As most new medications, Cabotegravir (CAB) was recently approved as an antiretroviral treatment of HIV infection without in-depth safety information on in utero exposure. Although no developmental toxicity in rats and rabbits was reported, recent studies demonstrated that CAB decreases pluripotency of human embryonic stem cells. CAB exposure effects during development were assessed in zebrafish embryos by the Fish Embryo Toxicity test after exposure at subtherapeutic concentrations up to 25× the human Cmax. Larvae behavior was assessed by the light–dark locomotion test. The expression of factors involved in neurogenesis was evaluated by whole-mount in situ hybridization. CAB did not cause gross morphological defects at low doses, although pericardial edema, uninflated swim bladder, decreased heartbeats, growth delay, and decreased hatching rate were observed at the highest concentrations. Decreased locomotion was observed even at the subtherapeutic dose, suggesting alterations of nervous system integrity. This hypothesis was supported by the observation of decreased expression of crucial factors involved in early neuronal differentiation in diencephalic and telencephalic dopaminergic areas, midbrain/hindbrain boundary, and craniofacial ganglia. These findings support CAB effects on neurogenesis in zebrafish embryos and suggest long-term follow-up of exposed infants to provide data on drug safety during pregnancy.

/pdf/cabotegravir-exposure-of-zebrafish-danio-rerio-embryos-1o2dqyac.pdf

Cabotegravir Exposure of Zebrafish (Danio rerio) Embryos Impacts on Neurodevelopment and Behavior

Cognitive decline of aging is modulated by chronic inflammation and comorbidities. In people with HIV-infection (PWH) it may also be affected by HIV-induced inflammation, lifestyle and long-term effects of antiretroviral therapies (ART). The role of genetics in the susceptibility to HIV-associated neurocognitive disorders (HAND) is not fully understood. Here we explored the possible relations among variants in 3 genes involved in inflammation and neurodegenerative disorders (APOE: ε2/ε3/ε4; HFE: H63D; C9ORF72: hexanucleotide expansions ≥ 9 repeats), cognitive/functional impairment (MiniMental State Examination MMSE, Clock Drawing Test CDT, Short Physical Performance Battery SPPB), comorbidities and HIV-related variables in a cohort of > 50 years old PWH (n = 60) with at least 10 years efficient ART. Patients with diabetes or hypertension showed significantly lower MMSE (p = .031) or SPPB (p = .010) scores, respectively, while no relations between HIV-related variables and cognitive/functional scores were observed. Patients with at least one APOEε3 allele had higher CDT scores (p = .019), APOEε2/ε4 patients showing the lowest scores in all tests. Patients with HFE-H63D variant showed more frequently hypertriglyceridemia (p = .023) and those harboring C9ORF72 expansions > 9 repeats had higher CD4+-cell counts (p = .032) and CD4% (p = .041). Multiple linear regression analysis computed to verify possible associations among cognitive/functional scores and all variables further suggested positive association between higher CDT scores and the presence of at least one APOEε3 allele (2,2; 95% CI [0,03 0,8]; p = .037), independent of other variables, although the model did not reach the statistical significance (p = .14). These data suggest that in PWH on efficient ART cognitive abilities and physical performances may be partly associated with comorbidities and genetic background. However, further analyses are needed to establish whether they could be also dependent and influenced by comorbidities and genetic background. 

https://link.springer.com/content/pdf/10.1007/s11011-022-00975-w.pdf

An exploratory pilot study on the involvement of APOE, HFE, C9ORF72 variants and comorbidities in neurocognitive and physical performance in a group of HIV-infected people

Isabella Zanella

Papers

Iron and Neurodegeneration

causes an iron-deficient phenotype with mitochondrial iron overload RNA silencing of the mitochondrial ABCB7 transporter in HeLa cells

An HIV elite controller patient carrying the homozygous H63D variant in the homeostatic iron regulator gene: A case report.

Cabotegravir Exposure of Zebrafish (Danio rerio) Embryos Impacts on Neurodevelopment and Behavior

An exploratory pilot study on the involvement of APOE, HFE, C9ORF72 variants and comorbidities in neurocognitive and physical performance in a group of HIV-infected people