Performance evaluation of five ELISA kits for detecting anti-SARS-COV-2 IgG antibodies

Objectives: To evaluate and compare the performances of five commercial ELISA assays (EDI, AnshLabs, Dia.Pro, NovaTec, and Lionex) for detecting anti-SARS-CoV-2 IgG. / Methods: Seventy negative control samples (collected before the COVID-19 pandemic) and samples from 101 RT-PCR-confirmed SARS-CoV-2 patients (collected at different time points from symptom onset: ≤7, 8–14 and >14 days) were used to compare the sensitivity, specificity, agreement, and positive and negative predictive values of each assay with RT-PCR. A concordance assessment between the five assays was also conducted. Cross-reactivity with other HCoV, non-HCoV respiratory viruses, non-respiratory viruses, and nuclear antigens was investigated. / Results: Lionex showed the highest specificity (98.6%; 95% CI 92.3–99.8), followed by EDI and Dia.Pro (97.1%; 95% CI 90.2–99.2), NovaTec (85.7%; 95% CI 75.7–92.1), then AnshLabs (75.7%; 95% CI 64.5–84.2). All ELISA kits cross-reacted with one anti-MERS IgG-positive sample, except Lionex. The sensitivity was low during the early stages of the disease but improved over time. After 14 days from symptom onset, Lionex and NovaTec showed the highest sensitivity at 87.9% (95% CI 72.7–95.2) and 86.4% (95% CI 78.5–91.7), respectively. The agreement with RT-PCR results based on Cohen's kappa was as follows: Lionex (0.89) > NovaTec (0.70) > Dia.Pro (0.69) > AnshLabs (0.63) > EDI (0.55). / Conclusion: The Lionex and NovaLisa IgG ELISA kits, demonstrated the best overall performance

IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen's elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction

Rab6 and Rab11 Regulate Chlamydia trachomatis Development and Golgin-84-Dependent Golgi Fragmentation

Many intracellular pathogens that replicate in special membrane bound compartments exploit cellular trafficking pathways by targeting small GTPases, including Rab proteins. Members of the Chlamydiaceae recruit a subset of Rab proteins to their inclusions, but the significance of these interactions is uncertain. Using RNA interference, we identified Rab6 and Rab11 as important regulators of Chlamydia infections. Depletion of either Rab6 or Rab11, but not the other Rab proteins tested, decreased the formation of infectious particles. We further examined the interplay between these Rab proteins and the Golgi matrix components golgin-84 and p115 with regard to Chlamydia-induced Golgi fragmentation. Silencing of the Rab proteins blocked Chlamydia-induced and golgin-84 knockdown-stimulated Golgi disruption, whereas Golgi fragmentation was unaffected in p115 depleted cells. Interestingly, p115-induced Golgi fragmentation could rescue Chlamydia propagation in Rab6 and Rab11 knockdown cells. Furthermore, transport of nutrients to Chlamydia, as monitored by BODIPY-Ceramide, was inhibited by Rab6 and Rab11 knockdown. Taken together, our results demonstrate that Rab6 and Rab11 are key regulators of Golgi stability and further support the notion that Chlamydia subverts Golgi structure to enhance its intracellular development

Haptoglobin and Sickle Cell Polymorphisms and Risk of Active Trachoma in Gambian Children

BACKGROUND: Susceptibility and resistance to trachoma, the leading infectious cause of blindness, have been associated with a range of host genetic factors. In vitro studies of the causative organism, Chlamydia trachomatis, demonstrate that iron availability regulates its growth, suggesting that host genes involved in regulating iron status and/or availability may modulate the risk of trachoma. The objective was to investigate whether haptoglobin (Hp) haplotypes constructed from the functional polymorphism (Hp1/Hp2) plus the functional promoter SNPs -61A-C (rs5471) and -101C-G (rs5470), or sickle cell trait (HbAS, rs334) were associated with risk of active trachoma when stratified by age and sex, in rural Gambian children. METHODOLOGY AND PRINCIPAL FINDINGS: In two cross sectional surveys of children aged 6-78 months (n = 836), the prevalence of the clinical signs of active trachoma was 21.4%. Within boys, haplotype E (-101G, -61A, Hp1), containing the variant allele of the -101C-G promoter SNP, was associated with a two-fold increased risk of active trachoma (OR = 2.0 [1.17-3.44]). Within girls, an opposite association was non-significant (OR = 0.58 [0.32-1.04]; P = 0.07) and the interaction by sex was statistically significant (P = 0.001). There was no association between trachoma and HbAS. CONCLUSIONS: These data indicate that genetic variation in Hp may affect susceptibility to active trachoma differentially by sex in The Gambia

[18F]2-Fluoro-2-deoxy-D-glucose incorporation by AGS gastric adenocarcinoma cells in vitro during response to epirubicin, cisplatin and 5-fluorouracil

Decreased tumour [18F]2-fluoro-2-deoxy-D-glucose (18FDG) incorporation is related to response however its significance at the cell level in gastro-oesophageal cancer and how it relates to cell death is unknown. Here human gastric adenocarcinoma (AGS) cells were treated with lethal dose 10 and 50 (LD10 and LD50), determined by using the MTT assay, of the three drugs, epirubicin, 5-fluorouracil and cisplatin, commonly used in the treatment of patients with gastro-oesophageal cancer. 18FDG incorporation was determined after 48 and 72 h of treatment with each drug and related to drug-induced changes in glucose transport, hexokinase activity, cell cycle distribution and annexin V-PE binding (a measure of apoptosis). Treatment of cells for 48 and 72 h with LD50 doses of cisplatin resulted in reductions in 18FDG incorporation of 27 and 25% respectively and of 5-fluorouracil reduced 18FDG incorporation by 34 and 33% respectively: epirubicin treatment reduced incorporation by 30 and 69% respectively. Cells that had been treated for 72 h with each drug were incubated in drug-free media for a further 6 days to determine their ability to recover. Comparison of the ability to recover from the chemotherapy agent, with 18FDG incorporation before the recovery period allowed an assessment of the predictive ability of 18FDG incorporation. Cells treated with either 5-fluorouracil or cisplatin demonstrated recovery on removal of the drug. In contrast, cells treated with epirubicin did not recover corresponding with the greatest 72 h treatment decrease in 18FDG incorporation. In contrast to adherent cells treated with cisplatin or 5-fluorouracil, adherent epirubicin-treated cells also exhibited very high levels of apoptosis. Glucose transport was decreased after each treatment whilst hexokinase activity was only decreased after 72 h of treatment with each drug. There was no consistent relationship observed between 18FDG incorporation and cell cycle distribution. Our results show that at the tumour cell level in gastric tumour cells, decreased 18FDG incorporation and glucose transport, accompanies therapeutic growth inhibition. 18FDG incorporation is particularly diminished in cells exhibiting apoptosis

Chlamydia species-dependent differences in the growth requirement for lysosomes

Genome reduction is a hallmark of obligate intracellular pathogens such as Chlamydia, where adaptation to intracellular growth has resulted in the elimination of genes encoding biosynthetic enzymes. Accordingly, chlamydiae rely heavily on the host cell for nutrients yet their specific source is unclear. Interestingly, chlamydiae grow within a pathogen-defined vacuole that is in close apposition to lysosomes. Metabolically-labeled uninfected host cell proteins were provided as an exogenous nutrient source to chlamydiae-infected cells, and uptake and subsequent labeling of chlamydiae suggested lysosomal degradation as a source of amino acids for the pathogen. Indeed, Bafilomycin A1 (BafA1), an inhibitor of the vacuolar H(+)/ATPase that blocks lysosomal acidification and functions, impairs the growth of C. trachomatis and C. pneumoniae, and these effects are especially profound in C. pneumoniae. BafA1 induced the marked accumulation of material within the lysosomal lumen, which was due to the inhibition of proteolytic activities, and this response inhibits chlamydiae rather than changes in lysosomal acidification per se, as cathepsin inhibitors also inhibit the growth of chlamydiae. Finally, the addition of cycloheximide, an inhibitor of eukaryotic protein synthesis, compromises the ability of lysosomal inhibitors to block chlamydial growth, suggesting chlamydiae directly access free amino acids in the host cytosol as a preferred source of these nutrients. Thus, chlamydiae co-opt the functions of lysosomes to acquire essential amino acids