Previous COVID-19 Infection and Antibody Levels After Vaccination.

Background: The emergence of new COVID-19 variants of concern coupled with a global inequity in vaccine access and distribution has prompted many public health authorities to circumvent the vaccine shortages by altering vaccination protocols and prioritizing persons at high risk. Individuals with previous COVID-19 infection may not have been prioritized due to existing humoral immunity. Objective: We aimed to study the association between previous COVID-19 infection and antibody levels after COVID-19 vaccination. Methods: A serological analysis to measure SARS-CoV-2 immunoglobulin (Ig)G, IgA, and neutralizing antibodies was performed on individuals who received one or two doses of either BNT162b2 or ChAdOx1 vaccines in Kuwait. A Student t-test was performed and followed by generalized linear regression models adjusted for individual characteristics and comorbidities were fitted to compare the average levels of IgG and neutralizing antibodies between vaccinated individuals with and without previous COVID-19 infection. Results: A total of 1,025 individuals were recruited. The mean levels of IgG, IgA, and neutralizing antibodies were higher in vaccinated subjects with previous COVID-19 infections than in those without previous infection. Regression analysis showed a steeper slope of decline for IgG and neutralizing antibodies in vaccinated individuals without previous COVID-19 infection compared to those with previous COVID-19 infection. Conclusion: Previous COVID-19 infection appeared to elicit robust and sustained levels of SARS-CoV-2 antibodies in vaccinated individuals. Given the inconsistent supply of COVID-19 vaccines in many countries due to inequities in global distribution, our results suggest that even greater efforts should be made to vaccinate more people, especially individuals without previous COVID-19 infection.

Correlation between heat shock proteins, adiponectin, and T lymphocyte cytokine expression in type 2 diabetics.

Type 2 diabetes mellitus (T2DM) features insulin resistance, hyperglycemia, dyslipidemia, overproduction of inflammatory cytokines, and systemic oxidative stress. Here, heat shock proteins Hsp70 and Hsp 90, adiponectin, and heme oxygenase-1 (HO-1, Hsp32) are profiled in peripheral blood mononuclear cells (PBMC) and serum from 25 T2DM patients and 25 healthy control subjects. Cells cultured with phorbol 12-myristate 13-acetate/ionomycin were evaluated by three-color flow cytometry for immunophenotypic biomarkers. Plasma HO-1, Hsp, and adiponectin levels were assayed by enzyme-linked immunosorbent assay (ELISA). Relative to healthy controls, T2DM patients exhibited significantly elevated plasma Hsp70, and representation of T helper immunophenotypes activated to express inflammatory cytokines, including CD4+ IFN-γ+, CD4+ TNF-α+, CD4+ IL-6+, CD4+ IL-1ß+ T cells, significantly lower representation of CD4+ IL-10+ T cells, plasma adiponectin and cell-associated HO-1 expression-with no significant differences in plasma Hsp90 between T2DM and healthy controls. Plasma HO-1 and adiponectin in T2DM patients inversely correlated with TNF-α and showed inverse correlation between serum LDL and plasma HO-1. Moreover, TNF-α and Hsp90 in T2DM patients correlated positively with fasting blood glucose (FBG). These results demonstrate correlation between potentially pathogenic T cells, HO-1, and adiponectin, additionally revealing a T helper (Th)1-related character of T2DM immunopathogenesis, suggesting potential for novel T cell-related management strategies for T2DM and related co-morbidities.

Molecular epidemiology and characterization of multiple drug-resistant (MDR) clinical isolates of Acinetobacter baumannii.

OBJECTIVES: The aim of this study was to identify the genetic relatedness of multiple drug-resistant (MDR) Acinetobacter baumannii clinical isolates recovered from a hospital in Los Angeles. METHODS: Twenty-one MDR A. baumannii isolates were collected and their antibiotic susceptibilities determined according to Clinical and Laboratory Standards Institute guidelines. Genes coding for antibiotic resistance were identified by PCR, and their identities were confirmed by DNA sequencing. Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: MDR consistently correlated with the presence of oxacillinases, mostly in the form of the plasmid-mediated OXA-23 enzyme, which was detected in 12 (57.1%) isolates. GES-type carbapenemases were found in 20 (95.2%) strains, AAC in all 21 (100%) strains, and PER in seven (33.3%) strains, and ISAba1 was detected in 16 (76.2%) isolates. The association between ISAba1 and resistance genes confirms insertion elements as a source of ß-lactamase production. Of the 21 clinical isolates, five were found to be related to sequence type 1 (ST1) and 16 to ST2, as analyzed by MLST. PFGE demonstrated that the majority of clinical isolates were highly related (>85%). CONCLUSIONS: This study supports a more complete understanding of genotyping of antibiotic resistance for better assessment of MDR strain transmission.

The emergence of plasmid mediated quinolone resistance qnrA2 in extended spectrum ß-lactamase producing Klebsiella pneumoniae in the Middle East.

BACKGROUND: Klebsiella pneumoniae is one of the most important opportunistic pathogens causing serious complications in patients in hospitals and community. The clinical significance of K. pneumoniae is mainly due to its ability to acquire multiple antibiotic resistance genes. In this study we report the findings of a survey of plasmid mediated quinolone resistance in Extended-Spectrum ß-lactamase (ESBL)-producing K. pneumoniae in Kuwait. METHODS: Clinical samples were collected from the microbiology laboratories of three major hospitals. Isolates were confirmed as ESBL-producers by disc diffusion method and PCR for the presence of bla genes. Antimicrobial susceptibility testing and genetic analysis were performed to detect the presence of a number of genes conferring resistance to ß-lactam and fluoroquinolone antimicrobial agents including bla SHV, bla TEM, aac (6')-Ib-cr, qnrA, qnrB and qnrS. Pulsed-field gel electrophoresis (PFGE) was used for typing the isolates. RESULTS: In total 173 ESBL-producing K. pneumoniae were detected. qnr genes were identified in 27 (15.6 %) isolates and aac(6')-Ib Ib-cr gene in 26 (96 %). One (3.7 %) contained qnrA2, 21 harbored qnrB1 (78 %) and 5 (18.5 %) contained qnrS. Twenty one (78 %) isolates contained all three bla genes. PFGE showed diverse profiles. CONCLUSION: We identified for the first time the emergence of the mobile fluoroquinolone resistance qnrA2 in a clinical isolate in the middle east and also showed the dissemination of aac (6')-Ib-cr, qnrB, and qnrS genes among ESBL-producing K. pneumoniae in Kuwait. The abundance of plasmid mediated resistance to fluoroquinolones among ESBL-producing K. pneumoniae is alarming as it facilitates therapy failure. Preventing the spread of these isolates is crucial if we are to sustain the effectiveness of the limited choices we have left in antimicrobial therapy.

Klebsiella oxytoca with reduced sensitivity to chlorhexidine isolated from a diabetic foot ulcer.

In most hospitals, chlorhexidine is used as skin antiseptic prior to clinical procedures, in dressings and when bathing patients. We hereby report, for the first time, the isolation of a clinical Klebsiella oxytoca isolate with reduced sensitivity to chlorhexidine from a foot ulcer of a diabetic patient, which is a common and serious complication associated with diabetes. The Minimum Inhibitory Concentration of the K. oxytoca isolate to chlorhexidine was found to be 30 mg/L and the Minimum Bactericidal Concentration was 60 mg/L. An increased resistance to ethidium bromide (MIC 200 mg/ L) was also observed. Molecular tests revealed that the isolate contained blaCTXM15, blaT(EM-1) and bla(SHV). The other resistant genes detected were qnrB1 and aac(6')-Ib-cr. The resistant determinants were located on a class I integron integrase (intI1) containing qacE gene. DNA sequencing showed homology to K. oxytoca plasmid pACM1. Identification of K. oxytoca with reduced sensitivity to chlorhexidine raises concern regarding dilution standards in hospitals. Adherence to the hospitals' infection control policies should be strictly monitored to avoid continuous low level exposure of bacteria to biocides, specifically in developing countries.

The characterization and antibiotic resistance profiles of clinical Escherichia coli O25b-B2-ST131 isolates in Kuwait.

BACKGROUND: Escherichia coli O25b-B2-ST131 are considered virulent extra-intestinal pathogens causing serious clinical complications such as urinary tract infection and bacteraemia. Our main objectives in this study were to characterise the multi-drug resistant (MDR) isolates of this lineage in Kuwait, and to demonstrate whether reduced susceptibility is spread clonally. RESULTS: A subset of 83 (10%) non-duplicate and non-selective E. coli O25b-B2-ST131 out of 832 MDR E. coli was identified and collected. Minimum inhibitory concentrations of the isolates were determined and pulsed-field gel electrophoresis was used for typing.The majority (95.2%) of the 83 E. coli O25b-B2-ST131 harboured at least one bla gene with blaCTX-M-15 being the most prevalent. blaCTX-M-2 was present in one isolate. Also one isolate harboured blaCTX-M-56, qnrB1 and blaCMY-2 genes and carried IncF1 plasmids of about 97 kb and160 kb. qnrB and qnrS were found in 8 other blaCTX-M-15 containing isolates. The blaNDM, blaIMP, blaVIM and qnrA were not detected, however, the blaOXA-48 was present in two (2.4%). CONCLUSIONS: The majority of isolates harbouring qnr genes demonstrated relatedness (≥85%) by PFGE. However, the diversity in PFGE profiles for the other MDR isolates reflected the changes in population genetics of E. coli O25b-B2-ST131. We identified for the first time the appearance of blaCTX-M-2 in the Middle East and blaCTX-M-56 outside the Latin American countries. The isolate harbouring blaCTX-M-56 also contained qnrB1 and blaCMY-2 genes and carried IncF1 plasmids. The appearance of a highly virulent E. coli O25b-ST131 that is resistant to penicillins, most cephalosproins, ß-lactamase inhibitors as well as fluoroquinolones is a cause for concern.

Internalization by HeLa cells of latex beads coated with mammalian cell entry (Mce) proteins encoded by the mce3 operon of Mycobacterium tuberculosis.

The mammalian cell entry (Mce) operon 3 (mce3) is one of four homologous mce operons of Mycobacterium tuberculosis, encoding six (Mce3A-F) invasin-like membrane-associated proteins. Previous studies have shown that recombinant expression of Mce1A encoded by the mce1 operon in Escherichia coli allows this non-pathogenic bacterium to invade and survive inside macrophages, and latex beads coated with Mce1A are internalized by non-phagocytic HeLa cells. However, the role of other mce1 operon proteins (Mce1B-F) and proteins encoded by the operons mce2-4 in facilitating the internalization of M. tuberculosis in mammalian cells has not been studied. This study was carried out to determine whether Mce proteins encoded by the mce3 operon also facilitated the internalization of latex beads by HeLa cells. Recombinant pure Mce3A and lipoprotein LprM (Mce3E) were expressed and purified from E. coli cells. Mce1A expressed as a fusion protein with glutathione S-transferase (GST-Mce1A) and GST alone, purified similarly from E. coli cells, were used as control proteins. Fluorescent latex beads coated with purified proteins were used to study their uptake by HeLa cells using fluorescence microscopy, flow cytometry and electron microscopy. Fluorescence microscopy and flow cytometry showed an association of HeLa cells with beads coated with both Mce3A and LprM, whilst GST-Mce1A and GST yielded the expected results. Transmission electron microscopy confirmed the uptake of beads coated with Mce3A or LprM by HeLa cells. The data showed that Mce3A encoded by the mce3 operon facilitated the uptake and internalization of latex beads by HeLa cells. The data also showed, for the first time, the role of another Mce protein (LprM/Mce3E) in facilitating the interaction and internalization of M. tuberculosis by mammalian cells.

Increased expression of pro-inflammatory cytokines in placentas of women undergoing spontaneous preterm delivery or premature rupture of membranes.

PROBLEM: The objective of this study was to determine the levels of cytokines in the placentas of women undergoing preterm delivery (PTD) or premature rupture of membranes (PROM) as compared with women undergoing normal delivery at term. METHOD OF STUDY: Placentas were obtained from 30 subjects with spontaneous PTD, 30 women with PROM and 30 women with a history of normal delivery at term. Levels of interleukin (IL)-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta, IL-4, IL-5, IL-6 and IL-10 and IL-12 were estimated by ELISA in detergent lysates of placentas from the subjects. RESULTS: We found significantly increased levels of the Th1 cytokines IL-2 and IFN gamma and of the Th1-inducing cytokine IL-12 in placentas from the PTD and PROM groups as compared with those delivering at term. In contrast, the levels of the Th2 cytokines IL-4, IL-6 and IL-10 were significantly higher in placentas from term pregnancy. CONCLUSIONS: These data support our observation of a pro-inflammatory cytokine bias in women with PTD and PROM.