Mycoplasma DnaK increases DNA copy number variants in vivo.

The human microbiota affects critical cellular functions, although the responsible mechanism(s) is still poorly understood. In this regard, we previously showed that Mycoplasma fermentans DnaK, an HSP70 chaperone protein, hampers the activity of important cellular proteins responsible for DNA integrity. Here, we describe a novel DnaK knock-in mouse model generated in our laboratory to study the effect of M. fermentans DnaK expression in vivo. By using an array-based comparative genomic hybridization assay, we demonstrate that exposure to DnaK was associated with a higher number of DNA copy number variants (CNVs) indicative of unbalanced chromosomal alterations, together with reduced fertility and a high rate of fetal abnormalities. Consistent with their implication in genetic disorders, one of these CNVs caused a homozygous Grid2 deletion, resulting in an aberrant ataxic phenotype that recapitulates the extensive biallelic deletion in the Grid2 gene classified in humans as autosomal recessive spinocerebellar ataxia 18. Our data highlight a connection between components of the human urogenital tract microbiota, namely Mycoplasmas, and genetic abnormalities in the form of DNA CNVs, with obvious relevant medical, diagnostic, and therapeutic implications.

Mucosal and Systemic Responses to Severe Acute Respiratory Syndrome Coronavirus 2 Vaccination Determined by Severity of Primary Infection.

With much of the world infected with or vaccinated against severe acute respiratory syndrome coronavirus 2 (commonly abbreviated SARS-CoV-2; abbreviated here SARS2), understanding the immune responses to the SARS2 spike (S) protein in different situations is crucial to controlling the pandemic. We studied the clinical, systemic, mucosal, and cellular responses to two doses of SARS2 mRNA vaccines in 62 individuals with and without prior SARS2 infection that were divided into three groups based on antibody serostatus prior to vaccination and/or degree of disease symptoms among those with prior SARS2 infection: antibody negative (naive), low symptomatic, and symptomatic. Antibody negative were subjects who were antibody negative (i.e., those with no prior infection). Low symptomatic subjects were those who were antibody negative and had minimal or no symptoms at time of SARS2 infection. Symptomatic subjects were those who were antibody positive and symptomatic at time of SARS2 infection. All three groups were then studied when they received their SARS2 mRNA vaccines. In the previously SARS2-infected (based on antibody test) low symptomatic and symptomatic groups, reactogenic symptoms related to a recall response were elicited after the first vaccination. Anti-S trimer IgA and IgG titers, and neutralizing antibody titers, peaked after the 1st vaccination in the previously SARS2-infected groups and were significantly higher than for the SARS2 antibody-negative group in the plasma and nasal samples at most time points. Nasal and plasma IgA antibody responses were significantly higher in the low symptomatic group than in the symptomatic group at most time points. After the first vaccination, differences in cellular immunity were not evident between groups, but the activation-induced cell marker (AIM+) CD4+ cell response correlated with durability of IgG humoral immunity against the SARS2 S protein. In those SARS2-infected subjects, severity of infection dictated plasma and nasal IgA responses in primary infection as well as response to vaccination (peak responses and durability), which could have implications for continued protection against reinfection. Lingering differences between the SARS2-infected and SARS2-naive up to 10 months postvaccination could explain the decreased reinfection rates in the SARS2-infected vaccinees recently reported and suggests that additional strategies (such as boosting of the SARS2-naive vaccinees) are needed to narrow the differences observed between these groups. IMPORTANCE This study on SARS2 vaccination in those with and without previous exposure to the virus demonstrates that severity of infection dictates IgA responses in primary infection as well as response to vaccination (peak responses and durability), which could have implications for continued protection against reinfection.

Performance of nucleocapsid and spike-based SARS-CoV-2 serologic assays.

There is an urgent need for an accurate antibody test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have developed 3 ELISA methods, trimer spike IgA, trimer spike IgG, and nucleocapsid IgG, for detecting anti-SARS-CoV-2 antibodies. We evaluated their performance along with four commercial ELISAs, EDI™ Novel Coronavirus COVID-19 ELISA IgG and IgM, Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA, and one lateral flow assay, DPP® COVID-19 IgM/IgG System (Chembio). Both sensitivity and specificity were evaluated and the probable causes of false-positive reactions were determined. The assays were evaluated using 300 pre-epidemic samples and 100 PCR-confirmed COVID-19 samples. The sensitivities and specificities of the assays were as follows: 90%/100% (in-house trimer spike IgA), 90%/99.3% (in-house trimer spike IgG), 89%/98.3% (in-house nucleocapsid IgG), 73.7%/100% (EDI nucleocapsid IgM), 84.5%/95.1% (EDI nucleocapsid IgG), 95%/93.7% (Euroimmun S1 IgA), 82.8%/99.7% (Euroimmun S1 IgG), 82.0%/91.7% (Chembio nucleocapsid IgM), 92%/93.3% (Chembio nucleocapsid IgG). The presumed causes of false positive results from pre-epidemic samples in commercial and in-house assays were mixed. In some cases, assays lacked reproducibility. In other cases, reactivity was abrogated by competitive inhibition (spiking the sample with the same antigen that was used for coating ELISAs prior to performing the assay), suggesting positive reaction could be attributed to the presence of antibodies against these antigens. In other cases, reactivity was consistently detected but not abrogated by the spiking, suggesting positive reaction was not attributed to the presence of antibodies against these antigens. Overall, there was wide variability in assay performance using our samples, with in-house tests exhibiting the highest combined sensitivity and specificity. The causes of "false positivity" in pre-epidemic samples may be due to plasma antibodies apparently reacting with the corresponding antigen, or spurious reactivity may be directed against non-specific components in the assay system. Identification of these targets will be essential to improving assay performance.

The silent presence of Mycoplasma hominis in patients with prostate cancer.

BACKGROUND: Mycoplasma hominis, an opportunistic pathogen in human genitourinary tract, can cause chronic infection in the prostate. Intracellular survival of M. hominis leads to a prolonged presence in the host cells that can affect the cell's biological cycle. In the present study, we aimed to evaluate the frequency of M. hominis DNA in prostate tissue of Iranian patients with prostate cancer (PCa) in comparison to a control group with benign prostatic hyperplasia (BPH). METHODS: This research was a retrospective case-control study using 61 archived formalin-fixed paraffin-embedded (FFPE) blocks of prostate tissue from patients with PCa and 70 FFPE blocks of patients with BPH. Real-time PCR, targeting two different genes, 16S rRNA and yidC, in the M. hominis genome was performed for all specimens. RESULTS: Out of 61 blocks of prostate biopsy from patients with PCa, eight samples (13%) were positive for M. hominis, while the bacterium was not detected in any of the 70 blocks of patients with BPH (P value, 0.002). CONCLUSIONS: The high frequency of M. hominis in patients with PCa likely shows a hidden role of the organism in prostate cancer during its chronic, apparently silent and asymptomatic colonization in prostate.

Specificity and Performance of Nucleocapsid and Spike-based SARS-CoV-2 Serologic Assays.

There is an urgent need for an accurate antibody test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we have developed 3 ELISA methods, trimer spike IgA, trimer spike IgG, and nucleocapsid IgG, for detecting anti-SARS-CoV-2 antibodies. We evaluated their performance in comparison with four commercial ELISAs, EDI™ Novel Coronavirus COVID-19 ELISA IgG and IgM, Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA, and one lateral flow assay, DPP® COVID-19 IgM/IgG System (Chembio). Both sensitivity and specificity were evaluated and the causes of false-positive reactions were determined. The assays were compared using 300 pre-epidemic samples and 100 PCR-confirmed COVID-19 samples. The sensitivities and specificities of the assays were as follows: 90%/100% (in-house trimer spike IgA), 90%/99.3% (in-house trimer spike IgG), 89%/98.3% (in-house nucleocapsid IgG), 73.7%/100% (EDI nucleocapsid IgM), 84.5%/95.1% (EDI nucleocapsid IgG), 95%/93.7% (Euroimmun S1 IgA), 82.8%/99.7% (Euroimmun S1 IgG), 82.0%/91.7% (Chembio nucleocapsid IgM), 92%/93.3% (Chembio nucleocapsid IgG). The presumed causes of positive signals from pre-epidemic samples in commercial and in-house assays were mixed. In some cases, positivity varied with assay repetition. In other cases, reactivity was abrogated by competitive inhibition (spiking the sample with analyte prior to performing the assay). In other cases, reactivity was consistently detected but not abrogated by analyte spiking. Overall, there was wide variability in assay performance using our samples, with in-house tests exhibiting the highest combined sensitivity and specificity. The causes of "false positivity" in pre-epidemic samples may be due to plasma antibodies apparently reacting with the analyte, or spurious reactivity may be directed against non-specific components in the assay system. Identification of these targets will be essential to improving assay performance.

Production of a chimeric protein and its potential application in sero-diagnosis of Mycoplasma hominis infection.

INTRODUCTION: Mycoplasma hominis is an opportunistic pathogen of the human genital tract. Detection of antibodies against this organism in human serum or plasma is theoretically unreliable because of high variation in bacterial surface antigens. In this study, we applied the bioinformatics tools to design a chimeric protein constructed of specific, conserved and predicted immuno-dominant epitopes from two different membrane proteins, P120 and P80. MATERIAL AND METHODS: Linear B-cell epitopes of P120 and P80 were predicted and evaluated by bioinformatics tools and the designed chimeric protein was expressed in Escherichia coli. The chimeric protein, Mh128, was further analyzed in terms of immuno-reactivity by western blotting and enzyme immuno-sorbent assay (ELISA). RESULTS: We found eight specific, conserved and immuno-dominant epitopes within P120 and P80 based on the bioinformatic studies. The constructed chimeric protein showed immuno-reaction in both western-blotting and ELISA tests. DISCUSSION: Because of extensive variation of genomic and antigenic structure, diagnosis of M. hominis infection is difficult. Mh128 as a predicted specific and conserved recombinant protein can be potentially used for sero-diagnosis of M. hominis infection. We plan to develop an immuno-assay based on Mh128 and further evaluate the clinical specificity and sensitivity of the method.

Long-term follow-up of elite controllers: Higher risk of complications with HCV coinfection, no association with HIV disease progression.

To estimate the effect of hepatitis C virus (HCV) coinfection on the development of complications and progression of human immunodeficiency virus (HIV) disease among HIV-infected elite controllers.Single-center retrospective cohort. Kaplan-Meier methods, prevalence ratios, and Cox proportional-hazards models were used.In all, 55 HIV-infected elite controllers were included in this study. Among them, 45% were HIV/HCV coinfected and 55% were HIV mono-infected. Median follow-up time for the cohort was 11 years. Twenty-five patients experienced a complication and 16 lost elite controller status during the study period. HCV coinfected patients were 4.78 times (95% confidence interval 1.50-15.28) more likely to develop complications compared with HIV mono-infected patients. There was no association between HCV coinfection status and loss of elite control (hazard ratio 0.75, 95% confidence interval 0.27-2.06).Hepatitis C virus coinfection was significantly associated with the risk of complications even after controlling for sex, injecting drug use, and older age. HCV coinfected patients had higher levels of cellular activation while also having similar levels of lipopolysaccharide and soluble CD14. HCV coinfection was not associated with loss of elite controller status. Taken together, this suggests that HCV coinfection does not directly affect HIV replication dynamics or natural history, but that it may act synergistically with HIV to produce a greater number of associated complications. Continued follow-up will be needed to determine whether HCV cure through the use of direct-acting antivirals among HIV/HCV coinfected elite controllers will make the risk for complications among these patients similar to their HIV mono-infected counterparts.