DNA Load Research Articles

A proof-of-concept study to investigate the efficacy of heat-inactivated autovaccines in Mycobacterium caprae experimentally challenged goats

This study aimed to assess the efficacy of a heat-inactivated Mycobacterium caprae (HIMC) vaccine in goats experimentally challenged with the same strain of M. caprae. Twenty-one goats were divided into three groups of seven: vaccinated with heat-inactivated Mycobacterium bovis (HIMB), with HIMC and unvaccinated. At 7 weeks post-vaccination all animals were endobronchially challenged with M. caprae. Blood samples were collected for immunological assays and clinical signs were recorded throughout the experiment. All goats were euthanized at 9 weeks post-challenge. Gross pathological examination, analysis of lung pathology using computed tomography, and bacterial load quantification in pulmonary lymph nodes (LN) by qPCR were carried out. Only HIMC vaccinated goats showed a significant reduction of lung lesions volume and mycobacterial DNA load in LN compared to unvaccinated controls. Both vaccinated groups showed also a significant reduction of the other pathological parameters, an improved clinical outcome and a higher proportion of IFN-γ-producing central memory T cells after vaccination. The results indicated that homologous vaccination of goats with HIMC induced enhanced protection against M. caprae challenge by reducing lung pathology and bacterial load compared to the heterologous vaccine (HIMB). Further large-scale trials are necessary to assess the efficacy of autovaccines under field conditions.

Different local, innate and adaptive immune responses are induced by two commercial Mycoplasma hyopneumoniae bacterins and an adjuvant alone.

Enzootic pneumonia still causes major economic losses to the intensive pig production. Vaccination against its primary pathogen, Mycoplasma hyopneumoniae, is carried out worldwide to control the disease and minimize clinical signs and performance losses. Nonetheless, the effects of both infection with, and vaccination against Mycoplasma hyopneumoniae on the innate and adaptive immune responses remain largely unknown. Therefore, we conducted a study in which piglets were injected once with a commercial bacterin V1 or V2, or the adjuvant of V1 (A) to investigate their effect on local, innate and adaptive immune responses. Three weeks after vaccination, piglets were challenge infected with M. hyopneumoniae and euthanized four weeks later to assess vaccine efficacy via macroscopic and microscopic evaluation of lung lesions. Blood and broncho-alveolar lavage fluid (BAL) samples were collected to measure antibody responses, cellular immunity, BAL cytokine levels and BAL M. hyopneumoniae DNA load as well as cytokine secretion by monocytes. After vaccination, proliferation of antigen-specific CD3+ T cells and a higher percentage of TNF-α+ CD8+, and TNF-α+ and TNF-α+IFN-γ+ CD4+CD8+ T cells was seen in V1, while proliferation of or a significant increase in cytokine production by different T cell subsets could not be observed for animals from V2. Interestingly, LPS-stimulated blood monocytes from V1 and A secreted less IL-10 on D7. After challenge, higher levels of IgA, more IL-10 and less IL-1β was detected in BAL from V1, which was not observed in V2. Animals from A had significantly more IL-17A in BAL. The macroscopic lung lesion score and the M. hyopneumoniae DNA load at euthanasia was lower in V1, but the microscopic lung lesion score was lower in both vaccinated groups. In conclusion, these results indicate that the two commercial bacterins induced different local and adaptive immune responses, that the adjuvant alone can reduce anti-inflammatory innate immune responses, and that both vaccines had a different efficacy to reduce Mycoplasma-like lung lesions and M. hyopneumoniae DNA load in the lung.

Comparison of the protective efficacy between the candidate vaccine ZJ01R carrying gE/gI/TK deletion and three commercial vaccines against an emerging pseudorabies virus variant

Pseudorabies virus (PRV) is a swine alpha-herpesvirus that mainly causes reproductive disorders in sows and neurological diseases in piglets. Vaccination is the most efficient method to prevent the disease. In China, since the emergence of PRV mutant strains in late 2011, the traditional commercial vaccines have not been providing complete protection. Our previous studies have demonstrated that PRV ZJ01 is a highly virulent strain, and its derivative, ZJ01R, which carries the gE/gI/TK gene deletion, could provide protection against the variant PRV challenge. However, the difference in immune efficacy between ZJ01R and other commercial vaccines remains unclear. In this study, the immune protection efficacy between ZJ01R and three commercial PRV vaccines (Bartha-K61, HB2000, and SA215) was evaluated in piglets. The safety of ZJ01R was shown to be equivalent to that of the three commercial vaccines. The titers of the neutralizing antibodies against the PRV classical strain LA in the four vaccine groups were similar, while the anti-PRV variant neutralizing antibody titers in the ZJ01R group were significantly higher than those in the Bartha-K61, HB2000, and SA215 strain groups. After the PRV challenge, ZJ01R, HB2000, and SA215 vaccinations could provide complete protection, whereas the Bartha-K61 vaccination could only provide 60 % protection. Importantly, the rectal viral excretion and PRV DNA loads in the lung tissues in the ZJ01R group were significantly lower than those in the Bartha-K61, HB2000, and SA215 groups. Altogether, these results indicated that ZJ01R could provide higher protection efficacy against the PRV virulent ZJ01 challenge than the three commercial PRV gene-deleted live vaccines derived from the classical vaccine strains, providing the potential to develop a new PRV vaccine to control the epidemic PRV variant strains in the future.

Bovine Papillomavirus Type 1 or 2 Virion-Infected Primary Fibroblasts Constitute a Near-Natural Equine Sarcoid Model.

Equine sarcoids are common, locally aggressive skin tumors induced by bovine papillomavirus types 1, 2, and possibly 13 (BPV1, BPV2, BPV13). Current in vitro models do not mimic de novo infection. We established primary fibroblasts from horse skin and succeeded in infecting these cells with native BPV1 and BPV2 virions. Subsequent cell characterization was carried out by cell culture, immunological, and molecular biological techniques. Infection of fibroblasts with serial 10-fold virion dilutions (2 × 106-20 virions) uniformly led to DNA loads settling at around 150 copies/cell after four passages. Infected cells displayed typical features of equine sarcoid cells, including hyperproliferation, and loss of contact inhibition. Neither multiple passaging nor storage negatively affected cell hyperproliferation, viral DNA replication, and gene transcription, suggestive for infection-mediated cell immortalization. Intriguingly, extracellular vesicles released by BPV1-infected fibroblasts contained viral DNA that was most abundant in the fractions enriched for apoptotic bodies and exosomes. This viral DNA is likely taken up by non-infected fibroblasts. We conclude that equine primary fibroblasts stably infected with BPV1 and BPV2 virions constitute a valuable near-natural model for the study of yet unexplored mechanisms underlying the pathobiology of BPV1/2-induced sarcoids.

Monitoring monkeypox virus in saliva and air samples in Spain: a cross-sectional study

The transmission of monkeypox virus occurs through direct contact, but transmission through saliva or exhaled droplets and aerosols has not yet been investigated. We aimed to assess the presence of monkeypox virus DNA and infectious virus in saliva samples and droplets and aerosols exhaled from patients infected with monkeypox virus. We did a cross-sectional study in patients with monkeypox confirmed by PCR who attended two health centres in Madrid, Spain. For each patient, we collected samples of saliva, exhaled droplets within a mask, and aerosols captured by air filtration through newly developed nanofiber filters. We evaluated the presence of monkeypox virus in the samples by viral DNA detection by quantitative PCR (qPCR) and isolation of infectious viruses in cell cultures. Between May 18 and July 15, 2022, 44 patients with symptomatic monkeypox attended two health centres in Madrid and were included in the study. All were cisgender men, with a median age of 35·0 years (IQR 11·3). We identified high loads of monkeypox virus DNA by qPCR in 35 (85%) of 41 saliva samples. Infectious monkeypox virus was recovered from 22 (67%) of 33 saliva samples positive for monkeypox virus DNA. We also found a significant association between the number of affected cutaneous areas or general symptoms and the viral load present in saliva samples. Droplets exhaled from patients with monkeypox, detected inside a mask, contained monkeypox virus DNA in 32 (71%) of 45 samples, with two of the 32 positive samples showing the presence of the infectious virus. Monkeypox virus DNA in aerosols, collected from the medical consultation room, were detected in 27 (64%) of 42 samples, despite patients wearing an FFP2 mask during the visit. Infectious virus was not recovered from aerosol samples. High levels of monkeypox virus DNA were identified in aerosols collected from a hospital isolation room housing a patient with monkeypox. The identification of high viable monkeypox virus loads in saliva in most patients with monkeypox and the finding of monkeypox virus DNA in droplets and aerosols warrants further epidemiological studies to evaluate the potential relevance of the respiratory route of infection in the 2022 monkeypox virus outbreak. EU, Consejo Superior de Investigaciones Científicas, and Ciberinfec.

LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples.

Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21st century and was more recently documented in humans. Whole genome sequencing (WGS) of this parasite can provide insight into gene content, provide novel surveillance targets, and elucidate the origin and range expansion. However, past attempts of whole genome sequencing of other Onchocerca species reported a substantial portion of their data unusable due to the variable over-abundance of host DNA in samples. Here, we have developed a method to determine the host-to-parasite DNA ratio using a quantitative PCR (qPCR) approach that relies on two standard plasmids each of which contains a single copy gene specific to the parasite genus Onchocerca (major body wall myosin gene, myosin) or a single copy gene specific to the canine host (polycystin-1 precursor, pkd1). These plasmid standards were used to determine the copy number of the myosin and pkd1 genes within a sample to calculate the ratio of parasite and host DNA. Furthermore, whole genome sequence (WGS) data for three O. lupi isolates were consistent with our host-to-parasite DNA ratio results. Our study demonstrates, despite unified DNA extraction methods, variable quantities of host DNA within any one sample which will likely affect downstream WGS applications. Our quantification assay of host-to-parasite genome copy number provides a robust and accurate method of assessing canine host DNA load in an O. lupi specimen that will allow informed sample selection for WGS. This study has also provided the first whole genome draft sequence for this species. This approach is also useful for future focused WGS studies of other parasites.

Functional molecular expression of nature killer cells correlated to HBsAg clearance in HBeAg-positive chronic hepatitis B patients during PEG-IFN α-2a therapy.

To explore whether the frequencies and functional molecules expression of Natural Killer cells (NK cells) are related to hepatitis B surface antigen (HBsAg) disappearance in hepatitis B e envelope antigen (HBeAg)-positive patients with chronic hepatitis B (CHB) throughout peginterferon alpha-2a (PEG-IFN α-2a) treatment. In this prospective research, HBeAg-positive patients with CHB received PEG-IFN α-2a treatment, completing 4-year follow-up. After PEG-IFN α-2a treatment, undetectable HBV DNA, HBsAg loss, and HBeAg disappearance were defined as functional cure. Proportions of NK, CD56dim, CD56bright, NKp46+, NKp46dim, NKp46high, and interferon alpha receptor 2 (IFNAR2)+ NK cells, and the mean fluorescence intensity (MFI) of NK cell surface receptors IFNAR2 and NKp46 were detected. 66 patients were enrolled into the study in which 17 patients obtained functional cure. At baseline, hepatitis B virus desoxyribose nucleic acid (HBV DNA) titer in patients with functional cure was remarkably lower than that in Non-functional cure group. Compared with baseline, HBV DNA levels, HBsAg levels, and HBeAg levels significantly declined at week 12 and 24 of therapy in patients with functional cure. At baseline, the negative correlation between CD56bright NK% and HBV DNA and the negative correlation between CD56dim NK% and HBV DNA was showed; CD56bright NK% and IFNAR2 MFI in patients with functional cure were remarkably higher than those in patients without functional cure. After therapy, CD56bright NK% and NKp46high NK% in patients with functional cure were higher than those in patients without functional cure. In Functional cure group, after 24 weeks of treatment NK%, CD56bright NK%, IFNAR2 MFI weakly increased, and NKp46high NK% and NKp46 MFI significantly increased, meanwhile, CD56dim NK% and NKp46dim NK% decreased. Only NKp46 MFI increased after therapy in patients without functional cure. The lower HBV DNA load and the higher CD56bright NK% before therapy, and the higher the post-treatment CD56bright NK%, IFNAR2 MFI, NKp46high NK%, the easier to achieve functional cure.

Whole Blood versus Plasma Samples-How Does the Type of Specimen Collected for Testing Affect the Monitoring of Cytomegalovirus Viremia?

Viral infections, or their reactivations, are one of the most important groups of transplantation complications that can occur among recipients of both hematopoietic cells and solid organ transplants. They are the most commonly caused by cytomegalovirus (CMV). Currently, the use of whole blood or plasma samples is recommended for CMV viral load monitoring. The aim of the study was to assess and compare the level of CMV DNA, depending on the type of clinical material—whole blood or plasma fraction derived from the same patient. The studies were carried out on 156 whole blood samples in which the presence of CMV genetic material was confirmed and the corresponding plasma samples from the same rounds of sampling. CMV DNA was not present in 59 (37.8%) of plasma samples compared to whole blood-positive counterparts. Of the samples positive in both types of clinical specimen, 77 (79.4%) had higher viral DNA levels in the whole blood samples. There were statistically significant differences in the detected CMV DNA load in the whole blood compared to plasma fraction counterparts (p < 0.001). The detected CMV DNA value is usually higher in whole blood compared to plasma samples of the same patient. Due to the variability in CMV viral load depending on the clinical material used for a particular patient, one type of specimen should be always used consequently for CMV viremia monitoring.

Detection of human papillomavirus, viral load and risk factors in patients with precancerous diseases and malignant neoplasms in St. Petersburg

Molecular genetic studies have revealed the involvement of different genotypes of human papillomavirus (HPV) in the carcinogenesis of cervical cancer and malignant lesions of other localizations. It is reported that patients with HPV-positive cancer have a better prognosis of the disease and survival than patients with unconfirmed HPV infection or with a low viral load. The objective was to identify the detectability, viral load, genotypes of human papillomavirus in HPV-associated precancerous and malignant neoplasia of various localization and to determine risk factors for their occurrence in the metropolis of St. Petersburg at the present time.&#x0D; Materials and methods. A total of 80 samples taken from morphologically confirmed tissues of oropharyngeal and anal cancer, malignant tumors of vulva, vagina, cervix and cervical intraepithelial neoplasia were studied in St. Petersburg Clinical Oncologic Center. Detection, quantification and genotyping of HPV DNA were carried out by real-time PCR at the St. Petersburg Pasteur Institute.&#x0D; Results. HPV was detected in 89.7% (61/68) of patients with malignant tumors and 83.3% (10/12) with severe cervical dysplasia. The vast majority (85.9%) of HPV-positive patients were infected with HPV genotype 16; papillomavirus mixed infection (genotypes 16, 18, 31, 33, 35, 39, 45) was detected in anal cancer, cancer and severe cervical dysplasia. The average viral load in stages IIIIV of anal cancer, cervical cancer and severe cervical dysplasia exceeded 5.7 lg HPV DNA/105 cells. Among patients with oropharyngeal cancer, men predominated (85.7%); anal cancer was detected in women (90.0%). No statistically significant risk factors (smoking and alcohol consumption) for the occurrence of HPV-associated malignancies were identified.&#x0D; Conclusions. The detection of HPV, mainly of genotype 16, varied depending on the location of the neoplasia: anal cancer 100%, cancer of the female genitalia 94% (in case of cancer of the vagina and cervix 100.0%), head and neck cancer 76.2%. The highest HPV DNA load in the tumor tissue was found in IIIIV stages of the cervical and anal cancer.

Detection of Torquetenovirus and Redondovirus DNA in Saliva Samples from SARS-CoV-2-Positive and -Negative Subjects.

Torquetenovirus (TTV) and Redondovirus (ReDoV) are the most prevalent viruses found in the human respiratory virome in viral metagenomics studies. A large-scale epidemiological study was performed to investigate their prevalence and loads in saliva samples according to SARS-CoV-2 status. Saliva samples from 448 individuals (73% SARS-CoV-2 negative and 27% SARS-CoV-2 positive) aged 23-88 years were tested. SARS-CoV-2 and TTV were determined in saliva by specific qualitative and quantitative real-time PCRs, respectively. A sub-cohort of 377 subjects was additionally tested for the presence and load of ReDoV in saliva, and a different sub-cohort of 120 subjects for which paired saliva and plasma samples were available was tested for TTV and ReDoV viremia at the same timepoints as saliva. TTV in saliva was 72% prevalent in the entire cohort, at a mean DNA load of 4.6 log copies/mL, with no difference regardless of SARS-CoV-2 status. ReDoV was found in saliva from 61% of the entire cohort and was more prevalent in the SARS-CoV-2-negative subgroup (65% vs. 52%, respectively). In saliva, the total mean load of ReDoV was very similar to the one of TTV, with a value of 4.4 log copies/mL. The mean viral loads in subjects infected with a single virus, namely, those infected with TTV or ReDoV alone, was lower than in dually infected samples, and Tukey's multiple-comparison test showed that ReDoV single-infected samples resulted in the only true outlier (p = 0.004). Differently from TTV, ReDoV was not detected in any blood samples. This study establishes the prevalence and mean value of TTV and ReDoV in saliva samples and demonstrates the existence of differences between these two components of the human virome.

Association of delayed chemoradiotherapy with elevated Epstein-Barr virus DNA load and adverse clinical outcome in nasopharyngeal carcinoma treatment during the COVID-19 pandemic: a retrospective study

Background:To summarize the impact of radiotherapy (RT) and chemotherapy delays on patients with nasopharyngeal carcinoma (NPC) during the COVID-19 pandemic.Methods:We retrospectively included 233 patients with stage II-IVa NPC treated with RT and chemotherapy between December 11, 2019 and March 11, 2020. The outcomes were elevation in the EBV DNA load between two adjacent cycles of chemotherapy or during RT, and 1-year disease-free survival (DFS).Results:RT delay occurred in 117 (50%) patients, and chemotherapy delay occurred in 220 (94%) patients. RT delay of ≥ 6 days was associated with a higher EBV DNA elevation rate (20.4% vs. 3.6%, odds ratio [OR] = 6.93 [95% CI = 2.49–19.32], P < 0.001), and worse 1-year DFS (91.2% vs. 97.8%, HR = 3.61 [95% CI = 1.37–9.50], P = 0.006), compared with on-schedule RT or delay of < 6 days. Chemotherapy delay of ≥ 10 days was not associated with a higher EBV DNA elevation rate (12.5% vs. 6.8%, OR = 1.94 [95% CI = 0.70–5.40], P = 0.20), or worse 1-year DFS (93.8% vs. 97.1%, HR = 3.73 [95% CI = 0.86–16.14], P = 0.059), compared with delay of < 10 days. Multivariable analyses showed RT delay of ≥ 6 days remained an independent adverse factor for both EBV DNA elevation and DFS.Conclusion:To ensure treatment efficacy for patients with nonmetastatic NPC, initiation of RT should not be delayed by more than 6 days; the effect of chemotherapy delay requires further investigation.

Sustained Virologic Suppression Reduces HIV-1 DNA Proviral Levels and HIV Antibodies in Perinatally HIV-Infected Children Followed from Birth.

The extent to which perinatally HIV-infected children, following cART initiation, develop a low proviral reservoir burden over time, as measured by HIV DNA droplet-digital polymerase chain reaction (ddPCR) and the effect on HIV antibody is not well characterized. We measured proviral HIV DNA and plasma RNA virus load (VL) in 37 perinatally HIV-infected children at 6 months of age who initiated stable cART. At 6-11 years of age, HIV proviral DNA, HIV VL (RNA), and HIV antibody by Western Blot (WB) were assessed. CART was initiated before 6 months of age in 13 children and after 6 months in 24. At school age, the HIV DNA levels did not differ by the timing of cART, and the HIV DNA levels were lower in children with negative/indeterminate WB (p = 0.0256). Children with undetectable HIV RNA VL > 50% of the time since cART initiation had lower median DNA VL than children with undetectable VL < 50% of the time (p = 0.07). Long-term viral suppression in perinatally HIV-infected children is associated with a decrease in HIV antibodies and reduced HIV reservoirs.

Clinical Significance and Properties of IFN-γ+IL-17+ Th17 Cells in Liver Injury Associated with Chronic Hepatitis B Virus Infection

Introduction: Our previous study demonstrated that intrahepatic Th17 cells exacerbated the progression of chronic hepatitis B virus (HBV) infection. Meanwhile, we found a small group of IFN-γ and IL-17 double-positive Th17 cells (IFN-γ⁺IL-17⁺ Th17 cells) in liver tissues. This study aimed to investigate the clinical significance and properties of IFN-γ⁺IL-17⁺ Th17 cells in liver injury associated with chronic HBV infection. Methods: The frequencies of CD4⁺ Th cells, Tregs, and CD4⁺ T cells expressing specific chemokine receptors in the blood and liver tissues were detected using flow cytometry. The chemotaxis of C C chemokine receptor 5 (CCR5) and C-X-C chemokine receptor 3 (CXCR3) toward IFN-γ⁺IL-17⁺ Th17 cells and Tregs was evaluated by transwell chemotactic assay. Analyses of different variables were performed using GraphPad Prism v 5.01 and IBM SPSS Statistics 23.0. HBV-specific IFN-γ⁺IL-17⁺ Th17 cells were investigated using a cell stimulation assay with HBV antigens in vitro. Results: The frequencies of IFN-γ⁺IL-17⁺ Th17, Th17 cells, and Tregs in the blood were increased from normal controls to chronic hepatitis B (CHB) and acute-on-chronic liver failure (ACLF). The same trend could also be observed in CHB liver tissues compared to those in CHB blood specimens. Furthermore, the frequencies of IFN-γ⁺IL-17⁺ Th17 cells were positively associated with Th17 cells, Th17 cell-related cytokines (IL-17 and IL-6), HBV DNA load, and the levels of HBsAg, HBeAg, and ALT. The ratios of IFN-γ⁺IL-17⁺ Th17 cells to Tregs extremely decreased in ACLF blood specimens compared with those in CHB blood specimens. Additionally, CCR5 and CXCR3 were conducive to the recruitment of IFN-γ⁺IL-17⁺ Th17 cells and Tregs to liver tissue. Conclusions: IFN-γ⁺IL-17⁺ Th17 cells have Th17 cell-like properties in the progression of chronic HBV infection. CCR5 and CXCR3 facilitated the recruitment of IFN-γ⁺IL-17⁺ Th17 cells and Tregs to the liver. Importantly, the ratio of IFN-γ⁺IL-17⁺ Th17 cells to Tregs might be an effective assessment indicator of the severity of liver injury.

Risk factors for the long-term prognosis and recurrence of HIV-negative cytomegalovirus retinitis in North China.

To demonstrate the clinical features, the risk factors, the visual prognosis and the recurrence of cytomegalovirus (CMV) retinitis (CMVR) in HIV-negative patients. HIV-negative patients with CMVR were involved in this study. Best corrected visual acuity (BCVA), intraocular pressure (IOP), CMV-DNA load in aqueous and/or serum samples, treatment, follow-up time, recurrence and complications were recorded. Ocular characteristics were evaluated by fundus photographs. Association between ocular factors and visual prognosis were analyzed by regression analysis. Twenty-five eyes of 16 patients were included. All 25 eyes underwent intravitreal injections of anti-viral agents. The mean logMAR BCVA improved from 0.94±0.98 (0.98-0.78) initially to 0.77±0.73 (0.82-0.68) at last visit, but not significantly. After antiviral treatment, the aqueous CMV DNA load significantly reduced to (3.42±1.47)×102 copies/mL (P=0.001), compared with (2.51±3.11)×105 copies/mL at baseline. Macular involvement (R 2=0.475, P=0.049) and initial visual acuity (R 2=0.475, P=0.017) were significantly associated with the poor visual prognosis (BCVA<20/400). The extent of retinal lesions (R 2=0.064, P=0.04) was significant associated with the risk of recurrence of CMVR. Intravitreal injection of anti-viral agents offers a safe and effective treatment for CMVR. Macular involvement and initial visual acuity significantly associate with visual prognosis. The extent of retinal lesions is significantly associated with the recurrence of CMVR. These ocular factors can be used as predictive risk factors for long term visual prognosis in HIV-negative CMVR patients.

The Incidence and the Clinical Significance of the Gray Zone in the Clinical Phases of Chronic Hepatitis B Virus Infection

Background: five clinical phases were described in patients with chronic (HBV) infection: HBeAg- positive HBV infection, HBeAg- positive CHB, HBeAg negative HBV infection, HBeAg-negative CHB and occult HBV infection.&#x0D; Aim: This study aimed to determine the incidence of the unclassified phase (gray zone) in chronic hepatitis B patients and its significant in the clinical practice. &#x0D; Patients and methods: The study was conducted retrospectively on 109 patients' who have HBsAg positive for more than 6 months. The data recorded include; HbeAg and anti-HBe Ab, ultrasound of the abdomen, HBV DNA load and alanine aminotransferase (ALT), accordingly; we classify the patients to known clinical phases. Patients who were unfit one of these phases considered to be in the gray zone and subsequently sent for liver fibroscan to determine the fibrosis stage. &#x0D; Results: The mean age of our patients was 34.25 (±13.9) years with 54.12% being males. The mean viral load was 5,885,490 IU/ml and mean ALT was 56.22 (±89.88) U/L. eight patients (7.3%) were in the HBeAg+ve HBV infection, 13 patients (11.9%) were in HBeAg+ve CHB . Thirty four patients (31.1%) were in the HBeAg-ve HBV infection and 23 (21.1%) were in HBeAg-ve CHB phase, both were showed a significant statistical relationship with age&gt; 35 years. Thirty one (28.4%) further patients failed to identify with any of the four phases (normal ALT with HBV load &gt; 2000 IU/ml), this group also showed significant relation to age above 35 years and 12 patients (38.8%) had significant fibrosis on fibroscan.&#x0D; Conclusions: A considerable number of patients with chronic HBV infection have persistently normal ALT levels, despite elevated levels of viral load; this is known as the "grey zone" phase. These patients merit close follow up with short-interval measurement of liver enzymes, liver fibroscan and biopsy may be considered.

Repeat infections with chlamydia in women may be more transcriptionally active with lower responses from some immune genes.

Chlamydia trachomatis, the most common bacterial sexually transmitted infection worldwide, is responsible for considerable health burden due to its significant sequelae. There are growing concerns about chlamydial treatment and management due to widely documented increasing burden of repeat infections. In the current study, a cohort study design of 305 women with urogenital chlamydial infections demonstrated that 11.8% of women experienced repeat infections after treatment with azithromycin. The chlamydial DNA load measured by quantitative PCR was higher in women who experienced a repeat infection (p = 0.0097) and repeat infection was associated with sexual contact. There was no genomic or phenotypic evidence of azithromycin resistance within the chlamydial isolates. During repeat infection, or repeat positive tests during follow up, vaginal chlamydial gene expression (ompA, euo, omcB, htrA, trpAB) was markedly higher compared to baseline, and two of the selected immune genes analyzed had significantly lower expression at the time of repeat infection. Overall, there are two implications of these results. The results could be generalized to all recent infections, or repeat positive events, and indicate that chlamydial infections are have higher transcriptional activity of select genes early in the infection in women. Alternatively, after azithromycin treatment, repeat infections of Chlamydia may be more transcriptionally active at certain genes, and there may be post-treatment immunological alterations that interplay into repeat exposures establishing an active infection. The potential that recent infections may involve a higher level of activity from the organism may have implications for management by more regular testing of the most at risk women to reduce the risk of sequelae.

Toward efficient and high-fidelity metagenomic data from sub-nanogram DNA: evaluation of library preparation and decontamination methods

BackgroundShotgun metagenomic sequencing has greatly expanded the understanding of microbial communities in various biological niches. However, it is still challenging to efficiently convert sub-nanogram DNA to high-quality metagenomic libraries and obtain high-fidelity data, hindering the exploration of niches with low microbial biomass.ResultsTo cope with this challenge comprehensively, we evaluated the performance of various library preparation methods on 0.5 pg–5 ng synthetic microbial community DNA, characterized contaminants, and further applied different in silico decontamination methods. First, we discovered that whole genome amplification prior to library construction led to worse outcomes than preparing libraries directly. Among different non-WGA-based library preparation methods, we found the endonuclease-based method being generally good for different amounts of template and the tagmentation-based method showing specific advantages with 0.5 pg template, based on evaluation metrics including fidelity, proportion of designated reads, and reproducibility. The load of contaminating DNA introduced by library preparation varied from 0.01 to 15.59 pg for different kits and accounted for 0.05 to 45.97% of total reads. A considerable fraction of the contaminating reads were mapped to human commensal and pathogenic microbes, thus potentially leading to erroneous conclusions in human microbiome studies. Furthermore, the best performing in silico decontamination method in our evaluation, Decontam-either, was capable of recovering the real microbial community from libraries where contaminants accounted for less than 10% of total reads, but not from libraries with heavy and highly varied contaminants.ConclusionsThis study demonstrates that high-quality metagenomic data can be obtained from samples with sub-nanogram microbial DNA by combining appropriate library preparation and in silico decontamination methods and provides a general reference for method selection for samples with varying microbial biomass.

Pathogen load and species monitored by droplet digital PCR in patients with bloodstream infections: A prospective case series study

Background and objectives:Bloodstream infection (BSI) is a life-threatening condition in critically ill patients, but pathogen quantification techniques during treatment are laborious. This study aimed to explore the impact of monitoring pathogen DNA load changes and polymicrobial infection in blood by droplet digital polymerase chain reaction (ddPCR) on the prognosis of patients with BSIs.MethodsThis prospective case series study was conducted in the general intensive care unit of the Zhejiang Provincial People’s Hospital and included patients with BSIs from May 2020 to January 2021. Pathogens DNA load and presence of polymicrobial BSIs were dynamically monitored by ddPCR.ResultsSixteen patients with BSIs proven by blood culture were recruited (87.5% men; mean age, 69.3 ± 13.7 years). All pathogens identified by blood culture were Gram-negative bacteria, among which seven were multidrug-resistant strains. The 28-day mortality rate was 62.5%. Compared to the 28-day survivors, the non-survivors were older (P = 0.04), had higher pathogen DNA load on the second (day 3–4) and third (day 6–7) ddPCR assay (P < 0.01 in both cases). In addition, the changes of pathogen DNA load in the 28-day survivors had a downward trend in the first three ddPCR assay, whereas stable load or an upward trend was observed in the 28-day non-survivors. Moreover, the number of pathogen species in patients with BSIs in the 28-day survivors decreased during the period of effective antibiotic treatment.ConclusionThe changes of pathogen DNA load and species monitored in blood by ddPCR may be used to determine antibiotic efficacy and make a more accurate prognostic assessment in patients with BSIs.

Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells

SummaryLong-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

A Prospective Study of Plasma and Bronchoalveolar Lavage Fluid CMV DNA Load Quantification for the Diagnosis and Outcome of CMV Pneumonitis in Immunocompromised Hosts.

Molecular testing has been utilized for cytomegalovirus (CMV) pneumonitis (CMVP) diagnosis, although its validity and optimal cut-off values remain limited. A prospective study of CMVP diagnosis among immunocompromised patients was conducted by measuring quantitative CMV DNA polymerase chain reaction in plasma and bronchoalveolar lavage fluid (BALF). Forty-five adult immunocompromised patients were investigated. Thirty-two patients (71%) received immunosuppressive therapy. Eleven patients (24%) were confirmed to have CMVP. Of those, three and eight patients were classified as proven and probable CMVP, respectively. Median (IQR) plasma CMV DNA loads in CMVP and non-CMVP were 41,939 (4,424-122,608) and 0 (0-44) IU/mL, respectively (p<0.001). Median (IQR) BALF CMV DNA loads in CMVP and non-CMVP were 379,652 (163,800-1,254,000) and 0 (0-1,348) IU/mL, respectively (p<0.001). A significant correlation was observed between plasma and BALF CMV DNA loads (r=0.887, p<0.001). Plasma CMV DNA load of 831 IU/mL was established as a cut-off value for diagnosing CMVP (AUC 0.9987, sensitivity 100%, specificity 94.1%, positive predictive value 84.5%, negative predictive value 100%). A strongly positive correlation was observed between CMV DNA loads measured in plasma and BALF. CMV DNA load quantification could potentially assist in diagnosing CMVP in immunocompromised patients, although bronchoscopy remains encouraged for a definitive diagnosis.