DNA Load Research Articles

Incidence of Occult Hepatitis B Infection (OBI) and hepatitis B genotype characterization among blood donors in Cameroon.

Occult hepatitis B infection (OBI) is characterized by the presence of hepatitis B virus (HBV) DNA at low levels in serum (<200 IU/mL) with a negative hepatitis B surface antigen (HBsAg) test. OBI remains a major challenge to blood safety, particularly in HBV-endemic regions like Cameroon, where HBV detection relies solely on HBsAg testing. This cross-sectional study aimed to investigate the actual incidence and genotype characteristics of OBI in Cameroonian blood donors. Between March and June 2023, samples were collected from 288 HBsAg-negative blood donors aged 18 to 55 years and analysed for antibodies against the HBV core (anti-HBc) and surface antigens (anti-HBs). Following DNA extraction from the serum samples, qualitative nested PCR and quantitative real-time PCR were used to detect HBV viral DNA and viral load respectively. For positive samples, sequencing of a fragment of the S gene was performed to identify the circulating HBV genotypes. The findings revealed that 58% (n = 167/288) of blood donors tested positive for anti-HBc, 29% (n = 83/288) tested positive for anti-HBs, and 26% (n = 75/288) being positive for both anti-HBc and anti-HBs. Occult hepatitis was confirmed in 4.5% of the blood donors, all of whom belonged to either HBV genotypes A or E, which are predominant in Cameroon. The amino acid substitution sA184V associated with HBsAg detection failure in genotype E was observed in 70% of OBI sequences, and the HBsAg immune escape variants (sT131N and sS143L) implicated in OBI were also observed. The mutation rtN139K in the reverse transcriptase (RT) domain of the overlapping HBV polymerase (P) gene was present in 17% of OBI-positive sequences of genotype E, likely contributing to masking HBsAg secretion. The results suggest a considerable risk of transfusion-transmitted HBV in this region. Therefore, to ensure blood safety, nucleic acid testing (NAT) is recommended, as relying solely on HBsAg assays is insufficient to eliminate this risk.

Development of a droplet digital PCR assay for the detection of BK polyomavirus.

The objective of this study was to establish a more sensitive and specific diagnostic method for detecting plasma BK polyomavirus (BKPyV) DNA load in patients after renal transplantation using droplet digital polymerase chain reaction (ddPCR) and to validate the methodology. The linear range, lower limit of detection, accuracy, precision, and specificity of the detection system were evaluated by using the WHO BKPyV standard (7.2 log10 IU/mL) as a reference, in accordance with the relevant documents of the Clinical and Laboratory Standards Institute. Plasma samples were collected from 74 renal transplantation patients with urinary BKPyV-DNA levels exceeding 7 log10 copies/mL. Quantitative PCR (qPCR) and ddPCR were performed, and their diagnostic efficacy for BKPyV-DNA in the diagnosis of BK polyomavirus-associated nephropathy was evaluated using a receiver operating characteristic (ROC) curve. The coefficients of variation for the repeated detection of BKPyV standard DNA were 2.55 and 4.71 at concentrations of 6.2 and 3.2 log10 IU/mL, respectively. The linear range was 2.2-6.2 log10 IU/mL, and the lowest detection limit was 100 IU/mL. By utilizing histopathological examination of renal biopsy as the gold standard for BKPyV diagnosis, the area under the ROC curve of 74 post-transplantation plasma samples detected by the ddPCR system was found to be 0.875 (95% CI: 0.797-0.953, P < 0.01). The optimal threshold was 512.86 copies/mL, with a sensitivity of 90.0% and a specificity of 67.6%. In comparison, the area under the ROC curve for qPCR was 0.668 (95% CI: 0.583-0.752, P < 0.01), with an optimal threshold of 11,481.54 copies/mL, a sensitivity of 35.0%, and a specificity of 100.0%. Pairwise comparison (Delong test) of the ROC curves of the two systems showed a significant difference in the area under the curve, with a difference of 0.207 and a P-value <0.01. The BKPyV nucleic acid detection system, based on ddPCR, is appropriate for the regular monitoring of the BK polyomavirus, specifically in plasma samples containing low viral DNA loads while it provides the benefits of both absolute quantification and high sensitivity.IMPORTANCEIt was previously believed that droplet digital polymerase chain reaction had limitations, including high cost, limited throughput, and cumbersome operation, which hindered its widespread application in clinical practice. However, the current fully automated digital PCR platform, combined with streamlined operations, can detect 96 samples at once, and the entire process can be completed within an hour, laying a solid foundation for its extensive use.

Isolation and pathogenicity of a fowl adenovirus 8b (FAdV-8b) strain in Cherry Valley ducks

ABSTRACT Inclusion body hepatitis (IBH) is an economically important viral disease primarily affecting the poultry industry. In this study, we isolated a strain of FAdV-8b (strain SDYT) from naturally infected ducks and the hexon and fiber gene sequences were analysed by polymerase chain reaction (PCR) amplification. In order to study the pathogenicity of FAdV-8b in Cherry Valley ducks, we inoculated 10- and 20-day-old ducks with 0.3 ml of FAdV-4 virus (TCID50 of 105.5/0.1 ml) either orally or intramuscularly. Clinical signs, gross lesions and histopathological changes, cytokines, viral load and antibody levels were observed and recorded within 15 days after infection. Pathomorphological investigations revealed that ducks in the experimental group exhibited hepatitis. Histopathology showed multiple organ damage, including serious liver and kidney lesions. Furthermore, elevated levels of inflammatory cytokines and antibodies were noticed, due to the infection and innate immune response. At a later stage of infection, immunosuppression occurred, resulting in decreased levels of cytokines. Determination of viral load showed that the virus was present in several organs, with the highest viral DNA load found in the liver, followed by the kidney. Compared to birds infected orally, the intramuscular group exhibited the highest viral load. In summary, this study increases our understanding of the pathogenicity of FAdV-8b in ducks and establishes a model that will inform antiviral drug testing and vaccine evaluation for IBH, thereby preventing and reducing the spread of IBH in the poultry industry. RESEARCH HIGHLIGHTS A strain (SDYT) of fowl adenovirus 8b (FAdV-8b) was successfully isolated from ducks. Cherry Valley ducks were successfully infected with FAdV-8b. Different routes of infection can lead to duck mortality, more pronounced when birds are injected intramuscularly. FAdV-8b (SDYT) was distributed in various tissues and organs of ducks, causing different degrees of lesions.

Risk factors for significant histological changes in both HBeAg positive and negative treatment-naive chronic hepatitis B with persistently normal alanine aminotransferase level

BackgroundChronic hepatitis B virus (HBV) infection remains a serious health issue, and determining the optimal time for antiviral therapy is challenging. We aimed to assess liver histological changes in patients with HBeAg-positive chronic hepatitis B (CHB) and those with HBeAg-negative CHB who had persistently normal alanine aminotransferase and to determine the association between significant liver injury and various clinical parameters.MethodsWe retrospectively included, in this study, 339 treatment-naïve patients with chronic HBV infections who had persistently normal alanine aminotransferase and underwent liver biopsy from 2013 to 2023. Histologic assessment was based on the Metavir scoring system to evaluate the association between clinical characteristics and the severity of liver inflammation and fibrosis.ResultsAmong the included participants, 138 were HBeAg-positive and 201 were HBeAg-negative. Lower hepatitis B surface antigen (HBsAg) (P = 0.003) and higher aspartate aminotransferase (AST) (P = 0.002) levels were associated with significant necroinflammation, whereas increasing age (P = 0.004) and lower HBV DNA (P < 0.001) levels were associated with significant fibrosis in HBeAg-positive patients with normal ALT levels. Higher HBV-DNA (P = 0.001) and AST levels(P < 0.001) were associated with significant necroinflammation, and higher AST(P < 0.001) levels were associated with significant fibrosis in HBeAg-negative patients.ConclusionsA substantial proportion of patients with HBV infection who had normal ALT presented significant liver injury. HBsAg and AST were independent predictive factors for evaluating inflammation, while HBV DNA load and age were independent predictive factors for evaluating fibrosis in the HBeAg-positive group. HBV DNA load and AST were independent predictive factors for evaluating inflammation, while AST were independent predictive factors for evaluating fibrosis in the HBeAg-negative group.

Prognostic significance of plasma cytomegalovirus (CMV) DNA load in immunocompetent patients with CMV pneumonia: A retrospective cohort study.

Cytomegalovirus (CMV) pneumonia, often presented as pneumonitis, is characterized by respiratory failure and large interstitial infiltrates visible on chest radiographs. This retrospective cohort study investigates the predictive significance of plasma CMV DNA load on the short- and long-term mortality among immunocompetent patients diagnosed with CMV pneumonia. The study included 61 immunocompetent patients suspected of having CMV pneumonia, treated with intravenous ganciclovir after positive CMV DNA results from bronchoalveolar lavage or plasma. Our multivariate Cox regression analysis identified several independent predictors of mortality. Having idiopathic pulmonary fibrosis (IPF) significantly increased the risk of in-hospital mortality (HR: 7.27, 95% CI: 1.62-32.52, p = 0.009), as did shorter durations of antiviral therapy (HR: 0.90, 95% CI: 0.84-0.97, p = 0.005) and higher CMV DNA levels (>3870 IU/mL; HR: 9.63, 95% CI: 2.32-39.98, p = 0.002). High CMV DNA levels (>5154 IU/mL) were also predictors of 30-day mortality (HR: 9.39, 95% CI: 2.20-40.01, p = 0.002). For 1-year mortality, the presence of IPF (HR: 2.96, 95% CI: 1.08-8.06, p = 0.034), hypersensitivity pneumonia (HP) (HR: 4.30, 95% CI: 1.57-11.78, p = 0.005), shorter duration of total antiviral therapy (HR: 0.95, 95% CI: 0.93-0.99, p = 0.010), and higher CMV DNA levels (>327 IU/mL) (HR: 3.36, 95% CI: 1.33-8.47, p = 0.010) were identified as independent determinants. The study reveals that IPF increases short and long-term mortality risks, while HP increases long-term mortality. Extended antiviral treatment duration results in a 10% reduction in in-hospital mortality for each additional day of treatment. Furthermore, elevated viral loads are associated with higher mortality rates, highlighting the necessity for careful monitoring.

Unraveling the role of hepatitis B virus DNA integration in B-cell lymphomagenesis.

Studies have shown that hepatitis B virus (HBV)-associated B-cell non-Hodgkin lymphoma (NHL) constitutes a unique subgroup with distinct clinical features. It still leaves open the question of whether the integration of HBV DNA into the B-cell genome is a causal mechanism in the development of lymphoma. Using the hybridisation capture-based next generation sequencing and RNA sequencing, we characterised the HBV integration pattern in 45 HBV-associated B-cell NHL tumour tissues. A total of 354 HBV integration sites were identified in 13 (28.9%) samples, indicating the relatively low integration frequency in B-cell NHLs. High plasma HBV DNA loads were not associated with the existence of HBV integration. The insertion sites distributed randomly across all the lymphoma genome without any preferential hotspot neither at the chromosomal level nor at the genetic level. Intriguingly, most HBV integrations were nonclonal in B-cell NHLs, implying that they did not confer a survival advantage. Analysis of the paired diagnosis-relapse samples showed the unstable status of HBV integrations during disease progression. Furthermore, transcriptomic analysis revealed the limited biological impact of HBV integration. Our study provides an unbiased HBV integration map in B-cell NHLs, revealing the insignificant role of HBV DNA integration in B-cell lymphomagenesis.

The plasma EBV DNA load with IL-6 and VEGF levels as predictive and prognostic biomarker in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is often diagnosed at a very advanced stage due to its location and non-specific initial symptoms. Moreover, no clinically useful serological marker has been established so far for early detection of NPC. In this study, we have investigated the clinical significance of plasma Epstein–Barr virus DNA load along with interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) levels to evaluate if these three all together can be useful as a strong serological marker for early detection and prediction of treatment response in patients with NPC. Plasma EBV DNA load, IL-6 level, VEGF expressions were measured in 24 patients with NPC at presentation and various time points during and after treatment. There was a positive correlation between high plasma EBV DNA load with higher IL-6 and VEGF expression, which was closely associated with therapeutic response as well. Persistent or recurrent plasma EBV load with higher IL-6 and VEGF levels can potentially predict disease progression and may be useful to select patients for additional therapy and longer follow-up.

Disparities in the Levels of Whole-Blood Epstein-Barr Virus between the Cancer and Non-Cancer Populations in Zhejiang, China

ObjectiveThis study aimed to investigate the prevalence of Epstein-Barr virus (EBV) infection in patients with and without cancer. MethodsA total of 26,648 participants who underwent whole-blood EBV DNA (WBEBV) assays between January 1, 2020, and August 31, 2023, were included. The chi-square test was used for categorical data analysis, and R software was used to analyze the differences in EBV DNA load levels and the diagnostic capabilities of WBEBV. ResultsPositive rates were 10.2% and 25.4% for healthy controls (HC) and patients, respectively. The positivity rate for EBV-associated neoplasms (EN) was the highest at 7.53%, followed by leukemia (Le) at 5.49%. The subgroup analysis showed that the positivity rate for abnormal proliferation or hyperplasia (APH) was 31.9%, followed by 30.5% for Le. The WBEBV of patients with transplants (TP), especially living-related transplants (LT), was the highest among all subgroups. WBEBV at diagnosis was used to differentiate between infectious mononucleosis (IM) and chronic active Epstein-Barr virus (CAEBV), with a sensitivity of 67.4% (95% confidence interval [CI]: 57.6–75.8) and specificity of 72% (95% CI: 63.3–79.3). We conclude that the prevalence of EBV infection is low in the healthy population in this region and that a high EBV load at baseline is more common in LT, IM, and Lymphocyte Leukemia (LL). ConclusionThis study used a large-sample survey to characterize the prevalence of whole-blood EBV levels in various diseases, including the stages and subtypes. The EBV detection rate was higher in patients with malignant disease than in those with benign disease. Our study provides clinicians with baseline information regarding EBV-associated diseases.

Breaking Latent Infection: How ORF37/38-Deletion Mutants Offer New Hope against EHV-1 Neuropathogenicity.

Equid alphaherpesvirus 1 (EHV-1) has been linked to the emergence of neurological disorders, with the horse racing industry experiencing significant impacts from outbreaks of equine herpesvirus myeloencephalopathy (EHM). Building robust immune memory before pathogen exposure enables rapid recognition and elimination, preventing infection. This is crucial for effectively managing EHV-1. Removing neuropathogenic factors and immune evasion genes to develop live attenuated vaccines appears to be a successful strategy for EHV-1 vaccines. We created mutant viruses without ORF38 and ORF37/38 and validated their neuropathogenicity and immunogenicity in hamsters. The ∆ORF38 strain caused brain tissue damage at high doses, whereas the ∆ORF37/38 strain did not. Dexamethasone was used to confirm latent herpesvirus infection and reactivation. Dexamethasone injection increased viral DNA load in the brains of hamsters infected with the parental and ∆ORF38 strains, but not in those infected with the ∆ORF37/38 strain. Immunizing hamsters intranasally with the ∆ORF37/38 strain as a live vaccine produced a stronger immune response compared to the ∆ORF38 strain at the same dose. The hamsters demonstrated effective protection against a lethal challenge with the parental strain. This suggests that the deletion of ORF37/38 may effectively inhibit latent viral infection, reduce the neuropathogenicity of EHV-1, and induce a protective immune response.

Variant load of mitochondrial DNA in single human mesenchymal stem cells

Heteroplasmic mitochondrial DNA (mtDNA) variants accumulate as humans age, particularly in the stem-cell compartments, and are an important contributor to age-related disease. Mitochondrial dysfunction has been observed in osteoporosis and somatic mtDNA pathogenic variants have been observed in animal models of osteoporosis. However, this has never been assessed in the relevant human tissue. Mesenchymal stem cells (MSCs) are the progenitors to many cells of the musculoskeletal system and are critical to skeletal tissues and bone vitality. Investigating mtDNA in MSCs could provide novel insights into the role of mitochondrial dysfunction in osteoporosis. To determine if this is possible, we investigated the landscape of somatic mtDNA variation in MSCs through a combination of fluorescence-activated cell sorting and single-cell next-generation sequencing. Our data show that somatic heteroplasmic variants are present in individual patient-derived MSCs, can reach high heteroplasmic fractions and have the potential to be pathogenic. The identification of somatic heteroplasmic variants in MSCs of patients highlights the potential for mitochondrial dysfunction to contribute to the pathogenesis of osteoporosis.

Investigation of Mother-to-Child Transmission of Hepatitis B in Yinchuan, China: Cross-Sectional Survey Study.

Hepatitis B poses a significant global public health challenge, with mother-to-child transmission (MTCT) being the primary method of hepatitis B virus (HBV) transmission. The prevalence of HBV infection in China is the highest in Asia, and it carries the greatest burden globally. This study aims to critically evaluate the existing local strategies for preventing MTCT and the proposed potential enhancements by analyzing the prevalence of hepatitis B among pregnant women and their neonates in Yinchuan. From January 2017 to December 2021, 37,557 prenatal screening records were collected. Among them, 947 pregnant women who tested positive for hepatitis B surface antigen (HBsAg) near delivery and their 960 neonates were included in an HBV-exposed group, while 29 pregnant women who tested negative and their 30 neonates were included in an HBV-nonexposed group. HBV markers in maternal peripheral blood and neonatal cord blood were analyzed using the least absolute shrinkage and selection operator (LASSO) regression, logistic regression, chi-square test, t-test, and U-test. Additionally, to further evaluate the diagnostic value of HBsAg positivity in cord blood, we conducted an additional follow-up study on 103 infants who tested positive for HBsAg in their cord blood. The prevalence of HBV among pregnant women was 2.5% (947/37,557), with a declining trend every year (χ²4=19.7; P=.001). From 2018 to 2020, only 33.0% (35/106) of eligible pregnant women received antiviral medication treatment. Using LASSO regression to screen risk factors correlated with HBsAg positivity in cord blood (when log [λ] reached a minimum value of -5.02), 5 variables with nonzero coefficients were selected, including maternal hepatitis B e-antigen (HBeAg) status, maternal hepatitis B core antibody (HBcAb) status, maternal HBV DNA load, delivery method, and neonatal birth weight. Through univariate and multivariate logistic regression, delivery by cesarean section (adjusted odds ratio [aOR] 0.52, 95% CI 0.31-0.87), maternal HBeAg positivity (aOR 2.05, 95% CI 1.27-3.33), low maternal viral load (aOR 2.69, 95% CI 1.33-5.46), and high maternal viral load (aOR 2.69, 95% CI 1.32-5.51) were found to be strongly associated with cord blood HBsAg positivity. In the additional follow-up study, 61 infants successfully completed the follow-up, and only 2 were found to be infected with HBV. The mothers of both these infants had detectable HBV DNA levels and should have received standard antiviral therapy. The results of the hepatitis B surface antibody (HBsAb) positivity rate and titer test indicated a gradual decline in the immunity of vaccinated infants as the interval after vaccination increased. The clinical relevance of HBV marker detection in cord blood is restricted within the current prevention measures for MTCT. There is an emphasis on the significance of public education regarding hepatitis B and the reinforcement of postnatal follow-up for the prevention of MTCT.

Update on Cytomegalovirus Infection Management in Allogeneic Hematopoietic Stem Cell Transplant Recipients. A Consensus Document of the Spanish Group for Hematopoietic Transplantation and Cell Therapy (GETH-TC).

Cytomegalovirus (CMV) infection is a common complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and in patients receiving novel hematological therapies. Its impact on morbidity and mortality necessitates effective management strategies. Despite recent advances in diagnostics and treatment, unresolved questions persist regarding monitoring and treatment, prompting the need for updated recommendations. A consensus was reached among a panel of experts selected for their expertise in CMV research and clinical practice. Key clinical areas and questions were identified based on previous surveys and literature reviews. Recommendations were formulated through consensus and graded using established guidelines. Recommendations were provided for virological monitoring, including the timing and frequency of CMV DNAemia surveillance, especially during letermovir (LMV) prophylaxis. We evaluated the role of CMV DNA load quantification in diagnosing CMV disease, particularly pneumonia and gastrointestinal involvement, along with the utility of specific CMV immune monitoring in identifying at-risk patients. Strategies for tailoring LMV prophylaxis, managing breakthrough DNAemia, and implementing secondary prophylaxis in refractory cases were outlined. Additionally, criteria for initiating early antiviral treatment based on viral load dynamics were discussed. The consensus provides updated recommendations for managing CMV infection in hematological patients, focusing on unresolved issues in monitoring, prophylaxis, treatment, and resistance. These recommendations aim to guide clinical practice and improve outcomes in this high-risk population. Further research is warranted to validate these recommendations and address ongoing challenges in CMV management with emerging antiviral combinations, particularly in pediatric populations.

Efficacy of NKG2D CAR-T cells with IL-15/IL-15Rα signaling for treating Epstein-Barr virus-associated lymphoproliferative disorder

Epstein-Barr virus (EBV) related post-transplant lymphoproliferative disorder (EBV-PTLD) is a life-threatening complication after hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT), for which no standard therapeutic means have been developed. Significant increase expression of natural killer group 2 member D ligands (NKG2DLs) was observed on B-lymphoblastoid cells of EBV-PTLD, indicating NKG2DLs as potential therapeutic targets for treatment of EBV-PTLD. In this study, the recombinant constructs of NKG2D CAR and IL-15/IL-15Rα-NKG2D CAR were generated with a retroviral vector and then transduced to human T cells to produce NKG2D CAR-T and IL-15/IL-15Rα-NKG2D CAR-T cells, respectively. B-lymphoblastoid cell lines (B-LCLs) and the xenografted mouse models were established to evaluate the efficacy of these CAR-T cells. IL-15/IL-15Rα-NKG2D CAR-T cells exhibited superior proliferation and antigen-specific cytotoxic effect compared to NKG2D CAR-T, as IL-15/IL-15Rα signaling promoted the expansion of less differentiated central memory T cells (TCM) and increased expression of CD107a and IFN-γ. Moreover, EBV DNA load was dramatically reduced, and 80% B-LCL cells were eliminated by IL-15/IL-15Rα-NKG2D CAR-T cells after co-culturing. In-vivo study confirmed that IL-15/IL-15Rα-NKG2D CAR-T cell therapy significantly enhanced antiviral efficacy in mice, as the serum load of EBV after IL-15/IL-15Rα-NKG2D CAR-T cell infusion was 1500 times lower than the untreated control (P < 0.001). The enhanced efficacy of IL-15/IL-15Rα-NKG2D CAR T cells was probably due to the IL-15/IL-15Rα signaling improved homing and persistence of NKG2D CAR-T cells in vivo, and increased the production of IFN-γ, Perforin, and Granulysin. In conclusion, NKG2D CAR-T cells co-expressing IL-15/IL-15Rα promoted the central memory CAR T cell proliferation and improved the homing and persistence of CAR T cells in vivo, resulting in enhanced anti-tumor and anti-viral effects in treating EBV-PTLD.

Cytokine analysis of aqueous humor in patients with cytomegalovirus corneal endotheliitis.

To evaluate cytokine levels of aqueous humor in patients with cytomegalovirus (CMV) corneal endotheliitis and their relationships with CMV DNA load. 44 aqueous humor samples were obtained from 26 patients with CMV corneal endotheliitis at various stages of treatment. 33 samples obtained from cataract patients during the same period were selected as a control group. Each sample was used to measure the concentration of the CMV DNA load using real-time quantitative polymerase chain reaction, and to examine the levels of IL-6, IL-8, IL-10, MCP-1, VCAM-1, VEGF, IP-10, G-CSF, ICAM-1 and IFN-γ using a cytometric bead array. All 10 cytokines were found to have statistically significant differences between the CMV endotheliitis and cataract groups. The Spearman correlation test showed that the concentration of CMV DNA load was significantly associated with the levels of IL-6 (P = 0.005, r = 0.417), IL-8 (P < 0.001, r = 0.514), IL-10 (P < 0.001, r = 0.700), MCP-1 (P = 0.001, r = 0.487), VEGF (P < 0.001, r = 0.690), IP-10 (P = 0.001, r = 0.469), G-CSF (P < 0.001, r = 0.554) and ICAM-1 (P < 0.001, r = 0.635), but not significantly associated with VCAM-1 (P = 0.056) and IFN-γ (P = 0.219). There was a combined innate and adaptive immune response in aqueous humor in patients with CMV endotheliitis. Levels of multiple cytokines were significantly correlated with viral particle. Cytokines are potential indicators to help diagnose CMV endotheliitis, evaluate disease activity and assess treatment response.

Cryptococcosis in wait-listed liver transplant candidates: Prevalence, manifestations, and risk factors

BackgroundLiver cirrhosis compromises immunity against cryptococcosis, and liver transplant recipients tend to develop the disease earlier after transplantation, possibly due to unrecognized pretransplant infection. We assessed the prevalence and characteristics of cryptococcosis among liver transplant candidates and whether pre-transplant cryptococcal antigen (CrAg) can detect the disease before transplantation. MethodsWe retrospectively included liver transplant candidates in a tertiary hospital during 2017–2022. Serum CrAg and pulmonary computed tomography were incorporated in routine transplant evaluation. Other investigations were done if indicated. Cryptococcosis was diagnosed by positive culture or CrAg. Risk factors for cryptococcosis were also assessed. ResultsOf the 377 candidates with a median MELD-Na score of 18, 84.4% had hepatitis B virus (HBV) infection. Cryptococcosis was diagnosed in 10 (2.6%) candidates, by CrAg in 6, culture in 2, or both in 2. Only 3 had fever, and 3 were asymptomatic; 7 had pulmonary cryptococcosis. Of the 10 candidates with cryptococcosis, one underwent transplantation after 143-day antifungals. Of the 87 candidates undergoing liver transplantation, one (1.2%) recipient developed cryptococcosis 14 days post-transplant with negative CrAg three weeks before transplantation. HBsAg-positive chronic HBV infection with HBV DNA loads <2000 IU/mL was significantly associated with cryptococcosis (odds ratio 4.4, 95% confidence interval 1.2–16.5, p = 0.03) after the adjustment of MELD-Na score. ConclusionsThe prevalence of cryptococcosis was 2.6% among our liver transplant candidates and CrAg detected 80% of the cases. Disease presentation was mild and pulmonary disease predominated. HBsAg-positive chronic HBV infection with HBV DNA loads <2000 IU/mL was significantly associated with cryptococcosis.

CSF1R inhibition depletes brain macrophages and reduces brain virus burden in SIV-infected macaques.

Perivascular macrophages (PVMs) and, to a lesser degree, microglia are targets and reservoirs of HIV and simian immunodeficiency virus (SIV) in the brain. Previously, we demonstrated that colony-stimulating factor 1 receptor (CSF1R) in PVMs was upregulated and activated in chronically SIV-infected rhesus macaques with encephalitis, correlating with SIV infection of PVMs. Herein, we investigated the role of CSF1R in the brain during acute SIV infection using BLZ945, a brain-penetrant CSF1R kinase inhibitor. Apart from three uninfected historic controls, nine Indian rhesus macaques were infected acutely with SIVmac251 and divided into three groups (n = 3 each): an untreated control and two groups treated for 20-30 days with low- (10 mg/kg/day) or high- (30 mg/kg/day) dose BLZ945. With the high-dose BLZ945 treatment, there was a significant reduction in cells expressing CD163 and CD206 across all four brain areas examined, compared with the low-dose treatment and control groups. In 9 of 11 tested regions, tissue viral DNA (vDNA) loads were reduced by 95%-99% following at least one of the two doses, and even to undetectable levels in some instances. Decreased numbers of CD163+ and CD206+ cells correlated significantly with lower levels of vDNA in all four corresponding brain areas. In contrast, BLZ945 treatment did not significantly affect the number of microglia. Our results indicate that doses as low as 10 mg/kg/day of BLZ945 are sufficient to reduce the tissue vDNA loads in the brain with no apparent adverse effect. This study provides evidence that infected PVMs are highly sensitive to CSF1R inhibition, opening new possibilities to achieve viral clearance.

Effectiveness of vaginal probiotics Lactobacillus crispatus chen-01 in women with high-risk HPV infection: a prospective controlled pilot study.

Female genital tract infection with high-risk human papilloma virus (HR-HPV) has the risk of developing into cervical cancer, and there is still a lack of effective therapeutic strategies. Probiotic intervention is considered as a potential intervention for HR-HPV, while exploration into living probiotic preparations for specific diseases remains limited and insufficient. This prospective controlled pilot study was conducted to observe the effect of intravaginal transplantation of a vaginal isolated natural probiotic strain, Lactobacillus crispatus chen-01, on the clearance of high-risk HPV infection. 100 women with high-risk HPV infection were enrolled and randomly divided into placebo group and probiotic treatment group, which received intravaginal transplantation of L. crispatus chen-01. Cervical exfoliated cells were collected 6 months later for detecting DNA load, typing of HPV, and cytological analysis. Our results showed that vaginal transplantation with L. crispatus chen-01 significantly reduced viral load of HPV, ameliorated HPV clearance rate, and improved vaginal inflammation state without causing obvious adverse reactions. Analysis of 16S rRNA sequencing revealed that L. crispatus chen-01 could effectively reconstitute the vaginal microbiota in women with high-risk HPV, which might be one of the underlying mechanisms of the beneficial effect of L. crispatus chen-01 transplantation. Our results suggested that vaginal transplantation of L. crispatus chen-01 might be a promising treatment for patients with high-risk HPV infection.

Multi-centric evaluation of Truenat MTB and MTB-RIF Dx assays for diagnosis of extrapulmonary tuberculosis

Extra-pulmonary TB (EPTB) is difficult to diagnose due to paucibacillary nature of disease. Current study evaluated accuracy of Truenat MTB and MTB-Rif Dx (TN), for detection of Mycobacterium tuberculosis and resistance to rifampicin. Samples were collected from 2103 treatment naive adults with presumptive EPTB, and tested by smear microscopy, liquid culture (LC) (MGIT-960) and GeneXpert MTB/RIF (GX) (Microbiological Reference Standards, MRS). TN results were compared to MRS and Composite Reference Standards (CRS, Microbiology, histopathology, radiology, clinical features prompting decision to treat, response to treatment). CRS grouped patients into 551 confirmed, 1096 unconfirmed, and 409 as unlikely TB. TN sensitivity and specificity was 73.7% and 90.4% against GX. Against LC, Overall sensitivity of GX was 67.6%, while that of TN was 62.3%. Highest sensitivity by TN was observed in pus samples (89%) and highest specificity (92%) in CSF samples, similar to GX. TN sensitivity was better in fluid and biopsy samples and slightly inferior for lymph node aspirates compared to GX. TN sensitivity for RIF resistance detection was slightly superior to GX. TN and GX results were further compared to Clinical Reference Standards. TN detected 170 TB patients initiated on treatment missed by GX, while GX detected 113 such patients missed by TN. Of 124 samples with RIF resistance discordance between GX and TN, GX reported 103/124 as sensitive, 3/124 as indeterminate and 18 as resistant (13/18 samples had low/very low DNA load) while TN reported RIF resistance indeterminate in 103/111 low/very low DNA load samples. Due to paucibacillary nature of EPTB samples, culture yield was poor and phenotypic drug susceptibility testing failed to resolve the discordance. The study establishes TN at par with GX and can be utilized for quick and accurate diagnosis of EPTB.

Early change of plasma Epstein-Barr virus DNA load and the viral lytic genome level could positively predict clinical outcome in recurrent or metastatic nasopharyngeal carcinoma receiving anti-programmed cell death 1 monotherapy

PurposePatients with recurrent or metastatic nasopharyngeal carcinoma (RM-NPC) have proven benefit from anti-programmed cell death 1 (anti-PD-1) monotherapy. Here, we retrospectively analyze the association of plasma Epstein-Barr virus (EBV) DNA load and tumor viral lytic genome with clinical outcome from 2 registered phase I trials.MethodsPatients with RM-NPC from Checkmate 077 (nivolumab phase I trial in China) and Camrelizumab phase I trial between March 2016 and January 2018 were enrolled. Baseline EBV DNA titers were tested in 68 patients and EBV assessment was performed in 60 patients who had at least 3 post-baseline timepoints of EBV data and at least 1 post-baseline timepoint of radiographic assessment. We defined “EBV response” as 3 consecutive timepoints of load below 50% of baseline, and “EBV progression” as 3 consecutive timepoints of load above 150% of baseline. Whole-exome sequencing was performed in 60 patients with available tumor samples.ResultsWe found that the baseline EBV DNA load was positively correlated with tumor size (spearman p < 0.001). Both partial response (PR) and stable disease (SD) patients had significantly lower EBV load than progression disease (PD) patients. EBV assessment was highly consistent with radiographic evaluation. Patients with EBV response had significantly improved overall survival (OS) than patients with EBV progression (log-rank p = 0.004, HR = 0.351 [95% CI: 0.171–0.720], median 22.5 vs. 11.9 months). The median time to initial EBV response and progression were 25 and 36 days prior to initial radiographic response and progression, respectively. Patients with high levels of EBV lytic genomes at baseline, including BKRF2, BKRF3 and BKRF4, had better progression-free survival (PFS) and OS.ConclusionIn summary, early clearance of plasma EBV DNA load and high levels of lytic EBV genes were associated with better clinical outcome in patients with RM-NPC receiving anti-PD-1 monotherapy.

Performance evaluation of the Alinity m system for quantifying cytomegalovirus DNA in samples of the respiratory, gastrointestinal, and urinary tract.

Quantitation of cytomegalovirus (CMV) DNA load in specimens other than blood such as bronchoalveolar lavages, intestinal biopsies, or urine has become a common practice as an ancillary tool for the diagnosis of CMV pneumonitis, intestinal disease, or congenital infection, respectively. Nevertheless, most commercially available CMV PCR platforms have not been validated for CMV DNA detection in these specimen types. In this study, a laboratory-developed test based on Alinity m CMV ("Alinity LDT") was evaluated. Reproducibility assessment using spiked bronchial aspirate (BAS) or urine samples showed low standard deviations of 0.08 and 0.27 Log IU/mL, respectively. Evaluating the clinical performance of Alinity LDT in comparison to a laboratory-developed test based on RealTime CMV ("RealTime LDT") showed good concordance across 200 clinical specimens including respiratory specimens, intestinal biopsies, urine, and stool. A high Pearson's correlation coefficient of r = 0.92, a low mean bias of -0.12 Log IU/mL, a good qualitative agreement of 90%, and a Cohen's kappa value of 0.76 (substantial agreement) were observed. In separate analyses of the sample types BAS, tracheal aspirates, bronchoalveolar lavage, biopsies, and urine, the assay results correlated well between the two platforms with r values between 0.88 and 0.99 and a bias <0.5 Log IU/mL. Overall, the fully automated, continuous, random access Alinity LDT yielded good reproducibility, high concordance, and good correlation to RealTime LDT in respiratory, gastrointestinal, and urine samples and may enhance patient management with rapid result reporting.IMPORTANCEIn transplant recipients, a major cause for morbidity and mortality is end-organ disease by primary or secondary CMV infection of the respiratory or gastrointestinal tract. In addition, sensorineural hearing loss and neurodevelopmental abnormalities are frequent sequelae of congenital CMV infections in newborns. Standard of care for highly sensitive detection and quantitation of the CMV DNA load in plasma and whole blood specimens is real-time PCR testing. Beyond that, there is a need for quantitative determination of CMV DNA levels in respiratory, gastrointestinal, and urinary tract specimens using a highly automated, random access CMV PCR assay with a short turnaround time to enable early diagnosis and treatment. In the present study, clinical performance of the fully automated Alinity m analyzer in comparison to the current RealTime LDT assay was evaluated in eight different off-label sample types.