Hu14.18K.322A Causes Direct Cell Cytotoxicity and Synergizes with Induction Chemotherapy in High-Risk Neuroblastoma.

The disialoganglioside, GD2, is a promising therapeutic target due to its overexpression in certain tumors, particularly neuroblastoma (NB), with limited expression in normal tissues. Despite progress, the intricate mechanisms of action and the full spectrum of the direct cellular responses to anti-GD2 antibodies remain incompletely understood. In this study, we examined the direct cytotoxic effects of the humanized anti-GD2 antibody hu14.18K322A (hu14) on NB cell lines, by exploring the associated cell-death pathways. Additionally, we assessed the synergy between hu14 and conventional induction chemotherapy drugs. Our results revealed that hu14 treatment induced direct cytotoxic effects in CHLA15 and SK-N-BE1 cell lines, with a pronounced impact on proliferation and colony formation. Apoptosis emerged as the predominant cell-death pathway triggered by hu14. Furthermore, we saw a reduction in GD2 surface expression in response to hu14 treatment. Hu14 demonstrated synergy with induction chemotherapy drugs with alterations in GD2 expression. Our comprehensive investigation provides valuable insights into the multifaceted effects of hu14 on NB cells, shedding light on its direct cytotoxicity, cell-death pathways, and interactions with induction chemotherapy drugs. This study contributes to the evolving understanding of anti-GD2 antibody therapy and its potential synergies with conventional treatments in the context of NB.

SARS-CoV-2 vaccination response in pediatric oncology patients.

BACKGROUND: There remains limited knowledge about the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in pediatric oncology patients, which is essential to provide counseling and risk adaptation in this vulnerable population. The goal of this study was to understand immunogenicity after vaccination in pediatric oncology patients, and determine if certain clinical factors impacted response. METHODS: Patients 0-25 years of age with a diagnosis of cancer and actively receiving therapy were enrolled on study. We excluded patients who were completely vaccinated prior to their cancer diagnosis. Blood samples were collected pre-vaccination, as well as 2, 4-6, and 8-12 weeks after vaccination. Healthy children who were fully vaccinated enrolled as controls. Clinical data and complete blood counts around time of vaccination were collected. To study B- and T-cell immunity, we measured neutralizing antibodies by enzyme-linked immunoassay and interferon gamma secretion by enzyme-linked immunospot, respectively. RESULTS: Twenty-six patients enrolled on study, for which 11 were evaluable oncology patients and seven were healthy controls. Adequate B-cell response was seen in 36.4% of patients, and adequate T-cell response in 77.8% of patients. Numbers were too small to detect differences based on malignancy type. There was no differences in immunity based on absolute lymphocyte count (ALC) or intensity of therapy. CONCLUSION: Pediatric oncology patients have a suboptimal immune response to SARS-CoV-2 vaccination. Booster doses will be imperative to provide optimal protection against COVID-19; however, blood counts may not be a useful guide to optimize the time of administration.

Second Generation Small Molecule Inhibitors of Gankyrin for the Treatment of Pediatric Liver Cancer.

Background: Gankyrin, a member of the 26S proteasome, is an overexpressed oncoprotein in hepatoblastoma (HBL) and hepatocellular carcinoma (HCC). Cjoc42 was the first small molecule inhibitor of Gankyrin developed; however, the IC50 values of >50 µM made them unattractive for clinical use. Second-generation inhibitors demonstrate a stronger affinity toward Gankyrin and increased cytotoxicity. The aim of this study was to characterize the in vitro effects of three cjoc42 derivatives. Methods: Experiments were performed on the HepG2 (HBL) and Hep3B (pediatric HCC) cell lines. We evaluated the expression of TSPs, cell cycle markers, and stem cell markers by Western blotting and/or real-time quantitative reverse transcription PCR. We also performed apoptotic, synergy, and methylation assays. Results: The treatment with cjoc42 derivatives led to an increase in TSPs and a dose-dependent decrease in the stem cell phenotype in both cell lines. An increase in apoptosis was only seen with AFM-1 and -2 in Hep3B cells. Drug synergy was seen with doxorubicin, and antagonism was seen with cisplatin. In the presence of cjoc42 derivatives, the 20S subunit of the 26S proteasome was more available to transport doxorubicin to the nucleus, leading to synergy. Conclusion: Small-molecule inhibitors for Gankyrin are a promising therapeutic strategy, especially in combination with doxorubicin.

Evaluation of angiogenic signaling molecules associated with reactive thrombocytosis in an iron-deficient rat model.

BACKGROUND: Iron deficiency anemia (IDA)-induced reactive thrombocytosis can occur in children and adults. The underlying mechanism for this phenomenon is indeterminate. Traditional cytokines such as thrombopoietin (TPO), interleukin-6 (IL-6), and IL-11 involved in megakaryopoiesis have not been shown to be the cause. Recent studies suggest that growth factors and signaling molecules involved with angiogenesis influence the proliferation and differentiation of megakaryocytes. METHODS: We investigated the possible association between angiogenic cytokines with reactive thrombocytosis due to IDA in an iron-deficient (ID) rat model. Complete blood count, iron panels, and TPO levels were measured at baseline and 5 weeks later in both control (C) and ID rats. Angiogenic cytokines were evaluated in the bone marrow in all rats. RESULTS: We successfully induced IDA in our rats by phlebotomy and reduced iron diet. We did not find an increase of TPO in ID rats. A review of the bone marrow showed an increase in the number of megakaryocytes, vascular structures, as well as increased intensity of stain for vascular endothelial growth factor (VEGF), and CXC chemokine receptor 4 (CXCR4) in rats with IDA compared to controls. CONCLUSIONS: Our results of histological bone marrow data suggest an important role for angiogenesis in the development of IDA-induced thrombocytosis. IMPACT: Thrombocytosis is common with IDA in both children and adults, but the mechanism is unclear. We confirmed that TPO is not the major driver of iron deficiency-associated thrombocytosis. We confirmed the increase in the number of megakaryocytes in the bone marrow despite stable TPO levels. We provided evidence supporting an important role of angiogenesis in megakaryocytopoiesis/thrombopoiesis with increased vascular structures and angiogenic cytokines in the bone marrow of iron-deficient rats. The demonstration that angiogenesis may play an important role in secondary thrombocytosis could lead to a new approach in treating symptomatic reactive thrombocytosis by targeting angiogenesis.

Circular dumbbell miR-34a-3p and -5p suppresses pancreatic tumor cell-induced angiogenesis and activates macrophages.

Angiogenesis is a tightly regulated biological process by which new blood vessels are formed from pre-existing blood vessels. This process is also critical in diseases such as cancer. Therefore, angiogenesis has been explored as a drug target for cancer therapy. The future of effective anti-angiogenic therapy lies in the intelligent combination of multiple targeting agents with novel modes of delivery to maximize therapeutic effects. Therefore, a novel approach is proposed that utilizes dumbbell RNA (dbRNA) to target pathological angiogenesis by simultaneously targeting multiple molecules and processes that contribute to angiogenesis. In the present study, a plasmid expressing miR-34a-3p and -5p dbRNA (db34a) was constructed using the permuted intron-exon method. A simple protocol to purify dbRNA from bacterial culture with high purity was also developed by modification of the RNASwift method. To test the efficacy of db34a, pancreatic cancer cell lines PANC-1 and MIA PaCa-2 were used. Functional validation of the effect of db34a on angiogenesis was performed on human umbilical vein endothelial cells using a tube formation assay, in which cells transfected with db34a exhibited a significant reduction in tube formation compared with cells transfected with scrambled dbRNA. These results were further validated in vivo using a zebrafish angiogenesis model. In conclusion, the present study demonstrates an approach for blocking angiogenesis using db34a. The data also show that this approach may be used to targeting multiple molecules and pathways.

Structural modification of the aryl sulfonate ester of cjoc42 for enhanced gankyrin binding and anti-cancer activity.

Gankyrin is an oncogenic protein involved in various biological processes, such as cellular growth and proliferation. Its overexpression in certain cancers results in an increase of gankyrin-mediated protein-protein interactions (PPIs), leading to cancer proliferation. To date, only one small molecule (cjoc42) has been identified to bind gankyrin, which simultaneously inhibits its interaction with the 26S proteasome. Despite this advance, 2nd generation inhibitors are needed to improve gankyrin binding and cellular efficacy. To this end, an extensive SAR for the aryl sulfonate ester moiety of the cjoc42 scaffold was explored, and showed that substitutions at the 2-, 3-, and 4-positions manifested significant increases in gankyrin binding, resulting in the most potent binders of gankyrin to date. Subsequent cell-based assay evaluation of our derivatives demonstrated antiproliferative activity against pediatric liver cancer cell lines Hep3B and HepG2, which was not previously observed for cjoc42.

Evaluating the PGMY-centre hospitalier universitaire vaudois assay as a cost-effective tool for human papillomavirus genotyping in HIV-infected women.

Aims: Cervical cancer is one of the leading causes of cancer among women, worldwide. HIV-positive women tend to have persistent infection and infection with multiple human papillomavirus (HPV) types. There is a need for affordable HPV DNA tests as viable alternatives to the existing costly commercial assays. The aim of the study was to establish PGMY-CHUV reverse hybridization assay as a cost-effective tool for HPV genotyping. Study Design: This was a prospective study conducted in a tertiary care centre from March 2011 to July 2012. Subjects and Methods: Fifty cervical brush samples from HIV-infected women and 43 WHO reference samples were tested by both the CHUV assay and linear array (LA). Results: The CHUV assay in comparison to the LA showed a sensitivity of 91%, specificity of 52% and a moderate agreement for all samples that were compared. However, most high-risk HPV types were identified amongst the clinical samples, and the entire range of genotypes in the WHO reference panel was detected. Statistical Analysis: The accuracy indices such as sensitivity, specificity, positive predictive value and negative predictive value were calculated. The level of agreement (kappa value) between the two assays was also calculated. Conclusion: The CHUV assay had an acceptable sensitivity, but it lacked specificity for HPV detection. Despite the lower rates of detection of multiple infections from clinical samples, better results were obtained with the WHO reference samples and the ability of the assay to identify the entire range of genotypes suggests that it can be an efficient tool for genotyping.

Targeting the Expression of Cathepsin B Using CRISPR/Cas9 System in Mammalian Cancer Cells.

Cathepsin B belongs to a family of cathepsins and plays an important role in normal physiological functions in the cell. However, overexpression of cathepsin B has been associated with different malignancies, and this has made it an attractive pharmacological target. The advent of CRISPR-Cas9 technology has allowed researchers to efficiently knock down genes with very less nonspecific activity compared to earlier methods. The protocol described below will enable investigators to develop cathepsin B knockdown stable cells and explains ways to study the knockdown.

Chronic radiation exposure of neuroblastoma cells reduces nMYC copy number.

Neuroblastoma accounts for >15% of cancer-associated mortalities of children in the USA. Despite aggressive treatment regimens, the long-term survival for these children remains <40%. The identification of v-Myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (nMYC) gene amplification during diagnosis is associated with poor prognosis in neuroblastoma. There are limited studies examining changes in nMYC copy numbers in response to therapy and its biological effect on cancer cells. The aim of the present study was to evaluate the effect of radiation on nMYC expression and amplification status in high-risk neuroblastoma. The effect of acute (5 Gy) and chronic (25 Gy) radiation on two nMYC-amplified cell lines, SK-N-BE (2) and NB-1691, was investigated. The results demonstrate that, following chronic but not acute radiation, the two cell lines regained their proliferation potential similar to the controls. This increased proliferation was characterized by loss of nMYC mRNA and protein expression. It was also revealed that nMYC loss was accompanied by nuclear localization of c-Myc. Using fluorescent in situ hybridization and quantitative polymerase chain reaction analysis, the results of the present study demonstrated that chronic radiation causes a severe loss of nMYC gene copy number. The present study is the first to provide experimental evidence that prolonged radiation therapy affects nMYC gene copy number in high-risk neuroblastoma but does not significantly improve the prognostic outlook.

SPARC overexpression suppresses radiation-induced HSP27 and induces the collapse of mitochondrial Δψ in neuroblastoma cells.

Neuroblastoma is the cause of >15% of cancer-associated mortality in children in the USA. Despite aggressive treatment regimens, the long-term survival rate for these children remains at <40%. The current study demonstrates that secreted protein acidic and rich in cysteine (SPARC) suppresses radiation-induced expression of heat shock protein 27 (HSP27) in vivo and suppresses mitochondrial membrane potential (Δψ) in neuroblastoma cells. In the present study, the overexpression of SPARC in SK-N-BE(2) and NB1691 neuroblastoma cell lines suppresses radiation-induced G2M cell cycle arrest, proliferation, HSP27 expression (in vitro and in vivo) and induces the collapse of the mitochondrial Δψ. Gene ontology analysis demonstrated that the overexpression of SPARC combined with irradiation, induces the expression of dissimilar molecular function genes in SK-N-BE(2) and NB1691 cells, providing evidence of a dissimilar response signaling pathway. These results demonstrate that overexpression of SPARC suppresses radiation-induced HSP27 expression in neuroblastoma cells and the combination of SPARC and radiation induces the expression of protein 21, but suppresses neuroblastoma tumor density in in vivo mouse models. SPARC also induces mitochondrial Δψ collapse in SK-N-BE(2) and NB1691 neuroblastoma cells.

Chemoresistance in pancreatic cancer: Emerging concepts.

Pancreatic cancer is one of the most lethal types of cancer in the world. The incidence of pancreatic cancer increases each year with no significant decrease in mortality. Pancreatic cancer is a complex disease, and this complexity is partly attributed to late diagnosis, an aggressive phenotype, environmental factors and lack of effective treatment options. Surgical resection followed by adjuvant chemotherapy is the treatment of choice for early stage cancer, whereas gemcitabine is the standard first line therapy for patients with advanced stage disease. Treatment regimens comprising folinic acid, 5-fluorouracil, irinotecan, oxaliplatin and nab-paclitaxel have demonstrated modest effects in improving median survival rates. A number of other chemotherapeutics are currently undergoing clinical trials as components of combination therapies with gemcitabine. An increasing number of novel molecular targets and cellular pathways are being identified, which highlights the complexity of this disease. The development of chemoresistance to gemcitabine is multifactorial and there exists an interplay between pancreatic cancer cells, the tumor microenvironment and cancer stem cells. These components appear to be governed by a complex network of non-coding RNAs such as micro RNAs and long non-coding RNAs. In the present study, studies describing previous research on the understanding of the factors associated with the development of chemoresistance to gemcitabine in pancreatic cancer are reviewed. A comprehensive understanding of the multiple pathways of chemoresistance is key to develop next generation therapeutics to pancreatic cancer.

Genital Human Papillomavirus Infection in Indian HIV-Seropositive Men Who Have Sex With Men.

BACKGROUND: The incidence of penile cancer in Indian men is high. Little is known about genital human papillomavirus (HPV) infection in Indian HIV-seropositive men who have sex with men (MSM), a population that may be at particularly high risk for genital HPV infection and, potentially, penile cancer. In this study, we assessed the prevalence and risk factors for genital HPV infection in this population. DESIGN AND METHODS: Three hundred HIV-seropositive MSM were recruited from 2 clinical sites in India. They were tested for genital HPV infection using L1 HPV DNA polymerase chain reaction with probes specific for 29 types and a mixture of 10 additional types. Participants received an interviewer-administered questionnaire that included questions on demographics and behaviors. RESULTS: Human papillomavirus data were available from 299 participants. The prevalence of any HPV type in the penis and scrotum was 55% and 54%, respectively. Human papillomavirus type 35 was the most common oncogenic HPV type followed by HPV-16. In multivariate analysis, being the insertive partner with 100+ male partners increased the odds of any penile HPV infection compared with not being insertive with any partners (odds ratio, 2.5; 95% confidence interval, 1.3-5.1). Circumcision was protective against penile HPV infection (odds ratio, 0.39; 95% confidence interval, 0.19-0.76). CONCLUSIONS: The prevalence of penile and scrotal HPV infection was high among Indian HIV-seropositive MSM. The most common oncogenic HPV type in this population, HPV-35, is not included in any currently available HPV vaccines. Insertive anal sex with men and lack of circumcision were the primary risk factors for penile HPV infection in this population.

Comparison of Three Different Hepatitis C Virus Genotyping Methods: 5'NCR PCR-RFLP, Core Type-Specific PCR, and NS5b Sequencing in a Tertiary Care Hospital in South India.

BACKGROUND: Based on genetic heterogeneity, hepatitis C virus (HCV) is classified into seven major genotypes and 64 subtypes. In spite of the sequence heterogeneity, all genotypes share an identical complement of colinear genes within the large open reading frame. The genetic interrelationships between these genes are consistent among genotypes. Due to this property, complete sequencing of the HCV genome is not required. HCV genotypes along with subtypes are critical for planning antiviral therapy. Certain genotypes are also associated with higher progression to liver cirrhosis. METHODS: In this study, 100 blood samples were collected from individuals who came for routine HCV genotype identification. These samples were used for the comparison of two different genotyping methods (5'NCR PCR-RFLP and HCV core type-specific PCR) with NS5b sequencing. RESULTS: Of the 100 samples genotyped using 5'NCR PCR-RFLP and HCV core type-specific PCR, 90% (κ = 0.913, P < 0.00) and 96% (κ = 0.794, P < 0.00) correlated with NS5b sequencing, respectively. Sixty percent and 75% of discordant samples by 5'NCR PCR-RFLP and HCV core type-specific PCR, respectively, belonged to genotype 6. All the HCV genotype 1 subtypes were classified accurately by both the methods. CONCLUSION: This study shows that the 5'NCR-based PCR-RFLP and the HCV core type-specific PCR-based assays correctly identified HCV genotypes except genotype 6 from this region. Direct sequencing of the HCV core region was able to identify all the genotype 6 from this region and serves as an alternative to NS5b sequencing.

SPARC overexpression combined with radiation retards angiogenesis by suppressing VEGF-A via miR­410 in human neuroblastoma cells.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N­BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the overexpression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410.

The 'SPARC' of life: Analysis of the role of osteonectin/SPARC in pancreatic cancer (Review).

Pancreatic ductal adenocarcinoma (PDAC) is one of the most clinically challenging cancers to manage. An estimated 48,960 people will be diagnosed with pancreatic cancer in 2015, of that population, 94% are projected to perish within 5 years. These dismal survival rates can be attributed, in part, to an advanced diagnosis occurring in 80% of cases. The heterogeneous and dynamic microenvironment of pancreatic cancer, and the lack of both specific risk factors and efficacious screening tools contribute to the challenge of diagnosing pancreatic cancer in its early stages. These clinical challenges have directed research into the unique characteristics that define PDAC. Recently, there has been an increased focus on the interaction of tumor cells with their microenvironment in the hope of identifying new therapeutic targets. One of the most promising avenues in this new vein of research is targeting protein communication between the cancer cells and the extracellular matrix. The secreted protein acidic and rich in cysteine (SPARC) is one such extracellular matrix protein that has shown potential as a therapeutic target due to its influence on PDAC invasion and metastasis. In this review, we discuss the complex interaction of SPARC with PDAC cells and its potential to guide treatment and eventually improve the survival of patients diagnosed with this devastating disease.

Prevalence of Anal HPV Infection Among HIV-Positive Men Who Have Sex With Men in India.

BACKGROUND: India has a large population of HIV-positive individuals, including men who have sex with men (MSM), and the incidence of human papillomavirus (HPV)-related cancers is high. In developed countries, HIV-positive MSM exhibit the highest prevalence of anal HPV infection and incidence of anal cancer. Little is known about anal HPV infection in HIV-positive Indian MSM. METHODS: We evaluated 300 HIV-positive MSM from 2 cities in India. Men were tested for anal HPV infection using L1-HPV DNA polymerase chain reaction with probes specific for 29 types and a mixture of 10 additional types. CD4 level and plasma HIV viral load were measured. Participants completed an interviewer-administered questionnaire including a sexual history. RESULTS: The prevalence of anal HPV was 95% (95% confidence interval: 91% to 97%). The 3 most common types were HPV 35 (20%), HPV 16 (13%), and HPV 6/11 (13%). History of taking antiretroviral medications decreased risk of anal HPV 16 infection [relative risk (RR): 0.6 (0.4-1.0)]. Having an increased number of vaginal sex partners lowered risk of any anal HPV infection. Ever having receptive sex increased risk of any anal HPV [RR: 1.2 (1.1-1.4)] and anal HPV 16 [RR: 6.5 (1.8-107)]. CONCLUSIONS: Almost all Indian HIV-positive MSM had anal HPV infection. The prevalence of HPV 16 was lower and the prevalence of other oncogenic HPV types was higher than in similar populations in North America and Europe. Vaccine-based prevention strategies for HPV infection in India should consider potential differences in HPV type distribution among HIV-infected MSM when designing interventions.

Performance characteristics and comparison of Abbott and artus real-time systems for hepatitis B virus DNA quantification.

Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log(10) IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log(10) IU/ml and limits of agreement of -0.91 to 1.11 log(10) IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B.

Detection and quantitation of HPV 16 and 18 in plasma of Indian women with cervical cancer.

OBJECTIVE: HPV infection is a necessary but insufficient cause of cervical cancer. The significance of HPV DNA in blood however is debatable because of variable detection rates due to the differences in the methodology used. The aim of this study was to detect and quantitate HPV 16 and 18 plasma viremia in women with cervical neoplasia. METHODS: HPV DNA was detected in cervical tissue using consensus PGMY primers and genotyped using reverse line blot hybridization. HPV 16 and 18 quantitation in tissue and detection and quantitation in plasma was performed using sensitive real time PCRs targeting E6/E7 region of HPV 16/18 genome respectively. Results were correlated with viral loads in corresponding tissue and with clinical disease stage. RESULTS: Viremia was detected in 56.4% of HPV 16 positive women and 20% of HPV 18 positive women. The prevalence of HPV 16 DNA in plasma increased with advancing disease stage (p=0.001), although HPV 16 absolute plasma viral load was not significantly associated with advancing disease stage (p=0.281). There was no correlation between absolute plasma viral load and viral load in corresponding cervical tissue (Spearman's rho=0.184, p=0.187). The prevalence of HPV 18 viremia and absolute HPV 18 plasma viral load were not associated with advancing disease stage (p=0.620, p=0.508). CONCLUSION: The presence of HPV 16 in plasma is a marker of advancing cervical disease.

Do we need an 'in-house' neutralization assay for confirmation of hepatitis B surface antigen? Answers from a tertiary care hospital in India.

BACKGROUND AND AIMS: Hepatitis B surface antigen (HBsAg) is an important serological marker for diagnosis of hepatitis B virus (HBV) infection. Commercial kits for detection of HBsAg emphasize confirmation by neutralization assays. In this study, we have standardized an 'in-house' neutralization test for HBsAg confirmation. METHODS: Among 6684 HBsAg-positive samples, 615 were subjected to an 'in-house' HBsAg neutralization test (NT). Of these, 91 (100%) high-reactive samples (optical density [OD] 1.000-3.000) and 286 (93%) of 289 low-reactive samples (OD < 1.000) were neutralized, and 235 (100%) grey-zone reactive samples were 'in-house' NT negative. Eighty-four samples of varying reactivities that were tested by the 'in-house' NT were compared with a commercial NT (AxSYM, Abbott). RESULTS: The 'in-house' NT showed an excellent agreement (kappa = 0.83, P < 0.001) with the commercial confirmatory assay. The sensitivity, specificity, positive and negative predictive values were 90%, 94%, 96% and 87%, respectively. CONCLUSION: The enzyme immunoassay-based 'in-house' HBsAg neutralization assay is a feasible alternative to the commercial HBsAg confirmatory assay. This technique is easily adaptable, cost-effective and reliable for the confirmation of HBsAg in a low resource setting, enhancing the overall quality of HBsAg screening.