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Objective(s): Suppression of viral replication in patients on antiretroviral therapy (ART) is determined by plasma viral load (pVL) measurement. Whenever pVL reaches values below the limit of quantification, the qualitative parameter 'target detected' or 'target not detected' is available but often not reported to the clinician. We investigated whether qualitative pVL measurements can be used to estimate the viral reservoir size. Design: The study recruited 114 people with HIV (PWH) who are stable on ART between 2016 and 2018. The percentage of pVL measurements qualitatively reported as 'target detected' (PTD) within a 2-year period was calculated. Methods: t-DNA and US-RNA were used to estimate viral reservoir size and were quantified on peripheral blood mononuclear cells (PBMCs) using droplet digital PCR. Results: A median of 6.5 pVL measurements over a 2-year period was evaluated for each participant to calculate PTD. A positive correlation was found between t-DNA and PTD (r = 0.24; P = 0.011) but not between US-RNA and PTD (r = 0.1; P = 0.3). A significantly lower PTD was observed in PWH with a small viral reservoir, as estimated by t-DNA less than 66 copies/10(6) PBMCs and US-RNA less than 10 copies/10(6) PBMCs, compared with PWH with a larger viral reservoir (P = 0.001). We also show that t-DNA is detectable whenever PTD is higher than 56% and that ART regimen does not affect PTD. Conclusion: Our study shows that PTD provides an efficient parameter to preselect participants with a small viral reservoir based on already available pVL data for future HIV cure trials.

Publisher's version (útgefin grein), It is valuable to establish a population‐based prevaccination baseline distribution of human papillomavirus (HPV) types among women with high‐grade cervical intraepithelial neoplasia (CIN) grade 2 or 3 and cervical cancer in order to assess the potential impact of HPV vaccination. In four countries (Denmark, Norway, Sweden, and Iceland), we collected consecutive series of cervical cancers (n = 639) and high‐grade precancerous cervical lesions (n = 1240) during 2004‐2006 before implementation of HPV vaccination and subjected the specimens to standardized HPV genotyping. The HPV prevalence was 82.7% (95% confidence interval [CI] 79.0‐86.4) in CIN2, 91.6% (95% CI 89.7‐93.5) in CIN3, and 86.4% (95% CI 83.7‐89.1) in cervical cancer. The most common HPV types in CIN2/3 were HPV16 (CIN2: 35.9%, 95% CI 31.2‐40.6; CIN3: 50.2%, 95% CI 46.8‐53.6) and HPV31 (CIN2: 10.9%, 95% CI 7.8‐13.9; CIN3: 12.1%, 95% CI 9.9‐14.3), while HPV16 and HPV18 were the most frequent types in cervical cancer (48.8%, 95% CI 44.9‐52.7 and 15.3%, 95% CI 12.5‐18.1, respectively). The prevalence of HPV16/18 decreased with increasing age at diagnosis in both CIN2/3 and cervical cancer (P < 0.0001). Elimination of HPV16/18 by vaccination is predicted to prevent 42% (95% CI 37.0‐46.7) of CIN2, 57% (95% CI 53.8‐60.5) of CIN3 and 64% (95% CI 60.3‐67.7) of cervical cancer. Prevention of the five additional HPV types HPV31/33/45/52/58 would increase the protection to 68% (95% CI 63.0‐72.2) in CIN2, 85% (95% CI 82.4‐87.2) in CIN3 and 80% (95% CI 77.0‐83.2) in cervical cancer. This study provides large‐scale and representative baselines for assessing and evaluating the population‐based preventive impact of HPV vaccination., We thank Cecilia Wahlström and Kia Sjölin for assistance with the HPV genotyping. SKK received lecture fee from Merck and Sanofi Pasteur MSD, scientific advisory board fee from Merck, and unrestricted research grants through her institution from Merck. JD reports having received research grants to his institution for the funding of the study. CM received lecture fees and travel grants from Sanofi Pasteur MSD. CL and MH report that their institution received a grant from Merck. KLL is a full‐time employee of Merck & Co. Inc and owns stocks and options of Merck. MN received research grants from MSD Norway/Merck through the affiliating institute. CDC, SG, LT, KS, and BTH report no conflicts of interest.

Rationale: Macrophage activation syndrome (MAS) is a rare life-threatening condition characterized by cytokine-mediated tissue injury and multiorgan dysfunction. Patient Concerns: We describe the unique case of young man who developed MAS as the sole manifestation of an otherwise paucisymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Diagnoses: Clinical and biological criteria led to the diagnosis of MAS; cytokine profile was highly suggestive reverse transcription polymerase chain reaction for SARS-CoV-2 in nasopharyngeal swabs was negative, but serum anti-SARS-CoV-2 immunoglobulin A and immunoglobulin G resulted positive leading to the diagnosis of SARS-CoV-2 infection. Interventions: The patient was treated with empiric antibiotic and hydroxychloroquine. Outcomes: Clinical improvement ensued. At follow-up, the patient is well. Lesson: SARS-CoV-2 infection may trigger develop life-threatening complications, like MAS. This can be independent from coronavirus disease 2019 gravity.

INTRODUCTION: As the coronavirus disease 2019 (COVID-19) epidemic evolves and test strategies change, understanding the concepts of testing (gold standard and test performance measures) becomes essential. The challenge of any novel disease is that the gold standard has yet to be defined.METHODS: We reanalysed published data on real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) of severe acute respiratory syndrome coronavirus-2 to illustrate how predictive values vary with disease prevalence, sensitivity (set to values between 30% and 95%) and specificity (set to 99% or 99.98%). We used published data on chest CT and RT-qPCR to examine the potential of latent class analysis to estimate the sensitivity and specificity of RT-qPCR when no single gold standard exists.RESULTS: For the various sensitivity values, the negative predictive value of a RT-qPCR test remained above 92% until a COVID-19 prevalence of > 10%. The positive predictive value (PPV) was more variable. For a sensitivity of 95% and a specificity of 99%, the PPV was less-than 10% at a prevalence of 0.1%, increasing to about 90% at a prevalence of 10%. This improved to a PPV of 85% and almost 100%, respectively, when specificity increased to 99.98%. In a restricted latent class analysis, the sensitivity was 97.1% and the specificity was 99.9%, which is similar to figures from the Danish Health Authority. However, derived predictive values depended on model specification.CONCLUSIONS: A high risk of false-positives should be considered when extending the testing strategy, whereas false-negatives may occur during local outbreaks. This may have consequences for, e.g., containment strategies and research. A confirmatory test (e.g., demonstrating seroconversion or repeated RT-qPCR) may be warranted.FUNDING: none.TRIAL REGISTRATION: not relevant.

HIV-1 infection cannot be cured due to the presence of the latent reservoir (LR). Novel cure or treatment strategies, such as "shock and kill" or therapeutic vaccination, aim to reduce or eradicate the LR. Cure strategies utilise robust DNA quantification assays to measure the change in the LR in low copy scenarios. No standard assay exists, which impedes the reliable comparison of results from different therapy and vaccine trials and HIV-1 total DNA quantification methods have not been previously compared. The HIV-1 long terminal repeat (LTR) has been shown to be the best target for DNA quantification. We have analysed two HIV-1 quantification assays, both able to differentiate between the variant HIV-1 DNA forms via the use of pre-amplification and primers targeting LTR. We identify a strong correlation (r=0.9759, P

A recent study conducted in blood has proposed CD32 as the marker identifying the "elusive" HIV reservoir. We have investigated the distribution of CD32 + CD4 T cells in blood and lymph nodes (LNs) of HIV-1-uninfected subjects and viremic untreated and long-term-treated HIV-1-infected individuals and their relationship with PD-1 + CD4 T cells. The frequency of CD32 + CD4 T cells was increased in viremic compared to treated individuals in LNs, and a large proportion (up to 50%) of CD32 + cells coexpressed PD-1 and were enriched within T follicular helper (Tfh) cells. We next investigated the role of LN CD32 + CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32 + and PD-1 + CD4 T cells compared to CD32 - and PD-1 - cells in both viremic and treated individuals, but there was no difference between CD32 + and PD-1 + cells. There was no enrichment of latently infected cells with inducible HIV-1 in CD32 + versus PD-1 + cells in antiretroviral therapy (ART)-treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32 + PD-1 + CD4 T cells were significantly enriched in cell-associated HIV RNA compared to CD32 - PD-1 - (averages of 5.2-fold in treated individuals and 86.6-fold in viremics), CD32 + PD-1 - (2.2-fold in treated individuals and 4.3-fold in viremics), and CD32 - PD-1 + (2.2-fold in ART-treated individuals and 4.6-fold in viremics) cell populations. Similar levels of HIV-1 transcription were found in CD32 + PD-1 - and CD32 - PD-1 + CD4 T cells. Interestingly, the proportion of CD32 + and PD-1 + CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and coexpression of CD32 and PD-1, the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.IMPORTANCE The existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication-competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells coexpressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

We assessed the relation of inflammatory and coagulation biomarkers with electrocardiographic (ECG) evidence of myocardial ischemia. High-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and D-dimer levels were measured at study entry for 3,085 human immunodeficiency virus-infected participants (mean age 44 years; 26.4% women; 24.6% black) in the Strategies for Management of Antiretroviral Therapy trial. Logistic regression models were used to examine the associations of these biomarkers with prevalent and incident myocardial ischemia. The latter analyses were performed for 1,411 participants who were randomly assigned to receive continuous antiretroviral therapy during follow-up to suppress the human immunodeficiency virus viral load and had ≥1 ECG reading during the follow-up period. The median hsCRP, IL-6, and D-dimer level was 1.65 μg/ml (interquartile range 0.69 to 4.11), 1.60 pg/ml (interquartile range 1.00 to 2.75), and 0.18 μg/ml (interquartile range 0.11 to 0.32), respectively. At baseline, the prevalence of major or minor Q-QS or ST-T ECG abnormalities was 18.6%. The biomarker levels were associated with prevalent major or minor ischemic abnormalities on the univariate analyses; however, adjustment for traditional risk factors attenuated these associations. The adjusted odds ratio for major or minor ischemic abnormalities and 95% confidence intervals for the greatest versus lowest quartiles was 1.3 (95% confidence interval 0.9 to 1.7) for hsCRP, 1.0 (95% confidence interval 0.7 to 1.3) for IL-6, and 1.1 (95% confidence interval 0.9 to 1.5) for D-dimer. During a median follow-up of 2.3 years, new definite or probable ischemic ECG abnormalities developed in 11.7% of participants receiving continuous antiretroviral therapy. Biomarker levels were not associated with incident abnormalities on unadjusted or adjusted analyses. In conclusion, higher levels of hsCRP, IL-6, and D-dimer were not associated with ischemic ECG abnormalities. Elevated biomarker levels and ECG abnormalities indicating myocardial ischemia might reflect different risk pathways for cardiovascular disease.

Varicella zoster virus (VZV) is responsible for the primary infection chickenpox. After the initial infection, it remains latent but can reactivate, resulting in shingles (herpes zoster). Previous reports have implicated VZV in the pathogenesis of apical periodontitis, but the involvement of the virus has not been investigated fully. The present case describes a patient who suffered from a severe episode of shingles and subsequently developed periapical radiolucencies of all the teeth in the affected nerve distribution. Molecular and culture techniques showed the presence of VZV DNA in the root canal system in the absence of bacteria. This confirms that VZV can cause localized pulp necrosis and apical periodontitis. The lesions healed after endodontic treatment, implying chemomechanical debridement using sodium hypochlorite irrigation and a calcium hydroxide interim dressing may be effective against the virus.

We aimed to investigate the ability of HBsAg plasma level kinetics to predict therapy response by studying 23 children with infancy-acquired chronic hepatitis B (CHB) during combination sequential therapy with lead-in lamivudine (LAM) and add-on interferon-α (IFN-α) [5 responders (R = anti-HBs seroconversion) and 18 nonresponders (NR)] and to assess their relationship with pretreatment intrahepatic HBV-DNA and cccDNA and HBsAg and HBcAg liver expression. Plasma HBsAg levels were measured in samples before (treatment week 0 = TW0), during (TW9, TW28, TW52) and after (follow-up week = FUW24) therapy by Abbott ARCHITECT(®) assay [log10 IU/mL]. Baseline liver HBV-DNA and cccDNA were quantified by real-time TaqMan PCR [log10 copies/ng genomic DNA]. HBsAg and HBcAg liver expression was evaluated by immunostaining of formalin-fixed, paraffin-embedded specimens [number of positive cells/1000 hepatocytes]. All results are presented as medians. Plasma: at baseline, on-treatment and during follow-up, HBsAg levels were lower in R than NR (TW0: 4.36 vs 4.75;TW28: 2.44 vs 4.35;TW52: 0 vs 4.08 and FUW24: 0.17 vs 4.35, all P < 0.05). Liver: baseline HBV-DNA (3.82 vs 4.71, P = 0.16) and cccDNA (1.98 vs 2.26, P = 0.18) tended to be lower in R than NR, HBsAg expression was lower in R than NR (0.5 vs 4.7, P = 0.03), and HBcAg expression was similar between R and NR. There were positive correlations between plasma HBsAg levels and liver HBV-DNA (r = 0.44, P = 0.04), cccDNA (r = 0.41, P = 0.04) and HBsAg liver expression (r = 0.38, P = 0.05). Lower baseline HBsAg plasma levels, lower HBsAg expression in liver and on-treatment decline of plasma HBsAg levels heralds HBsAg clearance and response to treatment in tolerant children with CHB.

BACKGROUND: Human Papilloma Virus (HPV) infection is the most common sexually transmitted viral infection worldwide. HPV is highly prevalent in sexually active men who have sex with men (MSM) and has been associated with anal cancer, penile cancer, and oropharyngeal cancer. METHODS: From March to September 2011, we conducted a cross-sectional study of HPV prevalence among MSM above age 18 years. Participants were recruited using respondent driven sampling at Clinica Cayetano Heredia. All participants provided anal, genital, and oral samples for HPV DNA testing, and blood for HIV and HPV antibody testing. RESULTS: A total of 200 MSM were recruited in the study. The mean age was 34 years (range 18-59 years, SD = 9.4) and101 participants were HIV negative (99 HIV positive). HPV 6/11/16/18 or quadrivalent HPV vaccine (HPV4) genotype seroprevalence among HIV negative and positive MSM was 64.3% (55%-75.9%) and 93.8% (87.6%-99.2%) respectively (p

This overview presents the up-to-date evidence on colposcopy practice and other diagnostic modalities such as HPV DNA test and cytology for cervical intraepithelial neoplasia (CIN). Current evidence supports the use of colposcopy for the detection of intraepithelial lesions as a second line tool. CIN treatment involves either excisional or destructive techniques, usually performed under local anesthesia. Although a debate exists about the most efficient approach, the currently available evidence reveals no differences in efficacy among the available conservative methods of treatment. New evidence supports treatment by destructive rather than excisional techniques, at least for low grade lesions in women wishing future childbearing, as they appear to have no apparent pregnancy-related morbidity. Treatment failures rates might increase in cases of involved excision margins, older age or glandular involvement. There is no worldwide consensus on the optimal follow-up policy, interventions or frequency in surveillance after treatment. HPV DNA test combined with either colposcopy or cytology is a promising combination for the early detection of treatment failures due to residual disease. Existing guidelines should probably be updated incorporating the new information emerged from recently published work. Cancer Treat Rev

Nonbacterial arthritis is a rare but well-recognized complication of acute varicella in children. Reported cases usually described monoarthritis of the knee that occurs at the onset of the rash or shortly after. Herein, we describe a case of arthritis of the hip that occurred in a 4-year-old girl 6 days before the onset of rash. The presence of varicella-zoster virus DNA in synovial fluid was confirmed by real-time polymerase chain reaction. A review of the literature reported 26 previous cases of aseptic arthritis due to varicella infection among children.

Mouse mammary tumor virus is known to infect newborn mice via mother's milk. A proposed key step for viral spread to the mammary gland is by the infection of lymphocytes. We show here that although in suckling mice retroviral proteins are found in all epithelial cells of the gut, viral DNA is exclusively detectable in the Peyer's patches. As early as 5 d after birth the infection leads to a superantigen response in the Peyer's patches but not in other lymphoid organs draining the intestine. Viral DNA can be detected before the superantigen response and becomes first evident in the Peyer's patches followed by mesenteric lymph nodes and finally all lymphoid organs.

Using new sensitive quantitative polymerase chain reaction (PCR) assays, cytomegalovirus (CMV) DNA is often detectable in the plasma of immunosuppressed patients. We investigated the prognostic value of a positive CMV DNA test for the development of CMV end-organ disease, other AIDS-defining events and mortality.

It has been reported that human immunodeficiency virus type 1 (HIV-1) infection may exist in persons without specific antibodies for years. To measure the frequency of a silent carrier state, a study was conducted in a cohort of 124 intravenous drug users (IVDUs) without anti-HIV-1 antibodies. All the participants had engaged in high-risk behavior for HIV-1 transmission for a number of years until 1987 or later. Samples were analyzed at 6-month intervals for the presence of HIV-1 provirus using DNA amplification and for the appearance of anti-HIV-1 antibodies. HIV-1 provirus and antibodies were undetectable in 122 participants, whereas seroconversion was observed in 2. In one of these, both amplified HIV-1 pol gene segment and anti-HIV-1 antibodies were detected simultaneously, and in the other, provirus was detected 1 month before seroconversion. This study suggests that long-term HIV-1 infection without anti-HIV-1 antibodies is rare and that repeated antibody testing is sufficient to determine the HIV-1 status of a person no longer at high risk for HIV-1 infection.

Varicella zoster virus (VZV) is a well-known cause of infectious uveitis. The aim of this study was to characterize the VZV-specific T-cell response in eyes of patients with VZV-induced uveitis. T-cell lines (TCL) were generated by mitogenic stimulation of intra-ocular fluid (IOF) samples obtained from eight patients with VZV-induced uveitis. Two patients with herpes simplex virus (HSV)-induced uveitis were included as disease controls. Characterization of individual T-cells in the TCL was performed by stimulating the TCL with mock, HSV-1 and VZV antigen pulsed autologous B cells and subsequent flow cytometric analyses. Virus specificity and phenotype of the T-cells were identified by simultaneous detection of intracellular gamma interferon and cell surface markers CD4, CD8, CD3 or T-cell receptor (TCR) beta chain variable region (TCRBV) usage. The TCL obtained from patients with HSV-1-induced uveitis contained higher numbers of T-cells reactive to HSV-1 compared to VZV. VZV-specific T-cells were detected in all TCL of the patients diagnosed with VZV-induced uveitis. Four out of six TCL obtained from patients with VZV-induced uveitis that were assayed for both viruses, contained higher numbers of T-cells reactive to VZV compared to HSV-1. Detailed analyses of the TCL of two patients demonstrated that the VZV reactivity within the assayed TCL was dominated by T-cells expressing one specific TCRBV gene. The data implicate that VZV-reactive T-cells infiltrate and participate in the local inflammatory response in eyes of patients with VZV-induced uveitis.

This study reports the establishment of three major subtypes of human mesothelioma cells in tissue culture, i.e. the epithelioid, sarcomatoid and biphasic forms, and compares their phenotypic and biological characteristics. Primary cells isolated from biopsies or pleural exudates were subcultured for over 50 passages. We evaluated immunoreactivity using various mesothelial markers related to histological patterns of these cell lines. For epithelioid cells, calretinin and cytokeratin were found to be useful and easily interpretable markers as for control mesothelial cells. The biphasic form was only partially positive and the sarcomatoid type negative. Vimentin was expressed by all cell lines. BerEP4, a specific marker for adenocarcinoma, was negative. Interestingly, while the macrophage marker CD14 was negative, immunoreactivity for a mature macrophage marker (CD68) was expressed by all cell types, suggesting that this marker might constitute an additional tool useful in the differential diagnosis of mesothelioma. At the ultrastructural level, a cell surface rich in microvilli confirmed their mesothelial origin. PCR analysis revealed that none of the cell lines contained SV40 DNA. Karyotypic analyses showed more complex abnormalities in the epithelioid subtype than in the sarcomatoid form. These cell lines may be useful in the study of cellular, molecular and genetic aspects of the disease.

Tissue-invasive disease due to porcine cytomegalovirus (PCMV) has been demonstrated after pig-to-baboon solid-organ xenotransplantation. Porcine lymphotropic herpesvirus (PLHV)-1 is associated with B cell proliferation and posttransplant lymphoproliferative disorder after allogeneic bone marrow transplantation in swine but has not been observed in pig-to-primate xenotransplantation. Activation of PCMV and PLHV-1 was investigated in 22 pig-to-baboon xenotransplants by use of quantitative polymerase chain reaction. PCMV was found in all xenografts; increased viral replication occurred in 68% of xenografts during immunosuppression. PLHV-1 was found in 12 xenografts (55%); no increases in viral replication occurred during immunosuppression. Control immunosuppressed swine coinfected with PCMV and PLHV-1 had activation of PCMV but not PLHV-1. PCMV, but not PLHV-1, is activated in solid-organ xenotransplantation.

A rapidly progressive disorder termed consumptive coagulopathy (CC) has been observed frequently in pig-to-baboon renal xenotransplantation. CC may be initiated by endothelial activation and induction of procoagulant factors after immunologic injury or infection, or by molecular incompatibilities between porcine coagulation proteins and primate clotting factors. The activation of porcine (P) cytomegalovirus (PCMV) and baboon (B) CMV infections has been documented in pig-to-primate xenotransplantation. The purpose of this study was to determine the contribution of PCMV and BCMV to CC.Endothelial activation was assessed by means of measurement of porcine tissue factor (pTF) in a functional assay in primary porcine aortic endothelial cells (PAEC) in vitro. Renal xenografts and native kidneys were studied by immunohistochemistry in immunosuppressed swine and baboons. BCMV and PCMV DNA was measured by quantitative molecular assays using real-time polymerase chain reaction.In vitro, infection of PAEC with PCMV resulted in a significant increase of pTF expression. In vivo, pTF increase occurred without the activation of PCMV in two xenografts, and in four grafts no pTF was detected despite PCMV activation. All animals with graft pTF increase developed CC. BCMV activation in the baboon xenograft recipients did not correlate with CC or pTF increase. Control pigs and baboons had activation of PCMV and BCMV, respectively, but without coagulation abnormalities.PCMV induces endothelial cell activation in vitro with procoagulant expression. However, in vivo, CC and pTF induction has an uncertain relationship to increased replication of PCMV within a xenograft. Although the data do not exclude a contributory role of PCMV in CC, other mechanisms are also likely to contribute to coagulopathies observed in pig-to-primate xenotransplantation.

An HIV-1–infected patient had a rebound in viremia when antiviral therapy was discontinued as part of a vaccine research protocol. Careful studies showed that this patient was infected initially with subtype AE of the virus but then became infected with subtype B. There was no evidence of dual infection, so the data indicate that this is a case of superinfection with a different strain of HIV-1. Natural infection does not necessarily produce cross-clade protection, a finding that may have implications for the development of a vaccine against HIV-1.

BACKGROUND: Cervical carcinoma is the second most common malignancy among women worldwide. The highest incidence rates are observed in developing countries. The increased susceptibility to cervical carcinoma in high incidence populations may result from several factors including human papillomavirus exposure and both inherited and acquired genetic traits. Using comparative molecular analysis of cervical carcinomas from Surinam, a high incidence area, and the Netherlands, a low incidence area, distinct molecular genetic profiles were studied in two populations with contrasting risk for the disease. METHODS: In the two populations, the authors compared allelic loss as a marker for the involvement of putative tumor suppressor genes in 40 and 67 carcinoma specimens from Surinam and the Netherlands, respectively. Loss of heterozygosity (LOH) analysis was performed using polymorphic microsatellite markers at sites of known tumor suppressor genes (17p [p53], 13q [Rb, BRCA2], 16q [E-cadherin], and 17q [BRCA1]) and at chromosomes 3p, 6p, 6q, and 11q, which frequently are lost in cervical carcinoma. RESULTS: Remarkable differences in LOH were found between both populations. The most prominent observation was the extremely high frequency of LOH, up to 72%, in the region of the major histocompatibility complex on chromosome 6p in specimens from Surinam. In the group of specimens from the Netherlands, only 45% of LOH was observed at this locus. In addition, LOH was detected significantly more frequently at 6q and 13q in the cases from Surinam whereas LOH was found more frequently at 17p in cases from the Netherlands. CONCLUSIONS: The results of the current study show that heterogeneity exists in tumor-associated somatic genetic alterations between these two populations that may be indicative of the existence of multiple genetic pathways in cervical tumorigenesis.

BACKGROUND: Large simple trials which aim to study therapeutic interventions and epidemiological associations of human immunodeficiency virus (HIV) infection, including perinatal transmission, in Africa may have substantial rates of loss to follow-up. A better understanding of the characteristics and the impact of women and children lost to follow-up is needed. METHODS: We studied predictors and the impact of losses to follow-up of infants born in a large cohort of delivering women in urban Malawi. The cohort was established as part of a trial of vaginal cleansing with chlorhexidine during delivery to prevent mother-to-infant transmission of HIV. RESULTS: The HIV infection status could not be determined for 797 (36.9%) of 2156 infants born to HIV-infected mothers; 144 (6.7%) with missing status because of various sample problems and 653 (30.3%) because they never returned to the clinic. Notably, the observed rates of perinatal transmission were significantly lower in infants who returned later for determination of their infection status (odds ratio = 0.94 per month, P = 0.03), even though these infants must have had an additional risk of infection from breastfeeding. In multivariate models, infants of lower birthweight (P = 0.003) and, marginally, singletons (P = 0.09) were less likely to return for follow-up. The parents of infants lost to follow-up tended to be less educated (P < 0.001) and more likely to be in farming occupations, although one educated group, teachers and students, were also significantly less likely to return. Of these variables, infant birthweight, twins versus singletons, and maternal education were also associated with significant variation in the observed risk of perinatal transmission among infants of known HIV status. CONCLUSIONS: Several predictors of loss to follow-up were identified in this large HIV perinatal cohort. Losses to follow-up can impact the observed transmission rate and the risk associations in different studies. Int J Epidemiol

Background/Aims: Hepatitis B virus (HBV) disease on a liver graft is associated with florid viral replication and graft failure. The aim of this study performed between 1992 and 1995 was to investigate the safety and efficacy of long-term intravenous ganciclovir for HBV infection in liver transplant recipients. Methods: Twelve patients with HBV re-infection and four with de novo HBV infection were studied. HBV DNA was positive in all (median titer: 437.5 pg/ml) and HBeAg was positive in seven. Intravenous ganciclovir was started after a median of 14.5 months from HBsAg positivation and continued for a median of 10 months. Results: A complete response with HBV DNA negativation was seen in ten cases, a partial response with a decrease of more than 50% of initial HBV DNA levels in four and no response in two. Overall tolerance was good. Among the ten complete responders, two seroconverted for both HBsAg and HBeAg and one for HBsAg alone. Among these ten patients, three were re-transplanted for liver failure: two of them are alive; three had a viral breakthrough during treatment; and four remained HBV DNA negative: two are alive and two died. Partial responders and nonresponders were treated with other antiviral agents and three were re-transplanted, two of them are alive. Overall 12 out of 16 patients (75%) survived with a median follow up of 46 months. Conclusions: Long-term intravenous ganciclovir can persistently inhibit HBV DNA replication in liver transplant recipients and is well tolerated. Further evaluation should be encouraged, especially for HBV recurrence after first-line treatments.

Mouse mammary tumor virus (MMTV) is a B type retrovirus transmitted to the suckling offspring through milk. MMTV crosses the intestinal barrier of neonates, initially infects the lymphoid cells of the Peyer's patches, and later spreads to all lymphoid organs and to the mammary gland. Adult mice can be infected systemically, but not by oral MMTV administration. In this study, we show that nasal administration of infected milk induces the infection of adult mice. Nasal MMTV infection shared the main features of systemic and neonatal intestinal MMTV infections: deletion of the superantigen (SAg)-reactive T cell subset from the peripheral T cell population, presence of viral DNA in lymphoid cells, and transmission of MMTV from mother to offspring. Viral DNA was restricted to the lungs and nasal-associated lymphoid tissue (NALT) 6 d after nasal infection. Furthermore, SAg-induced T cell proliferation was only detected in NALT. These results demonstrate that MMTV crosses the intact epithelium of the upper respiratory tract of adult mice and infects the lymphoid follicles associated with these structures.

A type D-related retrovirus has been demonstrated in enzootic nasal tumors (ENTs) of sheep and goats. This retrovirus, ENT virus (ENTV), has antigenic cross-reactivity with the jaagsiekte sheep retrovirus (JSRV), which is associated with a contagious lung tumor of sheep (sheep pulmonary adenomatosis). Here, we present the first report of nucleic acid sequence from ENTV which confirms, at the nucleic acid level, that this retrovirus is related to JSRV yet apparently distinct from it. Reverse transcription-PCR followed by restriction enzyme digestion specifically identified ENTV. By this technique, ENTV was demonstrated exclusively in tumor tissues and exudates of animals with ENT. Thus, there is a unique and consistent association between ENT and the retrovirus, just as there is between JSRV and sheep pulmonary adenomatosis. This gives further weight to the hypothesis that these retroviruses are the etiologic agents of the tumors.

A prospective study was undertaken to determine the prevalence and histological correlates of human papillomavirus infection in the head and neck epithelium. Oral, pharyngeal, and laryngeal paraffin-embedded samples were analyzed by polymerase chain reaction with use of human papillomavirus E6 consensus sequence primers. Human papillomavirus infection was detected in 20 of 126 (15.9%) patients. Twenty-five of 230 (10.9%) samples contained human papillomavirus DNA. Papillomaviruses were detected in 15 of 131 (11.4%) head and neck squamous cell carcinomas, in 3 of 32 (9.4%) dysplasias, and 2 of 19 (10.5%) keratoses. The most commonly identified human papillomavirus in cancerous, precancerous, and keratotic lesions was type 16 (80.0% of the isolates). Five papillomas were shown to contain human papillomavirus type 6. No other lesion in 42 samples contained human papillomavirus. Our data are consistent with the hypothesis that infection with certain types of human papillomavirus contributes to head and neck cancer although this may be so only in a minority of cases. Human papillomavirus infection may play a role in the earliest stages of tumorigenesis, since papillomaviruses can be found in laryngeal premalignant and keratotic lesions, which are closely linked to tobacco use.

Mouse mammary tumor virus (MMTV) encodes a superantigen (SAg) that promotes stable infection and virus transmission. Upon subcutaneous MMTV injection, infected B cells present SAg to SAg-reactive T cells leading to a strong local immune response in the draining lymph node (LN) that peaks after 6 d. We have used the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) to dissect in more detail the mechanism of SAg-dependent enhancement of MMTV infection in this system. Our data show that no detectable B or T cell response to SAg occurs in AZT pretreated mice. However, if AZT treatment is delayed 1-2 d after MMTV injection, a normal SAg-dependent local immune response is observed on day 6. Quantitation of viral DNA in draining LN of these infected mice indicates that a 4,000-fold increase in the absolute numbers of infected cells occurs between days 2 and 6 despite the presence of AZT. Furthermore MMTV DNA was found preferentially in surface IgG+ B cells of infected mice and was not detectable in SAg-reactive T cells. Collectively our data suggest that MMTV infection occurs preferentially in B cells without SAg involvement and is completed 1-2 d after virus challenge. Subsequent amplification of MMTV infection between days 2 and 6 requires SAg expression and occurs in the absence of any further requirement for reverse transcription. We therefore conclude that clonal expansion of infected B cells via cognate interaction with SAg-reactive T cells is the predominant mechanism for increasing the level of MMTV infection. Since infected B cells display a memory (surface IgG+) phenotype, both clonal expansion and possibly longevity of the virus carrier cells may contribute to stable MMTV infection.

Recent evidence has shown that most nasal lymphomas (NL) are associated with a T-cell phenotype and are thus called nasal T-cell lymphomas (NTCL), but little information is available about the T-cell receptor (TCR) expression. The presence of Epstein-Barr virus (EBV) genome has been recently reported in NTCL in Oriental populations in which NL and EBV-associated tumors are more common and in occasional Occidental cases. This prompted us to investigate lymphoma biopsies from 7 non- Oriental patients with NTCL for the expression of natural killer (NK) and T-cell antigens, including TCR proteins, for the presence of EBV- encoded latent membrane protein (LMP) using immunohistochemistry and for the presence of EBV DNA and Epstein-Barr early region (EBER) RNA using in situ hybridization (ISH). Six cases displayed a CD3-, TCR alpha beta-, TCR gamma delta-, CD2+, CD7+, CD5-, CD4-, CD8-, CD56+ phenotype, suggesting that these tumors may be peripheral T-cell lymphomas (PTCL) with extensive loss of T-cell antigens and expression of the NK-cell (CD56) antigen or, alternatively, NK-cell neoplasias. The remaining case was a gamma delta PTCL, as shown by the CD3+, TCR gamma delta+ phenotype and the biallelic gamma and delta TCR gene rearrangements. Using ISH, EBER RNA transcripts were detected in tumor cells in all cases and EBV DNA was shown in the 6 tested cases. In all cases, tumor cells expressed LMP. These findings support the concept that NTCL constitute a distinct group of lymphomas that, in addition to their peculiar clinical features, exhibit an unusual TCR “silent” CD56+ or TCR gamma delta+ phenotype and harbor the EBV. In view of the LMP transforming potential, these data suggest that EBV may play a role in the pathogenesis of NTCL.

AIMS: To evaluate the expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells in Hodgkin's disease, especially in relation to Epstein-Barr virus infection and expression of EBV encoded latent membrane protein (LMP). METHODS: Tissues from 33 cases of Hodgkin's disease were studied for the presence of EBV DNA by polymerase chain reaction (PCR) and DNA in situ hybridisation (DISH), for the presence of EBER-1 and EBER-2 EBV RNA by RNA in situ hybridisation (RISH); and for the presence of LMP, bcl-2, and c-myc proteins by immunohistochemical staining. RESULTS: A substantial number of Reed-Sternberg cells expressed bcl-2 in 20 of 29 (69%) and c-myc in 30 of 32 (94%) Hodgkin's disease samples. In 18 of the 25 (72%) cases Reed-Sternberg cells expressed both oncogene products. Of these 18 cases, 10 (56%) were EBV-PCR positive; eight (44%) were EBV-PCR negative. CONCLUSIONS: Reed-Sternberg cells in Hodgkin's disease frequently express both bcl-2 and c-myc oncogene products, suggesting that these oncogenes may act in concert in the pathogenesis of the disease. Moreover, the expression of c-myc and bcl-2 proteins in Reed-Sternberg cells is independent of EBV and LMP status. J Clin Pathol

Forty-four cases of Hodgkin's disease (HD), mostly of the nodular sclerosing type, were investigated for the presence of Epstein-Barr virus (EBV) by polymerase chain reaction (PCR) and DNA and RNA in situ hybridization (DISH, RISH), as well as by immunohistochemistry for the detection of latent membrane protein-1 (LMP-1) of EBV. In situ hybridization (ISH) was combined with immunohistochemistry to correlate the presence and activity of the virus at the cellular level. In 18/34 (53 per cent) cases, EBV-DNA sequences could be detected with the PCR method. In 12/18 positive cases, DISH and RISH were also positive. In the remaining six EBV-PCR positive cases, two were also positive with RISH and LMP-1, whereas no positive signal with DISH could be obtained. All DISH and/or RISH positive cases were also positive for LMP-1. With RISH, not only the Reed-Sternberg cells and their mononuclear variants (RS cells) stained positive, but also small and intermediate cells frequently reacted with the EBV-specific probes (EBER-1 and -2). Double staining with cellular markers (CD3, CD20, CD45, CD45RO, CD68, and the lectin PNA) revealed that most of the smaller EBER-positive cells frequently did not express T, B, or histiocytic markers, but that they, as well as the RS cells, showed cytoplasmic and membranous staining with PNA. These smaller EBER-positive cells were not found in EBV-PCR negative HD. EBER-positive RS cells were almost always LMP-1 positive, as well as a substantial proportion of the intermediate-sized cells, whereas the majority of the small EBER-positive cells remained LMP-1 negative.(ABSTRACT TRUNCATED AT 250 WORDS) J Pathol

The presence of Epstein Barr virus (EBV) DNA in biopsies from the post-nasal space (PNS) of patients suspected of nasopharyngeal carcinoma (NPC) was detected by in situ cytohybridization with an EBV DNA probe labelled with the novel labelling compound digoxigenin. The digoxigenin probe was hybridised to cryostat sections of NPC biopsies and subsequently detected by an enzyme immunoassay procedure. It was found that in situ cytohybridization using the digoxigenin probe was much more rapid and sensitive (96 h compared to five weeks) than the current method of using 3H-labelled probe. Using the digoxigenin EBV probe, it was found that in all the eighteen NPC biopsies tested, EBV DNA was detected in malignant epithelial cells and infiltrating lymphocytes. EBV DNA was also detected in some normal epithelial cells in these NPC biopsies. EBV DNA was not detected in epithelial cells of non-malignant biopsies.

Fourteen patients previously treated with zidovudine were monitored for laboratory parameters and clinical events during 1 year after introduction of didanosine (ddI) monotherapy. Proviral human immunodeficiency virus type 1 (HIV-1) copy numbers (cell-associated DNA) and concentration of free virions (viremia) were determined using a semiquantitative polymerase chain reaction (PCR). High levels of circulating virus were detected in all patients (range, 17 to 5,934 x 10(3)/ml of serum). Within 4 weeks of therapy, a decrease of viremia (60 to 98%) was observed in nine patients. After 1 year of treatment, eight of these nine patients still had decreased viremia when proviral HIV DNA was decreased or stable, and CD4+ lymphocytes were stable or higher in seven of these eight patients. Antiviral effect was more pronounced in the six patients with CD4+ > 100/mm3 at entry, five of them belonging to the subgroup of the seven responding patients as compared to two of eight patients with CD4+ < 100/mm3. Clinical events in this small group were not statistically correlated with virologic parameters; however, responding patients had a tendency to stabilize or gain weight. This study suggests that measurement of viremia deserves further study as a marker of antiviral efficacy and might predict, even at 4 weeks, the beneficial potential of ddI.

A previously healthy 9-year-old boy died from acute liver failure during an acute Epstein-Barr virus infection. Epstein-Barr virus DNA could be demonstrated in the liver by Southern blot — and by in situ hybridization techniques. The identification of the virus in the liver suggests a causal relation between the Epstein-Barr virus and the acute massive liver cell necrosis.

The molecular epidemiology of human papillomavirus (HPV) infection in a sample of Greek women (n = 2952, mean age 42.2 +/- 13.3 years) was examined. HPV prevalence was 50.7% (95% confidence interval, 48.8-52.6). The most frequent HPV types were HPV 53, 51 and 66 (10.2%, 9.4% and 9.3%, respectively). HPV positivity was associated with age, age of sexual debut, number of sexual partners and duration of sexual relationship, while marriage or multiparity protected against infection (all p

Epstein-Barr Virus is an important aetiological factor in the development of nasopharyngeal carcinoma (NPC). Serology suggested that besides NPC some other tumours located within the lymphoepithelial ring of the throat (Waldeyer's ring) might be associated with EBV. Nucleic-acid hybridizations were carried out to detect the presence of EBV-DNA in tumour biopsies derived from tonsillar carcinomas. We were able to demonstrate the presence of EBV-DNA in the tumour cells of two poorly differentiated carcinomas. We think that only a small fraction of tonsillar carcinomas is positive for EBV. The aetiologic relationship of EBV with tumours of Waldeyer's ring is discussed.

Molecular biology allowed considerable improvements in diagnostic procedures by production of new and more specific sonds for the detection of traces of viruses, both on the nucleic acid and protein levels, and by determining the immune response of the host to specific antigens. Improvements in sensitivity and improved correlation to the stage of viral disease are already evident from several applications and strongly suggest a broad application of these approaches.

Evidence for the pathogenicity of pseudorabies virus for the auditory and vestibular organs of experimentally infected mice is presented. We demonstrate viral genomes in cells of the peripheral sensory organs, the nerve structures, and the affected areas of the brain in single sections from an entire cranium of an adult mouse. The data were obtained by an in situ hybridization technique adapted for use with fixed, plastic-embedded materials. In contrast to conventional methods which use frozen sections, we were able to analyze cartilaginous and bony materials with high resolution.

The effects of heat treatment of CV1 cells infected with simian virus 40 (SV40) on viral and cellular protein synthesis were investigated by one-dimensional and two-dimensional polyacrylamide gel electrophoresis. A 12-h heat treatment during the late phase of the viral life-cycle inhibits VP1 synthesis. No inhibition of normal cellular proteins is apparent, but heat-shock proteins are strongly induced and accumulate in the cells. Inhibition of VP1 synthesis in infected cells is demonstrated to occur also after arsenite treatment, another agent known to induce heat-shock proteins. Northern blot analysis of cytoplasmic RNA demonstrated a decrease in the abundance of late SV40 mRNAs thus showing that the inhibition occurs at the transcriptional or immediately post-transcriptional level. Cumulative labeling with [3H]thymidine of viral DNA showed that the decrease in the abundance of late mRNAs is not due to a blocking of viral DNA synthesis. Immunofluorescence microscopy and immunoprecipitation analysis show that heat and arsenite treatments also affect the synthesis of T antigen. These results suggest that heat-shock proteins may play a role in the inhibition of SV40 virus gene functions. Eur J Biochem

Nucleic acid hybridization is widely used for scientific applications but essentially restricted to specialized laboratories. The use of recombinant m 13 phages as hybridization probes (Hu and Messing (1980) Gene 17, 271; Messing (1983) Methods Enzymol. 101, 20) offers a considerable advantage over the commonly used recombinant plasmids as the preparation of the DNA probe is very simple and it can easily be labeled directly, e.g. with isotopes with long half-life like 125I (Commerford (1971) Biochemistry 10, 11 (1983); Gu et al. (1983) Cancer (China) 2, 129; Han and Harding (1983) Nucleic Acids Res. 11, 14) and used for hybridization. However, as the application of nucleic acid hybridization for diagnostic and epidemiological purposes becomes almost unavoidable, the logistic problems of keeping numerous individually labeled hybridization probes increase considerably and may reach prohibitory levels in less well-equipped laboratories. In a new sandwich technique, the first step involves hybridization with an unlabeled recombinant m 13 DNA carrying an insert of the desired specificity. In a second step a universally usable labeled probe directed against the m 13 part of the recombinant phage DNA is applied. This reduces considerably the problems of preparing and keeping multiple labeled probes in stock.

This investigation is concerned with the possibility of identifying viral DNA using the in situ DNA hybridization method in methylmethacrylate-embedded material. As an experimental model we chose viral labyrinthitis produced by intranasal infection of the mouse with pseudorabies virus. Fixation and embedding methods specially adapted to this procedure and bony histology preparation technique (specimens by grinding or micromilling) made it possible to identify viral DNA directly morphologically and virologically in the inner ear. Quantitative microphotometric analyses of trans-sagittal sections of the entire skull after in situ DNA hybridization are presented and discussed here as an explicit method of investigating the path of distribution of viral DNA in the brain and the inner ear.

Se-Ax is a continuous mature T-cell line that we have established from a patient with Sézary's syndrome. An important finding was that the Se-Ax cell line required human serum for initial growth. Here we show that transfer of the permanent cell line to a medium deficient of human serum induces production of C-type retroviral-like particles with a unique morphology. Ultrastructurally, these particles are 120 nm in diameter with hexagonal shape, and show a small, centrally located round core of 30 nm. They are observed only extra-cellularly; typical budding, however, is not found. Both by serological testing and molecular analysis we substantiate the morphological conclusion that these particles do not represent common human or animal retroviruses. The inducibility of the particles may be a hint as to a possible endogenous origin.

Two independent techniques, in situ hybridization on frozen sections and reassociation kinetics, have been used to localize Epstein-Barr virus genomes in tissue samples from healthy human adults. Whereas specimens taken from the palatine tonsils were invariably negative, all samples from the parotid gland were positive when tested with either technique. This observation suggests that the parotid gland is, besides the peripheral lymphocytes, a site of lifelong persistence of Epstein-Barr virus and probably the site of low-level virus production which may be the source of virus found in the oropharynx.

BACKGROUND: Little is known about the natural history and the malignant potential of low-grade cervical intraepithelial neoplasia associated with human papillomavirus (HPV) infection. Cervical cancer remains the second most frequent cause of death in women across the world. Epidemiologic and molecular studies have shown that human papillomavirus (HPV) is associated with cervical carcinogenesis. In this prospective study we examined the behavior of low-grade cervical intraepithelial lesions associated with HPV infection over a 6-year period. MATERIAL AND METHODS: During 1992, women with Papanicolaou smears reporting koilocytotic atypia (HPV effect) with or without grade 1 cervical intraepithelial neoplasia (HPV +/- CINI, low grade squamous intraepithelial lesions, LgSIL), along with colposcopic impression of LgSIL were included to the study. Between 1992 and 1998 all women underwent repeat Papanicolaou smears, colposcopic evaluation and HPV DNA testing every six months. HPV typing of cervical scrapes was done by PCR. RESULTS: A total of 330 women completed at least 6 years of follow up. Among women with high-risk HPV types (16/18), the presence of dysplasia (grade 1 cervical intraepithelial neoplasia) was significantly correlated with the progression of the lesion; 29% of cases with HPV + CINI (15 out of 75) progressed to more severe lesions versus only 9% of cases with HPV infection (18 out of 225), P < 0.001, chi-square test. The incidence of histologically confirmed progressive lesions was significantly greater in women with mild dysplasia (18 out of 75, 24%) compared to women without dysplasia (13 out of 255, 5%), P < 0.001, chi-square test. CONCLUSIONS: In our study HPV typing was not predictive of the evolution of low-grade intraepithelial lesions associated with HPV infection. Standard cytologic screening and colposcopy are the most effective means of monitoring low-grade lesions. Anticancer Research