Detection of antibodies to Kaposi's sarcoma-associated herpesvirus: a new approach using K8.1 ELISA and a newly developed recombinant LANA ELISA.

Detection of antibodies to Kaposi's sarcoma-associated herpesvirus (KSHV or Human herpesvirus 8) is a topic of ongoing controversy. KSHV expresses multiple antigens and host responses are highly variable. We have previously described an algorithm for determining KSHV infection based on K8.1 ELISA and LANA immunofluorescence assay (IFA). Here we describe the development of a recombinant ELISA for LANA and an improved testing strategy using ELISAs for LANA and K8.1. We assessed mammalian and baculovirus expression systems for the production of full-length recombinant LANA. We evaluated the performance of LANA ELISAs using human serum samples from several sources including blood donors and clinical patients diagnosed with Kaposi's sarcoma and compared them to LANA IFA. Both LANA ELISAs exhibited comparable sensitivity and specificity to LANA IFA but showed considerably greater reliability. The LANA ELISA can thus be used in conjunction with the previously described K8.1 ELISA to enable the highly sensitive and specific detection of antibodies to KSHV. Use of this testing strategy will provide a more accurate and reliable diagnostic assessment of KSHV status.

Human papillomavirus false positive cytological diagnosis in low grade squamous intraepithelial lesion

The purpose of this study was to investigate the number of Human Papillomavirus false positive cytological diagnosis in low grade squamous intraepithelial lesions (LSIL). Three hundred and two women who assisted to an Out-Patient Gynecologic Clinic in Maracaibo, Venezuela, were recruited for this study. Each patient had the Pap smear and a cervical swab for Hybrid Capture 2 (HC2). Three cytotechnologists reviewed the Pap smears and two pathologists rescreened all of them. The cytotechnologists reported 161 (53.3 percent) Pap smears negatives for intraepithelial lesion (IL) or malignancy, and 141 cases (46.7 percent) with epithelial abnormalities. They reported 46 percent of 302 patients with HPV infection in Pap smear slides. The pathologists found that 241 (79.8 percent) Pap smears were negatives for IL or malignancy and 61 (20.2 percent), with abnormal Pap smears. They found 14.6 percent HPV infection in all Pap smears (p<0.0001; 46 percent vs 14.6 percent). The HC2 study showed that 47 samples (15.6 percent) were positive for HPV. The study found that 114 Pap smears (False Positive: 85 percent) of 134 reported by the cytotechnologists and 24 (False Positive: 43 percent) of 56 cytologies reported by the pathologists as LSIL, were negative for HPV infection determined by HC2 (p<0.00003). The present study suggests that the cytotechnologists overdiagnosed cellular changes associated with HPV infection in the Pap smear, increasing the FP cytological diagnosis of LSIL.

El presente trabajo tuvo por objeto el investigar el número de falsos positivos reportados en la citología cervicovaginal (CCV) de la presencia del Virus del Papiloma Humano (VPH) con diagnóstico de Lesión Intraepitelial Escamosa de bajo grado (LIE-BG). Se estudiaron 302 mujeres que asistieron a la Consulta de Patología de Cuello Uterino del Hospital Manuel Noriega Trigo, en Maracaibo, Venezuela. A cada paciente se le practicaron una CCV y muestra para la captura de híbridos 2 (CH2). Tres citotecnólogos y 2 patólogos estudiaron las CCV. Los citotecnólogos reportaron 161(53,3 por ciento) de CCV negativas para lesión intraepitelial o malignidad y 141 casos (46,7 por ciento) con anomalías epiteliales. Éstos encontraron 46 por ciento de presencia de VPH en las 302 CCV. Los patólogos reportaron 241 CCV (79,8 por ciento) negativas y 61 CCV (20,2 por ciento) anormales. Estos encontraron en 14,6 por ciento de las CCV, la presencia de VPH (p < 0, 0001; 46 por ciento vs 14,6 por ciento). La CH2 mostró que 47 muestras (15, 6 por ciento) fueron positivas a VPH. Esta investigación mostró que 112 CCV de 134 (Falso Positivo: 85 por ciento) reportados por los citotecnólogos y 24 de 56 CCV (Falso Positivo: 43 por ciento) reportados por los patólogos como LIE-BG, fueron negativos a la infección del VPH determinados por la CH2 (p < 0,00003). La investigación sugiere un sobrediagnóstico de la presencia de cambios celulares debidos al VPH en la CCV, por parte de los citotecnólogos, incrementando los falsos positivos de la presencia del VPH en CCV con diagnóstico de LIE-BG.

Prevalence and risk factors of human papillomavirus infection in asymptomatic women in a Venezuelan urban area.

The purpose of this study was to investigate the prevalence and risk factors of genital human papillomavirus (HPV) infection in asymptomatic women, using the HPV DNA Hybrid Capture 2 (HC2) test. Three hundred and two women who attended the Out-Patient Gynecological Clinic of a tertiary level hospital, in a Venezuelan urban area, were selected for the study. A pap smear, a cervical swab for HC2 and gynecological exam were performed to each patient. The HC2 testing showed that 47 samples (15.6%) were positive to HPV. Forty patients (13.2%) were positive to high risk-HPV (HR-HPV) and 11 (3.6%) were positive to low-risk-HPV (LR-HPV). The prevalence of HPV infections was higher for women under 35 years (51.1%; p < 0.02), and decreased to 6.4% for women > or =65 years old. Women who had not finished high school had a higher prevalence of HPV infection (p < 0.035). Twenty six (42.6%) of 61 pathological Pap smears were positives to HPV infection. A statistically significant difference was found when HPV infection was compared in normal and abnormal Pap smear (HSIL+LSIL; p < 0.0001). Twenty four of 56 (43%) women with diagnosis of LSIL, and 2 (40%) of 5 with diagnosis of HSIL were positive for HPV infection. A statistically significant difference was found when we compared HPV infection in negative Pap smears and those with LSIL (p < 0.001). The present study found that the prevalence of HPV infection in asymptomatic Venezuelan women who attended a tertiary level hospital was 15.6%. HPV infection was more frequent in young adult, and in women with low educational level.

A prospective study of serum soluble CD30 concentration and risk of non-Hodgkin lymphoma.

Prediagnostic serum concentration of soluble CD30 (sCD30), a marker for chronic B-cell stimulation, has been associated with increased risk of developing AIDS-related non-Hodgkin lymphoma (NHL) in a recent study of HIV(+) patients. To investigate among healthy persons whether serum sCD30 is associated with NHL risk, we carried out a nested case-control study within the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. There was a strong dose-response relationship between prediagnostic sCD30 concentration and NHL risk among 234 cases and 234 individually matched controls (odds ratio [95% confidence interval] for second, third, and fourth quartiles vs first quartile: 1.4 [0.8-2.6], 2.2 [1.2-4.1], 4.1 [2.2-7.8]; P(trend) < .001), which persisted among cases diagnosed 6 to 10 years after providing a blood sample. Given that a similar relationship has been observed among HIV(+) patients, our findings suggest that chronic B-cell stimulation may be an important mechanism involved in B-cell lymphomagenesis among severely immunocompromised and healthy populations alike.

Prevalence and risk factors of human papillomavirus infection in asymptomatic women in a Venezuelan urban area

The purpose of this study was to investigate the prevalence and risk factors of genital human papillomavirus (HPV) infection in asymptomatic women, using the HPV DNA Hybrid Capture 2 (HC2) test. Three hundred and two women who attended the Out-Patient Gynecological Clinic of a tertiary level hospital, in a Venezuelan urban area, were selected for the study. A pap smear, a cervical swab for HC2 and gynecological exam were performed to each patient. The HC2 testing showed that 47 samples (15.6%) were positive to HPV. Forty patients (13.2%) were positive to high risk-HPV (HR-HPV) and 11 (3.6%) were positive to low-risk-HPV (LR-HPV). The prevalence of HPV infections was higher for women under 35 years (51.1%; p < 0.02), and decreased to 6.4% for women 65 years old. Women who had not finished high school had a higher prevalence of HPV infection (p < 0.035). Twenty six (42.6%) of 61 pathological Pap smears were positives to HPV infection. A statistically significant difference was found when HPV infection was compared in normal and abnormal Pap smear (HSIL+LSIL; p<0.0001). Twenty four of 56 (43%) women with diagnosis of LSIL, and 2(40%) of 5 with diagnosis of HSIL were positive for HPV infection. A statistically significant difference was found when we compared HPV infection in negative Pap smears and those with LSIL (p<0.001). The present study found that the prevalence of HPV infection in asymptomatic Venezuelan women who attended a tertiary level hospital was 15.6%. HPV infection was more frequent in young adult, and in women with low educational level.

El propósito de este estudio fue investigar la prevalencia y factores de riesgo que influencia la presencia de la infección por el virus del papiloma humano (VPH) en pacientes asintomáticas que asistieron a un hospital nivel 3 en un área urbana venezolana. Se estudiaron las pacientes que acudieron a la Consulta de Patología del Cuello Uterino del Hospital Manuel Noriega Trigo. A cada paciente se le realizó una historia clínica, toma de citología cervico-vaginal y una muestra del cérvix para captura de híbridos 2(CH2). Se incluyeron 302 pacientes. La CH2 mostró 47 muestras (15,6%) positivas al VPH. Cuarenta mujeres (13,2%) fueron positivas a VPH de alto riesgo (VPH-AR) y 11 (3,6%) a VPH de bajo riesgo (VPH-BR). La prevalencia de la infección por VPH fue más alta en mujeres 35 años (51,1%; p < 0,02) y disminuyó a un 6,4% en mujeres 65 años. Las pacientes que no habían terminado los estudios de bachillerato presentaron un prevalencia más elevada del VPH (p < 0,035). Veinte y seis (42,6%) de 61 CCV patológicas fueron positivas a la infección del VPH. Una diferencia estadísticamente significativa fue encontrada cuando se comparó la presencia del VPH en las CCV normales con las CCV anormales (Lesión Intraepitelial Escamosa de Alto y Bajo Grado-LIE-AG y LIE-BG; p < 0,0001). Veinte y cuatro de 56 (43%) mujeres con diagnostico de LIE-BG, y 2(40%) de 5 con diagnóstico de LIE-AG fueron positivos a la presencia del VPH. Se encontró una diferencia estadísticamente significativa cuando se comparó la presencia de infección por el VPH en CCV normales y CCV con LIE-BG (p < 0,001). El presente estudio encontró una prevalencia de la infección por el VPH en mujeres asintomáticas que asisten a un hospital nivel 3 de 15,6% en área urbana venezolana. Fue más frecuente en mujeres jóvenes y de bajo nivel educacional.

Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study.

Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase-green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by fluorescence-activated cell sorter. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models.

Human papillomavirus false positive cytological diagnosis in low grade squamous intraepithelial lesion.

The purpose of this study was to investigate the number of Human Papillomavirus false positive cytological diagnosis in low grade squamous intraepithelial lesions (LSIL). Three hundred and two women who assisted to an Out-Patient Gynecologic Clinic in Maracaibo, Venezuela, were recruited for this study. Each patient had the Pap smear and a cervical swab for Hybrid Capture 2 (HC2). Three cytotechnologists reviewed the Pap smears and two pathologists rescreened all of them. The cytotechnologists reported 161 (53.3%) Pap smears negatives for intraepithelial lesion (IL) or malignancy, and 141 cases (46.7%) with epithelial abnormalities. They reported 46% of 302 patients with HPV infection in Pap smear slides. The pathologists found that 241 (79.8%) Pap smears were negatives for IL or malignancy and 61 (20.2%), with abnormal Pap smears. They found 14.6% HPV infection in all Pap smears (p<0.0001; 46% vs 14.6%). The HC2 study showed that 47 samples (15.6%) were positive for HPV. The study found that 114 Pap smears (False Positive: 85%) of 134 reported by the cytotechnologists and 24 (False Positive: 43%) of 56 cytologies reported by the pathologists as LSIL, were negative for HPV infection determined by HC2 (p<0.00003). The present study suggests that the cytotechnologists overdiagnosed cellular changes associated with HPV infection in the Pap smear, increasing the FP cytological diagnosis of LSIL.

Hepatitis B and C virus infection and the risk of biliary tract cancer: a population-based study in China.

Emerging data suggest that chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections may also play a role in extrahepatic bile duct cancers. To test the HBV hypothesis, we examined the relationship of HBV/HCV infection with risks of biliary tract cancer and biliary stones in a population-based case-control study conducted in Shanghai, China. Standard assays were used to detect HBV surface antigen (HBsAg) and antibodies against HBV core antigen (anti-HBc) and hepatitis C virus (anti-HCV) in sera from 417 patients with biliary tract cancers, 517 with biliary stones, and 762 healthy controls randomly selected from the population. Unconditional logistic regression was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) for each disease type. HBsAg seroprevalence was 7.3% among population controls and 14.2% among patients with extrahepatic bile duct cancer, resulting in a 2.4-fold risk of extrahepatic bile duct cancer (95% CI 1.2-4.5). No association was found for cancers of the gallbladder (prevalence 8.2%) or the ampulla of Vater (6.1%), or for stones in the gallbladder (10.1%) or bile duct (9.3%). Further adjustment for education, smoking, body mass index, diabetes and gallstones did not materially change the results. Prevalence of HCV infection in this population was low (2%), limiting our ability to detect an association with biliary diseases. In Shanghai, an HBV endemic area, chronic HBV infection was associated with a 2.4-fold risk of extrahepatic bile duct cancer. These results should be confirmed in other populations with varying risks of HBV and HCV infection.

Seroprevalence of hepatitis C virus and hepatitis B virus among San Francisco injection drug users, 1998 to 2000.

UNLABELLED: Previous studies suggest that most injection drug users (IDUs) become infected with hepatitis C virus (HCV) and hepatitis B virus (HBV) soon after initiating drug use. The Urban Health Study (UHS) recruited serial cross-sections of IDUs in the San Francisco Bay area from 1986 to 2005. In the current study, we determined the prevalence of antibody to HCV and HBV (core) among UHS participants during 1998 to 2000. To examine whether the time from onset of injection to acquisition of viral hepatitis has increased, we also compared the findings among recent (<10 years) initiates to drug use who participated during 1998-2000 with those who participated in 1987. Of 2,296 IDUs who participated during 1998-2000, 91.1% had antibody to HCV and 80.5% to HBV. The number of years a person had injected drugs strongly predicted infection with either virus (P(trend) < 0.0001). HCV seroprevalence among recent initiates in 1998-2000, by years of injection drug use, was:

Reactivation of Kaposi's sarcoma-associated herpesvirus by natural products from Kaposi's sarcoma endemic regions.

Kaposi's sarcoma (KS) and its causative agent, Kaposi's sarcoma associated herpesvirus (KSHV/HHV-8), a gamma2 herpesvirus, have distinctive geographical distributions that are largely unexplained. We propose the "oncoweed" hypothesis to explain these differences, namely that environmental cofactors present in KS endemic regions cause frequent reactivation of KSHV in infected subjects, leading to increased viral shedding and transmission leading to increased prevalence of KSHV infection as well as high viral load levels and antibody titers. Reactivation also plays a role in the pathogenesis of KSHV-associated malignancies. To test this hypothesis, we employed an in vitro KSHV reactivation assay that measured increases in KSHV viral load in KSHV infected primary effusion lymphoma (PEL) cells and screened aqueous natural product extracts from KS endemic regions. Of 4,842 extracts from 38 countries, 184 (5%) caused KSHV reactivation. Extracts that caused reactivation came from a wide variety of plant families, and extracts from Africa, where KSHV is highly prevalent, caused the greatest level of reactivation. Time course experiments were performed using 28 extracts that caused the highest levels of reactivation. The specificity of the effects on viral replication was examined using transcriptional profiling of all viral mRNAs. The array data indicated that the natural extracts caused an ordered cascade of lytic replication similar to that seen after induction with synthetic activators. These in vitro data provide support for the "oncoweed" hypothesis by demonstrating basic biological plausibility.

Hepatitis C virus genotype 4 in Ugandan children and their mothers.

In Kampala, Uganda, in 2001, hepatitis C virus antibodies were found in 27 (4%) of 603 children and in 62 (12%) of 525 of their mothers. However, only approximately 10% of positive results were confirmed by reverse transcription-PCR, which suggests frequent false-positive results or viral clearance. All sequenced types were genotype 4.

Real-time PCR assay for detection and quantification of hepatitis B virus genotypes A to G.

The detection and quantification of hepatitis B virus (HBV) DNA play an important role in diagnosing and monitoring HBV infection as well as assessing therapeutic response. The great variability among HBV genotypes and the enormous range of clinical HBV DNA levels present challenges for PCR-based amplification techniques. In this study, we describe the development, evaluation, and validation of a novel real-time PCR assay designed to provide accurate quantification of DNA from all eight HBV genotypes in patient plasma specimens. A computer algorithm was used to design degenerate real-time PCR primers and probes based upon a large number (n = 340) of full-length genomic sequences including HBV genotypes A to H from Europe, Africa, Asia, and North and South America. Genotype performance was tested and confirmed using 59 genotype A to G specimens from two commercially available worldwide genotype panels. This assay has a dynamic range of at least 8 log(10) without the need for specimen dilution, good clinical intra- and interassay precision, and excellent correlation with the Bayer Diagnostics VERSANT HBV DNA 3.0 (branched DNA) assay (r = 0.93). Probit analysis determined the 95% detection level was 56 IU/ml, corresponding to 11 copies per PCR well. The high sensitivity, wide linear range, good reproducibility, and genotype inclusivity, combined with a small sample volume requirement and low cost, make this novel quantitative HBV real-time PCR assay particularly well suited for application to large clinical and epidemiological studies.

Serum concentrations of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) and risk of primary liver cancer.

BACKGROUND: 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) exposure has been demonstrated to cause liver tumors in laboratory rodents. DDT's persistent metabolite and environmental degradation product, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), has also been associated with liver tumors in laboratory animals. Whether DDT and DDE are associated with hepatocarcinogenesis in humans is not clear. METHODS: We carried out a nested case-control study among the participants of the Nutritional Intervention Trials in Linxian, China. The case group included 168 individuals who developed liver cancer during the trials, and the control group included 385 individuals frequency-matched on age and sex who were alive and well at the end of the study. Serum concentrations of DDT and DDE were measured by gas chromatography-mass spectrometry. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using multivariable analysis. RESULTS: In multivariable-adjusted models, the risk of developing liver cancer increased with increased serum DDT concentration (OR for quintile 1 versus quintile 5 = 3.8, 95% CI = 1.7 to 8.6, P(trend) = .0024). In contrast, there was no statistically significant association between liver cancer and serum DDE concentration. The association between high serum DDT concentration and liver cancer was stronger among individuals with DDE concentrations below the median value (odds ratio for tertile 3 versus tertile 1 = 3.55, 95% CI = 1.45 to 8.74) than those with concentrations above the median (OR = 1.70, 95% CI = 0.97 to 2.98). A calculation of crude liver cancer risk found that there would be 26 liver cancers per 100 000 persons per year in the lowest quintile of DDT exposure versus 46 liver cancers per 100 000 persons per year in the highest quintile of DDT exposure. CONCLUSIONS: DDT may be a risk factor for liver cancer, particularly among persons with lower DDE concentrations. Risk may be particularly increased among persons exposed directly to DDT (resulting in a higher ratio of DDT to DDE) or, alternatively, risk may be associated with individual ability to metabolize DDT to DDE.

Distribution of Epstein-Barr viral load in serum of individuals from nasopharyngeal carcinoma high-risk families in Taiwan.

The utility of EBV load as a tumor marker in nasopharyngeal carcinoma (NPC) patients suggests that it might also serve as a screening test for individuals who are at high risk for developing NPC. We previously demonstrated that unaffected individuals from high-risk families had elevated anti-EBV antibody levels compared to community controls. In this study, we measured EBV load using 2 different real-time PCR assays (targeting BamH1W and polymerase gene sequences, respectively) carried out in 2 independent research labs in serum samples from 19 untreated NPC cases, 11 healthy community controls and 100 unaffected individuals from families in which 2 or more individuals were affected with NPC. EBV genomes were detectable in 68% of NPC cases by the EBV BamH1W assay and in 74% by the EBV polymerase assay (kappa = 0.64). Patients with stage III or IV disease had significantly higher EBV load compared to those with stage I or II disease (p = 0.008). EBV DNA was detected in a single community control sample by the EBV BamH1W assay and in none of the samples by the EBV polymerase assay. Only one of 100 unaffected family members tested positive by both assays. An additional 14 were positive by only one of the 2 EBV load assays used and usually in only one of the duplicate wells tested, all with very low viral loads (3-50 copies/ml). In addition, EBV load did not correlate with EBV serology results (anti-VCA, anti-DNase, anti-EBNA-1) among these unaffected family members. In conclusion, our study suggests limited clinical utility of the EBV load test, in its current configuration, to screen individuals from high-risk families. Should a more sensitive or specific molecular assay be developed that is capable of detecting and distinguishing tumor-derived EBV genomes or gene products from true negatives, it could be evaluated as a possible screening tool for asymptomatic and early-stage NPC.