Journal of Clinical Virology - Articles in Press

Human cytomegalovirus preferentially infects the neoplastic epithelium of colorectal cancer: A quantitative and histological analysis - Corrected Proof

2012-05-17

Abstract: Background: It has long been suggested that human cytomegalovirus (HCMV) might be involved in human oncogenesis. However, whether HCMV was associated with colorectal cancer (CRC) was still controversial.Objective: To clarify whether HCMV specifically infects the tumorous tissue of CRC.Study design: Paired tumor and adjacent non-neoplastic CRC specimens were collected from 163 patients. HCMV DNA was detected and quantified through PCR and quantitative real-time PCR. Virus location was determined by in situ hybridization (ISH) of formalin-fixed paraffin-embedded tissue sections with an HCMV-specific probe.Results: By PCR, HCMV DNA was detected in 42.3% (69/163) of the tumor specimens, while only 5.6%(14/163) samples of adjacent non-neoplastic tissue were positive for HCMV (p<0.0001). Quantitative real-time PCR in 54 sample pairs revealed significantly higher viral copies in the tumor specimens than the adjacent non-neoplastic tissue specimens (p<0.001). By ISH, the nucleic acids of HCMV were detected in the cytoplasm of neoplastic epithelium. No hybridization was detected in the inflammatory infiltrates, submucosa, or other stromal tissues.Conclusions: HCMV preferentially infects the tumor epithelium of CRC. How the virus subsists in and interacts with the microenvironment of tumor epithelium of CRC should be studied.

Despite triple vaccination - Corrected Proof

2012-05-14

In January 2011, a 51-year-old male patient presented to our Accident and Emergency Department in Hamburg, Germany, because of shortness of breath. The clinical examination found high temperature (38°C), cough and wheezing in both basal lungs. This obese man (175cm, 147kg, BMI 48) had a past medical history of alcohol and nicotine abuse, COPD, disturbed glucose tolerance and hypertension. The patient denied taking regular medication and recent international travelling. He had been yearly vaccinated against influenza, including vaccination against influenza A(H1N1)pdm09 twice with the non-adjuvanted whole-virion inactivated influenza vaccine Celvapan® (Baxter AG) in January 2010 and February 2010 and with the trivalent split influenza vaccine Begrivac 2010/2011® (Novartis Vaccines and Diagnostics Ltd.) in October 2010. The laboratory findings revealed a slightly elevated CRP of 24.9mg/l (<5mg/l), normal white blood count with regular differentiation. Pro-calcitonin 0.17ng/ml (0.10ng/ml) was slightly increased. On the chest X-ray, we noted right-sided peribronchitis, delicate infiltrations near the lower right hilar pole and a right-sided interlobular bruise (A), presenting as insufficiency with severe hypercapnia (pH 7.44, 36mmHg, 62mmHg, HCO3− 42.1mmol/l, base excess 12.6mmol/l, blood oxygenation 72%). In light of his acute chronic respiratory failure, he was immediately put on non-invasive ventilation (CPAP) and intravenous antibiotic treatment with ceftriaxone and clarithromycin plus steroids was initiated. After a temporary recovery, the patient's condition deteriorated; he developed respiratory failure with peak CO2 retention of 90mmHg requiring intubation and mechanical ventilation. Catecholamine treatment was started to treat cardiac failure. Bronchoscopy showed obstructed, inflammatory, swollen right lung segments 8 and 9 with thick, glassy mucus. Three days after admission, a follow-up chest X-ray demonstrated new total atelectasis in the right lower lung field and streaky atelectasis in the left lower lung field (B).Infectious exacerbation of the patient's COPD was considered here.What is your most likely clinical diagnosis and how do you confirm it?How do you explain the discrepancy between history of vaccinations and clinical and laboratory findings?How do you explain the serological results?What are your consequences concerning therapy?

Pattern recognition receptor responses in children with chronic hepatitis B virus infection - Corrected Proof

Ida Louise Heiberg, Thilde Nordmann Winther, Søren Riis Paludan, Birthe Hogh — 2012-05-14

Abstract: Background: Several studies have demonstrated that hepatitis B virus (HBV) affects the expression and function of Toll like receptors (TLRs), but data on TLR function in HBV infection are mainly from adult patients. The natural history of chronic hepatitis B (CHB) infection is distinctly different in children, since 90% of children become chronic carriers compared to 5% of adults when infected with HBV.Objectives: We wanted to study the function of TLRs and cytosolic DNA receptors in children with CHB infection compared to healthy children.Study design: PBMCs from 19 children with CHB and 19 healthy children were stimulated with ligands for TLR 2, 3, 4, 7 and 9 for 24h. For activation of cytosolic DNA receptors, cells were transfected with a double-stranded DNA using Lipofectamine 2000. Supernatants were analyzed for levels of IFN-α, TNF-α, IL-6, CXCL10 and CCL3 by Luminex.Results: Stimulation with ligands for TLR2, TLR3 and TLR9 induced IL-6, CCL3 and CXCL10 to a significantly higher level in children with CHB compared to healthy children. CHB patients displayed significantly lower IFN-α production compared to healthy children after stimulation with ligands for TLR2, TLR3 and TLR4. Stimulation of intracellular DNA sensors with synthetic double-stranded DNA elicited significantly higher induction of the inflammatory cytokines and chemokines IL-6, TNF-α and CCL3 in the CHB patients as compared to the healthy children.Conclusions: Our results indicate a TLR-mediated inflammatory response in children with CHB infection. Furthermore, our study is the first to show that the responses of intracellular DNA receptors are affected in CHB.

Epidemiological and genetic analyses of a diffuse outbreak of hepatitis A in Japan, 2010 - Corrected Proof

2012-05-07

We thank Dr. Escobar-Gutiérrez and colleagues for their interest in our recent article “Epidemiological and genetic analyses of a diffuse outbreak of hepatitis A in Japan, 2010” and welcome the opportunity to respond to their comments.

Field-based performance of three pre-market rapid hepatitis C virus antibody assays in STAHR (Study to Assess Hepatitis C Risk) among young adults who inject drugs in San Diego, CA - Corrected Proof

A. Jewett, B.D. Smith, R.S. Garfein, J. Cuevas-Mota, E.H. Teshale, C.M. Weinbaum — 2012-05-07

Abstract: Background: Approximately 4.1 million Americans are estimated to have been infected with hepatitis C virus (HCV), 45–85% of whom are unaware of their infection. Persons who inject drugs (PWID) account for 55.8% of all persons with HCV antibody (anti-HCV) in the U.S. PWID have limited access to healthcare and are infrequently tested for anti-HCV using conventional laboratory assays.Objective: To evaluate performance characteristics (sensitivity and specificity) of three, pre-market rapid point-of-care tests (one oral fluid and two finger-stick assays) from two manufacturers (Chembio and MedMira) in settings providing services to young adult PWID in San Diego, CA.Study design: Behavioral risk assessment surveys and testing for HCV were conducted among persons who reported injection drug use (IDU) within the past 6 months as part of the Study to Assess Hepatitis C Risk (STAHR) among PWID aged 18–40 years in 2009–2010. Sensitivity and specificity of the rapid anti-HCV assays were evaluated among STAHR participants, using two commonly used testing algorithms.Results: Variability in sensitivity (76.6–97.1%) and specificity (99.0–100.0%) was found across assays. The highest sensitivity achieved for the Chembio finger-stick blood, Chembio oral fluid and MedMira finger-stick blood tests was 97.1%, 85.4% and 80.0% respectively; the highest specificity was 99.0%, 100.0% and 100.0%, respectively. In multivariate analysis false negative anti-HCV results were associated with female sex for the MedMira blood assay.Conclusions: Sensitive anti-HCV rapid assays are appropriate and feasible for high-prevalence, high-risk populations such as young PWID.

Six fatal cases of classical rabies virus without biting incidents, Iran 1990–2010 - Corrected Proof

2012-05-03

Abstract: Background: Rabies is an endemic fatal zoonotic disease, commonly transmitted to humans through contact (bites and scratches) with infected animals.Objectives: During the years 1990–2010, six patients with the clinical symptoms of rabies (fever, tinnitus, buzzing, delirium and hydrophobia), with no history of a bite, were diagnosed by physicians in Iran. To obtain laboratory confirmation of rabies infection, different clinical specimens from each patient were sent to the World Health Organisation (WHO) Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran.The first case was a 39-year-old male veterinary technician who entered his uncovered scratched hand into the mouth of a rabid bovine and became infected. Two years later, a herd of sheep being tended by a shepherd and his two sons were attacked by a rabid wolf. All three individuals were infected when they applied burnt thorny wool to the sheep's wounds as a bandage. Their hands were scratched and then infected through contact with the remaining saliva of the rabid wolf on the sheep's wounds. In 1994, two other human cases occurred through corneal transplantation from the same donor who had died with the clinical signs of food poisoning (according to his hospital record), which probably was a misdiagnosis of rabies infection.Study design: This is a case series study that describes human rabies cases without biting incidents. According to the WHO recommendation, human rabies cases are notifiable, therefore, in Iran, a rabies surveillance system has been established to follow these cases. During the last decade, six patients with no ‘history of a bite’ were hospitalised with growing symptoms of rabies. The data were collected from each patient by the physicians and transferred to the Ministry of Health and Medical Education of Iran, and to the WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran as the only testing laboratory. Thus, they came to the attention of the surveillance system. Ante-mortem diagnosis was performed on saliva, cerebrospinal fluid and blood samples that were collected from the first patient by the physicians. Fresh brain specimens from all patients were kept in a mixture of 50% glycerol in phosphate-buffered saline and transported on ice to the WHO Collaborating Center for Reference and Research on Rabies.Results: For the first patient, rabies virus was investigated in saliva using the rapid tissue cell inoculation test (RTCIT) and the mouse inoculation test (MIT). Anti-rabies antibodies in this patient's serum and cerebrospinal fluid (CSF) were examined using the mouse neutralisation test (MNT). Fresh brain specimens from all patients were examined using the fluorescence antibody test (FAT) as recommended by the WHO laboratory manual in rabies as the post-mortem diagnostic test for rabies. Rabies infection was confirmed in all of the deceased patients. Anti-rabies antibodies were identified only in one patient's serum specimen. Testing also showed that the rabies virus isolated was the classic rabies virus (serotype 1), which is widespread in Iran.Conclusions: Prevention and control of this fatal disease require a sensitive surveillance system to follow ‘suspected’ animal and human rabies cases thoroughly through the improved reporting system, which contains the history of exposure, clinical examinations, symptoms and laboratory results. This study describes some notable human rabies infections and their transmission modes to prevent occupational accidents.

Development of a proficiency testing program for molecular diagnosis of influenza viruses - Corrected Proof

Michael D. Popowich, Scott J. Brunt, Ryan T. Bennett, Kirsten St. George — 2012-04-26

Abstract: Background: The Molecular Virology Proficiency Testing Program at the Wadsworth Center began the assembly and distribution of influenza virus panels to US public health labs (PHLs) in 2008. The program was created to assist PHLs in assessing their performance and in meeting CLIA regulations for mandated proficiency testing (PT).Objectives: To design and distribute proficiency testing panels containing influenza A virus subtypes H1N1 and H3N2, and influenza B; when H1N1pdm09 emerged it also was incorporated into the panels. A secondary objective was to determine the best matrix for long term storage of the molecular PT samples.Study design: Viruses were quantitated using TCID50 and quantitative real-time RT-PCR. Reference laboratories were enlisted to verify viral identity in the panels and to help determine viral titers to be used in the PT panels sent to PHLs.Results and conclusions: Of the 29 laboratories that participated the first year, 27 were able to correctly identify all of the virus types in the panel. Fifty-one PHLs participated in the program the second year when pandemic H1N1 was added, and 45 were able to correctly detect, type and subtype all of the viruses in the panel. In the program's third year, 60 laboratories participated; 58 correctly detected and subtyped all of the viruses in the panel. Annual surveys of assay techniques showed that the PHLs had shifted their extraction methods and PCR-thermocycler instrumentation to meet FDA-approved methods. The degradation study revealed that frozen viral stocks were stable for at least 30months, thus allowing ample time to prepare and pre-test panels.

A 3-year-old girl with vomiting and diarrhea - Corrected Proof

Virginia M. Pierce, Richard L. Hodinka — 2012-04-25

A 3-year-old female presented to the Emergency Department at Children's Hospital of Philadelphia (CHOP) in December 2010 with a one week history of fever (maximum temperature 103°F [39.4°C]), emesis, and diarrhea. The patient's vomitus had been neither bloody nor bilious, and the stool was described as watery, yellow, seedy, and foul-smelling without mucous or blood. Vomiting and loose bowel movements had each occurred multiple times per day, including seven episodes of emesis and six episodes of diarrhea on the day of presentation. The patient had also intermittently complained of periumbilical abdominal pain, especially prior to defecation, and was less active than usual. Her oral intake of both fluids and food was diminished compared to baseline and her urine had decreased in amount and had become dark yellow in color. On evaluation by her primary pediatrician, the patient was noted to have lost 1.8kg since her most recent office visit and was referred to the Emergency Department due to concern for dehydration.

Genetic relatedness among Japanese HAV isolates, 2010 - Corrected Proof

2012-04-23

We have read with interest the recent article by Ishii et al. In the manuscript the authors describe the partial molecular characterization of Japanese HAV isolates. While the information presented in the article is relevant, several issues are of concern. Here, we point out some of the shortcomings and interpretation of the data per se.

Extraction of viral nucleic acids: Comparison of five automated nucleic acid extraction platforms - Corrected Proof

2012-04-16

Abstract: Background: Nucleic acid extraction has a major impact on the reliability of results in routine molecular diagnostics. Optimal isolation of nucleic acids and removal of inhibitors are essential.Objectives: This study compares five different automated extraction platforms for the extraction of norovirus RNA from stool and cytomegalovirus (CMV) DNA from plasma samples.Study design: Norovirus positive stool samples and CMV positive plasma samples were aliquoted and analyzed using five different automated platforms (easyMAG, bioMerieux; m2000sp, Abbott; MagNA Pure LC 2.0, Roche; QiaSymphony, Qiagen; sample preparation module of the VERSANT kPCR Molecular System, Siemens). Similar sample input and output volumes, the identical real-time PCR cycler, and the identical assays for amplification and detection for norovirus RNA and CMV DNA, respectively, were chosen.Results: Of 39 stool samples, 36 tested positive for norovirus RNA with all extraction platforms. The three discrepant samples showed inhibition after extraction with at least one platform. Only with the VERSANT platform all samples tested positive for both the target RNA and the internal controls. Of 42 plasma samples, 27 gave quantifiable results for CMV DNA with all extraction platforms. There was significant variance between viral concentrations when different extraction platforms were compared. The majority of the 15 discrepant samples showed low viral concentrations. The internal control of the CMV assay gave positive results for all samples tested below the limit of quantification.Conclusions: The five automated extraction platforms yielded comparable results. However, the extraction performance was found to be impaired by inhibitory substances in stool samples.

Evaluation of a BK virus viral load assay using the QIAGEN Artus BK Virus RG PCR test - Corrected Proof

Hanna Rennert, Stephen G. Jenkins, Carmen Azurin, John Sipley — 2012-04-11

Abstract: Background: Viral load testing for BK Virus (BKV) has become the standard of care for the diagnosis of infection and monitoring of therapy of kidney transplant patients infected with BKV. However, there are currently no FDA-approved BKV quantification assays and no standardization among available tests.Objective and study design: This study evaluated the performance of the Artus BK Virus RG PCR (RUO) assay (QIAGEN) for accuracy, linearity, precision, analytical sensitivity, specificity, and correlation with a referral laboratory test in patient samples.Results: Linear regression analysis of the quantitative results demonstrated a linear range of quantification from 192 to 194 million (2.28 to 8.29 log10) DNA copies/mL and a coefficient of determination (R2) of 0.994. A dilution series demonstrated a limit of detection and a limit of quantification of 2.00 log10, and 2.30 log10 copies/mL (>95% positivity rate), respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.2% to 7.0%. A comparison of 34 matched samples showed a good agreement (R2=0.983) between the Artus BK test and the referral laboratory results, with an average positive bias (0.39 log10 copies/mL). Genotyping analysis using large-T antigen sequences demonstrated that 90% of the positive samples were BKV type I, and that there was no significant difference in quantification between the referral laboratory and Artus BK Virus tests.Conclusions: The Artus BK Virus RG PCR test is a reliable and sensitive assay for BKV DNA quantification as compared to the referral laboratory test.

A comparison of viral fitness and virulence between emergent adenovirus 14p1 and prototype adenovirus 14p strains - Corrected Proof

2012-04-09

Abstract: Background: Epidemiological studies from the last decade have suggested that the morbidity and mortality associated with a newly emergent strain of human adenovirus (HAdV-14p1) is greater than other, more prevalent, adenovirus strains. Recent molecular analysis identified very minor genetic differences in HAdV-14p1 compared to prototype HAdV-14p. No studies have evaluated how these differences may affect virulence.Objective: To compare HAdV-14p1 and HAdV-14p strains for competitive fitness and virulence.Study design: We performed in vitro and molecular assays to evaluate growth kinetics, cellular infectivity, cytotoxicity, and plaque morphology of the two strains.Results: Growth kinetic data showed no viral replication at 30°C and minimal differences at 37°C for both strains. Cellular infectivity data showed propagation capabilities for both strains in a diverse array of cell lines, with human lung and kidney cells having the highest propagation potential. Cytotoxicity data indicated cellular distress differences induced by both strains of virus in the first 12h, but similar distress levels between 12 and 48h. Plaque morphology assays showed some differences in average plaque diameter.Conclusions: These data suggest that the increase in morbidity and mortality observed in recent HAdV-14p1 infections is not due to viral growth or cellular infectivity differences from the prototypic HAdV-14 strain. While there were some statistically important differences detected between strains in cytotoxicity and plaque morphology assays, it seems more likely that other factors, such as environmental stressors, co-infections, or individual host response are likely contributing to the increase in morbidity.

Successful treatment of acyclovir-resistant herpes simplex virus type 2 proctitis with leflunomide in an HIV-infected man - Corrected Proof

Andrés F. Henao-Martínez, Adriana Weinberg, W. James Waldman, Marilyn E. Levi — 2012-04-05

Abstract: Human herpes simplex virus infections are very common and represent significant morbidity in the immunocompromised host. Patients with acyclovir resistant strains of HSV based on viral thymidine kinase gene mutations need alternative therapeutic approaches. Leflunomide has been shown to possess antiviral activity against several viruses. Herein we describe a case of acyclovir resistant HSV-2 proctitis in an HIV patient successfully treated with leflunomide without significant side effects.

Two laboratory-confirmed cases of Japanese encephalitis imported to Germany by travelers returning from Southeast Asia - Corrected Proof

2012-04-05

Abstract: Japanese encephalitis virus is the leading cause of encephalitis in Asia and parts of the Pacific. Despite the high number of symptomatic infections in endemic countries, clinical disease in travelers is rare. However, an increasing number of imported infections from popular holiday destinations in Southeast Asia have been recorded in the past few years, including serious disease courses in short-term travelers. Here we report two severe, non-fatal cases in tourists, who returned from a long-time stay in Thailand and a short-term trip to Bali, Indonesia, respectively. Recommendations for vaccination and pre-travel advice are discussed.

Value of herpes simplex virus type-specific serology: A case report - Corrected Proof

J. Bentley, A.P. Neubauer, A. Sauerbrei — 2012-04-02

Abstract: Genital herpes, usually caused by herpes simplex virus type 2 (HSV-2), is one of the most common sexually transmitted diseases in humans. By contrast, intrauterine HSV-2 infections have been described rarely in the literature. Our report describes a case of neonate who was delivered after 30+2 gestational weeks by cesarean section. He presented with a respiratory distress syndrome resulting in broncho-pulmonary dysplasia. At the age of 6 weeks, a chorioretinal scar was detected. During the 4th month of age, the infant developed recurrent HSV-2 infection with nasal lesions. The retrospective type-specific serologic diagnosis revealed previous HSV-2 infection of the mother resulting in prenatal HSV-2 infection of the infant. In conclusion, intrauterine HSV-2 infections may be underrepresented since they may not be associated with severe congenital malformations and the diagnosis requires the use of HSV type-specific serologic methods not widely applied in microbiological laboratories.

Hepatitis C virus genotype distribution varies by underlying disease status among patients in the same geographic region: A retrospective multicenter study - Corrected Proof

2012-03-30

Abstract: Background: Hepatitis C virus (HCV) is a known carcinogen with considerable genetic heterogeneity: six different genotypes have been identified. HCV genotype distribution varies from country to country. In the United States, the most prevalent genotypes are 1a, and 1b followed by genotypes 2, and 3.Objectives: To examine whether the distribution of HCV genotypes differed by cancer status among patients in the same area.Study design: We reviewed epidemiologic and virological data of 636 patients with HCV infection evaluated at 3 institutions in Houston, Texas, in 2008 and 2009.Results: We included 129 cancer patients (53 with hematologic malignancies and 76 with solid tumors), 333 immunocompetent patients, and 102 HIV-co-infected patients. The prevalence of genotype 1 (G-1) was 66% among cancer patients, 84% among immunocompetents (P=0.00004), and 99% among HIV-co-infected patients (P<0.00001). G-2 and G-3 were more common in cancer patients than other patients. Demographics, risk factors, and duration of HCV infection were similar between cancer and immunocompetent patients. G-1 was more prevalent in immunocompetents (84%) than in patients with hepatocellular carcinoma (74%, P=0.08) or lymphoma (59%, P=0.001). G-2 was more prevalent in lymphoma patients (24%) than in immunocompetents (8%, P=0.003); cancer risk was 3 times as great with G-2 as with other genotypes (OR 3.72, 95% CI 1.38–9.76).Conclusions: This multicenter retrospective study provides evidence of differences in HCV genotype distribution by underlying disease among geographically related patients and suggests a possible greater carcinogenic potential of some variants. Large-scale prospective studies are warranted to investigate HCV genotype distribution in other regions.

Chikungunya virus as a causative agent of fever of unknown origin in Finnish travellers to tropics - Corrected Proof

2012-03-29

Many travellers presenting with fever after a trip to tropics are left without a microbiological diagnosis. Of tropical illnesses, Chikungunya virus (CHIKV) infection has become increasingly common among European travellers. However, the infection is not well recognised among European clinicians. In Finland, prior to this systematic study, only six imported laboratory-confirmed CHIKV cases had been identified, and one previously unpublished CHIKV strain isolated (also described in this paper). We aimed to identify CHIKV infections in Finnish travellers presenting with fever after returning from tropics to estimate the extent of missed CHIKV infections in this population. Our further aim was to isolate and characterise novel CHIKV strains from these patients.

Cytomegalovirus-specific T-cell immunity to assign the infection status in individuals with passive immunity: A proof of principle - Corrected Proof

2012-03-29

Abstract: Background: Serological analysis of the infection status with the human cytomegalovirus (CMV) may be inaccurate after transfusion of blood products due to the variable content of CMV-specific antibodies.Objectives: In this situation, analysis of cellular immunity may represent a more accurate parameter to assign the individual CMV-infection status. This hypothesis was assessed in a sequence of clinically defined events where a CMV-seronegative patient received human immunoglobulins before AB0 incompatible transplantation of a graft from his CMV-seropositive mother and developed CMV-primary infection thereafter.Study design: Humoral immunity was analyzed using ELISA, and CMV-specific CD4 T-cells were flow-cytometrically quantified using intracellular cytokine staining after a 6h-stimulation with a CMV-antigen lysate.Results: Prior to transplantation, both CMV-specific antibody-titers and T-cell frequencies were below detection limit. After plasma infusion, the patient was temporarily seropositive but remained T-cell negative indicating passive immunity. CMV-specific T-cells became stably detectable after graft-related primary infection, thereby confirming a truly positive infection status.Conclusion: This case provides an instructive proof of principle to show that CMV-specific CD4 T-cells may serve as an accurate marker to define the true CMV-infection status in situations where serological testing is limited by the presence of passively administered antibodies.

WITHDRAWN: Viral Hepatitis: global goals for vaccination - Corrected Proof

Daniel Lavanchy — 2009-07-06

This article has been withdrawn at the request of the author. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.