From a case-control survey to a diagnostic viral gastroenteritis panel for testing of general practitioners' patients.

OBJECTIVE: To evaluate the pathogenicity of a broad range of 11 possible gastroenteritis viruses, by means of statistical relationships with cases vs. controls, or Ct-values, in order to establish the most appropriate diagnostic panel for our general practitioner (GP) patients in the Netherlands (2010-2012). METHODS: Archived stool samples from 1340 cases and 1100 controls were retested using internally controlled multiplex real-time PCRs for putative pathogenic gastroenteritis viruses: adenovirus, astrovirus, bocavirus, enterovirus, norovirus GI and GII, human parechovirus, rotavirus, salivirus, sapovirus, and torovirus. RESULTS: The prevalence of any virus in symptomatic cases and asymptomatic controls was 16.6% (223/1340) and 10.2% (112/1100), respectively. Prevalence of astrovirus (adjusted odds ratio (aOR) 10.37; 95% confidence interval (CI) 1.34-80.06) and norovirus GII (aOR 3.10; CI 1.62-5.92) was significantly higher in cases versus controls. Rotavirus was encountered only in cases. We did not find torovirus and there was no statistically significant relationship with cases for salivirus (aOR 1,67; (CI) 0.43-6.54)), adenovirus non-group F (aOR 1.20; CI 0.75-1.91), bocavirus (aOR 0.85; CI 0.05-13.64), enterovirus (aOR 0.83; CI 0.50-1.37), human parechovirus (aOR 1.61; CI 0.54-4.77) and sapovirus (aOR 1.15; CI 0.67-1.98). Though adenovirus group F (aOR 6.37; CI 0.80-50.92) and norovirus GI (aOR 2.22, CI: 0.79-6.23) are known enteropathogenic viruses and were more prevalent in cases than in controls, this did not reach significance in this study. The Ct value did not discriminate between carriage and disease in PCR-positive subjects. CONCLUSIONS: In our population, diagnostic gastroenteritis tests should screen for adenovirus group F, astrovirus, noroviruses GI and GII, and rotavirus. Case-control studies as ours are lacking and should also be carried out in populations from other epidemiological backgrounds.

Symptoms and treatment of Dientamoeba fragilis infection in children, a retrospective study.

INTRODUCTION: Dientamoeba fragilis infection in children is common, and its incidence has increased since the introduction of more sensitive molecular techniques. There is no consensus on the optimal treatment. Current medical practice in the Netherlands is to treat symptomatic children with clioquinol or metronidazole. This study attempts to obtain more information about the clinical picture of D. fragilis infection in children and to evaluate responses to both antiparasitic drugs. METHODS: Children <18 years of age with a positive stool polymerase chain reaction test for D. fragilis infection were retrospectively evaluated. Clinical data and effectiveness of treatment were analyzed by examining patient's hospital records from the Medical Centre Leeuwarden by repeated analysis of stool samples by the Centre for Infectious Diseases in Friesland. RESULTS: We analyzed 238 patients with an average age of 8.5 years (±4.2 years). Most patients were symptomatic (95.8%) and presented with abdominal pain (72.7%), loose stools (32.8%) and hard stools (24.8%). Coinfection with other gastrointestinal pathogens was present in 29 patients (12.2%). A higher incidence of infection was found in the winter. Clioquinol had a higher clinical success rate than metronidazole (74.7% versus 55.2%, P = 0.047). CONCLUSION: These results suggest that clioquinol could be more effective than metronidazole in alleviating symptoms of D. fragilis infection in children, but double-blind prospective placebo-controlled studies should be performed before final conclusions can be made.

A comparison of an interferon-gamma release assay and tuberculin skin test in refractory inflammatory disease patients screened for latent tuberculosis prior to the initiation of a first tumor necrosis factor α inhibitor.

Treatment with TNFα inhibitors increases risk of reactivating a latent tuberculosis\infection (LTBI). Therefore screening, prior to therapy with TNFα inhibitors, has been recommended, even in low-endemic areas such as well-developed Western Europe countries. We evaluated interferon-gamma release assay (IGRA), as opposed to tuberculin skin test (TST), for detection of LTBI in refractory inflammatory disease patients prior to the initiation of a first TNFα inhibitor. In addition, we evaluated the impact of impaired cellular immunity on IGRA. Patients starting on TNFα inhibition were screened for LTBI by TST and IGRA (Quantiferon-TB Gold). Data on tuberculosis exposure and Bacillus Calmette-Guérin (BCG) vaccination were obtained. Cellular immunity was assessed by CD4(+) T lymphocyte cell count. Nine out of 56 patients (16.1%) tested positive for LTBI. A concordant positive result was present in three patients with a medical history of tuberculosis exposure. Six patients with discordant test results had either: (1) a negative TST and positive IGRA in combination with a medical history of tuberculosis exposure (n = 1) or (2) a positive TST and negative IGRA in combination with BCG vaccination (n = 3) or a medical history of tuberculosis exposure (n = 2). CD4(+) T lymphocyte cell counts were within normal limits, and no indeterminate results of IGRA were present. IGRA appears reliable for confirming TST and excluding a false positive TST (due to prior BCG vaccination) in this Dutch serie of patients. In addition, IGRA may detect one additional case of LTBI out of 56 patients that would otherwise be missed using solely TST. Immune suppression appears not to result significantly in lower CD4(+) T lymphocyte cell counts and indeterminate results of IGRA, despite systemic corticosteroid treatment in half of the patients. Confirmation in larger studies, including assessment of cost-effectiveness, is required.

Clinical validation of a new real-time PCR assay for detection of enteroviruses and parechoviruses, and implications for diagnostic procedures.

BACKGROUND: Enteroviruses (EV) and parechoviruses (HPeV) are the most common causes of aseptic meningitis, encephalitis and sepsis-like syndrome in neonates. Detection by nucleic acid amplification methods improves patient management. OBJECTIVE: Development of a real-time PCR assay on a LightCycler for simultaneous detection of EV, HPeV and an internal control to monitor inhibition. STUDY DESIGN: We investigated the value of the new assay, prospectively, in a variety of samples from patients suspected of having viral meningitis or sepsis-like syndrome. RESULTS: The assay detected 64 EV serotypes and HPeV types 1-4. Of 186 patients, 63 (33.9%) were EV positive and 18 (9.7%) HPeV positive in one or more samples. In 43 of 159 feces and 6 of 57 throat samples viral culture and PCR were positive. With real-time PCR 27 extra EV and 19 HPeV positives were found. Blood and CSF were present from 33 patients. In 19 patients blood and CSF were positive, one was only positive in CSF, two were only positive in blood, 11 were negative. From 96 patients CSF and/or blood samples were tested and compared to results in throat and/or feces samples. Forty patients were EV-PCR and 14 HPeV-PCR positive in blood and/or CSF. All of these were confirmed by a positive PCR for the respective virus in feces and/or throat. CONCLUSIONS: Simultaneous detection of EV and HPeV with this two-step real-time PCR is specific, faster and more sensitive than viral culture. All systemic infections (blood or CSF positive) were confirmed in feces. Culture is no longer necessary for clinical diagnosis and should only be performed on PCR-positive samples to obtain isolates for typing purposes. Application of this assay is an important improvement for patient management since the outcome of the analysis is available within the time frame of clinical decision-making.

Detailed kinetics of EBV-specific CD4+ and CD8+ T cells during primary EBV infection in a kidney transplant patient.

The etiology of infectious mononucleosis is poorly understood and usually detected many weeks after infection. Here, we present a unique case of primary symptomatic EBV infection after kidney transplantation, in whom we analyzed both EBV-specific CD4+ and CD8+ T cells in detail from the moment of infection up to latency. We show that EBV-specific T-cell responses in peripheral blood during primary EBV infection after kidney transplantation peaked early after the appearance of viral load, but well before onset of IM symptoms, suggesting that IM in this case is not caused by high numbers of CD8+ T cells per se but may be caused by lack of homing to lymph nodes or tonsils.

Development of virus-specific CD4+ T cells on reexposure to Varicella-Zoster virus.

Immunity to childhood diseases is maintained for decades by mechanisms that, at present, are still unclear. We longitudinally studied immune responses in 16 adults exposed to children experiencing varicella (chicken pox). None of the individuals showed clinical signs of infection, and varicella-zoster virus (VZV) DNA could not be detected in peripheral blood or cultured from nasopharyngeal swabs. Exposure to VZV, however, induced expansion of antigen-specific CD4(+) T cells in peripheral blood, with concomitant changes in cytotoxic CD8(+) T cells and natural killer cells. VZV-specific memory CD4(+) T cells were uniformly CD45RA(-) and enriched for CD27(-) cells. The virus-specific cells produced interferon- gamma, tumor necrosis factor- alpha, and interleukin-2. These memory responses to VZV were compared with the primary immune responses of children experiencing varicella. VZV-specific memory CD4(+) T cell responses largely resemble the primary immune response to VZV.

Pattern of white matter abnormalities at MR imaging: use of polymerase chain reaction testing of Guthrie cards to link pattern with congenital cytomegalovirus infection.

PURPOSE: To define a magnetic resonance (MR) imaging pattern suggestive of congenital cytomegalovirus (CMV) infection by using polymerase chain reaction (PCR) testing to detect CMV DNA in neonatal blood on Guthrie cards for validation. MATERIALS AND METHODS: On the basis of findings in eight patients with documented congenital CMV infection, the authors developed MR imaging inclusion criteria, including multifocal lesions predominantly located in the deep parietal white matter. If gyral abnormalities were present, white matter lesions were either multifocal or diffuse. The criteria were applied to 152 patients with static leukoencephalopathy of unknown etiology. Guthrie cards for 22 of the 43 patients fulfilling the MR imaging criteria, 20 patients not fulfilling them, and 300 control subjects were analyzed. Fisher exact testing was used to evaluate the association between MR imaging characteristics and CMV status, and backward elimination linear discriminant analysis was used to identify MR imaging characteristics predictive of CMV infection in addition to the initial criteria. RESULTS: PCR test results were positive in 12 of 22 patients suspected of having congenital CMV infection, in no patient not suspected of having infection (P <.001), and in two of 300 control subjects (negative predictive value [NPV] of MR imaging criteria, 100% [95% CI: 83%, 100%]; positive predictive value [PPV], 55% [95% CI: 32%, 76%]). The most important additional MR imaging finding predicting a positive PCR result was abnormality of the anterior part of the temporal lobe, including abnormal white matter, cysts, and enlargement of inferior horns. Including this finding in the MR imaging criteria enhanced the PPV (89%; 95% CI: 52%, 99%) at the expense of the NPV (88%; 95% CI: 72%, 97%). CONCLUSION: In patients with static encephalopathy, an MR imaging pattern of multifocal lesions predominantly involving deep parietal white matter, with or without gyral abnormalities, is predictive of congenital CMV infection. When gyral abnormalities are present, leukoencephalopathy may also be diffuse. The presence of abnormalities in the anterior part of the temporal lobe increases the likelihood that CMV infection is present.

Spontaneous outgrowth of EBV-transformed B-cells reflects EBV-specific immunity in vivo; a useful tool in the follow-up of EBV-driven immunoproliferative disorders in allograft recipients.

During immunosuppressive medication, Epstein-Barr virus (EBV) infection is associated with a risk of developing posttransplant lymphoproliferative disease (PTLD). The appropriateness of a spontaneous EBV B-cell transformation (SET) assay as a monitor of EBV-specific immunity was evaluated to investigate if it safely allows reducing immunosuppressive medication, thereby decreasing the risk of developing PTLD. PBMC were isolated longitudinally from 20 pediatric renal allograft recipients treated with prednisone and cyclosporine combined with either azathioprine or mycophenolate mofetil. Most significantly, EBV-peptide-specific CD8+ T cells were detectable in the blood of patients with negative SET assays, coinciding with significantly lower EBV loads, whereas these cells were less frequent in the blood of patients with positive SET assays. Reducing the levels of immunosuppression resulted in normalization of the SET assays. Therefore, the SET assay is a reflection of the interaction between viral replication, transformation of B cells, and EBV-specific immunity in vivo and hence a valuable screening test for EBV-driven lymphoproliferative phenomena in allograft recipients.

Human cytomegalovirus DNA in plasma and serum specimens of renal transplant recipients is highly fragmented.

Quantitation of cytomegalovirus (CMV) DNA in plasma and serum by PCR is increasingly used to identify patients at risk for developing CMV disease and to monitor the efficacy of antiviral therapy. Although CMV DNA levels are generally interpreted as viral loads, the exact nature of the viral DNA in these specimens is unknown. We studied the state of CMV DNA in plasma and serum specimens obtained from three renal transplant recipients at peak viral DNA levels during primary CMV infection. For this purpose, DNA isolated from these specimens was fractionated by size, and CMV DNA levels in the resulting DNA fractions were measured by quantitative PCR targeted at large (578-bp) and small (134-bp) amplicons. These experiments showed that the molecular sizes of DNA fragments from which CMV DNA is amplified were small (<2,000 bp), indicating that CMV DNA in plasma and serum is highly fragmented. Furthermore, CMV DNA levels were consistently higher when targeted at the smaller amplicon, providing additional evidence for the fragmentation of viral DNA. In conclusion, the first results with three patients have shown that CMV DNA in plasma and serum is highly fragmented and does not necessarily reflect the amount of infectious virus. These observations have potential consequences for understanding CMV pathogenesis and interpreting CMV DNA levels in individual patient management.