Cytomegalovirus prophylaxis in pediatric kidney transplantation: the Dutch experience.

Many children receiving a kidney transplant are seronegative for CMV and therefore, highly susceptible to a primary CMV infection. This study aims at evaluating incidence, time of occurrence, and severity of CMV infection in the first year post-transplantation in relation to different types of CMV prophylaxis. Transplantations in three centers in the Netherlands between 1999 and 2010 were included. Retrospective, observational, multicenter study. Clinical data and PCR measurements of CMV were collected. Prophylaxis in high-risk patients (CMV serostatus D+R-) consisted of (val)ganciclovir during three months, or acyclovir plus CMV immunoglobulin at a former stage. Intermediate-risk patients (R+) received (val)acyclovir, or acyclovir plus CMV immunoglobulin at a former stage. Low-risk patients (D-R-) did not receive prophylaxis. Infection was defined as CMV PCR above 50 geq/mL plasma or whole blood, a clinically relevant infection above 1000 geq/mL. One hundred and fifty-nine transplantations were included. CMV infection was documented for 41% of high-risk, 24% of intermediate-risk, and 13% of low-risk patients, in the latter two groups typically during the first three months. The infection rate was highest in the high-risk group after cessation of valganciclovir prophylaxis. Valganciclovir provided better protection than did acyclovir + CMV immunoglobulin. Adding an IL2-receptor blocker to the immunosuppressive regimen did not affect the infection rate. Acute graft rejection was not related with CMV infection. Valganciclovir prophylaxis effectively prevents CMV infection in high-risk pediatric kidney recipients, but only during prophylaxis. Valacyclovir prophylaxis in intermediate-risk patients is less effective.

P2 domain profiles and shedding dynamics in prospectively monitored norovirus outbreaks.

BACKGROUND: Norovirus P2 domain is commonly used to extrapolate transmission within an outbreak (OB) setting. The current definition is that transmission among cases is considered to be proven when no sequence variation is found. OBJECTIVES: Previous studies have shown a high mutation rate and errors during replication of the norovirus genome, therefore the validity of this criterion must be evaluated. STUDY DESIGN: Sequences of the P2 domain were obtained from patients and health care workers sampled during 4 prospectively GII.4 outbreaks. Fecal samples were tested by RT-PCR for presence of norovirus RNA against a standard control preparation to allow quantification. Estimated time of onset of shedding was derived from shedding kinetics modeled on data from sequential sampling. Thereby P2 sequence variation could be linked to estimated total virus excretion in individual subjects. RESULTS: In all the outbreaks, P2 domain variation was found that resulted in unique codon changes in some patients. Mutations were found in 14% of initial samples and >50% of follow-up samples taken from patients involved in an outbreak. In three patients, aa mutations was observed in or near sites involved in host or antigen binding. CONCLUSIONS: We concluded that P2 domain variation increases with duration of virus shedding, but was unrelated to total amounts of virus shed. Therefore, we propose that cluster identification based on identical sequences should be relaxed to accommodate minor sequence variation. When using sequence data to support outbreak investigations, sequence diversity should be interpreted in relation to timing of sampling since onset of illness.

Imported scrub typhus in The Netherlands.

Two cases of travel-acquired scrub typhus imported in the Netherlands are described. The characteristic eschar was absent in both cases. One case acquired scrub typhus in non-rural surroundings in India, highlighting that scrub typhus must also be considered a (sub) urban zoonosis.

Unrecognized norovirus infections in health care institutions and their clinical impact.

Noroviruses (NoVs) have emerged as the leading cause of acute viral gastroenteritis (GE) in humans. Although diagnostic facilities have greatly improved, significant underdiagnosis of NoV in hospitals may still occur, thereby increasing clinical burden and nosocomial spread. We evaluated the underdiagnosis of sporadic NoV infections in a tertiary care hospital and estimated its clinical impact. From December 2008 until July 2009, fecal samples specifically referred for bacterial but not viral examination were retrospectively tested for NoV by real-time PCR. The clinical and virological data from patients with undiagnosed NoV infection (missed patients) were evaluated and compared with those from patients with recognized NoV. During the study period, 45 patients with undiagnosed NoV were detected, whereas 50 patients were regularly diagnosed. The missed NoV cases were more frequently adults than children (80% versus 46%; P < 0.001). The viral load levels did not differ between the diagnosed and missed patients, but missed patients more frequently presented without diarrhea (20% versus 4%; P < 0.07). The newly admitted missed NoV cases with GE underwent more diagnostic imaging (24% versus 4%; P < 0.01) and tended to be hospitalized longer. When missed-NoV patients were included, the number of nosocomial clusters doubled and missed patients were index cases in 5 of the 6 clusters. These data indicate that NoV infections are frequently missed despite routine laboratory testing and demonstrate that underdiagnosis of NoV patients is associated with costly abdominal imaging and nosocomial clustering. Awareness of NoV infection in adult patients and education about the importance of viral GE should be increased.

Nosocomial transmission of norovirus is mainly caused by symptomatic cases.

BACKGROUND: Nosocomial norovirus (NoV) infection is common and may increase the burden of disease in healthcare settings, particularly in vulnerable hospitalized patients. Implementing effective infection control during and after admission may limit further spread, but evidence-based measures are lacking. METHODS: In this study, we performed a systematic evaluation of sources and modes of transmission during NoV outbreaks within 2 types of healthcare facilities. An outbreak protocol was developed to sample all patients and healthcare workers (HCWs) with and without symptoms on wards involved in outbreaks. Data on clinical history and possible high-risk exposures were collected. Five outbreaks were investigated, involving 28 patients with recognized symptomatic NoV infection. RESULTS: Enhanced sampling, however, yielded 65 additional cases, of whom 14% (n = 9) were asymptomatic patients, 57% (n = 37) were symptomatic HCWs, and 17% (n = 11) were asymptomatic HCWs. For 12% (n = 8), clinical data were not provided (2 HCWs and 6 patients). On the basis of the shedding kinetics, the onset of infection was estimated for each case. The generation interval was then used to construct plausible transmission pathways and reproduction numbers for symptomatic and asymptomatic patients and HCWs. CONCLUSIONS: We found that symptomatic patients and HCWs were more often involved in transmission events than asymptomatic shedders. Asymptomatic HCWs rarely contributed to transmission, despite high levels of fecal virus shedding.

A comparison of two assays for quantification of Hepatitis B surface Antigen in patients with chronic hepatitis B.

BACKGROUND AND OBJECTIVES: Serum Hepatitis B surface Antigen (HBsAg) levels correlate with hepatitis B virus intrahepatic covalently closed circular DNA and may predict response to treatment. Currently, 2 commercial platforms are available for HBsAg quantification in clinical practice, the Architect HBsAg QT and the Elecsys HBsAg. We aimed to directly compare the results of these assays. STUDY DESIGN: HBsAg levels were measured in 1427 serum samples from HBeAg-positive chronic hepatitis B patients who participated in a randomized trial of peginterferon alfa-2b±lamivudine. Samples were extracted from our serum bank, thawed, and subsequently analysed for HBsAg levels using both assays. RESULTS: Of 1427 samples, 242 (17%) were taken before and 1185 during the treatment phase of the study. Distribution of HBV genotypes was 447 (31%) genotype A, 125 (9%) B, 210 (15%) C and 534 (37%) D. Correlation between Architect and Elecsys results was high (r=0.96, p<0.001). By Bland-Altman analysis, agreement between the two assays was close (mean difference between Architect and Elecsys: -0.01logIU/mL, 95% CI: -0.55-0.52logIU/mL), also when analysed separately for HBV genotypes A-D. Additionally, the performance of our recently published stopping rule for HBeAg-positive patients treated with peginterferon was comparable: the negative predictive values were 96% and 98% for Elecsys and Architect, respectively. CONCLUSIONS: There is a high correlation and close agreement between quantitative HBsAg measurements conducted with the Architect and the Elecsys. Clinical prediction rules derived from data from one platform can be applied on the other; both can therefore be used in clinical practice.

Using molecular epidemiology to trace transmission of nosocomial norovirus infection.

Nosocomial norovirus (NoV) infection is common and may lead to complications in vulnerable hospitalized patients. Understanding sources and modes of transmission of noroviruses within health care settings will support the design of evidence-based strategies for reducing introduction and further spread. We sequenced a highly variable segment of the genome to identify possible clusters in patients with and without acute gastroenteritis who were hospitalized in the period 2002-2007. Admission and sampling dates were used to separate patients with nosocomial infection from those without nosocomial infection. Epidemiological clustering retrieved 22 clusters, defined as ≥ 2 patients with nosocomial infection on the same ward within 5 days. In total, 264 patients (of 2,458 tested) were diagnosed with NoV infection, and 61% of the patient strains could be genotyped. Of those, 51% (n = 82) belonged to GII.4, 34% (n = 54) belonged to GII.3, and 15% (n = 24) belonged to other genotypes (GI.6B, GII.17, GII.7, and GII.2). In children's wards, GII.3 strains were associated with nosocomial spread more often than other viruses were, whereas in adults this was the case for GII.4 strains. Sequence alignment recognized 11 new clusters based on identical P2 domains (4 GII.3 and 7 GII.4 clusters), involving patients in different wards. This increased the total number of recognized clusters by 50%. Five of these clusters involved at least one outpatient, providing a possible target for improvement of infection control. We concluded that the use of sequence-based typing should be considered for identifying hidden nosocomial clusters of NoV infections within health care settings.

Chronic shedders as reservoir for nosocomial transmission of norovirus.

Norovirus (NoV) infection in immunocompromised patients may lead to prolonged norovirus shedding. Here, we demonstrate the involvement of three chronic shedders in hospital outbreaks. Combined epidemiological and molecular evidence suggests that in one case, NoV transmission occurred at least 17 days after the first diagnosis.

The value of Chlamydia trachomatis-specific IgG antibody testing and hysterosalpingography for predicting tubal pathology and occurrence of pregnancy.

We assessed two diagnostic methods, Chlamydia trachomatis-specific IgG and hysterosalpingography (HSG), as a screening test for the likelihood of tubal damage or occurrence of pregnancy before laparoscopy in 178 subfertile women who were randomly assigned to HSG followed by laparoscopy or immediate laparoscopy. The diagnostic accuracy and prognostic value of both C. trachomatis-specific IgG antibody testing and HSG are comparable but show poor performance.

Parvovirus B19 infection in pregnancy studied by maternal viral load and immune responses.

OBJECTIVES: Facilitate risk assessment of vital complications in fetuses of pregnancies affected by acute parvovirus B19 (B19V) infection. DESIGN: Study of the natural course of maternal B19V infection in four cases, from early pregnancy on. SETTING: University Medical Center in the Netherlands. POPULATION: Pregnant mothers attending obstetric services. METHODS: Serial measurements of the maternal and fetal or neonatal viral load and antibody responses. MAIN OUTCOME MEASURES: Maternal and fetal/neonatal serum B19V viral DNA load and specific IgM and IgG antibodies in maternal serum. RESULTS: Peak viral load levels occurred within 1 week after maternal infection and peak IgM levels were observed 1 week after the peak viral load levels. Approximation of IgG and IgM ratios usually took place 4 weeks after infection. Vertical transmission occurred 1-3 weeks after maternal infection, suggesting that fetal infection occurs during the maternal peak viral load. CONCLUSIONS: Maternal B19V DNA load levels and IgM responses are useful to estimate the risk of parvovirus B19-associated fetal complications. The maternal peak viral load directly precedes the onset of fetal infection and may be used to indicate the stage of intrauterine B19V infection.

Fourth human parechovirus serotype.

We identified a novel human parechovirus (HPeV) type (K251176-02) from a neonate with fever. Analysis of the complete genome showed K251176-02 to be a new HPeV genotype. Since K251176-02 could not be neutralized with antibodies against known HPeV serotypes 1-3, it should be classified as a fourth HPeV serotype.

Fetal stroke and congenital parvovirus B19 infection complicated by activated protein C resistance.

UNLABELLED: Parvovirus B19 infection in gestation has been associated with severe fetal complications such as anaemia, hydrops and fetal demise. Fetal infection in the first trimester poses the greatest risk for these complications, but infection during the third trimester is more common than previously appreciated and can be associated with severe complications, i.e. fetal death, in the absence of hydrops or classical clinical symptoms. Parvovirus B19 infection has been associated with vasculitis and pathological changes in the central nervous system, which may cause stroke. We report a newborn infant with a rare combination of a recent central nervous system infection with parvovirus B19 and a factor V Leiden mutation, who developed fetal stroke. CONCLUSION: Factor V Leiden mutation leads to activated protein C resistance and increases the risk of thromboembolism. Thromboembolism occurs rarely in newborns with activated protein C resistance, but can be precipitated by dehydration, asphyxia and infection. Although parvovirus B19 infection of the central nervous system may be a precipitant in neonatal and/or fetal stroke, it can also cause stroke independent of a thrombophilic mutation. In this case, both causative factors may have coincided.

Parvovirus B19 infection in pregnancy.

Parvovirus B19 is a small single-stranded DNA virus and a potent inhibitor of erythropoiesis, due to its cytotoxicity to erythroid progenitor cells. Infection with parvovirus B19 during pregnancy can cause several serious complications in the fetus, such as fetal anemia, neurological anomalies, hydrops fetalis, and fetal death. Early diagnosis and treatment of intrauterine parvovirus B19 infection is essential in preventing these fetal complications. Testing maternal serum for IgM antibodies against parvovirus B19 and DNA detection by PCR can confirm maternal infection. If maternal infection has occurred, ultrasound investigation of the fetus and measurement of the peak systolic flow velocity of the middle cerebral artery are sensitive non-invasive procedures to diagnose fetal anemia and hydrops. Intrauterine transfusion is currently the only effective treatment to alleviate fetal anemia, but if the fetus is (near) term, induction of delivery should be considered. Most maternal infections with parvovirus B19 occur through contact with infected children at home. Individual counseling of susceptible pregnant women will reduce unnecessary fetal deaths.

Parvovirus B19 viral loads in relation to VP1 and VP2 antibody responses in diagnostic blood samples.

BACKGROUND: Human parvovirus B19 infection is characterised by high peak viral load levels followed by episodes of prolonged viremia. The risk of transmission of parvovirus B19 by blood or blood products has been increasingly recognised and parameters that can predict the risk of transmission are subject of interest. OBJECTIVES: This study aimed to study correlations between B19 viral DNA loads and antibody responses to the viral antigens VP1 and VP2 in clinical serum samples. STUDY DESIGN: A panel of 1610 serum samples from patients clinically suspected from acute B19 infection were analysed. Antibodies were measured by the parvovirus anti-VP1 immuno-fluorescence assay (IFA) and the anti-VP2 enzyme immunoassay (EIA) from Biotrin. B19 viral loads were measured by a real-time PCR using the external WHO standard for DNA quantification. RESULTS: Positive IgM responses were found in 154 (9.6%) of the 1610 sera tested. Based on the PCR results in a subset of 312 sera, the anti-VP2 EIA IgM showed a better combination of sensitivity/specificity (91%/94%) compared to the anti-VP1 IFA (66%/97%). B19 DNA levels in the sera strongly correlated with the levels of IgM antibodies, all sera with high viral loads (>10(6)IU/ml) having VP2 EIA IgM ratios above 3.0. CONCLUSIONS: The B19 VP2 IgM ELISA is superior to the B19 VP1 IgM IFA if verified by PCR. Anti-VP2 IgM antibodies in sera are indicative for the presence B19 DNA and can be used to predict high levels of B19 DNA in diagnostic sera.

Evaluation of 12 commercial tests and the complement fixation test for Mycoplasma pneumoniae-specific immunoglobulin G (IgG) and IgM antibodies, with PCR used as the "gold standard".

Serology and nucleic acid amplification are the main diagnostic tools for the diagnosis of Mycoplasma pneumoniae infection. Since no reference standard is generally accepted, serologic assays for M. pneumoniae have not been evaluated on a broad scale. In this study, 12 commercially available serologic assays (for immunoglobulin G [IgG] and IgM) and the complement fixation test (CFT) were evaluated by using M. pneumoniae DNA detection by real-time PCR as the "gold standard." The assays tested were Platelia EIA (Bio-Rad), SeroMP EIA (Savyon), Serion classic EIA (Virion/Serion), Biotest EIA (Biotest), Ridascreen EIA (r-Biopharm), AniLabsystems EIA (Labsystems), Novum EIA (Novum Diagnostica), Diagnosys EIA (MP products), Genzyme/Virotech EIA, ImmunoWell EIA (Genbio), ImmunoCard EIA (Meridian), and SerodiaMycoII microparticle agglutination (Fujirebio). Serum samples (n = 46) from 27 PCR-positive patients with a known first day of disease and sera (n = 33) from PCR-negative controls were obtained from prospective studies of acute lower respiratory tract infections. Additionally, control sera (n = 63) from patients with acute viral or bacterial respiratory infections other than those caused by M. pneumoniae were tested. The results showed low specificities for both the Novum and the ImmunoCard IgM assays. The IgM assays with the best performances in terms of sensitivity and specificity were AniLabsystems (77% and 92%, respectively), SeroMP (71% and 88%, respectively), and CFT (65% and 97%, respectively). Good receiver operating characteristic areas under the curve were found for CFT (0.94), the Platelia assay (0.87), and the AniLabsystems assay (0.85). We conclude that there are few commercial serologic assays for the detection of M. pneumoniae infections with appropriate performances in terms of sensitivity and specificity and that PCR has become increasingly important for the diagnosis of M. pneumoniae infections in defined groups of patients.

Immune reconstitution and clearance of human adenovirus viremia in pediatric stem-cell recipients.

BACKGROUND: Human adenovirus (HAdV) infections are increasingly frequent complications of allogeneic stem-cell transplantation (SCT), especially in children. Only a few data on the correlation between immune recovery and the course of HAdV infection are available, and data on HAdV-specific responses are lacking. METHODS: In a prospective study, we determined the correlation between the HAdV DNA load in plasma and lymphocyte reconstitution in 48 children after allogeneic SCT. Additionally, HAdV-specific humoral and cellular immune responses were investigated. RESULTS: HAdV infection occurred in 21 patients (44%), and, in 6 of these patients, the infection progressed to viremia, as demonstrated by the presence of HAdV DNA in plasma. Low lymphocyte counts at the onset of infection were predictive of HAdV viremia. Survival of patients with HAdV viremia was associated with an increase in lymphocyte counts during the first weeks after infection. In these patients, HAdV-specific CD4+ T cell responses, as well as increases in titers of neutralizing antibody, were detected after clearance of HAdV DNA from plasma. CONCLUSIONS: Lymphocyte reconstitution appears to play a crucial role in clearance of HAdV viremia and survival of the host, warranting further development of therapeutic interventions aimed at improving immune recovery.

Genotypic and phenotypic characterization of acyclovir-resistant herpes simplex viruses isolated from haematopoietic stem cell transplant recipients.

Thirty-one herpes simplex virus type one (HSV-1) isolates from 12 haematopoietic stem cell transplant recipients with persistent HSV infections despite acyclovir (ACV) prophylaxis or treatment, were genotypically and phenotypically characterized. The relationship between drug susceptibility of the isolates and mutations in thymidine kinase (TK) and DNA polymerase (DNA pol) genes was examined. In all 12 patients, HSV infections were due to ACV-resistant, foscarnet-sensitive viruses. Out of 31 isolates examined, 23 were resistant and eight were sensitive to ACV; eight patients carried viruses with frameshift mutations in the TK gene (due to addition or deletion of single nucleotides in homopolymeric repeats). These mutations were found at codon 61 (G deletion, one patient), 146 (G insertion, five patients) and 153 or 185 (C deletion, one patient each). In four patients, viruses were selected during ACV therapy that contained novel amino acid substitutions in the TK gene (H58R, G129D, A189V, R216H, R220C). Their possible role in ACV resistance was further confirmed phenotypically and by the absence of any resistance-associated mutations in the DNA pol gene. These substitutions were located in ATP- or nucleoside-binding sites or in conserved regions of the TK gene. In addition, a single mutation, Q570R, in the delta-region C of the DNA pol gene, was identified in an isolate from a single patient with resistance to ACV. Our study confirms and expands previous data on genotypic changes associated with ACV resistance of HSV-1 clinical isolates.

Validation of clinical application of cytomegalovirus plasma DNA load measurement and definition of treatment criteria by analysis of correlation to antigen detection.

Successful preemptive cytomegalovirus (CMV) therapy in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infections. The pp65 antigenemia assay has been used for this purpose with considerable success. Quantification of CMV DNA is currently regarded to be an alternative diagnostic approach. The precise relationship between these two methods has still to be defined, but is essential to compare diagnostic results. This study compared the results of both assays with a large series of transplant recipients in different categories. An internally controlled quantitative real-time CMV DNA PCR was used to test 409 plasma samples from solid organ transplant (SOT) and stem cell transplant (SCT) patients. Levels of CMV DNA in plasma correlated well with classified outcomes of the pp65 antigenemia test. Despite this correlation, the quantitative CMV PCR values in a class of antigen test results were within a wide range, and the definition of an optimal cutoff value for initiating treatment required further analysis by a receiver-operating characteristic curve analysis. This is essential for reactivating infections in particular. For the SCT patients the optimal cutoff value of CMV DNA load defining relevant viral reactivation (in this assay, 10,000 copies/ml) was slightly higher than that for the SOT patients (6,300 copies/ml). Based on a comparison with the established pp65 antigenemia assay, quantification of CMV DNA in plasma appeared to be capable of guiding the clinical management of transplant recipients. This approach may have important advantages, which include a superior reproducibility and sensitivity, allowing the inclusion of kinetic criteria in clinical guidelines.

Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza a and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4.

Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory RNA viruses. A rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, human respiratory syncytial virus (RSV), parainfluenza virus 1 (PIV1), PIV2, PIV3, and PIV4 in a two-tube multiplex reaction which used molecular beacons to discriminate the pathogens. A total of 358 respiratory samples taken over a 1-year period were analyzed by the multiplex assay. The incidence of respiratory viruses detected in these samples was 67 of 358 (19%) and 87 of 358 (24%) by culture and real-time PCR, respectively. Culture detected 3 influenza A virus, 2 influenza B virus, 57 RSV, 2 PIV1, and 2 PIV3 infections. All of these culture-positive samples and an additional 5 influenza A virus, 6 RSV, 2 PIV1, 1 PIV2, 1 PIV3, and 3 PIV4 infections were detected by the multiplex real-time PCR. The application of real-time PCR to clinical samples increases the sensitivity for respiratory viral diagnosis. In addition, results can be obtained within 6 h, which increases clinical relevance. Therefore, use of this real-time PCR assay would improve patient management and infection control.

Sequential switching of DNA polymerase and thymidine kinase-mediated HSV-1 drug resistance in an immunocompromised child.

Sequential herpes simplex virus type 1 (HSV-1) isolates were obtained from a paediatric haematopoietic stem cell transplant (HSCT) patient who received prolonged therapy with acyclovir (ACV) followed by foscarnet (PFA) and topical cidofovir (HPMPC) for severe persistent mucocutaneous HSV-1 infection. The isolates were retrospectively studied for drug resistance. The first resistant isolate associated with clinical failure of antiviral therapy emerged 44 days post-ACV treatment initiation. Susceptibility testing revealed an ACV-resistant HSV strain that demonstrated cross resistance to PFA in the absence of any previous PFA treatment. The observed cross resistance was conferred by a single amino acid substitution, Ser724Asn, in the HSV DNA polymerase (DNA pol) gene. During the subsequent course of ACV therapy, the ACV/PFA-cross-resistant isolates were replaced by ACV-resistant, PFA-sensitive isolates. These isolates carried no DNA pol mutations, but had an Arg163His substitution in the thymidine kinase gene. Upon subsequent switching of antiviral therapy from ACV to PFA, the original ACV/PFA-cross-resistant DNA pol mutant re-appeared. Our study shows the emergence of different drug-resistant HSV variants during ongoing, unchanged ACV therapy. Furthermore, a rapid re-selection of the original resistant variant was observed after switch. For optimal antiviral management of HSV infections in HSCT recipients, therapeutic decisions should be guided by drug susceptibility results whenever therapeutic failure is observed and/or when changes in antiviral treatment are considered.