How much should we sequence? An analysis of the Swiss SARS-CoV-2 surveillance effort.

During the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent-something that is unknown at the time of sequencing-and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses.

[False positives in infectious serology : a rare issue that should not be ignored]. / Faux positifs en sérologie infectieuse : un problème rare à ne pas ignorer.

Although rare, false-positive results from infectious serology tests can mislead the practitioner and have harmful consequences for the patient. The causes are not always clear, but there are certain principles that are important to be aware of, and that help to interpret these diagnostic puzzles correctly. Similarities between different families of pathogens, the technical characteristics of the tests used, the use of therapeutic human immunoglobulins, the detection of vaccine-induced antibodies or even the detection of vaccine antigens themselves can cause non-specific reactions. This article uses examples from routine laboratory practice to illustrate the problem and draw the attention of the treating physician to this issue.

Bien que rares, les résultats faussement positifs des tests de sérologies infectieuses peuvent induire en erreur le praticien et entraîner des conséquences délétères pour le patient. Leurs causes ne sont pas toujours claires, mais certains principes sont importants à connaître et permettent d'interpréter correctement ces casse-têtes diagnostiques. Des similarités entre différentes familles de pathogènes, les caractéristiques techniques des tests utilisés, l'utilisation d'immunoglobulines humaines thérapeutiques, la détection d'anticorps induits par des vaccins ou même la détection des antigènes vaccinaux eux-mêmes peuvent causer des réactions non spécifiques. Cet article illustre ce problème par des exemples tirés de la routine du laboratoire afin d'attirer l'attention du médecin traitant sur ce problème.

IVDR: Analysis of the Social, Economic, and Practical Consequences of the Application of an Ordinance of the In Vitro Diagnostic Ordinance in Switzerland.

IVDR regulation represents a major challenge for diagnostic microbiology laboratories. IVDR complicates a broad range of aspects and poses a risk given the high diversity of pathogens (including rare but highly virulent microbes) and the large variety of samples submitted for analysis. The regular emergence of new pathogens (including Echovirus E-11, Adenovirus 41, Monkeypox virus, Alongshan virus, and Enterovirus D68, as recent examples in Europe in the post SARS-CoV-2 era) is another factor that makes IVDR regulation risky, because its detrimental effect on production of in-house tests will negatively impact knowledge and expertise in the development of new diagnostic tests. Moreover, such regulations negatively impact the availability of diagnostic tests, especially for neglected pathogens, and has a detrimental effect on the overall costs of the tests. The increased regulatory burden of IVDR may thereby pose an important risk for public health. Taken together, it will have a negative impact on the financial balance of diagnostic microbiology laboratories (especially small ones). The already-high standards of quality management of all ISO-accredited and Swissmedic-authorized laboratories render IVDR law of little value, at least in Switzerland, while tremendously increasing the regulatory burden and associated costs. Eventually, patients will need to pay for diagnostic assays outside of the framework of their insurance in order to obtain a proper diagnostic assessment, which may result in social inequity. Thus, based on the risk assessment outlined above, the coordinated commission for clinical microbiology proposes adjusting the IvDO ordinance by (i) introducing an obligation to be ISO 15189 accredited and (ii) not implementing the IvDO 2028 milestone.

Venous thromboembolism in critically Ill patients with COVID-19: Results of a screening study for deep vein thrombosis.

BACKGROUND: The rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and coronavirus disease 2019 (COVID-19), has caused more than 3.9 million cases worldwide. Currently, there is great interest to assess venous thrombosis prevalence, diagnosis, prevention, and management in patients with COVID-19. OBJECTIVES: To determine the prevalence of venous thromboembolism (VTE) in critically ill patients with COVID-19, using lower limbs venous ultrasonography screening. METHODS: Beginning March 8, we enrolled 25 patients who were admitted to the intensive care unit (ICU) with confirmed SARS-CoV-2 infections. The presence of lower extremity deep vein thrombosis (DVT) was systematically assessed by ultrasonography between day 5 and 10 after admission. The data reported here are those available up to May 9, 2020. RESULTS: The mean (± standard deviation) age of the patients was 68 ± 11 years, and 64% were men. No patients had a history of VTE. During the ICU stay, 8 patients (32%) had a VTE; 6 (24%) a proximal DVT, and 5 (20%) a pulmonary embolism. The rate of symptomatic VTE was 24%, while 8% of patients had screen-detected DVT. Only those patients with a documented VTE received a therapeutic anticoagulant regimen. As of May 9, 2020, 5 patients had died (20%), 2 remained in the ICU (8%), and 18 were discharged (72%). CONCLUSIONS: In critically ill patients with SARS-CoV-2 infections, DVT screening at days 5-10 of admission yielded a 32% prevalence of VTE. Seventy-five percent of events occurred before screening. Earlier screening might be effective in optimizing care in ICU patients with COVID-19.

[Management of acute diarrhea]. / Prise en charge ambulatoire de la diarrhée aiguë.

Acute diarrhea is one of the most common pathologies in resource-limited, as well as in industrialized countries. For the clinician the major challenge is to know when to perform diagnostic tests, how to interpret them, and particularly to recognize the situations where an antibiotic treatment is recommended. This will also avoid unnecessary treatments, costs, side effects and selection of resistant strains.

La diarrhée aiguë est l'une des pathologies les plus fréquentes, que ce soit dans les pays aux ressources limitées ou dans les pays industrialisés. Pour le clinicien, le défi majeur est de savoir quand il faut effectuer des tests diagnostiques, comment les interpréter, et surtout de reconnaître les situations pour lesquelles un traitement antibiotique est nécessaire. Cela permet aussi d'éviter des traitements inutiles, des coûts, des effets secondaires et la sélection de souches résistantes.

[Common causes of infectious mononucleosis]. / Causes fréquentes de mononucléose infectieuse.

Most cases of infectious mononucleosis are caused by Epstein-Barr virus (EBV). However, other rare but potentially serious etiologies need to be considered. Cytomegalovirus (CMV) and Toxoplasma gondii infections, although generally benign, can cause severe congenital infections. An acute infection by the human immunodeficiency virus (HIV) can also mimic infectious mononucleosis. Laboratory diagnostic of those infections relies primarily on the detection of specific antibodies and antigens. The interpretation of laboratory results can be impeded by cross-reactions or persistence over several months of markers of acute infection. This article reviews the most common causes of infectious mononucleosis and their diagnosis.

La majorité des cas de mononucléose infectieuse sont dus à une infection par le virus d'Epstein-Barr (EBV). D'autres étiologies plus rares peuvent cependant avoir des conséquences graves. L'infection primaire à cytomégalovirus (CMV) ou la toxoplasmose, généralement bénignes, peuvent causer des infections congénitales sévères. Une primo-infection par le virus de l'immunodéficience humaine (VIH) peut également se présenter sous forme de mononucléose. Le diagnostic de ces infections repose sur la détection d'anticorps et d'antigènes spécifiques. Les problèmes de réactions croisées ou de persistance des marqueurs d'infection aiguë durant plusieurs mois rendent parfois délicate l'interprétation des résultats de laboratoire. Cet article passe en revue les causes les plus fréquentes de mononucléose infectieuse et leur approche diagnostique.

[Mycoplasma pneumoniae: update and new challenges]. / Mycoplasma pneumoniae: connaissances actuelleset nouveaux défis.

Mycoplasma pneumoniae is a frequent cause of community acquired pneumonia, especially in children. Discovered by Eaton in the 1940s, it has long been considered as a virus, in part because of its difficult growth in cultures. M. pneumoniae can cause many complications, some of which are severe such as dermatological lesions or affections of the central nervous system. The laboratory diagnosis of M. pneumoniae is difficult, notably because of the fastidious growth conditions, the persistence of IgM antibody after acute infection and the debated existence of asymptomatic carriers. In recent years, the spread of mutants resistant to macrolides caused an additional challenge linked to this pathogen.

Mycoplasma pneumoniae est une cause fréquente de pneumonie communautaire, principalement chez l'enfant. Découvert par Eaton dans les années quarante, ce microorganisme a longtemps été considéré comme un virus, notamment de par la difficulté de le cultiver. M. pneumoniae peut causer de nombreuses complications, certaines graves, telles que des lésions dermatologiques et des atteintes du système nerveux central. Le diagnostic microbiologique est rendu difficile par des conditions de culture fastidieuses, la persistance des anticorps IgM après l'infection aiguë et l'existence de porteurs asymptomatiques actuellement débattue. Ces dernières années, la propagation de mutants résistant aux macrolides pose un défi supplémentaire lié à ce pathogène.

Subacute, tetracycline-responsive, granulomatous osteomyelitis in an adult man, consistent with Q fever infection.

Osteomyelitis due to Coxiella burnetii infection is a rare condition in adults. We report the case of a healthy young man presenting with subacute osteomyelitis of the left cheek bone, evolving gradually after an episode of acute febrile illness. Histological evaluation confirmed subacute granulomatous inflammation. Despite antibody titres not reaching the standard cut-off for chronic Q fever (phase I IgG 1/160, phase II IgG 1/2560), osteomyelitis was radiologically and histologically confirmed. A 6-month course of doxycycline/hydroxychloroquine brought clinical and radiological cure while various conventional antibiotic treatments had failed to improve the clinical condition. Currently, at 6-month follow-up, no relapse has occurred and antibody titres have declined. A shorter course of doxycycline/hydroxychloroquine than that used for chronic Q fever osteomyelitis may be sufficient to treat subacute Q fever osteomyelitis in some cases.

The human fetal glial cell line SVG p12 contains infectious BK polyomavirus.

UNLABELLED: The human fetal glial cell line SVG was generated in 1985 by transfecting primary fetal brain cells with a plasmid containing an origin-defective mutant of simian virus 40 (SV40). The cells, which express SV40 large T-antigen, support the replication of human JC polyomavirus (JCPyV) and have been used for JCPyV studies but also for other studies in which cells of neural origin were desirable. We intended to use the SVG p12 cells from ATCC for antiviral drug studies with JCPyV. However, during initial experiments, immunofluorescence microscopy controls unexpectedly revealed cells expressing the late viral proteins VP1, VP2/VP3, and agno. This was confirmed by Western blotting. Since our agnoprotein antiserum is specific for BKPyV agnoprotein, infection with BKPyV was suspected. Indeed, specific BKPyV PCR of SVG p12 supernatants revealed a viral load of >1 × 10(10) genomic equivalents/ml. Negative-staining electron microscopy showed characteristic polyomavirus virions, and infectious BKPyV was transmitted from SVG p12 supernatant to other cells. Long-range PCR covering the viral genome, followed by DNA sequencing, identified BKPyV strain UT as well as deletion derivatives. This was confirmed by next-generation sequencing. JCPyV (MAD-4) was found to infect apparently uninfected and BKPyV-infected SVG p12 cells. In total, 4 vials from 2 different ATCC lots of SVG p12 cells dating back to 2006 contained BKPyV, whereas the subclone SVG-A was negative. In conclusion, SVG p12 cells from ATCC contain infectious BKPyV. This may have affected results and interpretations of previous studies, and caution should be taken in future experiments. IMPORTANCE: This work reveals that one of the most frequently used cell lines for JC polyomavirus (JCPyV) research, the SV40-immortalized human fetal glial cell line SVG p12 obtained directly from ATCC, contains infectious BK polyomavirus (BKPyV) of strain UT and a spectrum of defective mutants. Strain UT has been previously found in urine and in tumors of different patients but is also frequently used for research. It is therefore not clear if BKPyV was present in the brain tissue used to generate the cell line or if this is a contamination. Although productive JCPyV infection of SVG cells was not dependent on prior BKPyV infection, the unnoticed presence of BKPyV may have influenced the results of studies using these cells. The interpretation of past results should therefore be reconsidered and cells tested for BKPyV before new studies are initiated. The frequently used subclone SVG-A did not contain BKPyV and could be a useful substitute.

Comparison of two quantitative real-time CMV-PCR tests calibrated against the 1st WHO international standard for viral load monitoring of renal transplant patients.

Cytomegalovirus (CMV) replication in organ transplant recipients is commonly diagnosed by quantitative PCR methods. However, there has been a poor inter-laboratory correlation of viral load values due to the lack of an international reference standard. In a recent study, the COBAS® AmpliPrep/COBAS® TaqMan® (CAP/CTM) CMV test calibrated to the 1st WHO CMV standard, showed good reproducibility in CMV load values across multiple laboratories. Fifty-seven follow-up plasma specimens from 10 kidney transplant recipients with CMV replication were examined using the new quantitative CAP/CTM CMV test and the "in-house" quantitative CMV real-time PCR method, also calibrated against the 1st WHO CMV standard for their clinical applicability for monitoring CMV load in renal transplant patients. By CAP/CTM CMV test 49/57 specimens were CMV-DNA positive compared to 44/57 by the "in-house" PCR test. The "in-house" PCR and CAP/CTM CMV test correlated well in monitoring individual kidney transplant patients. Conversion of the CMV-DNA copies to IUs made the results of the "in-house" PCR and CAP/CTM CMV test less uniform in analysis of the patient samples. In specimens of one patient, significant underquantification of CMV load with "in-house" PCR emerged during follow-up due to a point mutation in the "in-house" PCR primer sequence. The CAP/CTM CMV test was found suitable for diagnosing and monitoring CMV replication in renal transplant patients. Multicenter studies are needed to provide more information of the commutability of the 1st WHO CMV standard and to define the clinical thresholds.

BK polyomavirus-associated hemorrhagic cystitis among pediatric allogeneic bone marrow transplant recipients: treatment response and evidence for nosocomial transmission.

BACKGROUND: BK polyomavirus-associated hemorrhagic cystitis (BK-PyVHC) is a significant complication of allogenic hematopoietic stem cell transplantation (HSCT), but risk factors and treatment are currently unresolved. BK-PyVHC typically presents with clinical cystitis, macrohematuria, and increasing urine and blood BKV loads. OBJECTIVES: Characterization of children undergoing allogeneic HSCT with BK-PyVHC and their clinical and antibody response to cidofovir treatment. STUDY DESIGN: By prospective screening of urine and plasma in 50 pediatric allogenic HSCT performed between 2008 and 2010, we identified 6 (12%) children with BK-PyVHC. Cidofovir was administered intravenously to 5 patients and intravesically to 4 patients (3 double treatments). RESULTS: Decreasing BKV viremia of>2log(10)copies/mL and clinical resolution was seen in 4 patients over 5-12 weeks. Responses occurred only in patients mounting BKV-specific IgM and IgG responses. Epidemic curve plots, BKV genotyping and contact tracing provided evidence of transmission between 2 BKV-seronegative patients, but ruled out transmission among the remaining four patients CONCLUSIONS: The data suggest that BK-PyVHC may be the result of nosocomial transmission in children with low/undetectable BKV antibodies and raises urgent questions about appropriate infection control measures and the role of cidofovir.

A retrospective analysis of nosocomial viral gastrointestinal and respiratory tract infections.

BACKGROUND: Viral nosocomial infections (NIs) in children are common and most frequently affect the gastrointestinal or respiratory tract. Few studies are dedicated to this topic. We aimed to determine incidence and characteristics of these specific viral NIs at our hospital. METHODS: This was a retrospective analysis of nosocomial gastroenteritis and respiratory tract infections (RTIs) of hospitalized patients at the University Children's Hospital Basel over a 12-month period. Patients with new-onset gastroenteritis or RTI during hospitalization or a physician diagnosis of NI on discharge were included for analysis. NIs were defined by use of Centers for Disease Control and Prevention recommendations and specific agents' incubation periods. RESULTS: Overall, 5493 patients were hospitalized accounting for 22,251 hospital days. Forty-five (0.8%) patients acquired an NI: 15 cases of gastroenteritis (mean age, 9.9 months; range: 3-24; NI incidence: 0.7 per 1000 hospitalization days) and 30 cases of RTI (mean age, 63.7 months; range: 1-174; NI incidence: 1.3 per 1000 hospitalization days). Main agents were rotavirus (10/15 gastroenteritis, 67%) and rhinovirus (22/30 nosocomial RTI; 73%). Physicians reported 9 cases of NI, of which 2 (22%) did not fulfill the criteria for an NI, 3 were surgical site infections, 1 was a case of rotavirus gastroenteritis and 3 were RTIs by rhinovirus. CONCLUSIONS: Viral NIs, especially caused by rotavirus and rhinovirus, are frequent in children of all ages but underestimated if exclusively reported by physicians. Prospective studies should further investigate the role and epidemiology of rhinovirus in nosocomial RTI, ideally by use of automated information technology support.

Reactivation of hepatitis B virus with mutated hepatitis B surface antigen in a liver transplant recipient receiving a graft from an antibody to hepatitis B surface antigen- and antibody to hepatitis B core antigen-positive donor.

BACKGROUND: Fresh-frozen plasma (FFP) may contain antibodies to hepatitis B surface antigen (HBsAg, anti-HBs). These anti-HBs may lead to a misinterpretation of the actual hepatitis B immune status. Furthermore, they may not only confer protection against hepatitis B virus (HBV), but may also favor the selection of HBsAg mutants. CASE REPORT: We report a case of de novo HBV infection in a HBV-naïve recipient with alcoholic liver disease, who received a liver from a donor with antibodies to hepatitis B core antigen (HBcAg, anti-HBc) and anti-HBs. RESULTS: A lookback investigation revealed the following: 1) Due to anti-HBs passively acquired through FFP, the recipient was considered immune to HBV and did not receive anti-HBV prophylaxis. 2) Within 1 year after transplantation he developed hepatitis B in absence of any elevated liver enzymes after the anti-HBs by FFP declined. 3) Despite an infection with HBV-containing wild-type HBcAg, the patient did not seroconvert to anti-HBc positivity. 4) The replicating HBV encoded two HBsAg mutations, first sQ129R and 4 months later sP127S. They map to the highly conserved "α" determinant of the HBsAg loop. CONCLUSION: 1) Passive transfer of anti-HBs from FFP led to an erroneous pretransplant diagnosis of HBV immunity when the patient was in fact HBV-naïve. 2) HBsAg mutations might have been selected in escape from donor's actively produced anti-HBs and the recipient's anti-HBs by FFP might have favored this selection. 3) It is doubtful whether hepatitis B immunoglobulin could have prevented the reactivation. 4) Antiviral prophylaxis would have been crucial.

Comparison of a UL111a real-time PCR and pp65 antigenemia for the detection of cytomegalovirus.

Surveillance of cytomegalovirus (CMV) replication in transplant patients is crucial for the success of transplantation. To compare a CMV pp65 antigenemia (pp65Ag) and a quantitative real-time PCR targeting the CMV-UL111a (UL111aPCR), all whole blood samples taken between July 2008 and October 2009 were identified which had been analyzed prospectively by both assays in parallel. Discordant results were re-analyzed using a published CMV duplex PCR targeting regions UL55 and UL123exon4. Of 720 samples from 81 transplant patients, CMV replication was detected in 244 specimens (34%) by the UL111aPCR (median, 1,019 geq/ml), compared to 113 (16%) detected by the pp65Ag (median, 2/250,000 leukocytes). Concordant UL111aPCR/pp65Ag results were obtained in 561 (78%) samples, being positive in 99 (14%), and negative in 462 (64%). As a rule of thumb, 1 pp65Ag-positive cell per 250,000 leukocytes corresponded to 1,000 geq/ml CMV DNA of whole blood. Discordant results were found in 159 samples (22%), being UL111aPCR-positive/pp65Ag-negative in 145 (91%; median, 650 geq/ml), or UL111aPCR-negative/pp65Ag-positive in 14 (9%; median, 1/250,000 cells). Using the duplex PCR targeting the CMV UL55 and the UL123-exon4 genes, 131 of 139 (94%) discordant UL111aPCR-positives (median UL111aPCR, 639 geq/ml; median UL55PCR, 715 geq/ml; median UL123PCR, 1,103 geq/ml) were confirmed. Of 14 discordant pp65Ag-positives, duplex PCR was also negative in 8, and of low copy number in 6. Thus, CMV UL111aPCR provides more sensitive quantitation of CMV replication than pp65Ag, however, discordant results can occur at very low viral loads.

Prevalence of polyomavirus BK and JC infection and replication in 400 healthy blood donors.

BACKGROUND: The replication of BK virus (BKV) and JC virus (JCV) is linked to polyomavirus-associated nephropathy, hemorrhagic cystitis, and multifocal leukoencephalopathy in immunodeficient patients, but the behavior of these viruses in immunocompetent individuals has hardly been characterized. METHODS: We used EIA to study samples obtained from 400 healthy blood donors aged 20-59 years for BKV- and JCV-specific antibodies against virus-like particles. We also studied BKV and JCV loads in plasma and urine among these individuals by use of real-time polymerase chain reaction. RESULTS: IgG seroprevalence was 82% (328 of 400 donors) for BKV and 58% (231 of400) for JCV. As age increased (age groups were divided by decade), the seroprevalence of BKV decreased from 87% (87 of 100) in the youngest group (aged 20-29 years) to 71% (71 of 100) in the oldest group (aged 50-59 years) (P = .006), whereas the seroprevalence of JCV increased from 50% (50 of 100) in the youngest group to 68% (68 of 100) in the oldest group (P = .06). Asymptomatic urinary shedding of BKV and JCV was observed in 28 (7%) and 75 (19%) of 400 subjects, respectively, with median viral loads of 3.51 and 4.64 log copies/mL, respectively (P < .001). Unlike urinary BKV loads, urinary JCV loads were positively correlated with IgG levels. The shedding of JCV was more commonly observed among individuals who were seropositive only for JCV, compared with individuals who were seropositive for both BKV and JCV, suggesting limited cross-protection from BKV immunity. Noncoding control regions were of archetype architecture in all cases, except for 1 rearranged JCV variant. Neither BKV nor JCV DNA was detected in plasma. CONCLUSIONS: Our study provides important data about polyomavirus infection and replication in healthy, immunocompetent individuals. These data indicate significant differences between BKV and JCV with respect to virus-host interaction and epidemiology.

Pan-dengue virus detection by PCR for travelers returning from the tropics.

We performed dengue virus (DENV) serology and quantitative real-time pan-DENV reverse transcription-PCR (RT-PCR) on 186 sera of 171 patients returning from the tropics. DENV loads significantly decreased with increasing times of disease and were higher in immunoglobulin M-negative samples. In the first week of disease, pan-DENV RT-PCR is the test of choice.

Cytomegalovirus-specific T-cell responses and viral replication in kidney transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) seronegative recipients (R-) of kidney transplants (KT) from seropositive donors (D+) are at higher risk for CMV replication and ganciclovir(GCV)-resistance than CMV R(+). We hypothesized that low CMV-specific T-cell responses are associated with increased risk of CMV replication in R(+)-patients with D(+) or D(-) donors. METHODS: We prospectively evaluated 73 consecutive KT-patients [48 R(+), 25 D(+)R(-)] undergoing routine testing for CMV replication as part of a preemptive strategy. We compared CMV-specific interferon-gamma (IFN-gamma) responses of CD4+CD3+ lymphocytes in peripheral blood mononuclear cells (PBMC) using three different antigen preparation (CMV-lysate, pp72- and pp65-overlapping peptide pools) using intracellular cytokine staining and flow cytometry. RESULTS: Median CD4+ and CD8+T-cell responses to CMV-lysate, pp72- and pp65-overlapping peptide pools were lower in D(+)R(-) than in R(+)patients or in non-immunosuppressed donors. Comparing subpopulations we found that CMV-lysate favored CD4+- over CD8+-responses, whereas the reverse was observed for pp72, while pp65-CD4+- and -CD8+-responses were similar. Concurrent CMV replication in R(+)-patients was associated with significantly lower T-cell responses (pp65 median CD4+ 0.00% vs. 0.03%, p = 0.001; CD8+ 0.01% vs. 0.03%; p = 0.033). Receiver operated curve analysis associated CMV-pp65 CD4+ responses of > 0.03% in R(+)-patients with absence of concurrent (p = 0.003) and future CMV replication in the following 8 weeks (p = 0.036). GCV-resistant CMV replication occurred in 3 R(+)-patients (6.3%) with pp65- CD4+ frequencies < 0.03% (p = 0.041). CONCLUSION: The data suggest that pp65-specific CD4+ T-cells might be useful to identify R(+)-patients at increased risk of CMV replication. Provided further corroborating evidence, CMV-pp65 CD4+ responses above 0.03% in PBMCs of KT patients under stable immunosuppression are associated with lower risk of concurrent and future CMV replication during the following 8 weeks.

Cytomegalovirus and polyomavirus BK posttransplant.

Virus replication and progression to disease in transplant patients is determined by patient-, graft- and virus-specific factors. This complex interaction is modulated by the net state of immunosuppression and its impact on virus-specific cellular immunity. Due to the increasing potency of immunosuppressive regimens, graft rejections have decreased, but susceptibility to infections has increased. Therefore, cytomegalovirus (CMV) remains the most important viral pathogen posttransplant despite availability of effective antiviral drugs and validated strategies for prophylactic, preemptive and therapeutic intervention. CMV replication can affect almost every organ system, with frequent recurrences and increasing rates of antiviral resistance. Together with indirect long-term effects, CMV significantly reduces graft and patient survival after solid organ and hematopoietic stem cell transplantation. The human polyomavirus called BK virus (BKV), on the other hand, only recently surfaced as pathogen with organ tropism largely limited to the reno-urinary tract, manifesting as polyomavirus-associated nephropathy in kidney transplant and hemorrhagic cystitis in hematopoetic stem cell transplant patients. No licensed anti-polyoma viral drugs are available, and treatment relies mainly on improving immune functions to regain control over BKV replication. In this review, we discuss diagnostic and therapeutic aspects of CMV and BKV replication and disease posttransplantation.

Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity.

DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of -131 mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsbA.