The importance of effect sizes when comparing cycle threshold values of SARS-CoV-2 variants.

PURPOSE: We aimed to elaborate whether cycle threshold (Ct) values differ significantly between wild type SARS-CoV-2 (wtV) and certain viral variants and how strong or weak a potential significant effect might be. METHODS: In a retrospective study, we investigated 1873 SARS-CoV-2 positive samples for the occurrence of viral marker mutations. Age, gender, clinical setting, days after onset of symptoms, and Ct values were recorded. Statistical analysis was carried out with special consideration of effect sizes. RESULTS: During the study period wtV was detected in 1013 samples (54%), while 845 (45%) patients carried the Alpha variant of concern (VOC), and 15 (1%) the Beta VOC. For further analysis, only wtV and the Alpha VOC were included. In a multi-factor ANOVA and post-hoc test with Bonferroni-correction for the age groups we found significant main-effects for Ct values of the viral variant (wtV mean 26.4 (SD 4.27); Alpha VOC mean 25.0 (SD 3.84); F (1,1850) = 55.841; p < .001) and the clinical setting (outpatients: mean 25.7 (SD 4.1); inpatients: mean 27.0 (SD 4.2); F (1,1850) = 8.520, p = .004). However, since the effect sizes were very small (eta squared for the Alpha VOC = .029 and the clinical setting = .004), there was only a slight trend towards higher viral loads of the Alpha VOC compared to wtV. CONCLUSIONS: In order to compare different variants of SARS-CoV-2 the calculation of effect sizes seems to be necessary. A combination of p-values as estimates of the existance of an effect and effect sizes as estimates of the magnitude of a potential effect may allow a better insight into transmission mechanisms of SARS-CoV-2.

Where have the enteric viruses gone? - Differential effects on frequent causes of infectious diarrhoea by SARS-CoV-2 pandemic lockdown measures.

BACKGROUND: Measures of distancing, wearing face/medical masks and lockdown introduced in many countries to meet the challenges of the SARS-CoV-2 pandemic have led to gross changes in the epidemiology of important infections. The observation of decline of positive norovirus tests after introduction of lockdown in Germany led us to investigate changes in the detection of major causes of diarrhoea by comparing pre-pandemic quarters (PPQ: 1Q/17 through 1Q/20) since 2017 and pandemic quarters (PQ: 2Q/20 through 1Q/21). METHODS AND SETTING: Bioscientia Laboratory Ingelheim is a large regional clinical pathology laboratory serving > 50 hospitals and > 5000 general practitioners and specialist outpatient practices located in the federal states Hesse, Rhineland-Palatinate and North Rhine-Westphalia, Germany. Antigen detection assays were used for detection of astrovirus, adenovirus, rotavirus, and Campylobacter antigen and Clostridium difficile Toxin A/B, while norovirus was detected by qualitative RT-PCR. FINDINGS: The mean positivity-ratios of norovirus, adenovirus and astrovirus assays were 3-20 fold lower in periods PQ (2Q/20 through 1Q/21) compared to PPQ (1Q/17 through 1Q/20) (p<.01). The mean positivity-ratio was lower in PQ compared to PPQ for rotavirus (p=.31), but failed to reach statistical significance, while for campylobacter antigen (p=.91) and C. difficile Toxin A/B (p=.17) the mean positivity-ratio was even higher in PQ compared to PPQ. CONCLUSIONS: Apparently, hygienic measures used to contain the SARS-CoV-2 pandemic have differential effects on incidence of diarrhoea viruses as compared to bacterial gastrointestinal agents, particularly C. difficile, which may lead to re-evaluate measures implemented against this important cause of nosocomial diarrhoea.

Analysis of cycle threshold values in SARS-CoV-2-PCR in a long-term study.

BACKGROUND: Cycle threshold (Ct) values can be used in an attempt to semiquantify results in the qualitative real-time polymerase-chain-reaction (PCR) for the new coronavirus SARS-CoV-2. The significance of Ct values in epidemiological studies and large cohorts is still unclear. OBJECTIVE: To monitor Ct values in a long-term study and compare the results with demographic data of patients who tested positive for SARS-CoV-2 by real-time PCR. STUDY DESIGN: S gene SARS-CoV-2 Ct values were analyzed retrospectively from consecutive patients between March 15th to September 15th 2020 with special regard to age, gender, and in- or outpatient status. RESULTS: In total, 65,878 patients were tested, 1103 (1.7 %) of whom were positive for SARS-CoV-2. Twenty-six positive patients were excluded, because the respective PCR runs did not meet the stability requirements (Ct value of the positive controls between 26 and 29). Of the remaining 1077 patients, females (n = 566; 53 %) were significantly older than males (n = 511; 47 %) (50.9 versus 45.1 years; p = 0.006) and had slightly higher mean Ct values than males (25.4 vs. 24.8; p = 0.04). Patients in the age groups >80 years had significantly higher Ct values than the remaining age groups (p < 0.001). Children (0-19 years) showed Ct values in the range of those found in adults (25.2 vs. 25.1, p = 0.9). There were no statistically different Ct values between in- and outpatients (p = 0.1), however, SARS-CoV-2 positive inpatients were significantly older than outpatients (p < 0.0001). CONCLUSIONS: CT values are suitable for more detailed monitoring of the SARS-CoV-2 pandemic. Age is an important cofactor in SARS-CoV-2 positive patients and may have influence on Ct values in SARS-CoV-2-PCR.

[Infection of the orbita due to Delftia acidovorans after a cat scratch]. / Orbita-Infektion mit Delftia acidovorans nach Katzenkratzer.

HISTORY: A 60 years old woman experienced a cat scratch 34 months ago on the left eyelid. Chronic, progredient skin lesions and headache developed. Treatments with cortisone, pimecrolimus, pregabalin and metamizole were not successful. After 24 months the patient complained of severe bulbus pain in the left eye, increased eye movement pain, and high photosensitivity. There were granulomatous papules in the area of the eye. FINDINGS AND DIAGNOSIS: The interdisciplinary examination findings and clinical-chemical parameters were inconspicuous. A biopsy of the eyelid area revealed the detection of Delftia acidovorans by bacterial 16S-rRNA-PCR. THERAPY AND COURSE: Treatment with piperacillin/tazobactam 3 × 4.5 g/d IV for 10 days led to rapid clinical improvement, so that the patient could be discharged after 11 days. After additional 10 months, she had no relapse and was free of complaints. CONCLUSIONS: D. acidovorans has not yet appeared as a zoonotic pathogen but should be included in the case of injury by animals in the differential diagnostic considerations.

Selection and counterselection of the rtI233V adefovir resistance mutation during antiviral therapy.

Recently, we reported on three patients with chronic hepatitis B virus (HBV) infection for whom adefovir (ADF) therapy virologically failed, most likely due to a preexisting rtI233V HBV polymerase mutation. Here, we describe two further patients with chronic HBV infection who were found to develop the rtI233V mutation after initiation of ADF therapy. These patients represent the first cases known so far in which the rtI233V ADF resistance mutation evolved under persistent HBV replication during HBV therapy with ADF. Interestingly, one of the previously described patients, who was initially successfully switched from ADF to tenofovir (TDF) and became virologically suppressed subsequently, experienced a moderate but remarkable rebound of HBV viremia after switching from TDF to entecavir, due to the emergence of renal toxicity. Thus, we provide evidence for the selection and counterselection of the rtI233V ADF resistance mutation during antiviral therapy.

Temporary HBV resolution in an HIV-coinfected patient during HBV-directed combination therapy followed by relapse of HBV.

Coinfection of hepatitis B virus (HBV) and HIV is common due to overlapping routes of transmission accompanied by an increased risk for liver-related mortality. We report the case of a chronically infected hepatitis Be antigen positive patient, coinfected with HIV (CD4+ T-cell count > 500 cells/microl), with histological evidence of advanced liver disease. The patient developed anti-HBs (antibody to hepatitis B surface antigen [HBsAg]) seroconversion, a strong reduction of intrahepatic covalently closed circular DNA and a marked improvement of liver histology after 24 weeks of HBV-targeted combination therapy with adefovir dipivoxil and pegylated interferon-alpha2b followed by another 12 weeks of adefovir dipivoxil monotherapy. Antiviral therapy was stopped after the development of stable anti-HBs titres, and anti-HBs titres remained stable for additional 9 months post-treatment. A continuous decline of anti-HBs was observed during the next 6 months until anti-HBs disappeared despite a stable HIV infection. A triple course of therapeutic vaccination failed to re-establish anti-HBs antibodies, but reappearance of HBV DNA and HBsAg was detected. By enzyme-linked immunosorbent spot analyses, HBV-directed T-cell responses clearly increased during antiviral combination therapy followed by a reduction to pre-treatment levels in association with disappearance of anti-HBs antibodies despite therapeutic vaccination. The presented case highlights the volatile nature of chronic HBV infection even after a prolonged disease-free period in the setting of an underlying HIV coinfection in a patient with a stable and relatively high CD4+ T-cell count but nevertheless impaired immune system and calls for further investigation of probably temporary immunomodulatory effects of interferon-alpha and/or nucleoside analogues in immunocompromised patients.

Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is responsible for persistent infection of hepatocytes. The aim of this study was to determine changes in intrahepatic cccDNA in patients with chronic hepatitis B (CH-B) during 48 weeks of antiviral therapy and its correlation to virological, biochemical, and histological parameters. Twenty-six HBsAg-positive CH-B patients received combination treatment with pegylated interferon alpha-2b (peg-IFN) and adefovir dipivoxil (ADV) for 48 weeks. Paired liver biopsies from before and at the end of treatment were analyzed for intrahepatic HBV-DNA. Median serum HBV-DNA had decreased by -4.9 log10 copies/mL at the end of treatment and was undetectable in 13 individuals (54%). Median intrahepatic total HBV-DNA and cccDNA had decreased by -2.2 and -2.4 log10, respectively. Changes in intracellular HBV-DNA positively correlated with HBsAg serum reduction and were accompanied by a high number of serological responders. Eight of 15 HBeAg-positive patients lost HBeAg, and five developed anti-HBe antibodies during treatment. These eight patients exhibited lower cccDNA levels before and at the end of therapy than did patients without HBeAg loss. Four patients developed anti-HBs antibodies. ALT normalized in 11 patients. The number of HBs-antigen- and HBc-antigen-positive hepatocytes was significantly lower after treatment, suggesting the involvement of cytolytic mechanisms. In conclusion, combination therapy with peg-IFN and ADV led to marked decreases in serum HBV-DNA and intrahepatic cccDNA, which was significantly correlated with reduced HBsAg.

Tenofovir for patients with lamivudine-resistant hepatitis B virus (HBV) infection and high HBV DNA level during adefovir therapy.

Incomplete virological response to adefovir dipivoxil (ADV) has been observed in patients with lamivudine-resistant hepatitis B virus (HBV) infection and may be associated with developing resistance and disease progression. We therefore investigated whether the efficacy of viral suppression could be improved by replacing ADV with tenofovir disoproxil fumarate (TDF). Twenty patients with chronic HBV infection (18 HBeAg+), viral breakthrough during lamivudine therapy, and persistent viral replication (>10(4) copies/mL) after 15 months of ADV monotherapy (range 4-28 months) were treated with TDF 300 mg daily and were retrospectively analyzed. A screening for nucleoside/nucleotide analogue resistance mutations within the HBV polymerase gene was performed in all patients by direct sequencing. Within a median of 3.5 months, application of TDF led to undetectable HBV DNA in 19 of 20 patients, as demonstrated by suppression of HBV DNA below the detection limit of 400 copies/mL. Initially elevated ALT levels had normalized in 10 of 14 patients by the end of follow-up (median 12 months, range 3-24 months). Four patients lost HBeAg, after 3, 4, 5, and 16 months, and one patient seroconverted to anti-HBs after 16 months of TDF therapy. Lamivudine-associated mutations (rtV173L, rtL180M, rtM204V/I) could be detected in 6 patients at baseline of TDF, but this obviously did not influence the response. ADV-resistant mutations were not detected. No side effects were reported. In conclusion, these preliminary observations strongly suggest that TDF might be a highly effective rescue drug for HBV-infected patients with altered responsiveness to treatment with lamivudine and ADV.

Clinical reactivation after liver transplantation with an unusual minor strain of hepatitis B virus in an occult carrier.

Hepatitis B virus (HBV) DNA is detectable in a number of liver transplant candidates who are negative for hepatitis B surface antigen (HBsAg). After liver transplantation (LT), such patients may have molecular and/or serologic evidence of HBV replication. However, clinical disease from reactivation of occult HBV infection after LT has not been described. We report a patient who underwent LT for cryptogenic cirrhosis and had to be retransplanted twice for hepatic artery thrombosis. The patient was negative for HBsAg and positive for anti-hepatitis B core (HBc) and anti-HBs before all LT procedures and developed acute hepatitis B shortly after receiving the third graft. The HBV strain isolated at that time exhibited an unusual in frame insertion of a CAG motif within the HBV polymerase (HBV(INS+)). HBV(INS+) was detected retrospectively as a minor species in pretransplantation sera and the explanted native liver by insertion-specific polymerase chain reaction. This case in an occult HBV carrier shows that clinically apparent, endogenous reinfection of the graft may occur with minor HBV variants that are not detectable in pretransplantation samples by standard diagnostic procedures. This has implications for the analysis of sources of acute hepatitis B in patients after LT and possibly for consideration of antiviral prophylaxis in anti-HBc/anti-HBs/HBV DNA-positive patients.

Detection of minor populations of drug-resistant HIV-1 in acute seroconverters.

OBJECTIVE: The transmission of drug-resistant HIV-1 is a major health concern. To date, most clinical studies have relied on sequencing techniques for genotypic analyses which do not allow quantification of minority viral populations below 25%. As minor populations of drug-resistant HIV-1 could impact the efficiency of antiretroviral therapy, this study was performed to determine the prevalence of minor populations of drug-resistant HIV-1 in acute seroconverters. DESIGN AND METHODS: Forty-nine acute seroconverters from two clinical centers in Germany were included in the study. Individuals were identified between June 1999 and March 2003, and none had received antiretroviral therapy prior to sampling. Minor populations of drug-resistant variants were detected by quantitative real-time polymerase chain reaction using allele-discriminating oligonucleotides for three key resistance mutations: L90M (protease), K103N and M184V (reverse transcriptase). The approximate discriminative power was between 0.01 and 0.2%. RESULTS: Drug-resistant variants were detected in 10 of 49 patients (20.4%). The L90M mutation was found in one of 49 (2%), the K103N mutation in five of 49 (10.2%) and the M184V mutation in six of 49 (12.2%) patients, respectively. In five of the 10 individuals with detectable drug-resistant virus (50%), the detected population represented a minor viral quasi-species (< 25% of viruses) and was not detected by direct sequencing. CONCLUSIONS: The prevalence of minor populations of drug-resistant HIV-1 in acute seroconverters can be frequently detected and may impact the success of antiretroviral therapy.

Viral features of lamivudine resistant hepatitis B genotypes A and D.

Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo. Forty-one patients carrying lamivudine resistant HBV were enrolled. Twenty-six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D). The rate of reverse transcriptase 204I mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P =.006) and rt180M mutants (81% in group A vs 40% in group D, P =.015) prevailed in group A. The median time of shift from rt204I to rt204V mutants was significantly shorter in group A (4 months in group A, >12 months in group D, P <.001). Additional resistance associated mutations were detected exclusively in group D (P =.004). In a multivariate analysis, HBV genotype (P =.039) and pretreatment serum HBV DNA (P =.001) were independently associated with emerging rt204I or rt204V mutants, respectively. Serum HBV copy numbers after emergence of resistance were higher in group A (mean log(10) 6.99 copies/ml; range 3-9) compared with group D (mean log(10) 6.1 copies/ml; range 3.3-8; P =.04). There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow-up. In conclusion, the mutational pattern during selection of lamivudine resistant HBV strains differs between genotypes A and D. This may have consequences for a salvage regimen initiated for treatment of lamivudine resistant HBV.

Effective protection of allogeneic stem cell recipients against Aspergillosis by HEPA air filtration during a period of construction--a prospective survey.

High incidence of aspergillosis on transplant units or hematological wards without HEPA air conditioning during periods of demolishing or construction has been reported by several investigators. Here we report monitoring of fungal air contamination during a period of construction on a stem cell transplantation ward using the gravity air-setting plate (GASP) method. Fungal air contamination in HEPA-conditioned patient rooms was constantly low, independent from construction activity. Outside of the patient rooms at the ward's corridor, the fungal load was significantly higher with some peak values. Outside the transplant unit measures of construction led to a significant increase of fungal spore concentration in air. Transplant activity was not reduced during construction and patients were nursed strictly under HEPA conditions. Patients were monitored prospectively for incidence of infections since 1990 and data of patients grafted during construction (n = 28) were compared to those grafted outside building activity (n = 652). An increase of aspergillosis during construction could be clearly excluded. It can be concluded: Nursing of patients undergoing stem cell transplantation in HEPA-conditioned rooms is an effective protection against acquisition of aspergillus-infection, even under environmental conditions with increased air contamination by conidia. The gravity air-setting plate (GASP) method is not expensive and easy to use and allows reliable and quantitative aerobiological spore monitoring.

Multiple infections with different HCV genotypes: prevalence and clinical impact.

BACKGROUND: In a HCV genotype 3a-infected patient, viremia with a different genotype (1b) was detected after 16 weeks of ineffective therapy. Serological typing revealed that this genotype had already been present prior to therapy. OBJECTIVES: To investigate the epidemiology of multiple HCV infections and the therapeutical consequences for patients superinfected with a new HCV strain. METHODS: Sera of 600 patients were screened for infection with multiple genotypes by using sequencing and a serological assay in parallel. RESULTS: Infection with two different HCV types was detected in 13 patients. The prevailing strain was genotyped by sequencing. From two of these patients additional sera were available which had been drawn up to 24 and 28 months prior to the current sample, respectively. Those early samples showed viremia with a HCV subtype that could not be detected by PCR afterwards. Only antibodies to the initial strain were detectable in the later samples. CONCLUSION: In patients serially infected by different HCV strains, one strain will prevail as the viremic virus. Under antiviral therapy, the displaced strain may become viremic again and may influence the outcome of therapy. Detection of inferior strains by serological assays before antiviral therapy may be important for choosing the adequate regimen.

A novel DNA virus (SEN) among patients on maintenance hemodialysis: prevalence and clinical importance.

BACKGROUND: A recently discovered DNA virus (SEN) has been assumed to be responsible for posttransfusion hepatitis in humans. Phylogenetic analysis of SEN virus has revealed the existence of 8 different strains. Two of them (SEN virus strain H (SENV-H) and SENV-D) have been described as possible candidate viruses for inducing posttransfusion hepatitis. Until now, it is unclear whether patients on maintenance hemodialysis are on increased risk for acquiring SEN virus. OBJECTIVES: To investigate the prevalence of SENV-H among patients on maintenance hemodialysis and to examine whether special measures have to be taken to prevent nosocomial spreading of the virus. STUDY DESIGN: Serum samples derived from 78 chronically hemodialysed patients were examined for SENV-H viremia by seminested polymerase chain reaction. A panel of 226 samples from healthy blood donors served as a control group. RESULTS: The prevalence of SENV-H was determined to be 12.8% (n=10) among patients on maintenance hemodialysis. This is nearly the same prevalence as in healthy blood donors (16.8%; n=38). None of the solely SENV-H-viremic individuals had clinical or biochemical signs of liver disease. Enhanced severity of liver disease could not be observed in patients coinfected with hepatitis C virus and SENV-H. CONCLUSION: We conclude that SENV-H viremia is widespread among hemodialysis patients. Since no viremic patient had clinical or biochemical signs of liver disease, in our setting the hepatitis-inducing capacity of SENV-H remains unclear. On the basis of our results, at present, we do not regard it as necessary to dialyse SENV-H-viremic patients on separate machines.

Epidemiological dynamics of hepatitis C virus among 747 German individuals: new subtypes on the advance.

This study demonstrates the dynamics in the epidemiology of hepatitis C virus subtypes. Subtypes 3a and 4a have become increasingly prevalent in patients where an infection within recent years can be assumed. Evidence is presented that the subtypes observed among younger patients can spread rapidly and lead to significant changes in the subtype distribution.

Genotyping of hepatitis C virus types 1, 2, 3, and 4 by a one-step LightCycler method using three different pairs of hybridization probes.

Determination of hepatitis C virus (HCV) genotypes has become increasingly important during the last years for prediction of the clinical course and the outcome of antiviral therapy. Therefore, numerous different methods have been developed to enable HCV genotyping. However, many of them are very laborious and expensive, leading to limited usage in daily routine diagnostics. We have established a method which combines the speed of the new LightCycler technology with the use of amplification products generated for diagnostic quantitative HCV RNA determination. Differentiation of HCV genotypes is performed with these amplicons in a single step by using fluorophore-labeled hybridization probes. Although currently only two different acceptor fluorophores are available for the LightCycler, types 1, 2, 3, and 4, which are by far the prevailing HCV genotypes in Europe and the United States, can be distinguished. Genotypes of specimens from 190 chronically HCV-infected patients were determined by the LightCycler method and compared with the results of nucleotide sequencing. Concordant results were obtained for all samples. This new method offers a fast and convenient possibility to determine the quantitative HCV RNA load and the genotype in large-scale settings within about 4 h.

Subtype-dependent response of hepatitis B virus during the early phase of lamivudine treatment.

We conducted a 12-month longitudinal investigation of the subtype-dependent response of hepatitis B virus (HBV) to lamivudine treatment in 43 consecutive patients with chronic hepatitis B. HBV subtype ayw appears to respond better to lamivudine monotherapy than does HBV subtype adw (P=.005). This might be the reason for the lower incidence of lamivudine-resistant strains observed in persons infected with HBV subtype ayw during follow-up.

Reappearance of HIV multidrug-resistance in plasma and circulating lymphocytes after reintroduction of antiretroviral therapy.

BACKGROUND: After the discontinuation of antiretroviral therapy in HIV-infected patients with highly resistant virus, the detectability of viral resistance mutations quickly decreases. To which extent this represents a true loss of resistance or rather a detectability phenomenon remains unclear. OBJECTIVES: To monitor virologic response and resistance pattern during a non-strategic treatment interruption in the presence of highly drug-resistant viral strains. STUDY DESIGN: We performed serial genotypic resistance analyses on viral DNA isolated from a patient with a multidrug-resistant human immunodeficiency virus infection who discontinued and later on reintroduced antiretroviral therapy. Sequencing was performed on viral DNA from plasma as well as DNA from circulating leukocytes. RESULTS: While under combination antiretroviral therapy with two nucleosidic reverse transcriptase inhibitors, a non-nucleosidic reverse transcriptase inhibitor and a protease inhibitor, the viral load of the patient was around five logs. Genotypic resistance to all available agents was detected during this time. Antiretroviral therapy was then interrupted, and 14 weeks later an almost complete reversion of the virus to wild type was observed. After introduction of a new antiretroviral therapy regimen, the reappearance of nearly all of the formerly present resistance mutations had to be noted within 6 weeks, including mutations without known relation to any of the drugs in the new regimen. CONCLUSIONS: We obviously observed not the de novo appearance of a complex resistance pattern under just 6 weeks of potent antiretroviral therapy, but a reappearing archival strain of the virus. This finding provides evidence for subdetectable persistence of resistant variants during treatment interruptions. Therefore, resistance analyses from peripheral blood performed in times of treatment interruptions should be interpreted with caution as they may provide incomplete information about the resistance profile soon after reintroduction of therapy.