Impact of the COVID-19 pandemic on norovirus circulation in Germany.

Human norovirus is a major cause of viral gastroenteritis in all age groups. The virus is constantly and rapidly changing, allowing mutations and recombination events to create great diversity of circulating viruses. With the start of the COVID-19 pandemic in 2020, a wide range of public health measures were introduced worldwide to control human-to-human transmission of SARS-CoV-2. In Germany, control measures such as distance rules, contact restrictions, personal protection equipment as well as intensive hand hygiene were introduced. To better understand the effect of the measures to control the COVID-19 pandemic on incidence and the molecular epidemiological dynamics of norovirus outbreaks in Germany, we analyzed national notification data between July 2017 and December 2022 and characterized norovirus sequences circulating between January 2018 and December 2022. Compared to a reference period before the pandemic, the incidence of notified norovirus gastroenteritis decreased by 89.7% to 9.6 per 100,000 during the 2020/2021 norovirus season, corresponding to an incidence rate ratio (IRR) of 0.10. Samples from 539 outbreaks were genotyped in two regions of the viral genome from pre-pandemic (January 2018 to February 2020) and samples from 208 outbreaks during pandemic time period (March 2020 to December 2022). As expected, norovirus outbreaks were mainly found in child care facilities and nursing homes. In total, 36 genotypes were detected in the study period. A high proportion of recombinant strains (86%) was found in patients, the proportion of detected recombinant viruses did not vary between the pre-pandemic and pandemic phase. The proportion of the predominant recombinant strain GII.4 Sydney[P16] was unchanged before pandemic and during pandemic at 37.5%. The diversity of most common genotypes in nursing homes and child care facilities showed a different proportion of genotypes causing outbreaks. In nursing homes as well as in child care facilities GII.4 Sydney[P16] was predominant during the whole study period. Compared to the nursing homes, a greater variety of genotypes at the expense of GII.4 Sydney[P16] was detected in child care facilities. Furthermore, the overall proportion of recombinant strain GII.3[P12] increased during the pandemic, due to outbreaks in child care facilities. The COVID-19 pandemic had a high impact on the occurrence of sporadic cases and norovirus outbreaks in Germany, leading to a near suppression of the typical norovirus winter season following the start of the pandemic. The number of norovirus-associated outbreak samples sent to the Consultant Laboratory dropped by 63% during the pandemic. We could not identify a clear influence on circulating norovirus genotypes. The dominance of GII.4 Sydney recombinant strains was independent from the pandemic. Further studies are needed to follow up on the diversity of less predominant genotypes to see if the pandemic could have acted as a bottleneck to the spread of previously minoritized genotypes like GII.3[P12].

Transmitted drug resistance and subtype patterns of viruses from reported new HIV diagnoses in Germany, 2017-2020.

BACKGROUND: The transmission of resistant HIV variants jeopardizes the effective use of antiretrovirals for therapy and prophylaxis. Molecular surveillance of new HIV diagnoses with a focus on prevalence and type of resistance associated mutations and the subtype of circulating viruses is mandatory. METHOD: From 2017 to 2020, 11,527 new HIV diagnoses were reported in Germany to the Robert Koch Institute (RKI). Protease (PR) and reverse-transcriptase (RT) sequences were obtained from 4559 (39.6%) cases, and PR, RT and integrase (IN) sequences were obtained from 3097 (26.9%) cases. The sequences were analyzed with data from the national HIV reports. RESULTS: Among all cases in the analysis, the proportion of primary resistance was 4.3% for nucleoside reverse-transcriptase inhibitors (NRTIs), 9.2% for non-NRTI (NNRTIs), 3.3% for protease inhibitors (PIs) and 1.4% for integrase inhibitors (INIs). Dual-class resistance was highest for NRTIs/NNRTIs with 1.2%. There was no trend in the proportion of viruses resistant to drug classes. Most individual key mutations associated with relevant resistance had a prevalence below 1% including K65R (0.1%) and M184V (0.6%). A notable exception was K103NS, with a prevalence of 2.9% and a significant increase (pTrend=0.024) during 2017-2020. In this period, diagnoses of infections with HIV-1 subtype B were the most common at 58.7%, but its prevalence was declining (pTrend=0.049) while the frequency of minority subtypes (each < 1%) increased (pTrend=0.007). Subtype B was highest (75.6%) in men who have sex with men (MSM) and lowest in reported heterosexual transmissions (HETs, 22.6%). CONCLUSION: The percentage of primary resistance was high but at a stable level. A genotypic determination of resistance is therefore still required before the start of therapy. The subtype diversity of circulating HIV-1 is increasing.

Molecular characterisation of a rabbit Hepatitis E Virus strain detected in a chronically HEV-infected individual from Germany.

In immunocompromised individuals persisting viremia frequently leads to a chronic hepatitis E virus (HEV) infection. Zoonotic transmission of HEV from pigs and wild boar to humans is proven and sporadic infections with rabbit HEV (raHEV) have recently been reported. Here, the molecular characterisation of a raHEV strain isolated from an immunocompromised, chronically HEV-infected, heart-transplanted patient is described. After successful ribavirin (RBV) treatment of a HEV infection in 2019, the patient was again tested HEV positive in 2021 and received a second RBV therapy cycle. Full-length HEV genome amplification and next generation sequencing was performed on a plasma sample taken between first and second cycle of RBV therapy and a stool sample taken two months after starting the second cycle. The sequence of plasma (raHEV-83) and stool (raHEV-99) derived virus showed the highest nucleotide sequence identity to a Chinese raHEV and a phylogenetic relationship to a raHEV strain isolated from a French patient. Furthermore, sequence analysis revealed the presence of RBV-associated substitutions V1479I and G1634K in the HEV sequences from plasma and additionally K1398R from stool. The results underline the role of rabbits as putative sources of HEV infection and emphasize the need of a one health concept for a better understanding of HEV epidemiology and to develop tools for prevention and control of HEV infection.

Insertions and deletions in the hypervariable region of the hepatitis E virus genome in individuals with acute and chronic infection.

BACKGROUND AND AIMS: Hepatitis E virus is a major cause of acute hepatitis worldwide and can progress to chronicity in immunocompromised individuals. Various virus-host recombination events have been reported in the hypervariable region of the hepatitis E virus genome, but the patterns of assembly and selection remain unclear. METHODS: To gain further insight into viral evolution, we assessed the presence of low abundance variants in 16 samples from individuals with acute or chronic infection using a targeted next-generation sequencing approach. RESULTS: In seven samples, different variants with insertions and/or deletions were identified. Among them, eight insertions originating either from human genes or from the hepatitis E virus genome. Five different deletions could be identified. The amino acid composition of sequences with insertions showed a higher frequency of lysine and a lower abundance of proline, and additionally acetylation and ubiquitination sites were more frequent than in hepatitis E virus wild-type sequences. CONCLUSIONS: These findings suggest that the nucleotide composition of insertions and sites for post-translational modification may contribute to recombination events. Although the impact of low-level hepatitis E virus variants is uncertain, our results highlight the importance of a highly sensitive next-generation sequencing approach to capture the full diversity of hypervariable region.

Advanced sequencing approaches detected insertions of viral and human origin in the viral genome of chronic hepatitis E virus patients.

The awareness of hepatitis E virus (HEV) increased significantly in the last decade due to its unexpectedly high prevalence in high-income countries. There, infections with HEV-genotype 3 (HEV-3) are predominant which can progress to chronicity in immunocompromised individuals. Persistent infection and antiviral therapy can select HEV-3 variants; however, the spectrum and occurrence of HEV-3 variants is underreported. To gain in-depth insights into the viral population and to perform detailed characterization of viral genomes, we used a new approach combining long-range PCR with next-generation and third-generation sequencing which allowed near full-length sequencing of HEV-3 genomes. Furthermore, we developed a targeted ultra-deep sequencing approach to assess the dynamics of clinically relevant mutations in the RdRp-region and to detect insertions in the HVR-domain in the HEV genomes. Using this new approach, we not only identified several insertions of human (AHNAK, RPL18) and viral origin (RdRp-derived) in the HVR-region isolated from an exemplary sample but detected a variant containing two different insertions simultaneously (AHNAK- and RdRp-derived). This finding is the first HEV-variant recognized as such showing various insertions in the HVR-domain. Thus, this molecular approach will add incrementally to our current knowledge of the HEV-genome organization and pathogenesis in chronic hepatitis E.

A Recent Human Immunodeficiency Virus Outbreak Among People Who Inject Drugs in Munich, Germany, Is Associated With Consumption of Synthetic Cathinones.

BACKGROUND: Needle and syringe sharing among people who inject drugs (PWID) can result in a rapid regional spread of a human immunodeficiency virus (HIV) variant. Such outbreaks have been identified recently in several countries and have raised public health attention because of an association with new psychoactive substances (NPS). METHODS: Dried serum spots from approximately 60% of newly diagnosed HIV cases in Germany in 2013-2018 were received together with statutory notification data. Samples were sequenced in the pol-region, genotyped, and viral phylogenies were analyzed. For selected samples, the hepatitis C virus (HCV) status and the presence of NPS were determined. RESULTS: An outbreak of closely related 27 subtype C infections with a core of 11 cases with almost identical sequences was identified using phylogenetic analyses. The first case of the outbreak was diagnosed in 2015, and the last one was in 2018. With exception of 3 infections, all were reported from Munich, the capital of the federal state of Bavaria. Of 26 analyzed outbreak members, 24 (92.3%) had a resolved or viremic HCV coinfection. In 8 of 18 (44%) cases, α-pyrrolidinopentiothiophenone and/or the related substance α-pyrrolidinoheptiophenone was identified. CONCLUSIONS: Despite harm reduction services in place, HIV outbreaks of considerable size can occur in PWID. The establishment of a real-time molecular surveillance is advised to rapidly identify outbreaks and target prevention measures.

Increasing proportions of HIV-1 non-B subtypes and of NNRTI resistance between 2013 and 2016 in Germany: Results from the national molecular surveillance of new HIV-diagnoses.

BACKGROUND: Molecular surveillance of newly diagnosed HIV-infections is important for tracking trends in circulating HIV-variants, including those with transmitted drug resistances (TDR) to sustain ART efficacy. METHODS: Dried serum spots (DSS) are received together with the statutory notification of a new diagnosis. 'Recent infections' (<155 days) classified by a 'recent infection test algorithm' (BED-CEIA and clinical data) are genotyped in HIV-protease (PR), reverse transcriptase (RT) and integrase (INT) to determine the HIV-1 subtype, to calculate prevalence and trends of TDR, to predict baseline susceptibility and to identify potential transmission clusters for resistant variants. RESULTS: Between January 2013 and December 2016, 1,885 recent infections were analysed regarding the PR/RT genomic region, with 43.5% of these also being subjected to the analysis of INT. The proportion of HIV-1 non-B viruses (31.3%; 591/1,885) increased from 21.6% to 36.0%, particularly the subtypes A (5.0% to 8.3%) and C (3.2% to 7.7%; all ptrends < 0.01). The subtype A increment is mainly due to transmissions within men who have sex with men (MSM) while subtype C transmissions are associated with heterosexuals and people who inject drugs. The prevalence of TDR was stable at 11.0% (208/1,885) over the study period. Resistances to nucleotide RT inhibitors (NRTI) and PR inhibitors (PI) were 4.5% and 3.2%, respectively, without identifiable trends. In contrast, resistances to non-NRTIs (NNRTI, 4.7%) doubled between 2014 and 2016 from 3.2% to 6.4% (ptrend = 0.02) mainly due to the K103N mutation (from 1.7% to 4.1%; ptrend = 0.03) predominantly detected in recently infected German MSM not linked to transmission clusters. Transmitted INSTI mutations were present in only one case (T66I) and resistance to dolutegravir was not identified at all. Reduced susceptibility to recommended first-line therapies was low with 1.0% for PIs, 1.3% for NRTIs and 0.7% for INSTIs, but high for the NNRTIs efavirence (4.9%) and rilpivirine (6.0%) due to the K103N mutation and the polymorphic mutation E138A. These trends in therapy-naïve individuals impact current first-line regimens and require awareness and vigilant surveillance.

Decreased emergence of HIV-1 drug resistance mutations in a cohort of Ugandan women initiating option B+ for PMTCT.

BACKGROUND: Since 2012, WHO guidelines for the prevention of mother-to-child transmission (PMTCT) of HIV-1 in resource-limited settings recommend the initiation of lifelong antiretroviral combination therapy (cART) for all pregnant HIV-1 positive women independent of CD4 count and WHO clinical stage (Option B+). However, long-term outcomes regarding development of drug resistance are lacking until now. Therefore, we analysed the emergence of drug resistance mutations (DRMs) in women initiating Option B+ in Fort Portal, Uganda, at 12 and 18 months postpartum (ppm). METHODS AND FINDINGS: 124 HIV-1 positive pregnant women were enrolled within antenatal care services in Fort Portal, Uganda. Blood samples were collected at the first visit prior starting Option B+ and postpartum at week six, month six, 12 and 18. Viral load was determined by real-time RT-PCR. An RT-PCR covering resistance associated positions in the protease and reverse transcriptase HIV-1 genomic region was performed. PCR-positive samples at 12/18 ppm and respective baseline samples were analysed by next generation sequencing regarding HIV-1 drug resistant variants including low-frequency variants. Furthermore, vertical transmission of HIV-1 was analysed. 49/124 (39.5%) women were included into the DRM analysis. Virological failure, defined as >1000 copies HIV-1 RNA/ml, was observed in three and seven women at 12 and 18 ppm, respectively. Sequences were obtained for three and six of these. In total, DRMs were detected in 3/49 (6.1%) women. Two women displayed dual-class resistance against all recommended first-line regimen drugs. Of 49 mother-infant-pairs no infant was HIV-1 positive at 12 or 18 ppm. CONCLUSION: Our findings suggest that the WHO-recommended Option B+ for PMTCT is effective in a cohort of Ugandan HIV-1 positive pregnant women with regard to the low selection rate of DRMs and vertical transmission. Therefore, these results are encouraging for other countries considering the implementation of lifelong cART for all pregnant HIV-1 positive women.