An axillary skin lesion revealing disseminated paracoccidioidomycosis.

Paracoccidioidomycosis, uncommon in Europe, primarily affects South America travellers. We report a 58-year-old Colombian man, who has lived in France for 20 years, presented with an axillary skin lesion seven years after his last trip to Colombia. The diagnosis of paracoccidioidomycosis was established using histopathological, mycological, and molecular analyses.

Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics.

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Gynoid Fat Distribution and Adipocyte Trapping May Explain Virological Failure With Intramuscular Long-Acting Cabotegravir and Rilpivirine.

Intramuscular long-acting antiretroviral drugs can improve adherence to lifelong antiretroviral treatment. Nevertheless, adipose tissue thickness and distribution play a critical role with injectable drugs. We describe a virological failure with cabotegravir and rilpivirine in a Black African woman with human immunodeficiency virus type 1 with gynoid fat distribution (ie, adipose tissue prevailing in the pelvis and hip area) and body mass index <30 kg/m2.

Unknown Circovirus in Immunosuppressed Patient with Hepatitis, France, 2022.

Hepatitis of undetermined origin can be caused by a wide variety of pathogens, sometimes emerging pathogens. We report the discovery, by means of routine shotgun metagenomics, of a new virus belonging to the family Circoviridae, genus Circovirus, in a patient in France who had acute hepatitis of unknown origin.

Insights on 21 Years of HBV Surveillance in Blood Donors in France.

Hepatitis B virus (HBV) infection is the most frequent viral infection found in blood donors (BDs) in France. We analyzed the epidemiological and sero-molecular data on HBV infection gathered over the past two decades by the French haemovigilance surveillance network, blood screening laboratories, and the national reference center for transfusion infectious risks (NRC). Between 2000 and 2020, 6149 of the 58,160,984 donations (1.06/10,000) tested HBV positive, 98% of them from first-time blood donors (FTBDs). In addition, 2212 (0.0071%) of the 30,977,753 donations screened for HBV DNA tested DNA positive, of which 25 (1.1%) were positive only for this marker. HBV prevalence decreased by 2.8-fold and the residual risk for transfusion-transmitted HBV infection decreased 13-fold and was divided by 13. The major risk factor for HBV infection was the origin of donors (endemic country, 66.5%), followed by parenteral exposure (10.7%). In the whole HBV-positive BD population, genotype D was predominant (41.8%), followed by genotypes A (26.2%) and E (20.4%), reflecting the geographical origin of donors. The low and decreasing prevalence and incidence of HBV infection in French BDs, coupled with a screening strategy using three HBV markers (HBsAg, anti-HBc and DNA), ensures a high level of blood safety, further reinforced by the implementation of pathogen-reduction measures.

SARS-CoV-2 and post-donation information: a one-year experience of the French haemovigilance network.

BACKGROUND: There is growing evidence to support the hypothesis that SARS-CoV-2 is probably not transmissible by blood transfusion. In this study, we use the data gathered over one year by the French haemovigilance network on post-donation information related to SARS-CoV-2, and virological investigations on corresponding plasma to explore viral transmission by transfusion. MATERIALS AND METHODS: Whenever a donor reported COVID-19 symptoms and/or a positive SARS-CoV-2 nasopharyngeal (NP) PCR test, information regarding diagnosis and symptoms was collected using a specific questionnaire, and repository plasmas were screened using the SARS-COV-2 R-GENE® assay (Biomérieux). RNA sequencing (Sanger and deep sequencing) and virus isolation on Vero E6 cells were applied in plasma from donors testing positive. RESULTS: We investigated 1,092 SARS-CoV-2-related post-donation information (PDI) reports. PDI donors were younger than the global donor population and donated more often in the Paris region. Sixty-eight percent reported a positive NP real-time (RT)-PCR or antigenic testing and 22% of these also had symptoms at the time of testing. Thirty-seven (3.4%) donations tested positive for SARS-CoV-2 RNA, 11 (30%) were confirmed by another molecular assay, and 7 (19%) by sequencing, confirming low viral level. Most RNAemic blood donors donated in southern regions and in Paris. There was no difference in demographic data or duration parameter between RNAemic and non-RNAemic donors. Duration parameter was determined as the time elapsed between donation and: i) the onset of symptoms; ii) a positive NP RT-PCR; and iii) PDI. Cell culture experiments did not show any infectivity related to RNAemic plasmas. DISCUSSION: SARS-CoV-2 RNA can be detected in a small fraction of blood donors with PDI, reporting very low levels of RNA. The corresponding plasma is probably not infectious. These findings highlight the value of haemovigilance and PDI to guide blood safety strategies.

Low rate of RNAemia in blood donations collected during the first wave of COVID-19 in France.

BACKGROUND: To investigate the transmission of SARS-CoV-2 via blood, we conducted retrospective molecular screening in blood donated during the first pandemic peak in the two French regions with the highest community transmission. METHODS: Archived plasma samples randomly selected from donations collected between March 23 and 29, 2020, in Eastern and Northern regions of France were tested for SARS-CoV-2 RNA in minipools of 4 donations (MP4) using the Grifols ProcleixSARS-CoV-2 assay. Reactive MP4 and the four corresponding plasmas were further tested with alternative RT-PCRs and sequencing. Testing for SARS-CoV-2 antibodies and in vitro infectivity in cell culture were also performed. RESULTS: Among the 2818 MP4 (corresponding to 9672 donations) tested for viral RNA, 5 were weakly reactive. Among the 20 plasmas included in these five MP4, one presented low-level reactivity with RT-PCRs and Procleix SARS-CoV-2 and was confirmed on sequencing. The estimated prevalence was 1.03/10,000 (95% CI 0-3.1). The 20 plasmas were antibody nonreactive and none of them showed cytopathic effects in cell culture. When recalled, the index-donor declared having had symptoms compatible with SARS-CoV-2 infection a few days after donation. The two immunocompromised recipients transfused with red blood cells and an inactivated pooled platelet product did not develop COVID-19. CONCLUSION: Our results indicated a low prevalence of SARS-CoV-2 RNA in the plasma of asymptomatic blood donors during the pandemic peak and no evidence of infectivity in vivo and in vitro. The transfusion risk remains theoretical and does not justify the implementation of SARS-CoV-2 NAT for blood donations.

Effectiveness of the HCV blood screening strategy through eighteen years of surveillance of HCV infection in blood donors in France.

BACKGROUND: The question of maintaining blood screening based on both Hepatitis C virus (HCV) infection antibodies (Ab) and Nucleic Acid Testing (NAT) has been raised in several countries. The French blood donor surveillance database was used to address this issue. MATERIALS AND METHODS: In France, HCV-NAT was implemented in mini pools (MP) in 2001 and in individual testing (ID) in 2010. HCV-positive donations are further investigated including detection of RNA with an alternative polymerase chain reaction assay: Amplicor HCV v2.0 (Roche; LOD95 50 IU/mL) from 2001 to 2006 and CobasTaqMan (CTM) HCV 2.0 assay (Roche; LOD95 9.3 IU/mL) since 2007. RESULTS: From 2001 to 2018, 3,058/48.8 million donations were confirmed HCV positive: 64.4% were Ab+/NAT+, 35.1% Ab+/NAT- and 0.5% Ab-/NAT+. From 2001 to 2018, the NAT yield decreased from 0.65 per million donations to 0, and NAT+ donations dropped from 77% to 46% of the total of HCV donations. 2,491/3,058 were further tested for HCV-RNA: 1,032 (816 NAT+, 216 NAT-) with Amplicor and 1,459 (897 NAT+, 562 NAT-) with CTM. Four (3 MP and 1 ID-NAT, 0.5%) of the 778 NAT negative donations had low viral loads. DISCUSSION: The decline in HCV-NAT yield cases raises the question of the relevance of NAT. Conversely, the increase in Ab+/NAT-donors, suggesting a growing number of resolved infections, argue for Ab discontinuation. In our experience, at least 0.5% of Ab+/NAT-donations had low RNA level when retested. Although the risk of viral transmission by such donations is probably low, the uncertainty associated with their infectivity goes against the removal of Ab in blood screening in our country.

HIV transmission network analysis allows identifying unreported risk factors in HIV-positive blood donors in France.

OBJECTIVES: As sex between men is a major route of human immunodeficiency virus (HIV) infection in most western countries, restrictive deferral rules for blood donation have largely been implemented regarding men having sex with men (MSM). Here, we sought here to assign unreported HIV risk factors in blood donors (BDs) and reevaluated the MSM-associated fraction of HIV transfusion residual risk (%RRMSM ). METHODS: We applied a genetic distance-based approach to infer an HIV transmission network for 384 HIV sequences from French BDs and 1337 HIV sequences from individuals with known risk factors (ANRS PRIMO primary HIV infection cohort). We validated the possibility of assigning a risk factor according to clustering using assortative mixing. Finally, we recalculated the %RRMSM . RESULTS: A total of 81 of 284 (28.5%) male and 5 of 100 (5%) female BDs belonged to a cluster; 72 (88.9%) of the 81 male BDs belonged to MSM clusters. After cluster correction, 8 of 67 (11.9%), 4 of 21 (19.0%), and 19 of 88 (21.6%) HIV-positive (HIV+) male BDs with heterosexual, other, or unknown risk factors could be reclassified as MSM, accounting for 10.9% of the total HIV+ male BDs. Overall, 139 of 284 HIV+ male donors (48.9%) could be considered MSM between 2000 and 2016 in France. Between 2005 and 2016, the %RRMSM increase varied from 0 to 19%, without differing significantly from the %RRMSM before reclassification. CONCLUSION: Network inference can be used to complement declaration data on risk factors for HIV infection in BDs. This approach, complementary to behavioral studies, is a valuable tool to evaluate the effect of changes in deferral criteria on BD compliance.

No Evidence of Hepatitis C Virus (HCV)-Assisted Hepatitis D Virus Propagation in a Large Cohort of HCV-Positive Blood Donors.

A study reported in 2019 showed that hepatitis C virus (HCV) could help disseminate hepatitis D virus (HDV). To test this finding, 2123 plasma samples positive for anti-HCV antibody were screened for anti-HDV antibodies, and HDV-RNA was searched for in samples positive for anti-HDV antibody. Of 41 samples (1.9%) that tested positive for anti-HDV antibody, 27 (65.9%) were positive and 14 (34.1%) negative for antibody to hepatitis B core antigen (anti-HBc). Anti-HDV antibodies were significantly more present in samples positive for anti-HBc (6.21% vs 0.8% in negative samples; P < .001) and in samples negative for HCV RNA (2.9% vs 1.5% for positive samples; P = .03). Serological ratios were significantly higher in samples positive for anti-HBc (P < .01). No anti-HDV-positive sample was HDV RNA positive. In conclusion, this study found no evidence suggesting a role for HCV in HDV dissemination in humans.

A Highly Prevalent Polymorphism in the Core Region Impairs Quantification of Hepatitis B Virus (HBV) by the cobas TaqMan HBV Assay.

The high genetic variability of hepatitis B virus (HBV) can impair DNA quantification. Here, we investigate a major underquantification of HBV by the cobas TaqMan HBV assay (CTM; Roche). In France, between 2005 and 2017, HBV DNA was detected in 3,102 blood donations by use of the CTM (95% limit of detection [LOD95], 4.8 IU/ml). HBV strains were sequenced in the S region (LOD95, ∼30 IU/ml). Concordant (n = 120) and discordant (n = 45) samples were identified according to the agreement between the plasma viral load (pVL) determined by the CTM and sequencing; all samples were also quantified using the RealTime HBV assay (RTH; Abbott). The viral signature, cloning, and mutagenesis were used to characterize the polymorphism responsible for CTM misquantification. A CTM-RTH discordance (>1 log IU/ml) was found in 14/45 samples that had low pVLs and were successfully genotyped (pVLlow genoS+). PreC/C clones of concordant (C1, C2) and discordant (D1, D2) strains were used to challenge the CTM. Strains D1 and D2 were highly underquantified (42- and 368-fold). In clones, mutating the region corresponding to the CTM reverse primer from a discordant sequence to a concordant sequence restored the levels of quantification to 24% (D1→C1) and 59% (D2→C1) of theoretical levels, while mutating the sequence of a concordant strain to that of a discordant strain led to 78-fold (C1→D1) and 146-fold (C1→D2) decreases in quantification. Moreover, mutating positions 1961 and 1962 was enough to induce a 5-fold underquantification. We conclude that the CTM underestimates pVLs for HBV strains with mutations in the reverse primer target. Specifically, the polymorphism at nucleotides 1961 and 1962 is naturally present in 4.79 and 4.22% of genotype A and D strains, which are highly frequent in Europe, leading to a 5-fold decrease in quantification. Quantification using the new-generation Roche C4800 assay is not affected by this polymorphism.

NKNK: a New Essential Motif in the C-Terminal Domain of HIV-1 Group M Integrases.

Using coevolution network interference based on comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four noncontiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3' processing and inefficient nuclear import of reverse-transcribed genomes. Solution of the crystal structures of wild-type (wt) and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K's could be permutated or additional K's could be inserted in the motif, generally without affecting integration per se Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions that integrase exerts. We propose that the existence of several amino acid arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution.IMPORTANCE Intensive studies of HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatment escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens up the possibility of adapting to the optimization of further functionalities exerted by the same protein. Such a property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.

High rate of hepatitis C virus and human immunodeficiency virus false-positive results in serologic screening in sub-Saharan Africa: adverse impact on the blood supply.

BACKGROUND: False positivity in blood screening may cause unnecessary deferral of healthy donors and exacerbate blood shortages. An international multicenter study was conducted to estimate the frequency of HCV and HIV false seropositivity in seven African countries (Burundi, Cameroon, Democratic Republic of Congo, Madagascar, Mali, Mauritania, and Niger). STUDY DESIGN AND METHODS: Blood donations were tested for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) with rapid detection tests (RDTs), third-generation enzyme immunoassays (EIAs), or fourth-generation EIAs. HCV (456/16,613 [2.74%]) and HIV (249/16,675 [1.49%]) reactive samples were then confirmed with antigen/antibody assays, immunoblots, and nucleic acid testing. Partial viral sequences were analyzed when possible. RESULTS: The HCV reactivity rate with RDTs was significantly lower than with EIAs (0.55% vs. 3.52%; p < 0.0001). The HIV reactivity rate with RDTs was lower than with third-generation EIAs (1.02% vs. 2.38%; p < 0.0001) but similar to a fourth-generation assay (1.09%). Only 16.0% (57/357) and 21.5% (38/177) of HCV and HIV initial reactive samples, respectively, were repeatedly reactive. HCV and HIV infections were confirmed in 13.2% and 13.7%, respectively, of repeated reactive donations. The predominant HCV genotype 2 and 4 strains in West and Central Africa showed high genetic variability. HIV-1 subtype CRF02_AG was most prevalent. CONCLUSION: High rates (>80%) of unconfirmed anti-HCV and anti-HIV reactivity observed in several sub-Saharan countries highlights the need for better testing and confirmatory strategies for donors screening in Africa. Without confirmatory testing, HCV and HIV prevalence in African blood donors has probably been overestimated.

Transfusion of HIV-infected blood products despite highly sensitive nucleic acid testing.

BACKGROUND: In France, the risk of HIV transmission by transfusion was reduced by implementing pooled nucleic acid testing (NAT) in 2001 and individual NAT in 2010. We report here the first case in France of transfusion of human immunodeficiency virus (HIV)-infected blood donated during HIV pre-ramp-up phase that tested individual NAT negative. METHODS: Blood donations are screened for HIV antibodies and HIV RNA (ProcleixUltrio, Grifols; limit of detection at 95%, 23 copies/mL). When a repeat donor tests positive for HIV, a repository sample from the previous donation is tested with the Cobas Taqman HIV-1 test (CTM, Roche; limit of detection at 95%, 17 copies/mL). RESULTS: In August 2017, a 57-year-old male repeat donor was screened positive for HIV antibodies and RNA (plasma viral load, 11,599 copies/mL). The previous donation had tested negative with Ultrio in March 2017 but was positive with an unquantifiable plasma viral load when tested with CTM. Sequencing showed no mismatch between Ultrio primers/probes and the target sequence. HIV transmission was excluded by lookback studies in the recipient of platelets, which had been pathogen reduced, but not in the recipient of RBCs due to premature death. CONCLUSION: This case demonstrates that the risk of contaminated donations due to the early HIV infection phase going undetected by highly sensitive NAT is real but exceptional. The absence of transmission to the platelets recipient could be due to the very low viral inoculum and/or to the efficacy of the viral inactivation. This case also highlights the additional value of a systematic donation archiving and the importance of donor education and predonation selection.

Detection of numerous HIV-1/MO recombinants in France.

BACKGROUND: The broad genetic divergence of HIV-1/O relative to HIV-1/M has important implications for diagnosis, monitoring and treatment. Despite this divergence, some HIV-1/M+O dual infections and HIV-1/MO recombinant forms have been reported, mostly in Cameroon, where both groups are prevalent. Here, we describe the characteristics of such infections detected in France in 10 new patients, and discuss their implications for biological and clinical practice, owing to the presence of group O species. METHODS: The French National Reference Centre for HIV received samples within the framework of mandatory notification of HIV infections, and for expert analysis. A strategy combining serotyping, viral quantification, group-specific molecular amplification and whole-genome sequencing was used for strain characterization and complementary investigations. RESULTS: We identified one patient with M+O infection, three patients with M+O infection associated with an MO recombinant, and six patients with only an MO recombinant. These atypical infections were detected upon strain characterization (n = 4) or because of anomalies during patient monitoring (n = 6). We identified eight new URF_MO, all but one originating from Cameroon. Interestingly, two distinct recombinant strains were found in two unrelated patients, representing possible precursors of a CRF_MO. CONCLUSION: Our work highlights the fact that the continuous evolution of HIV can hinder diagnosis and complicate clinical practice. We stress that unexpected results during diagnosis or monitoring necessitate further serological and molecular exploration, these atypical infections influence biological and therapeutic management and necessitate appropriate tools, and specific surveillance is necessary, especially as the frequency of such infections may be underestimated.

HIV-1 sequences in the epidemic suggest an alternative pathway for the generation of the Long Terminal Repeats.

To generate the long-terminal repeats (LTR) that border the integrated viral genome, two-strand transfer steps must occur during reverse transcription. Analysis of the genetic polymorphisms that are present in the LTR of HIV-1 heterozygous virions in single infection cycle studies has revealed which of the two copies of genomic RNAs is used for each transfer event. Thus, the first event of strand transfer has been described to be either intra- or intermolecular, while the second event is generally intramolecular. Here, we repeated these analyses using sequences from HIV databases and extended the study to the regions surrounding the LTR. We observed a striking correlation between the pattern of recombination in the LTR and the phylogenetic origin of the surrounding sequences. This correlation suggests that the second-strand transfer can be either intra- or intermolecular and, interestingly, could reflect an effect of proximity between nucleic acids that would guide this transfer. This factor could be particularly relevant for heterozygous viruses containing highly divergent genomic RNAs, such as those considered in the present study.

A Multiplex PCR Approach for Detecting Dual Infections and Recombinants Involving Major HIV Variants.

The cocirculation of different HIV types and groups can lead to dual infections and recombinants, which hinder diagnosis and therapeutic management. We designed two multiplex PCRs (mPCRs) coupled with capillary electrophoresis to facilitate the detection of such infections. The first, MMO2, targets three variants (HIV-1/M, HIV-1/O, and HIV-2), and the second, MMO, targets HIV-1/M and HIV-1/O. These mPCRs were validated on DNA and RNA extracts from 19 HIV-1/M, 12 HIV-1/O, and 13 HIV-2 cultures and from mixtures simulating dual infections. They were then assessed with DNA and RNA extracts from samples of 47 clinical monoinfections and HIV-1/M+O dual infections or infections with HIV-1/MO recombinants. Both mPCRs had excellent specificity. Sensitivities ranged from 80 to 100% for in vitro samples and from 58 to 100% for clinical samples, with the results obtained depending on the material used and the region of the genome concerned. Sensitivity was generally lower for DNA than for RNA and for amplifications of the integrase and matrix regions. In terms of global detection (at least one target gene for each strain), both mPCRs yielded a detection rate of 100% for in vitro samples. MMO2 detected 100% of the clinical strains from DNA and 97% from RNA, whereas MMO detected 100% of the strains from both materials. Thus, for in vitro and clinical samples, MMO2 was a useful tool for detecting dual infections with HIV-1 and HIV-2 (referred to as HIV-1+HIV-2) and HIV-1/M+O, and MMO was useful for detecting both MO dual infections and MO mosaic patterns.

Viral etiology of influenza-like illnesses in Cameroon, January-December 2009.

BACKGROUND: No information is available on the viral etiology of upper respiratory tract infections in Cameroon. METHODS: We prospectively enrolled outpatients with influenza-like illness (ILI) presenting at 14 sentinel clinics located across the country from January through December 2009. The specimens were tested using real-time and multiplex reverse-transcription polymerase chain reaction methods for the detection of 15 RNA respiratory viruses. RESULTS: We detected at least 1 respiratory virus in 365 of 561 specimens (65.1%). Overall, influenza virus was the most commonly detected virus (28.2% of specimens), followed by human rhinovirus (17.8%); parainfluenza virus (PIV) types 1-4 (7.5%); enterovirus (5.9%); respiratory syncytial virus (RSV; 5.7%); human coronavirus (HCoV) OC43, 229E, NL63, and HKU1 (5.3%); and human metapneumovirus (HMPV; 5.0%). RSV (26 of 31 specimens [83.9%]), PIV (30 of 39 [76.9%]), and HRV (64 of 99 [64.6%]) were most common among children <5 years of age. Coinfections were found in 53 of 365 positive specimens (14.5%), and most (71.7%) were in children <5 years of age. While influenza virus, enterovirus, RSV, and HMPV had a defined period of circulation, the other viruses were detected throughout the year. CONCLUSIONS: We found that respiratory viruses play an important role in the etiology of ILI in Cameroon, particularly in children <5 years of age.