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1.
Microorganisms ; 9(4)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807490

RESUMO

Background: The investigation of the antibody response to SARS-CoV-2 represents a key aspect in facing the COVID-19 pandemic. In the present study, we compared the new Immundiagnostik IDK® anti-SARS-CoV-2 S1 IgG assay with four widely-used commercial serological assays for the detection of antibodies targeting S (spike) and NC (nucleocapsid) proteins. Methods: Serum samples were taken from an unbiased group of convalescent patients and from a negative control group. Sample were simultaneously analyzed by the new Immundiagnostik IDK® anti-SARS-CoV-2 S1 IgG assay, by the DiaSorin LIAISON® SARS-CoV-2 S1/S2 IgG assay, and by the Euroimmun anti-SARS-CoV-2 S1 IgG ELISA. Antibodies binding NC were detected by the Abbott SARS-CoV-2 IgG assay and by the pan-immunoglobulin immunoassay Roche Elecsys® anti-SARS-CoV-2. Moreover, we investigated samples of a group of COVID-19 convalescent subjects that were primarily tested S1 IgG non-reactive. Samples were also tested by live virus and pseudovirus neutralization tests. Results: Overall, the IDK® anti-SARS-CoV-2 S1 IgG assay showed the highest sensitivity among the evaluated spike (S) protein-based assays. Additionally, the Immundiagnostik assay correlated well with serum-neutralizing activity. Conclusions: The novel IDK® anti-SARS-CoV-2 S1 IgG assay showed high sensitivity and specificity, representing a valid option for use in the routine diagnostic.

4.
Vaccines (Basel) ; 9(3)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673566

RESUMO

Humanized mice are critical for HIV-1 research, but humanized mice generated from cord blood are inefficient at mucosal HIV-1 transmission. Most mucosal HIV-1 transmission studies in mice require fetal tissue-engraftment, the use of which is highly restricted or prohibited. We present a fetal tissue-independent model called CD34T+ with enhanced human leukocyte levels in the blood and improved T cell homing to the gut-associated lymphoid tissue. CD34T+ mice are highly permissive to intra-rectal HIV-1 infection and also show normal env diversification in vivo despite high viral replication. Moreover, mucosal infection in CD34T+ mice can be prevented by infusion of broadly neutralizing antibodies. CD34T+ mice can be rapidly and easily generated using only cord blood cells and do not require any complicated surgical procedures for the humanization process. Therefore, CD34T+ mice provide a novel platform for mucosal HIV-1 transmission studies as well as rapid in vivo testing of novel prevention molecules against HIV-1.

5.
Eur J Clin Microbiol Infect Dis ; 40(5): 1063-1071, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33534090

RESUMO

Evaluation and power of seroprevalence studies depend on the performed serological assays. The aim of this study was to assess four commercial serological tests from EUROIMMUN, DiaSorin, Abbott, and Roche as well as an in-house immunofluorescence and neutralization test for their capability to identify SARS-CoV-2 seropositive individuals in a high-prevalence setting. Therefore, 42 social and working contacts of a German super-spreader were tested. Consistent with a high-prevalence setting, 26 of 42 were SARS-CoV-2 seropositive by neutralization test (NT), and immunofluorescence test (IFT) confirmed 23 of these 26 positive test results (NT 61.9% and IFT 54.8% seroprevalence). Four commercial assays detected anti-SARS-CoV-2 antibodies in 33.3-40.5% individuals. Besides an overall discrepancy between the NT and the commercial assays regarding their sensitivity, this study revealed that commercial SARS-CoV-2 spike-based assays are better to predict the neutralization titer than nucleoprotein-based assays are.


Assuntos
/métodos , /epidemiologia , /isolamento & purificação , Adolescente , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , /normas , Busca de Comunicante , Feminino , Humanos , Imunoensaio , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Prevalência , Sensibilidade e Especificidade , Adulto Jovem
6.
Artigo em Inglês | MEDLINE | ID: mdl-33511435

RESUMO

OBJECTIVES: The aims of this study were to evaluate the isolated prevalence of real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 on the ocular surface without systemic infection in hospitalized asymptomatic patients and to determine the risk for ophthalmologists and medical staff to be infected by prescreened asymptomatic patients in a tertiary eye care center. METHODS: In this prospective, observational study, bilateral swaps of the conjunctiva in the lower fornices as well as nasopharyngeal swaps were collected in 1145 hospitalized asymptomatic patients of a tertiary eye care center. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed for each swap to evaluate the prevalence of SARS-CoV-2. Demographic data and potential risk factors for an isolated infection of the ocular surface were noted. RESULTS: Two thousand two hundred eighty-eight (99.9%) of all 2290 tested eyes had negative results in the RT-PCR analysis of the conjunctival swabs. One patient had bilateral false-positive results in the conjunctival swabs. None of the 1145 patients had any positive RT-PCR-confirmed result in the nasopharyngeal swabs. CONCLUSIONS: The risk for an isolated conjunctival viral activity in patients with a negative nasopharyngeal swab-based RT-PCR seems to be absent or extremely low, suggesting no need to perform additional conjunctival swabs in patients with negative nasopharyngeal swabs. Furthermore, the risk of a work-related SARS-CoV-2 infection due to direct contact with preselected asymptomatic patients in an eye care center is very low, especially when additional hygiene standards and safe distances are respected carefully. This might reassure medical staff and reduce the fear of SARS-CoV-2 infection.

7.
J Immunol Methods ; 490: 112958, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33412174

RESUMO

The current Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic is a public health emergency of international concern. Sensitive and precise diagnostic tools are urgently needed. In this study, we developed a SARS-CoV-2 spike (S1) protein enzyme-linked immunosorbent assay (ELISA) to detect SARS-CoV-2-specific antibodies. The SARS-CoV-2 S1 ELISA was found to be specific [97.8% (95% CI, 96.7% - 98.5%)], reproducible and precise (intra-assay coefficient of variability (CV) 5.3%, inter-assay CV 7.9%). A standard curve and the interpolation of arbitrary ELISA units per milliliter served to reduce the variability between different tests and operators. Cross-reactivity to other human coronaviruses was addressed by using sera positive for MERS-CoV- and hCoV HKU1-specific antibodies. Monitoring antibody development in various samples of twenty-three and single samples of twenty-nine coronavirus disease 2019 (COVID-19) patients revealed seroconversion and neutralizing antibodies against authentic SARS-CoV-2 in all cases. The comparison of the SARS-CoV-2 (S1) ELISA with a commercially available assay showed a better sensitivity for the in-house ELISA. The results demonstrate a high reproducibility, specificity and sensitivity of the newly developed ELISA, which is suitable for the detection of SARS-CoV-2 S1 protein-specific antibody responses.


Assuntos
/métodos , Células Epiteliais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticorpos Neutralizantes/sangue , Chlorocebus aethiops , Ensaios Enzimáticos , Ensaio de Imunoadsorção Enzimática , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soroconversão , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
8.
Gut ; 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33323394

RESUMO

OBJECTIVE: Neutralising antibodies are key effectors of infection-induced and vaccine-induced immunity. Quantification of antibodies' breadth and potency is critical for understanding the mechanisms of protection and for prioritisation of vaccines. Here, we used a unique collection of human specimens and HCV strains to develop HCV reference viruses for quantification of neutralising antibodies, and to investigate viral functional diversity. DESIGN: We profiled neutralisation potency of polyclonal immunoglobulins from 104 patients infected with HCV genotype (GT) 1-6 across 13 HCV strains representing five viral GTs. Using metric multidimensional scaling, we plotted HCV neutralisation onto neutralisation maps. We employed K-means clustering to guide virus clustering and selecting representative strains. RESULTS: Viruses differed greatly in neutralisation sensitivity, with J6 (GT2a) being most resistant and SA13 (GT5a) being most sensitive. They mapped to six distinct neutralisation clusters, in part composed of viruses from different GTs. There was no correlation between viral neutralisation and genetic distance, indicating functional neutralisation clustering differs from sequence-based clustering. Calibrating reference viruses representing these clusters against purified antibodies from 496 patients infected by GT1 to GT6 viruses readily identified individuals with extraordinary potent and broadly neutralising antibodies. It revealed comparable antibody cross-neutralisation and diversity between specimens from diverse viral GTs, confirming well-balanced reporting of HCV cross-neutralisation across highly diverse human samples. CONCLUSION: Representative isolates from six neutralisation clusters broadly reconstruct the functional HCV neutralisation space. They enable high resolution profiling of HCV neutralisation and they may reflect viral functional and antigenic properties important to consider in HCV vaccine design.

9.
EMBO J ; : e105594, 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33146906

RESUMO

Failures to produce neutralizing antibodies upon HIV-1 infection result in part from B-cell dysfunction due to unspecific B-cell activation. How HIV-1 affects antigen-specific B-cell functions remains elusive. Using an adoptive transfer mouse model and ex vivo HIV infection of human tonsil tissue, we found that expression of the HIV-1 pathogenesis factor NEF in CD4 T cells undermines their helper function and impairs cognate B-cell functions including mounting of efficient specific IgG responses. NEF interfered with T cell help via a specific protein interaction motif that prevents polarized cytokine secretion at the T-cell-B-cell immune synapse. This interference reduced B-cell activation and proliferation and thus disrupted germinal center formation and affinity maturation. These results identify NEF as a key component for HIV-mediated dysfunction of antigen-specific B cells. Therapeutic targeting of the identified molecular surface in NEF will facilitate host control of HIV infection.

10.
PLoS One ; 15(11): e0238612, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33137122

RESUMO

BACKGROUND: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose. METHODS: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). RESULTS: Whilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK. CONCLUSION: In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , RNA Viral/metabolismo , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/virologia , Humanos , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
12.
Viruses ; 12(9)2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32962125

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a global health emergency. To improve the understanding of the systemic component of SARS-CoV-2, we investigated if viral load dynamics in plasma and respiratory samples are associated with antibody response and severity of coronavirus disease 2019 (COVID-19). SARS-CoV-2 RNA was found in plasma samples from 14 (44%) out of 32 patients. RNAemia was detected in 5 out of 6 fatal cases. Peak IgG values were significantly lower in mild/moderate than in severe (0.6 (interquartile range, IQR, 0.4-3.2) vs. 11.8 (IQR, 9.9-13.0), adjusted p = 0.003) or critical cases (11.29 (IQR, 8.3-12.0), adjusted p = 0.042). IgG titers were significantly associated with virus Ct (Cycle threshold) value in plasma and respiratory specimens ((ß = 0.4, 95% CI (confidence interval, 0.2; 0.5), p < 0.001 and ß = 0.5, 95% CI (0.2; 0.6), p = 0.002). A classification as severe or a critical case was additionally inversely associated with Ct values in plasma in comparison to mild/moderate cases (ß = -3.3, 95% CI (-5.8; 0.8), p = 0.024 and ß = -4.4, 95% CI (-7.2; 1.6), p = 0.007, respectively). Based on the present data, our hypothesis is that the early stage of SARS-CoV-2 infection is characterized by a primary RNAemia, as a potential manifestation of a systemic infection. Additionally, the viral load in plasma seems to be associated with a worse disease outcome.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/sangue , Idoso , Betacoronavirus/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/sangue , Infecções por Coronavirus/patologia , Feminino , Alemanha/epidemiologia , Hospitalização , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/patologia , RNA Viral/análise , Sistema Respiratório/virologia , Índice de Gravidade de Doença , Carga Viral , Viremia/sangue , Viremia/patologia , Viremia/virologia
14.
EMBO J ; 39(20): e106230, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32876341

RESUMO

COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within 2 days of exposure. We identified that SARS-CoV-2 preferably targets neurons of brain organoids. Imaging neurons of organoids reveal that SARS-CoV-2 exposure is associated with altered distribution of Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Our studies, therefore, provide initial insights into the potential neurotoxic effect of SARS-CoV-2 and emphasize that brain organoids could model CNS pathologies of COVID-19.


Assuntos
Betacoronavirus/fisiologia , Encéfalo/virologia , Neurônios/virologia , Animais , Morte Celular , Chlorocebus aethiops , Humanos , Doenças do Sistema Nervoso/virologia , Organoides , Células Vero , Proteínas tau/metabolismo
15.
Clin Microbiol Infect ; 26(11): 1560.e5-1560.e8, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32835793

RESUMO

OBJECTIVES: To evaluate ocular symptoms in European non-hospitalized patients with severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and to investigate associations with the demographic data as well as nasal and general physical symptoms. METHODS: In this prospective, observational study, 108 non-hospitalized patients with PCR-confirmed SARS-CoV-2 infection not requiring intensive care were asked about disease-associated ocular symptoms, demographic data, as well as general physical and nasal symptoms using a standardized questionnaire. Total ocular symptom score (TOSS) was evaluated during and, retrospectively, before development of coronavirus disease 2019 (COVID-19). Associations between TOSS and demographic data as well as general and nasal symptoms were evaluated. RESULTS: Seventy-five of the 108 COVID-19 patients (69.4%) had at least one ocular symptom during COVID-19. The most common symptoms included burning sensations in 39 (36.1%), epiphora in 37 (34.3%) and redness in 28 (25.9%), compatible with conjunctivitis. These symptoms occurred 1.96 ± 3.17 days after the beginning of COVID-19 and were mild. TOSS was significantly higher during COVID-19 (1.27 ± 1.85) than before the infection (0.33 ± 1.04; p < 0.001). There were no significant associations between TOSS and gender (ß coefficient -0.108; p 0.302), age (-0.024; p 0.816), rhinorrhoea (-0.127; p 0.353), nasal itching (-0.026; p 0.803), sneezing (0.099; p 0.470), nasal congestion (-0.012; p 0.930), cough (-0.079; p 0.450), headache (0.102; p 0.325), sore throat (0.208; p 0.052), or fever (0.094; p 0.361). CONCLUSIONS: Ocular involvement in European non-hospitalized individuals with COVID-19 seems to be highly underestimated. Overall, these ocular symptoms, including burning sensations, epiphora and redness, seem to be mild and to not need treatment.


Assuntos
Infecções por Coronavirus/complicações , Oftalmopatias/etiologia , Oftalmopatias/patologia , Pneumonia Viral/complicações , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Europa (Continente)/epidemiologia , Oftalmopatias/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Prevalência , Estudos Prospectivos , Adulto Jovem
16.
BMC Bioinformatics ; 21(1): 290, 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32640981

RESUMO

BACKGROUND: Well-characterized biomaterials of high quality have great potential for acceleration and quality improvement in translational biomedical research. To improve accessibility of local sample collections, efforts have been made to create central biomaterial banks and catalogues. Available technical solutions for creating professional local sample catalogues and connecting them to central systems are cost intensive and/or technically complex to implement. Therefore, the Translational Thematic Unit HIV of the German Center for Infection Research (DZIF) developed a Laboratory Information and Management System (LIMS) called HIV Engaged Research Technology (HEnRY) for implementation into the Translational Platform HIV (TP-HIV) at the DZIF and other research networks. RESULTS: HEnRY is developed at the University Hospital of Cologne. It is an advanced LIMS to manage processing and storage of samples and aliquots of different sample types. Features include: monitoring of stored samples and associated information data selection via query tools or Structured Query Language (SQL) preparation of summary documents, including scannable search lists centralized management of the practical laboratory part of multicentre studies (e.g. import of drawing schemes and sample processing steps), preparation of aliquot shipments, including associated documents to be added to shipments unique and secure identification of aliquots through use of customizable Quick Response (QR) code labels directly from HEnRY support of aliquot data transmission to central registries. In summary, HEnRY offers all features necessary for a LIMS software. In addition, the structure of HEnRY provides sufficient flexibility to allow the implementation in other research areas. CONCLUSION: HEnRY is a free biobanking tool published under the MIT license. While it was developed to support HIV research in Germany, the feature set and language options, allow much broader applications and make this a powerful free research tool.


Assuntos
Bancos de Espécimes Biológicos , Software , Materiais Biocompatíveis , Sistemas Computacionais , Gerenciamento de Dados , Documentação , Humanos , Laboratórios , Estudos Multicêntricos como Assunto
17.
PLoS Pathog ; 16(7): e1008560, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667948

RESUMO

Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8+ and CD4+ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4+, liver B/IgA+ and spleen B/IgG+ cells as predictive biomarkers of immunization (≈87% accuracy). CD8+ and CD4+ T cell responses against gB were validated. Splenic gB-binding IgM-/IgG+ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Vacinas contra Citomegalovirus/imunologia , Imunização Passiva , Imunoglobulina G/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Antígenos Virais/imunologia , Citomegalovirus/imunologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Humanos , Imunoglobulina G/farmacologia , Camundongos
18.
Cell ; 182(4): 843-854.e12, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32673567

RESUMO

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and the world economy. Because approved drugs and vaccines are limited or not available, new options for COVID-19 treatment and prevention are in high demand. To identify SARS-CoV-2-neutralizing antibodies, we analyzed the antibody response of 12 COVID-19 patients from 8 to 69 days after diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days after diagnosis. Of these, 28 potently neutralized authentic SARS-CoV-2 with IC100 as low as 0.04 µg/mL, showing a broad spectrum of variable (V) genes and low levels of somatic mutations. Interestingly, potential precursor sequences were identified in naive B cell repertoires from 48 healthy individuals who were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2-neutralizing antibodies are readily generated from a diverse pool of precursors, fostering hope for rapid induction of a protective immune response upon vaccination.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Betacoronavirus/imunologia , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Memória Imunológica , Estudos Longitudinais , Pandemias , Hipermutação Somática de Imunoglobulina
19.
Eur J Haematol ; 105(4): 508-511, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32575156

RESUMO

The number of people suffering from the new coronavirus SARS-CoV-2 continues to rise. In SARS-CoV-2, superinfection with bacteria or fungi seems to be associated with increased mortality. The role of co-infections with respiratory viral pathogens has not yet been clarified. Here, we report the course of COVID-19 in a CLL patient with secondary immunodeficiency and viral co-infection with parainfluenza.

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