Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting.

BACKGROUND: Recombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subsequently obtain purified untagged rMVA upon loss of that marker by site-specific recombination. The standard RGSSM is quite costly in terms of bench work, reagents, and Sorting Facility fees. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product. METHODS: The direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads. RESULTS: Upon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days. CONCLUSIONS: Altogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases.

[Monkeypox: Epidemiological surveillance in the Muñiz Outpatient Monitoring Unit, Buenos Aires]. / Viruela símica: Vigilancia epidemiológica en la Unidad de Monitoreo Ambulatorio Muñiz, Buenos Aires.

INTRODUCTION: Monkeypox (SV) is a zoonotic viral disease. Monkeypox virus belongs to the Orthopoxvirus genus of the Poxviridae family. In 1970 it was detected for the first time in humans, in the Democratic Republic of the Congo. It is currently considered an endemic disease in central and western Africa. It can present with fever, malaise, painful lymphadenopathy and rash, and last between 2 and 4 weeks. It is usually selflimited, although severe cases have been described, mainly in immunocompromised people, with lethality varies between 3% and 6% in endemic countries. The objectives of epidemiological surveillance in suspected cases of SV are: to describe the personal variables, time and place of the reported cases, to track and follow up their contacts, to identify outbreaks and perform clinical epidemiological follow-up. METHODS: A retrospective cohort study was conducted from June 9 to September 15, 2022. RESULTS: 82 patients with compatible symptoms have been treated, 56 cases were positive and 26 negative. Of the 26 negative cases, 7 cases (27%) corresponded to chickenpox, 4 cases (15%) to syphilis, and 2 cases (8%) to herpes zoster. CONCLUSIONS: The first symptoms began two to five days prior to consultation, the most frequent being fever, myalgia, asthenia, headache, and painful lymphadenopathy. Epidemiological surveillance of SV provides timely detection of cases, continuous and systematic follow-up of contacts through an integrated process between surveillance and laboratory, to generate timely, valid and reliable information, which allows guiding prevention and control measures.

Introducción: La viruela símica (VS) es una enfermedad viral zoonótica debida a un virus del género Orthopoxvirus, familia Poxviridae. En 1970 se detectó por primera vez en humanos en la República Democrática del Congo. Actualmente es endémica en áfrica central y occidental. Puede presentarse con fiebre, malestar general, linfo-adenopatías dolorosas y exantema, y durar entre 2 y 4 semanas. Suele ser autolimitada, aunque se han descrito casos graves, principalmente en personas inmunocomprometidas, con una letalidad que varía entre 3% y 6% en países endémicos. Los objetivos de la vigilancia epidemiológica en los casos sospechosos de VS son: describir las variables personales, tiempo y lugar de los casos notificados, realizar un rastreo y seguimiento de sus contactos, identificar brotes y realizar seguimiento clínico epidemiológico. Métodos: Se realizó un estudio de cohorte retrospectivo durante el 9 de junio al 15 de septiembre de 2022. Resultados: Se han atendido 82 pacientes con sintomatología compatible 56 casos fueron positivos y 26 negativos. De los 26 casos negativos, 7 casos (27%) correspondían a varicela, 4 caso s (15%) a sífilis y 2 casos (8%) a herpes zoster. Los primeros síntomas se iniciaron dos a cinco días previos a la consulta, siendo los más frecuentes fiebre, mialgia, astenia, cefalea y linfoadenopatía dolorosa. Conclusiones: La vigilancia epidemiológica de VS permite la detección de casos, el seguimiento continuo y sistemático de los contactos mediante un proceso integrado entre vigilancia y laboratorio, para generar información oportuna, válida y confiable, que permite orientar medidas de prevención y control.

Viruela símica: vigilancia epidemiológica en la Unidad de Monitoreo Ambulatorio Muñiz, Buenos Aires / Monkeypox: epidemiological surveillance in the Muñiz Outpatient Monitoring Unit, Buenos Aires

Resumen Introducción: La viruela símica (VS) es una enfermedad viral zoonótica debida a un virus del género Orthopoxvirus, familia Poxviridae. En 1970 se detectó por primera vez en humanos en la República Democrática del Congo. Actualmente es endémica en África central y occidental. Puede presentarse con fiebre, malestar general, linfo-adenopatías dolorosas y exantema, y durar entre 2 y 4 semanas. Suele ser autolimitada, aunque se han descrito casos graves, principalmente en personas inmunocomprometidas, con una letalidad que varía entre 3% y 6% en países endémicos. Los objetivos de la vigilancia epidemiológica en los casos sospecho sos de VS son: describir las variables personales, tiempo y lugar de los casos notificados, realizar un rastreo y seguimiento de sus contactos, identificar brotes y realizar seguimiento clínico epidemiológico. Métodos: Se realizó un estudio de cohorte retrospectivo durante el 9 de junio al 15 de septiembre de 2022. Resultados: Se han atendido 82 pacientes con sintomatología compatible 56 casos fueron positivos y 26 negativos. De los 26 casos negativos, 7 casos (27%) correspondían a varicela, 4 caso s (15%) a sífilis y 2 casos (8%) a herpes zoster. Los primeros síntomas se iniciaron dos a cinco días previos a la consulta, siendo los más frecuentes fiebre, mialgia, astenia, cefalea y linfoadenopatía dolorosa. Conclusiones: La vigilancia epidemiológica de VS permite la detección de casos, el seguimiento continuo y sistemático de los contactos mediante un proceso integrado entre vigilancia y laboratorio, para generar información oportuna, válida y confiable, que permite orientar medidas de prevención y control.

Abstract Introduction: Monkeypox (SV) is a zoonotic viral disease. Monkeypox virus belongs to the Ortho poxvirus genus of the Poxviridae family. In 1970 it was detected for the first time in humans, in the Democratic Republic of the Congo. It is currently considered an endemic disease in central and western Africa. It can pres ent with fever, malaise, painful lymphadenopathy and rash, and last between 2 and 4 weeks. It is usually self-limited, although severe cases have been described, mainly in immunocompromised people, with lethality varies between 3% and 6% in endemic countries. The objectives of epidemiological surveillance in suspected cases of SV are: to describe the personal variables, time and place of the reported cases, to track and follow up their contacts, to identify outbreaks and perform clinical epidemiological follow-up. Methods: A retrospective cohort study was conducted from June 9 to September 15, 2022. Results: 82 patients with compatible symptoms have been treated, 56 cases were positive and 26 negative. Of the 26 negative cases, 7 cases (27%) corresponded to chickenpox, 4 cases (15%) to syphilis, and 2 cases (8%) to herpes zoster. Conclusions: The first symptoms began two to five days prior to consultation, the most frequent being fever, myalgia, asthenia, headache, and painful lymphadenopathy. Epidemiological surveillance of SV provides timely detection of cases, continuous and systematic follow-up of contacts through an integrated process between surveillance and laboratory, to generate timely, valid and reliable information, which allows guiding prevention and control measures.

[Infectiousness and virulence of multidrug-resistant and drug susceptible tuberculosis in adult contacts]. / Infecciosidad y virulencia de la tuberculosis multirresistente o sensible a drogas antituberculosas en contactos adultos.

Patients carrying multidrug-resistant (MDR) strains of Mycobacterium tuberculosis have been considered traditionally as presenting a diminished epidemiological risk according to animal experimentation results. The experience obtained from the MDRTB/AIDS related epidemics showed MDRTB transmission to immunocompetent health care workers and adults close contacts. In this retrospective study, the infectiousness (measured as the frequency of intradermal test with PPD 2TU over 10 mm) and virulence (cases bacteriologically confirmed) among close contacts of patients with MDR and susceptible TB were evaluated. A total of 97 contacts of 37 MDRTB patients vs. 356 contacts of 100 patients with susceptible TB were studied. No statistical differences were found in PPD 2 UT positivity and TB cases between both contact groups, nor in relation to HIV seropositivity of index cases. According to these observations, MDR strains of M. tuberculosis present similar infectiousness and virulence compared with susceptible strains.

Infecciosidad y virulencia de la tuberculosis multirresistente o sensible a drogas antituberculosas en contactos adultos / Infectiousness and virulence of multidrug-resistant and drug susceptible tuberculosis in adult contacts

Patients carrying multidrug-resistant (MDR) strains of Mycobacterium tuberculosis have been considered traditionally as presenting a diminished epidemiological risk according to animal experimentation results. The experience obtained from the MDRTB/AIDS related epidemics showed MDRTB transmission to immunocompetent health care workers and adults close contacts. In this retrospective study, the infectiousness (measured as the frequency of intradermal test with PPD 2TU over 10 mm) and virulence (cases bacteriologically confirmed) among close contacts of patients with MDR and susceptible TB were evaluated. A total of 97 contacts of 37 MDRTB patients vs. 356 contacts of 100 patients with susceptible TB were studied. No statistical differences were found in PPD 2 UT positivity and TB cases between both contact groups, nor in relation to HIV seropositivity of index cases. According to these observations, MDR strains of M. tuberculosis present similar infectiousness and virulence compared with susceptible strains (AU)#S#a

Infecciosidad y virulencia de la tuberculosis multirresistente o sensible a drogas antituberculosas en contactos adultos. / [Infectiousness and virulence of multidrug-resistant and drug susceptible tuberculosis in adult contacts]

Patients carrying multidrug-resistant (MDR) strains of Mycobacterium tuberculosis have been considered traditionally as presenting a diminished epidemiological risk according to animal experimentation results. The experience obtained from the MDRTB/AIDS related epidemics showed MDRTB transmission to immunocompetent health care workers and adults close contacts. In this retrospective study, the infectiousness (measured as the frequency of intradermal test with PPD 2TU over 10 mm) and virulence (cases bacteriologically confirmed) among close contacts of patients with MDR and susceptible TB were evaluated. A total of 97 contacts of 37 MDRTB patients vs. 356 contacts of 100 patients with susceptible TB were studied. No statistical differences were found in PPD 2 UT positivity and TB cases between both contact groups, nor in relation to HIV seropositivity of index cases. According to these observations, MDR strains of M. tuberculosis present similar infectiousness and virulence compared with susceptible strains.

Infecciosidad y virulencia de la tuberculosis multirresistente o sensible a drogas antituberculosas en contactos adultos / Infectiousness and virulence of multidrug-resistant and drug susceptible tuberculosis in adult contacts

Patients carrying multidrug-resistant (MDR) strains of Mycobacterium tuberculosis have been considered traditionally as presenting a diminished epidemiological risk according to animal experimentation results. The experience obtained from the MDRTB/AIDS related epidemics showed MDRTB transmission to immunocompetent health care workers and adults close contacts. In this retrospective study, the infectiousness (measured as the frequency of intradermal test with PPD 2TU over 10 mm) and virulence (cases bacteriologically confirmed) among close contacts of patients with MDR and susceptible TB were evaluated. A total of 97 contacts of 37 MDRTB patients vs. 356 contacts of 100 patients with susceptible TB were studied. No statistical differences were found in PPD 2 UT positivity and TB cases between both contact groups, nor in relation to HIV seropositivity of index cases. According to these observations, MDR strains of M. tuberculosis present similar infectiousness and virulence compared with susceptible strains (AU)#S#a