SARS-CoV-2 in an immunocompromised host: convalescent plasma therapy and viral evolution elucidated by whole genome sequencing.

The evolution of SARS-CoV-2 within immunocompromised hosts who fail to clear the virus over many months has been proposed as a route to the development of Variants of Concern (VoCs). We present a case of an immunocompromised male patient with a prolonged SARS-CoV-2 infection. During hospitalisation, 7 weeks after first diagnosis, his condition worsened to require continuous ventilation support. Resolution of symptoms was observed after convalescent plasma therapy. Whole genome sequencing of the virus showed Pango lineage B.1.221. Between the first sample and the second from bronchoalveolar lavage fluid 7 weeks later, we identified eight mutations, including minor variants, which could be used to estimate the chronology of mutations. This suggests an elevated mutation rate, in-host accumulation of mutations and further evidence for sources of VoCs. Prolonged SARS-CoV-2 infections in immunocompromised hosts increase the likelihood of hospital stays and morbidity, and also pose an increased risk to global public health.

Detection of Viral Infection and Bacterial Coinfection and Superinfection in Coronavirus Disease 2019 Patients Presenting to the Emergency Department Using the 29-mRNA Host Response Classifier IMX-BVN-3: A Multicenter Study.

Background: Identification of bacterial coinfection in patients with coronavirus disease 2019 (COVID-19) facilitates appropriate initiation or withholding of antibiotics. The Inflammatix Bacterial Viral Noninfected (IMX-BVN) classifier determines the likelihood of bacterial and viral infections. In a multicenter study, we investigated whether IMX-BVN version 3 (IMX-BVN-3) identifies patients with COVID-19 and bacterial coinfections or superinfections. Methods: Patients with polymerase chain reaction-confirmed COVID-19 were enrolled in Berlin, Germany; Basel, Switzerland; and Cleveland, Ohio upon emergency department or hospital admission. PAXgene Blood RNA was extracted and 29 host mRNAs were quantified. IMX-BVN-3 categorized patients into very unlikely, unlikely, possible, and very likely bacterial and viral interpretation bands. IMX-BVN-3 results were compared with clinically adjudicated infection status. Results: IMX-BVN-3 categorized 102 of 111 (91.9%) COVID-19 patients into very likely or possible, 7 (6.3%) into unlikely, and 2 (1.8%) into very unlikely viral bands. Approximately 94% of patients had IMX-BVN-3 unlikely or very unlikely bacterial results. Among 7 (6.3%) patients with possible (n = 4) or very likely (n = 3) bacterial results, 6 (85.7%) had clinically adjudicated bacterial coinfection or superinfection. Overall, 19 of 111 subjects for whom adjudication was performed had a bacterial infection; 7 of these showed a very likely or likely bacterial result in IMX-BVN-3. Conclusions: IMX-BVN-3 identified COVID-19 patients as virally infected and identified bacterial coinfections and superinfections. Future studies will determine whether a point-of-care version of the classifier may improve the management of COVID-19 patients, including appropriate antibiotic use.

Brote de tuberculosis resistente a isoniacida en una comunidad de inmigrantes en España / Outbreak of isoniazid-resistant tuberculosis in an immigrant community in Spain

Introducción: La tuberculosis (TB) continúa siendo un importante problema de salud pública. En 2013 se declararon 9 millones de casos nuevos de TB activa a nivel mundial, siendo la proporción de nuevos casos de TB multirresistente del 3,5%. Método: Se realizó un estudio de contactos de un caso de TB pulmonar en una paciente de nacionalidad boliviana. Las pruebas diagnósticas se realizaron según los protocolos establecidos a nivel nacional y local. Resultados: Se detectaron 5 casos a partir del caso índice y se constató la existencia de un brote de TB en una comunidad inmigrante. El resultado del genotipado y del antibiograma ampliado de las muestras de esputo fue crecimiento de Mycobacterium tuberculosis (KatG-msp no mutado/inhA C-T 5RBS) resistente a isoniacida. Se realizó la búsqueda activa de convivientes y contactos con un censo total de 39 personas. La incidencia de infección latente fue de 71,43%. Discusión: El estudio de este brote como otros en la literatura constata la importancia de la búsqueda activa de la localización de contactos y su estudio, de la investigación de laboratorio para lograr la mejora en la detección precoz de la TB. Un diagnóstico precoz del enfermo, el cumplimiento de un tratamiento adecuado y la vigilancia de la farmacorresistencia se consideran pilares fundamentales para la prevención y el control de la TB

Background: Tuberculosis (TB) remains a major public health problem. In 2013, 9 million new cases of active TB were estimated globally and the proportion of reported new cases with multi-drug resistance (MDR) was 3.5%. Methods: Contact tracing of a case of pulmonary tuberculosis was performed in a Bolivian patient. Diagnostic tests were performed according to national and local protocols. Results: An outbreak of tuberculosis in an immigrant community was detected, with 5 cases originating from one index case. Genotyping and drug susceptibility testing of the sputum samples determined Mycobacterium tuberculosis resistant to isoniazid (KatG-msp unmutated/inhA 5RBS CT). Active case finding revealed a total of 39 contacts with an incidence of latent infection of 71.43%. Conclusions: The present study confirms the importance of active case finding through contact tracing as well as rapid laboratory diagnosis to achieve improvements in early detection of TB. Early diagnosis of the patient, compliance with appropriate treatment protocols and monitoring of drug resistance are considered essential for the prevention and control of TB

Neuroleptic drugs in the treatment of tuberculosis: Minimal inhibitory concentrations of different phenothiazines against Mycobacterium tuberculosis.

Due to an increase of drug resistant TB, alternative drugs that are not currently listed in the WHO guidelines on MDR TB treatment are currently being evaluated. Our group tested 100 susceptible, 20 MDR and 2 XDR Mtb strains against the phenothiazine derivatives thioridazine, trifluoperazine and triflupromazine. MIC testing was performed on Middlebrook 7H10 agar and was defined as the lowest drug concentration that inhibits ≥99% of the bacterial population. We confirm very good in vitro activity of phenothiazines against Mycobacterium tuberculosis. In >77% of all strains MICs of ≤10 µg/ml were found.

Outbreak of isoniazid-resistant tuberculosis in an immigrant community in Spain. / Brote de tuberculosis resistente a isoniacida en una comunidad de inmigrantes en España.

BACKGROUND: Tuberculosis (TB) remains a major public health problem. In 2013, 9 million new cases of active TB were estimated globally and the proportion of reported new cases with multi-drug resistance (MDR) was 3.5%. METHODS: Contact tracing of a case of pulmonary tuberculosis was performed in a Bolivian patient. Diagnostic tests were performed according to national and local protocols. RESULTS: An outbreak of tuberculosis in an immigrant community was detected, with 5 cases originating from one index case. Genotyping and drug susceptibility testing of the sputum samples determined Mycobacterium tuberculosis resistant to isoniazid (KatG-msp unmutated/inhA 5RBS CT). Active case finding revealed a total of 39 contacts with an incidence of latent infection of 71.43%. CONCLUSIONS: The present study confirms the importance of active case finding through contact tracing as well as rapid laboratory diagnosis to achieve improvements in early detection of TB. Early diagnosis of the patient, compliance with appropriate treatment protocols and monitoring of drug resistance are considered essential for the prevention and control of TB.

Minimal inhibitory concentrations of first-line drugs of multidrug-resistant tuberculosis isolates.

CONTEXT: The treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) is consistently difficult. Besides resistances, drug availability can be problematic and costs for therapy are high. AIMS: Our aim was to evaluate alternatives in treatment of MDR and XDR TB other than using second-line drugs. MATERIALS AND METHODS: We analyzed retrospectively the minimal inhibitory concentrations (MICs) of first-line drugs for 44 multidrug-resistant Mycobacterium tuberculosis isolates determined in our institute over a period of 20 years (1990 - 2010, n = 44). Drug susceptibility testing (DST) was performed using the proportion method on Lowenstein-Jensen Medium or Middlebrook 7H10 agar. MICs were defined as the lowest drug concentration after two-fold serially diluted concentration of the drugs that inhibits growth of more than 99.0% of a bacterial proportion of the tested M. tuberculosis within 14 to 21 days of incubation at 37°C. STATISTICAL ANALYSIS USED: Summation. RESULTS: The MICs of isoniazid and ethambutol were equal or slightly above the critical concentration in most of the strains (92% and 84%, respectively), defined as "low-level resistance". Rifampicin and streptomycin exhibited very high MICs in most of the strains (100% and 77%, respectively), indicating a "high-level resistance". CONCLUSION: Our results indicate that isoniazid and ethambutol could still play a role in treating MDR and XDR TB patients if low-level resistance is detected. Quantitative DST seems to be promising for the recognition of residual drug activity, but has to be confirmed by clinical studies.

The use of interferon gamma release assays in the diagnosis of active tuberculosis.

Interferon gamma release assays (IGRAs) are in vitro immunologic diagnostic tests used to identify Mycobacterium tuberculosis infection. They cannot differentiate between latent and active infections. The cutoff suggested by the manufacturer is 0.35 IU/mL for latent tuberculosis. As IGRA tests were recently approved for the differential diagnosis of active tuberculosis, we assessed the diagnostic accuracy of the latest generation IGRA for detection of active tuberculosis in a low-incidence area in Germany. Our consecutive case series includes 61 HIV negative, Mycobacterium tuberculosis culture positive patients, as well as 234 control patients. The retrospective analysis was performed over a period of two years. In 11/61 patients with active tuberculosis (18.0%) the test result was <0.35 IU/mL, resulting in a sensitivity of 0.82. We recommend establishing a new cut-off value for the differential diagnosis of active tuberculosis assessed by prospective clinical studies and in various regions with high and low prevalence of tuberculosis.

Reassessment of the 2010-2011 Haiti cholera outbreak and rainfall-driven multiseason projections.

Mathematical models can provide key insights into the course of an ongoing epidemic, potentially aiding real-time emergency management in allocating health care resources and by anticipating the impact of alternative interventions. We study the ex post reliability of predictions of the 2010-2011 Haiti cholera outbreak from four independent modeling studies that appeared almost simultaneously during the unfolding epidemic. We consider the impact of different approaches to the modeling of spatial spread of Vibrio cholerae and mechanisms of cholera transmission, accounting for the dynamics of susceptible and infected individuals within different local human communities. To explain resurgences of the epidemic, we go on to include waning immunity and a mechanism explicitly accounting for rainfall as a driver of enhanced disease transmission. The formal comparative analysis is carried out via the Akaike information criterion (AIC) to measure the added information provided by each process modeled, discounting for the added parameters. A generalized model for Haitian epidemic cholera and the related uncertainty is thus proposed and applied to the year-long dataset of reported cases now available. The model allows us to draw predictions on longer-term epidemic cholera in Haiti from multiseason Monte Carlo runs, carried out up to January 2014 by using suitable rainfall fields forecasts. Lessons learned and open issues are discussed and placed in perspective. We conclude that, despite differences in methods that can be tested through model-guided field validation, mathematical modeling of large-scale outbreaks emerges as an essential component of future cholera epidemic control.

Cholera ante portas - The re-emergence of cholera in Kinshasa after a ten-year hiatus.

BACKGROUND: Cholera is an endemic disease in certain well-defined areas in the east of the Democratic Republic of Congo (DRC). The west of the country, including the mega-city Kinshasa, has been free of cases since mid 2001 when the last outbreak ended. METHODS AND FINDINGS: We used routinely collected passive surveillance data to construct epidemic curves of the cholera cases and map the spatio-temporal progress of the disease during the first 47 weeks of 2011. We compared the spatial distribution of disease spread to that which occurred in the last cholera epidemic in Kinshasa between 1996 and 2001. To better understand previous determinants of cholera spread in this region, we conducted a correlation analysis to assess the impact of rainfall on weekly health zone cholera case counts between December 1998 and March 2001 and a Generalized Linear Model (GLM) regression analysis to identify factors that have been associated with the most vulnerable health zones within Kinshasa between October 1998 and June 1999. In February 2011, cholera reemerged in a region surrounding Kisangani and gradually spread westwards following the course of the Congo River to Kinshasa, home to 10 million people. Ten sampled isolates were confirmed to be Vibrio cholerae O1, biotype El Tor, serotype Inaba, resistant to trimethoprim-sulfa, furazolidone, nalidixic acid, sulfisoxaole, and streptomycin, and intermediate resistant to Chloramphenicol. An analysis of a previous outbreak in Kinshasa shows that rainfall was correlated with case counts and that health zone population densities as well as fishing and trade activities were predictors of case counts. CONCLUSION: Cholera is particularly difficult to tackle in the DRC. Given the duration of the rainy season and increased riverine traffic from the eastern provinces in late 2011, we expect further increases in cholera in the coming months and especially within the mega-city Kinshasa. We urge all partners involved in the response to remain alert.Didier Bompangue and Silvan Vesenbeckh contributed equally to this work. *corresponding author: Silvan Vesenbeckh, Harvard School of Public Health (vesenbeckh@gmail.com)Didier Bompangue is Associate Professor in the Department of Microbiology (University of Kinshasa) andEpidemiologist in the DRC Ministry of Health. He was involved in the investigations of the described outbreak since February 2011.

Potential of gene therapy for restoration of endocrine thymic function in thymus-deficient animal models.

The aim of the present article is to discuss the potential of gene therapy for thymic hormones as a novel therapeutic strategy to treat dyshomeostatic conditions associated with congenital athymia or hypofunction of the endocrine thymus. Recent studies using an adenoviral vector harboring a synthetic gene for the thymic peptide thymulin are reviewed. This adenoviral vector was injected intramuscularly in thymectomized and nude mice as well as in thymectomized rats. Transduced myocytes acted as an ectopic source of thymulin thus restoring circulating thymulin levels to normal values. This restorative effect was long lasting (several months) even though an adenoviral vector was used. In the rat brain, adenovirally-mediated delivery of the synthetic gene for thymulin achieved longer expression than in the case of adenovirally-delivered reporter genes, which is consistent with the reported antiinflammatory activity of thymulin in the brain. Furthermore, neonatal thymulin gene therapy in nude female mice was able to prevent the pituitary and ovarian alterations that typically occur in this mutant after puberty. Neonatal thymulin gene therapy in nude mice was able to prevent some of the alterations in lipid metabolism that develop during adult life in congenitally athymic mice. We conclude that the availability of the above biotechnological tools should boost basic studies on the molecular biology of thymulin and should also allow an assessment of the potential of gene therapy to restore circulating thymulin levels in thymodeficient animal models and eventually, in humans.

Thymulin gene therapy prevents the reduction in circulating gonadotropins induced by thymulin deficiency in mice.

Integrity of the thymus during perinatal life is necessary for a proper maturation of the pituitary-gonadal axis in mice and other mammalian species. Thus congenitally athymic (nude) female mice show significantly reduced levels of circulating gonadotropins, a fact that seems to be causally related to a number of reproductive derangements described in these mutants. Interestingly, a number of in vitro studies suggest that the thymic peptide thymulin may be involved in thymus-pituitary communication. To determine the consequences of low serum thymulin in otherwise normal animals, we induced short (8 days)- and long (33 days)-term thymulin deficiency in C57BL/6 mice by neonatally injecting (intraperitoneally) an anti-thymulin serum and assessed their circulating gonadotropin levels at puberty and thereafter. Control mice received an irrelevant antiserum. Gonadotropins were measured by radioimmunoassay and thymulin by bioassay. Both long- and short-term serum thymulin immunoneutralization resulted in a significant reduction in the serum levels of gonadotropins at 33 and 45 days of age. Subsequently, we injected (intramuscularly) an adenoviral vector harboring a synthetic DNA sequence (5'-ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3') encoding a biologically active analog of thymulin, methionine-FTS, in newborn nude mice (which are thymulin deficient) and measured circulating gonadotropin levels when the animals reached 52 days of age. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels that typically emerges in these mutants after puberty. Our results indicate that thymulin plays a relevant physiological role in the thymus-pituitary-gonadal axis.

Morphometric assessment of the impact of serum thymulin immunoneutralization on pituitary cell populations in peripubertal mice.

Thymulin is a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Thymulin also possesses hypophysiotropic activity which suggests that this metallopeptide may play an important role in thymus-pituitary communication, particularly during early life. The aim of the present study was to evaluate the impact of serum thymulin suppression from birth to peripuberty on the morphology of different pituitary cell populations in prepubertal C57Bl/6 mice. Animals were submitted to immunoneutralization of circulating thymulin from postnatal day 1 to the end of the study (age 32 days). From their 1st day of life, the animals were submitted to a protocol of intraperitoneal injections of rabbit anti-thymulin serum (alpha-FTS) and normal rabbit serum (NRS) in the controls. On their 33rd day of life, the animals were killed and their pituitaries were immediately dissected, fixed and immunostained using the EnVision system with primary antibodies against growth hormone, thyrotropin, corticotropin, gonadotropins and prolactin. Morphometry was performed by means of an image analysis system. The following parameters were calculated: volume density = Sigma cell area/reference area (RA); cell density (CD) = number of cells/RA, and cell size (expressed in microm2). Serum thymulin was measured by a rosette bioassay while pituitary hormones were assayed by radioimmunoassay. Serum prolactin, luteinizing hormone, follicle-stimulating hormone, growth hormone and thyroid-stimulating hormone were significantly lower in the alpha-FTS animals of either sex compared with the corresponding NRS counterparts. The somatotrope, lactotrope and corticotrope populations showed a significant decrease in CD, while cell hypertrophy was observed in some of the pituitary cell populations of the alpha-FTS group compared to the NRS group. In the alpha-FTS group, there were sex differences in the morphometric changes observed. Our results suggest that serum thymulin plays a significant role during early life in the postnatal maturation of endocrine cells of the mouse anterior pituitary gland.