Multi-organ failure is one of the common causes of fatal outcome in COVID-19 patients. However, the pathogenetic association of the SARS-CoV-2 viral load (VL) level with fatal dysfunctions of the lungs, liver, kidneys, heart, spleen and brain, as well as with the risk of death in COVID-19 patients remains poorly understood. SARS-CoV-2 VL in the lungs, heart, liver, kidneys, brain, spleen and lymph nodes have been measured by RT qPCR using the following formula: NSARS-CoV−2/NABL1 × 100. Dissemination of SARS-CoV-2 in 30.5% of cases was mono-organ, and in 63.9% of cases, it was multi-organ. The average SARS-CoV-2 VL in the exudative phase of diffuse alveolar damage (DAD) was 60 times higher than in the proliferative phase. The SARS-CoV-2 VL in the lungs ranged from 0 to 250,281 copies. The “pulmonary factors” of SARS-CoV-2 multi-organ dissemination are the high level of SARS-CoV-2 VL (≥4909) and the exudative phase of DAD. The frequency of SARS-CoV-2 dissemination to lymph nodes was 86.9%, heart–56.5%, spleen–52.2%, liver–47.8%, kidney–26%, and brain–13%. We found no link between the SARS-CoV-2 VL level in the liver, kidneys, and heart and the serum level of CPK, LDH, ALP, ALT, AST and Cr of COVID-19 patients. Isolated detection of SARS-CoV-2 RNA in the myocardium of COVID-19 patients who died from heart failure is possible. The pathogenesis of COVID-19-associated multi-organ failure requires further research in a larger cohort of patients.

Lymphopenia is a frequent hematological manifestation, associated with a severe course of COVID-19, with an insufficiently understood pathogenesis. We present molecular genetic immunohistochemical, and electron microscopic data on SARS-CoV-2 dissemination and viral load (VL) in lungs, mediastinum lymph nodes, and the spleen of 36 patients who died from COVID-19. Lymphopenia <1 × 109/L was observed in 23 of 36 (63.8%) patients. In 12 of 36 cases (33%) SARS-CoV-2 was found in lung tissues only with a median VL of 239 copies (range 18–1952) SARS-CoV-2 cDNA per 100 copies of ABL1. Histomorphological changes corresponding to bronchopneumonia and the proliferative phase of DAD were observed in these cases. SARS-CoV-2 dissemination into the lungs, lymph nodes, and spleen was detected in 23 of 36 patients (58.4%) and was associated with the exudative phase of DAD in most of these cases. The median VL in the lungs was 12,116 copies (range 810–250281), lymph nodes—832 copies (range 96–11586), and spleen—71.5 copies (range 0–2899). SARS-CoV-2 in all cases belonged to the 19A strain. A immunohistochemical study revealed SARS-CoV-2 proteins in pneumocytes, alveolar macrophages, and bronchiolar epithelial cells in lung tissue, sinus histiocytes of lymph nodes, as well as cells of the Billroth pulp cords and spleen capsule. SARS-CoV-2 particles were detected by transmission electron microscopy in the cytoplasm of the endothelial cell, macrophages, and lymphocytes. The infection of lymphocytes with SARS-CoV-2 that we discovered for the first time may indicate a possible link between lymphopenia and SARS-CoV-2-mediated cytotoxic effect.

Background:Jak2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9 and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Phi‐negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases.Aims:To analyze cases with combined mutations in Russian cohort of MPN patientsMethods:Study includes 3267 samples, directed for genetic analysis to the National Research Center for Hematology between 2014 and 2019 from the patients with clinical evidence of MPN. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantitated by real‐time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity > = 3%). MPL W515L/K mutations were assessed by in‐house allele specific PCR. All cases were tested for phi‐negativity using BCR‐ABl p210 PCR kit from Interlabservice (Russia).Results:In 406 of 3267 cases (12.5%) CALR mutations were detected. In 24 of these 406 cases (6%) Jak2 V617F mutation was found in addition to CALR mutation. Matched measures were obtained at least twice at different time points during the course of disease for these cases. In 19 from 24 (80%) cases with combined CALR and Jak2 mutations V617F allele burden was lower than 3%. Also combination of Jak2 V617F with MPL W515L/K was observed in 3 out of 3267 cases only. Two of them had V617F allele burden lower than 3% (Tabl.1).Summary/Conclusion:Additional mutations may coexist over low background of JAK2 V617F allele. Therefore cases of MPNs with low initial JAK2 V617F allele should be further tested for CALR (and possibly MPL) mutations to ensure correct disease and treatment monitoring. Further dynamic observation studies are required to assess the value of additional mutations for MPN disease progression and treatment outcome.image

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Цель исследования: провести у умерших пациентов с COVID-19 количественный анализ уровня вирусной нагрузки (ВН) SARS-CoV-2 в тканях легких и оценку его связи с характером гистологических изменений в легких и продолжительностью пребывания в ОРИТ до наступления летального исхода.Материалы и методы. В качестве материала использованы срезы фиксированных в формалине и заключенных в парафин тканей легких 36 умерших пациентов с COVID-19. Количественная оценка уровня ВН SARS-CoV-2 проведена с использованием оригинального способа кПЦР РВ. ВН рассчитана по формуле: число копий SARS-CoV-2 /число копий ABL1 × 100, выражена отношением истинного числа копий кДНК SARS-CoV-2 на 100 копий кДНК гена ABL1.Результаты. В случаях с отсутствием комплекса гистологических изменений, характерных для диффузного альвеолярного повреждения легких (ДАП), частота выявления РНК SARS-CoV-2 и средний уровень ВН SARS-CoV-2 составили 62,5% (5 из 8 наблюдений) и 104,75 (диапазон 0-313) копий кДНК SARS-CoV-2 на 100 копий кДНК человеческой гена ABL1. Средний уровень ВН SARS-CoV-2 в легких с преобладанием гистологических изменений, характерных для пролиферативной и экссудативной фазы ДАП, отличался в 60 раз и составил 909 (18-2 657) и 54 924 (834-250 281) копий кДНК SARS-CoV-2 на 100 копий кДНК человеческой ABL1 соответственно. Среднее время, проведенное в отделении реанимации и интенсивной терапии у группы пациентов с экссудативной и пролиферативной фазами ДАП, составляло 10,64 (1-22) и 8,14 (1-21) койко-дня соответственно. Частота выявления РНК SARS-CoV-2 у пациентов с ДАП легких составила 100%.