Prevalence of co-existent COVID-19-associated pulmonary aspergillosis (CAPA) and its impact on early mortality in patients with COVID-19-associated pulmonary mucormycosis (CAPM).

BACKGROUND: Data on mixed mould infection with COVID-19-associated pulmonary aspergillosis (CAPA) and COVID-19-associated pulmonary mucormycosis (CAPM) are sparse. OBJECTIVES: To ascertain the prevalence of co-existent CAPA in CAPM (mixed mould infection) and whether mixed mould infection is associated with early mortality (≤7 days of diagnosis). METHODS: We retrospectively analysed the data collected from 25 centres across India on COVID-19-associated mucormycosis. We included only CAPM and excluded subjects with disseminated or rhino-orbital mucormycosis. We defined co-existent CAPA if a respiratory specimen showed septate hyphae on smear, histopathology or culture grew Aspergillus spp. We also compare the demography, predisposing factors, severity of COVID-19, and management of CAPM patients with and without CAPA. Using a case-control design, we assess whether mixed mould infection (primary exposure) were associated with early mortality in CAPM. RESULTS: We included 105 patients with CAPM. The prevalence of mixed mould infection was 20% (21/105). Patients with mixed mould infection experienced early mortality (9/21 [42.9%] vs. 15/84 [17.9%]; p = 0.02) and poorer survival at 6 weeks (7/21 [33.3] vs. 46/77 [59.7%]; p = 0.03) than CAPM alone. On imaging, consolidation was more commonly encountered with mixed mould infections than CAPM. Co-existent CAPA (odds ratio [95% confidence interval], 19.1 [2.62-139.1]) was independently associated with early mortality in CAPM after adjusting for hypoxemia during COVID-19 and other factors. CONCLUSION: Coinfection of CAPA and CAPM was not uncommon in our CAPM patients and portends a worse prognosis. Prospective studies from different countries are required to know the impact of mixed mould infection.

Toxoplasma gondii aspartic protease 5: structural basis of substrate binding and inhibition mechanism.

Toxoplasma gondii, a worldwide prevalent parasite is responsible for causing toxoplasmosis in almost all warm-blooded animals, including humans. Golgi-resident T. gondii aspartic protease 5 (TgASP5) plays an essential role in the maturation and export of the effector proteins those modulate the host immune system to establish a successful infection. Hence, inhibiting this enzyme can be a possible therapeutic strategy against toxoplasmosis. This is the first report of the detailed structural investigations of the TgASP5 mature enzyme using molecular modeling and an all-atom simulation approach which provide in-depth knowledge of the active site architecture of TgASP5. The analysis of the binding mode of the TEXEL (Toxoplasma EXport Element) substrate to TgASP5 highlighted the importance of the active site residues. Ser505, Ala776 and Tyr689 in the S2 binding pocket are responsible for the specificity towards Arg at the P2 position of TEXEL substrate. The molecular basis of inhibition by the only known inhibitor RRLStatine has been identified, and our results show that it blocks the active site by forming a hydrogen bond with a catalytic aspartate. Besides that, known aspartic protease inhibitors were screened against TgASP5 using docking, MD simulations and MM-PBSA binding energy calculations. The top-ranked inhibitors (SC6, ZY1, QBH) showed higher binding energy than RRLStatine. Understanding the structural basis of substrate recognition and the binding mode of these inhibitors will help to develop potent mechanistic inhibitors against TgASP5. This study will also provide insights into the structural features of pepsin-like aspartic proteases from other apicomplexan parasites for developing antiparasitic agents.Communicated by Ramaswamy H. Sarma.

Inhibition of Plasmodium falciparum plasmepsins by drugs targeting HIV-1 protease: A way forward for antimalarial drug discovery.

Plasmodium species are causative agents of malaria, a disease that is a serious global health concern. FDA-approved HIV-1 protease inhibitors (HIV-1 PIs) have been reported to be effective in reducing the infection by Plasmodium parasites in the population co-infected with both HIV-1 and malaria. However, the mechanism of HIV-1 PIs in mitigating Plasmodium pathogenesis during malaria/HIV-1 co-infection is not fully understood. In this study we demonstrate that HIV-1 drugs ritonavir (RTV) and lopinavir (LPV) exhibit the highest inhibition activity against plasmepsin II (PMII) and plasmepsin X (PMX) of P. falciparum. Crystal structures of the complexes of PMII with both drugs have been determined. The inhibitors interact with PMII via multiple hydrogen bonding and hydrophobic interactions. The P4 moiety of RTV forms additional interactions compared to LPV and exhibits conformational flexibility in a large S4 pocket of PMII. Our study is also the first to report inhibition of P. falciparum PMX by RTV and the mode of binding of the drug to the PMX active site. Analysis of the crystal structures implies that PMs can accommodate bulkier groups of these inhibitors in their S4 binding pockets. Structurally similar active sites of different vacuolar and non-vacuolar PMs suggest the potential of HIV-1 PIs in targeting these enzymes with differential affinities. Our structural investigations and biochemical data emphasize PMs as crucial targets for repurposing HIV-1 PIs as antimalarial drugs.

Risk factors, mortality, and predictors of survival in COVID-19-associated pulmonary mucormycosis: a multicentre retrospective study from India.

OBJECTIVES: To compare COVID-19-associated pulmonary mucormycosis (CAPM) with COVID-19-associated rhino-orbital mucormycosis (CAROM), ascertain factors associated with CAPM among patients with COVID-19, and identify factors associated with 12-week mortality in CAPM. METHODS: We performed a retrospective multicentre cohort study. All study participants had COVID-19. We enrolled CAPM, CAROM, and COVID-19 subjects without mucormycosis (controls; age-matched). We collected information on demography, predisposing factors, and details of COVID-19 illness. Univariable analysis was used to compare CAPM and CAROM. We used multivariable logistic regression to evaluate factors associated with CAPM (with hypoxemia during COVID-19 as the primary exposure) and at 12-week mortality. RESULTS: We included 1724 cases (CAPM [n = 122], CAROM [n = 1602]) and 3911 controls. Male sex, renal transplantation, multimorbidity, neutrophil-lymphocyte ratio, intensive care admission, and cumulative glucocorticoid dose for COVID-19 were significantly higher in CAPM than in CAROM. On multivariable analysis, COVID-19-related hypoxemia (aOR, 2.384; 95% CI, 1.209-4.700), male sex, rural residence, diabetes mellitus, serum C-reactive protein, glucocorticoid, and zinc use during COVID-19 were independently associated with CAPM. CAPM reported a higher 12-week mortality than CAROM (56 of the 107 [52.3%] vs. 413 of the 1356 [30.5%]; p = 0.0001). Hypoxemia during COVID-19 (aOR [95% CI], 3.70 [1.34-10.25]) and Aspergillus co-infection (aOR [95% CI], 5.40 [1.23-23.64]) were independently associated with mortality in CAPM, whereas surgery was associated with better survival. DISCUSSION: CAPM is a distinct entity with a higher mortality than CAROM. Hypoxemia during COVID-19 illness is associated with CAPM. COVID-19 hypoxemia and Aspergillus co-infection were associated with higher mortality in CAPM.

Structures of plasmepsin X from Plasmodium falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme.

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.