Targeted next-generation sequencing of pathogens reveals the profile of secondary infections in COVID-19 patients (preprint)

PURPOSE: To use targeted next-generation sequencing (tNGS) of pathogens for analysing the etiological distribution of secondary infections in patients with severe and critical novel coronavirus pneumonia (COVID-19), to obtain microbial epidemiological data on secondary infections in patients with COVID-19, and to provide a reference for early empirical antibiotic treatment of such patients. METHODS: Patients with infections secondary to severe and critical COVID-19 and hospitalised at the First Affiliated Hospital of Shandong First Medical University between 1 December 2022 and 30 June 2023 were included in the study. The characteristics and etiological distribution of secondary infections in these patients were analysed using tNGS. RESULTS: A total of 95 patients with COVID-19 secondary infections were included in the study, of whom 87.37% had one or more underlying diseases. Forty-eight pathogens were detected, the most common being HSV-4, Candida albicans, Klebsiella pneumoniae, Enterococcus faecium, HSV-1, Staphylococcus aureus, Aspergillus fumigatus, Acinetobacter baumannii, HSV-5, and Stenotrophomonas maltophilia, with Pneumocystis jirovecii being detected in 14.29% of cases. The majority (76.84%) of COVID-19 secondary infections were mixed infections, with mixed viral-bacterial-fungal infections being the most common (28.42%). CONCLUSION: Most secondary infections in severe and critical COVID-19 patients are mixed, with high rates of viral and fungal infections. In clinical settings, monitoring for reactivation or secondary infections by Herpesviridae viruses is crucial; additionally, these patients have a significantly higher rate of P. jirovecii infection. tNGS testing on bronchoalveolar lavage fluid can help determine the aetiology of secondary infections early in COVID-19 patients and assist in choosing appropriate antibiotics.

Analysis of coinfections in patients with hematologic malignancies and COVID-19 by next-generation sequencing of bronchoalveolar lavage fluid (preprint)

Background Coinfections in patients with coronavirus disease 2019 (COVID-19) affect patient prognosis. Patients with hematologic malignancies (HMs) are usually immunosuppressed and may be at high risk of coinfection, but few related data have been reported. Here, we conducted a retrospective study to explore coinfections in patients with HMs and COVID-19 by next-generation sequencing (NGS) of bronchoalveolar lavage fluid (BALF).Methods The data of hospitalized patients with pneumonia who underwent NGS analysis of BALF were reviewed. COVID-19 patients with HMs were enrolled in the HM group, and those without HMs were enrolled in the non-HM group. The coinfections of the two groups identified by NGS were analyzed.Results Fifteen patients were enrolled in the HM group, and 14 patients were enrolled in the non-HM group. The coinfection rates in the HM group and non-HM group were 80.0% and 85.7%, respectively. The percentage of coinfected bacteria in the HM group was significantly lower than that in the non-HM group (20.0% vs 71.4%, p = 0.005). The coinfection rates of fungi and viruses were 60.0% and 35.7%, respectively, in the HM group and 35.7% and 78.6%, respectively, in the non-HM group, with no significant differences. The most common coexisting pathogen in patients with HMs was Pneumocystis jirovecii (33.3%), and the most common coexisting pathogen in patients without HMs was human gammaherpesvirus 4 (50%). Coinfection with herpesviruses occurred frequently in both groups.Conclusions Our study showed that hospitalized patients with COVID-19 had a high incidence of coinfection. Pneumocystis jiroveci and herpesvirus are commonly coinfected pathogens in patients with HMs. Bacterial coinfection is rare in patients with HMs but is more common in patients without HMs.

Pneumocystis jirovecii pneumonia among pediatric inpatients before and after COVID-19 pandemic: molecular diagnosis, predisposing factors, and outcomes in Northeastern Iran (preprint)

Background: Pneumocystis  pneumonia (PCP), caused by P. jirovecii, is one of the opportunistic fungal infections that can cause life-threatening pneumonia in children with underlying diseases. Due to the similarity of the symptoms of PCP with other lung infections, such as tuberculosis, differential and accurate diagnosis is necessary. The current study investigated the molecular diagnosis of P. jirovecii , predisposing factors, and the outcomes, among pediatric inpatients in Northeastern Iran. Method: In this study, 180 bronchoalveolar lavage (BAL) specimens were obtained from hospitalized children with respiratory disorders. The specimens were examined using Giemsa stain, and the genomic DNA was extracted according to the protocol of the AmpliSens® kit. Real-time polymerase chain reaction technique was used to detect P. jirovecii by the AmpliSens Pneumocystis jirovecii ( carinii )-FRT PCR kit. Results: : Among the patients studied, 34 (18.9%) were positive, and 8 (4.4%) were suspicious for the presence of P. jirovecii . Among the 34 positive cases, 12 (35%) were related to before, and 22 (65%) were affected during the COVID-19 pandemic. Only 2 cases (5.88%) of the positive cases detected by Real-time PCR method were observed using Giemsa staining. Also, no correlation was observed between positive cases of infection and the sex, the outcomes, and underlying diseases. Conclusion: The results showed that PCP has a relatively high prevalence among pediatric inpatients with respiratory disorders. Neutropenia is a significant predisposing factor in these patients. However, there is no correlation between PCP cases and the outcomes and underlying diseases. Most of patients with PCP were affected during the COVID-19 pandemic.

The Pathogenetic Dilemma of Post-COVID-19 Mucormycosis in India (preprint)

There has been a surge of mucormycosis cases in India in the wake of the second wave of COVID-19 with more than 14000 cases reported. Mucormycosis in patients of COVID-19 in India is at variance to other countries where Aspergillus, Pneumocystis, and Candida have been reported to be the major secondary fungal pathogens. We discuss the probable causes of the mucormycosis epidemic in India. Whereas dysglycaemia and inappropriate steroid use have been widely suggested as tentative reasons, we explore other biological, iatrogenic, and environmental factors. The likelihood of a two-hit pathogenesis remains strong. We propose that COVID-19 itself provides the predisposition to invasive mucormycosis (first hit), through upregulation of GRP78 and downregulation of spleen tyrosine kinase involved in anti-fungal defense, as also through inhibition of CD8+ T-cell mediated immunity. The other iatrogenic and environmental factors may provide the second hit which may have resulted in the surge.

Bronchoscopy in COVID19 ARDS patients on mechanical ventilation: a prospective study (preprint)

Background: Bronchoscopy has been done sparingly in COVID19 patients due to the risk of aerosol generation, with few reports describing its clinical utility. We describe a large case series of bronchoscopy in mechanically ventilated (MV) COVID-19 patients outlining the procedural, clinical, utilitarian and safety aspects. Methods: Bedside bronchoscopy was performed in suspected or confirmed COVID-19 cases on MV; only positive cases were included in the study. Demographic, clinical, bronchoscopic and laboratory findings were noted and analysed. Results: 98 procedures were performed on 61 patients, mean age of 62.1 years, 51 (83.6%) males. 42 patients (69%) had at least 1 co-morbidity. Major indications for bronchoscopy were new radiographic infiltrates with clinical deterioration, increased endotracheal tube (ETT) secretions and haemorrhagic secretions/hemoptysis. Common findings were copious secretions in 87 (88.8%), purulent in 61%, mucoid in 18%, haemorrhagic in 7% and frothy in 14% cases. Morphologically, hyperaemic airways were seen in 85 (86.7%) cases, ranging from mild (61%) to moderate-severe (39%). On the management front, antibiotics were changed in 31 (31.6%) cases based on bronchoscopic findings. Other significant changes included reduction or stopping of steroids and anticoagulation, fluid, and diuretic adjustment and ETT repositioning. The incidence of bacterial superinfection was also high (54% culture positivity for various bacteria), a significant number (94%) with multi-drug resistant organisms. Fungi were seen in 7 cases (7.1%). Pneumocystis jiroveci was not seen and cytology did not show any viral inclusions. Therapeutic mucus plug removal was done in 30 cases (30.6%), and hemoptysis control in 4% cases. The procedures were safe with no complications, and none of the HCW developed any COVID19 infection. Conclusion: Bronchoscopy in critically ill MV COVID-19 patients contributes on both diagnostic and therapeutic fronts and can significantly influence management decisions. With adequate precautions and standard protocols, it is safe for both HCW and patients.

Do COVID-19 patients admitted to the ICU require anti-Pneumocystis jirovecii prophylaxis? (preprint)

We are currently facing a frightening increase in COVID-19 patients admitted to the ICU. Aiming at screening for secondary pneumonia, we collected the data of our first twelve ICU patients who underwent bronchoalveolar lavage (BAL). Surprisingly, four were detected with Pneumocystis jirovecii (Pj) DNA and RNA, resulting in Pj prevalence of 17%. Pj is a ubiquitous ascomycetes fungus that thrives at the surface of type-I pneumocytes, specifically in human alveoli, leading to pneumocystosis in immunocompromised patients. Interestingly, none of our patients was immunocompromised per se before admission, while all presented the recognized risk factors for life-threatening COVID-19 infection. Observing such high prevalence in COVID-infected patients was unexpected. Almost all patients developed ARDS and received high-dose steroids to prevent worsening, as suggested by reports from China. In Pj-positive patients requiring steroids, prophylaxis was given to avoid the risk of pneumocystosis and increased lung inflammation that may compromise the outcome. We are strongly convinced that testing deep lung specimens for Pj in severe COVID-19 patients should be recommended and Pj-positive patients treated with steroids, and given anti-Pj prophylaxis. This message is important, given the high mortality rate of COVID-19 patients in the ICU.