Rapid Diagnosis of Pneumocystis jirovecii Pneumonia and Respiratory Tract Colonization by Next-Generation Sequencing.

OBJECTIVES: To describe the epidemiology of Pneumocystis jirovecii pneumonia and colonization diagnosed by next-generation sequencing (NGS) and explore the usefulness of the number of P. jirovecii sequence reads for the diagnosis of P. jirovecii pneumonia. METHODS: We examined the NGS results for P. jirovecii in respiratory samples collected from patients and analysed their clinical, radiological and microbiological characteristics. RESULTS: Among 285 respiratory samples collected over a 12-month period (January to December 2022), P. jirovecii sequences were detected in 56 samples from 53 patients. Fifty (94.3%) of the 53 patients were HIV-negative. Following our case definitions, 37 (69.8%) and 16 (30.2%) of the 53 patients had P. jirovecii infection and colonization respectively. P. jirovecii infection was associated with presence of underlying disease with immunosuppression (94.6% vs 18.8%, P < 0.05), positive serum 1,3-ß-D-glucan (41.2% vs 0%, P < 0.01) and higher number of P. jirovecii sequence reads (P < 0.005). In contrast, P. jirovecii colonization was associated with the male sex (93.8% vs 54.1%, P < 0.01), another definitive infectious disease diagnosis of the respiratory tract (43.8% vs 2.7%, P < 0.001) and higher survival (100% vs 67.6%, P < 0.01). Although P. jirovecii pneumonia was associated with higher number of P. jirovecii reads in respiratory samples, only a sensitivity of 82.14% and a specificity of 68.75% could be achieved. CONCLUSION: Detection of P. jirovecii sequences in respiratory samples has to be interpreted discreetly. A combination of clinical, radiological and laboratory findings is still the most crucial in determining whether a particular case is genuine P. jirovecii pneumonia.

Bats-The Magnificent Virus Player: SARS, MERS, COVID-19 and Beyond.

Irrespective of whether COVID-19 originated from a natural or a genetically engineered virus, the ultimate source of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is bats [...].

Differential innate immune responses of human macrophages and bronchial epithelial cells against Talaromyces marneffei.

Talaromyces marneffei is a thermally dimorphic fungal pathogen endemic in Southeast Asia. As inhalation of airborne conidia is believed as the major infection route, airway epithelial cells followed by pulmonary macrophages are the first cell types which the fungus encounters inside the host. In this study, we established an in vitro infection model based on human peripheral blood-derived macrophages (hPBDMs) cultured with the supplementation of autologous plasma. Using this model, we determined the transcriptomic changes of hPBDMs in response to T. marneffei infection by quantitative real-time reverse-transcription polymerase chain reaction as well as high-throughput RNA sequencing. Results showed that T. marneffei infection could activate hPBDMs to the M1-like phenotype and trigger a potent induction of chemokine and pro-inflammatory cytokine production as well as the expression of other immunoregulatory genes. In contrast to hPBDMs, there was no detectable innate cytokine response against T. marneffei in human bronchial epithelial cells (hBECs). Using a green fluorescent protein-tagged T. marneffei strain and confocal microscopy, internalization of the fungus by hBECs was confirmed. Live cell imaging further demonstrated that the infected cells exhibited normal cellular physiology, especially that the process of cell division could be observed. Moreover, T. marneffei also survived better inside hBECs than hPBDMs. Our results illustrated a potential role of hBECs to serve as reservoir cells for T. marneffei to evade immunosurveillance by phagocytes, from which the fungus reactivates when the host immunity is weakened and causes infection. Such immunoevasion and reactivation may also help explain the long incubation period observed for talaromycosis, in particular the travel-related cases. IMPORTANCE Talaromyces marneffei is an important fungal pathogen especially in Southeast Asia. To understand the innate immune response to talaromycosis, a suitable infection model is needed. Here, we established an in vitro T. marneffei infection model using human peripheral blood-derived macrophages (hPBDMs). We then examined the transcriptomic changes of hPBDMs in response to T. marneffei infection with this model. We found that contact with T. marneffei could activate hPBDMs to the M1-like phenotype and induced mRNA expressions of five cytokines and eight immunoregulatory genes. Contrary to hPBDMs, such immunoresponse was not elicited in human bronchial epithelial cells (hBECs), despite normal physiology observed in infected cells. We also found that infected hBECs did not eliminate T. marneffei as efficiently as hPBDMs. Our observation suggested that hBECs may potentially serve as reservoir cells for T. marneffei to evade immunosurveillance. When the host immunity deteriorates later, then the fungus reactivates and causes infection.

Usefulness of Next-Generation Sequencing in Excluding Bovine Leukemia Virus as a Cause of Adult Camel Leukosis in Dromedaries.

Adult camel leukosis is an emerging hematological and neoplastic disease in dromedaries. It has been hypothesized that bovine leukemia virus (BLV) or its genetic variants may be associated with adult camel leukosis. In this study, we used next-generation sequencing (NGS) to detect all possible viruses in five lung samples from five dromedaries with histopathological evidence of adult camel leukosis and four tissue samples from two control dromedaries. A total throughput of 114.7 Gb was achieved, with an average of 12.7 Gb/sample. For each sample, all the pair-end 151-bp reads were filtered to remove rRNA sequences, bacterial genomes and redundant sequences, resulting in 1-7 Gb clean reads, of which <3% matched to viruses. The largest portion of these viral sequences was composed of bacterial phages. About 100-300 reads in each sample matched "multiple sclerosis-associated retrovirus", but manual analysis showed that they were only repetitive sequences commonly present in mammalian genomes. All viral reads were also extracted for analysis, confirming that no BLV or its genetic variants or any other virus was detected in the nine tissue samples. NGS is not only useful for detecting microorganisms associated with infectious diseases, but also important for excluding an infective cause in scenarios where such a possibility is suspected.

Rapid diagnosis of fatal Nocardia kroppenstedtii bacteremic pneumonia and empyema thoracis by next-generation sequencing: a case report.

Nocardia species do not replicate as rapidly as other pyogenic bacteria and nocardial infections can be highly fatal, particularly in immunocompromised patients. Here, we present the first report of fatal Nocardia kroppenstedtii bacteremic pneumonia and empyema thoracis diagnosed by next-generation sequencing (NGS) using the Oxford Nanopore Technologies' MinION device. The bacterium was not identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Due to its low equipment cost, short turn-around-time, and portable size, the Oxford Nanopore Technologies' MinION device is a useful platform for NGS in routine clinical microbiology laboratories.

ICTV Virus Taxonomy Profile: Coronaviridae 2023.

The family Coronaviridae includes viruses with positive-sense RNA genomes of 22-36 kb that are expressed through a nested set of 3' co-terminal subgenomic mRNAs. Members of the subfamily Orthocoronavirinae are characterized by 80-160 nm diameter, enveloped virions with spike projections. The orthocoronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome-related coronavirus are extremely pathogenic for humans and in the last two decades have been responsible for the SARS and MERS epidemics. Another orthocoronavirus, severe acute respiratory syndrome coronavirus 2, was responsible for the recent global COVID-19 pandemic. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Coronaviridae which is available at www.ictv.global/report/coronaviridae.

Seasonal shift in gut microbiome diversity in wild Sichuan takin (Budorcas tibetanus) and environmental adaptation.

In this study, we investigated the change in microbiome composition of wild Sichuan takin (Budorcas tibetanus) during winter and spring and analyzed the physiological implications for such changes. Diversity analyses of the microbiome (average 15,091 high-quality reads per sample) in 24 fecal samples (15 from winter, 9 from spring) revealed that spring samples had higher species diversity and were compositionally different from winter samples (P < 0.05). Taxonomic composition analysis showed that the relative abundance increased in spring for Patescibacteria (2.7% vs. 0.9% in winter, P < 0.001) and Tenericutes (1.9% vs. 1% in winter, P < 0.05). Substantial increases in relative abundance of Ruminococcaceae and Micrococcaceae were identified in spring and winter, respectively. Mann-Whitney U and ANCOM identified seven differentially abundant genera: Enterococcus, Acetitomaculum, Blautia, Coprococcus 1, Lachnospiraceae UCG 008, Ruminococcus 2 and Ralstonia. All seven genera were significantly more abundant in spring (average 0.016-1.2%) than winter (average 0-0.16%), with the largest difference found in Ruminococcus (1.21% in spring vs. 0.16% in winter). The other six genera were undetectable in winter. Functional prediction and pathway analysis revealed that biosynthesis of cofactors (ko01240) had the highest gene count ratios in the winter, followed by the two-component system (ko02020). Seasonal variation affects the gut microbiomes in wild Sichuan takins, with winter associated with lower species diversity and spring with enrichment of cellulose-degrading genera and phytopathogens. Such changes were crucial in their adaptation to the environment, particularly the difference in food abundance.

A platform technology for generating subunit vaccines against diverse viral pathogens.

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.

Listeriosis in a Metropolitan Hospital: Is Targeted Therapy a Risk Factor for Infection?

Targeted therapies are widely used for treatment of autoimmune diseases as well as solid organ and hematological malignancies. Various opportunistic infections have been described in patients on targeted therapies. Although case reports or a few case series of listeriosis have been reported to be associated with targeted therapy, most of the cases were related to anti-tumor necrosis factor-α monoclonal antibody. In this study, we describe the epidemiological and clinical profiles of listeriosis in a tertiary hospital in Shenzhen, a Southern Chinese metropolitan city in China. During the 9-year-and-6-month study period, a total of five cases of listeriosis were recorded and all of them had Listeria monocytogenes bacteremia. All five patients had predisposing factors, including corticosteroid (n = 3), targeted therapy (n = 2), pregnancy (n = 2) and anti-interferon gamma autoantibody (n = 1). The two patients who had targeted therapy during their course of cancer treatment received inhibitors of the epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) pathway. The first one was a 52-year-old woman with metastatic adenocarcinoma of the lung. She was given gefitinib (EGFR tyrosine kinase inhibitor), osimertinib (third-generation EGFR tyrosine kinase inhibitor) and afatinib (tyrosine kinase inhibitor that can bind to EGFR, HER2 and HER4). The second one was a 40-year-old woman with carcinoma of the breast with brain metastasis. She was given trastuzumab (anti-HER2 monoclonal antibody) and lapatinib (dual tyrosine kinase inhibitor of the EGFR/HER2 pathway). These two patients represent the second and third reports of listeria infections associated with EGFR/HER2 pathway inhibitors in the literature. Targeted therapy is an important predisposing factor for listeriosis. Listeria infection is an important differential diagnosis in patients on targeted therapy who present with sepsis and/or central nervous system infection, and the use of antibiotic regimens that cover listeria is crucial for empirical treatment. Avoidance of high-risk food items in these patients is important for the prevention of listeriosis.

Streptococcus oriscaviae sp. nov. Infection Associated with Guinea Pigs.

Pet bite-related infections are commonly caused by the pet's oral flora transmitted to the animal handlers through the bite wounds. In this study, we isolated a streptococcus, HKU75T, in pure culture from the purulent discharge collected from a guinea pig bite wound in a previously healthy young patient. HKU75T was alpha-hemolytic on sheep blood agar and agglutinated with Lancefield group D and group G antisera. API 20 STREP showed that the most likely identity for HKU75T was S. suis I with 85.4% confidence while Vitek 2 showed that HKU75T was unidentifiable. MALDI-TOF MS identified HKU75T as Streptococcus suis (score of 1.86 only). 16S rRNA gene sequencing showed that HKU75T was most closely related to S. parasuis (98.3% nucleotide identity), whereas partial groEL and rpoB gene sequencing showed that it was most closely related to S. suis (81.8% and 89.8% nucleotide identity respectively). Whole genome sequencing and intergenomic distance determined by ANI revealed that there was <85% identity between the genome of HKU75T and those of all other known Streptococcus species. Genome classification using concatenated sequences of 92 bacterial core genes showed that HKU75T belonged to the Suis group. groEL gene sequences identical to that of HKU75T could be directly amplified from the oral cavities of the two guinea pigs owned by the patient. HKU75T is a novel Streptococcus species, which we propose to be named S. oriscaviae. The oral cavity of guinea pigs is presumably a reservoir of S. oriscaviae. Some of the reported S. suis strains isolated from clinical specimens may be S. oriscaviae. IMPORTANCE We reported the discovery of a novel Streptococcus species, propose to be named Streptococcus oriscaviae, from the pus collected from a guinea pig bite wound in a healthy young patient. The bacterium was initially misidentified as S. suis/S. parasuis by biochemical tests, mass spectrometry. and housekeeping genes sequencing. Its novelty was confirmed by whole genome sequencing. Comparative genomic studies showed that S. oriscaviae belongs to the Suis group. S. oriscaviae sequences were detected in the oral cavities of the two guinea pigs owned by the patient, suggesting that the oral cavity of guinea pigs could be a reservoir of S. oriscaviae. Some of the reported S. suis strains may be S. oriscaviae. Further studies are warranted to refine our knowledge on this novel Streptococcus species.

Diverse and atypical manifestations of Q fever in a metropolitan city hospital: Emerging role of next-generation sequencing for laboratory diagnosis of Coxiella burnetii.

Although Q fever has been widely reported in the rural areas of China, there is a paucity of data on the epidemiology and clinical characteristics of this disease in large metropolitan cities. In this study, we profile the epidemiology and clinical manifestations of Q fever from a tertiary hospital in Shenzhen, a Southern Chinese metropolitan city with a large immigrant population from other parts of China. A total of 14 patients were confirmed to have Q fever during a nine-year-and-six-month period, five of whom were retrospectively diagnosed during case review or incidentally picked up because of another research project on unexplained fever without localizing features. Some patients had the typical exposure histories and clinical features, while a few other patients had rare manifestations of Q fever, including one with heart failure and diffuse intracapillary proliferative glomerulonephritis, a patient presenting with a spontaneous bacterial peritonitis-like syndrome, and another one with concomitant Q fever and brucellosis. Using a combination of clinical manifestation, inflammatory marker levels, echocardiographic findings and serological or molecular test results, nine, three and two patients were diagnosed to have acute, chronic and convalescent Q fever, respectively. Seven, five and two patients were diagnosed to have Q fever by serological test, nested real-time PCR and next-generation sequencing respectively. Diverse and atypical manifestations are associated with Q fever. The incidence of Q fever is likely to be underestimated. Next-generation sequencing is becoming an important diagnostic modality for culture-negative infections, particularly those that the physicians fail to recognize clinically, such as Q fever.

Rapid Diagnosis of Mycobacterium marinum Infection by Next-Generation Sequencing: A Case Report.

We present the first report of histology- and culture-proven Mycobacterium marinum infection diagnosed by next-generation sequencing (NGS). It took <2 days to make a microbiological diagnosis using the Oxford Nanopore Technologies' MinION device, compared to 20 days for the mycobacterium to be isolated from the tissue biopsy. NGS is particularly useful for culture-negative and slow-growing microorganism infections, such as mycobacterial, fungal and partially treated pyogenic bacterial infections. Due to its low equipment cost, short turn-around-time and portable size, the Oxford Nanopore Technologies' MinION device is a useful platform for NGS in routine clinical microbiology laboratories.

In Vitro Susceptibility of Typhoidal, Nontyphoidal, and Extended-Spectrum-ß-Lactamase-Producing Salmonella to Ceftolozane/Tazobactam.

Both typhoidal and nontyphoidal salmonellae are included in the top 15 drug-resistant threats described by the U.S. Centers for Disease Control and Prevention. There is an urgent need to look for alternative antibiotics for the treatment of Salmonella infections. We used the broth microdilution test to examine the in vitro susceptibilities of typhoidal and nontyphoidal salmonellae, including isolates positive for extended-spectrum ß-lactamase (ESBL), to ceftolozane/tazobactam and six other antibiotics. Of the 313 (52 typhoidal and 261 nontyphoidal) Salmonella isolates tested, 98.7% were susceptible to ceftolozane/tazobactam. Based on the overall MIC50/90 values, Salmonella isolates were more susceptible to ceftolozane/tazobactam (0.25/0.5 mg/L) than all the comparator agents: ampicillin (≥64/≥64 mg/L), levofloxacin (0.25/1 mg/L), azithromycin (4/16 mg/L), ceftriaxone (≤0.25/4 mg/L), chloramphenicol (8/≥64 mg/L), and trimethoprim/sulfamethoxazole (1/≥8 mg/L). Comparison of the activities of the antimicrobial agents against nontyphoidal Salmonella isolates according to their serogroups showed that ceftolozane/tazobactam had the highest activity (100%) against Salmonella serogroup D, G, I, and Q isolates, whereas the lowest activity (85.7%) was observed against serogroup E isolates. All 10 ESBL-producing Salmonella isolates (all nontyphoidal), of which 8 were CTX-M-55 producers and 2 were CTX-M-65 producers, were sensitive to ceftolozane/tazobactam, albeit with MIC50/90 values higher (1/2 mg/L) than those for non-ESBL producers (0.25/0.5 mg/L). In summary, our data indicate that ceftolozane/tazobactam is active against most strains of both typhoidal and nontyphoidal salmonellae and also against ESBL-producing salmonellae.

Substantial Decline in Invasive Pneumococcal Disease During Coronavirus Disease 2019 Pandemic in Hong Kong.

Compared with other countries, a more substantial decrease in the incidence of invasive pneumococcal disease was observed in Hong Kong, which is most likely attributable to the proactive mass adoption of face masks by the public. Human behavioral changes, particularly mask wearing, should be considered as an additional preventive strategy against invasive pneumococcal disease.