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.

Rare/cryptic Aspergillus species infections and importance of antifungal susceptibility testing.

BACKGROUND: The number of patients infected with Aspergillus rose dramatically in recent years. However, studies on the clinical spectrum and antifungal susceptibilities of non-classical (non-fumigatus, non-flavus, non-niger and non-terreus) pathogenic Aspergillus species are very limited. OBJECTIVES: We examined the clinical spectrum and antifungal susceptibilities of 34 non-duplicated, non-classical Aspergillus isolates collected from Hong Kong, Shenzhen and Shanghai. METHODS: The Aspergillus isolates were identified by internal transcribed spacer, partial BenA and partial CaM sequencing and phylogenetic analyses. Susceptibility testing against eight antifungals was performed following the European Committee for Antimicrobial Susceptibility Testing's methodology. RESULTS: The 34 Aspergillus isolates were identified as 14 different rare/cryptic species of four sections (Flavi [n = 8], Nidulantes [n = 8], Nigri [n = 17] and Restricti [n = 1]). Except for one patient whose clinical history could not be retrieved, 72.7% of the remaining patients had underlying conditions predisposing them to Aspergillus infections. The most common diseases were pulmonary infections (n = 15), followed by skin/nail infections (n = 6), chronic otitis externa and/or media (n = 5), wound infections (n = 2) and mastoiditis/radionecrosis (n = 1), while three were colonisations. Five patients succumbed due to the infections during the admission, and another two died 5 years later because of chronic pulmonary aspergillosis. Antifungal susceptibility testing showed that they possessed different susceptibility profiles compared to the classical Aspergillus species. The majority of isolates characterised were sensitive or wild-type to amphotericin B. The minimum effective concentrations for all the three echinocandins were also low. CONCLUSION: Susceptibility testing should be performed for infections due to these rare/cryptic Aspergillus species to guide proper patient management.

Clinical characteristics, rapid identification, molecular epidemiology and antifungal susceptibilities of Talaromyces marneffei infections in Shenzhen, China.

Although case series of talaromycosis have been reported in China, their detailed clinical and microbiological characteristics have never been systematically profiled. In this study, we report the clinical characteristics, molecular epidemiology, rapid identification and antifungal susceptibilities of talaromycosis in The University of Hong Kong-Shenzhen Hospital in Shenzhen. Seven cases of talaromycosis were observed since commencement of hospital service in 2012. Three patients were local Shenzhen residents, whereas the other four were immigrants from other parts of China. Two patients were HIV-negative, but with underlying diseases requiring immunosuppressive therapy. Two of the seven patients succumbed. All the seven isolates were successfully identified as T. marneffei by MALDI-TOF MS using Bruker database expanded with in-house generated T. marneffei mass spectra. MLST showed that the seven strains belonged to six different, novel sequences types. Phylogenetic analyses of the concatenated five-locus sequence revealed that the seven strains were scattered amongst other T. marneffei strains. The MICs of itraconazole, isavuconazole, posaconazole and voriconazole against the seven clinical isolates were low but MICs of anidulafungin were high. Underlying diseases other than HIV infection are increasingly important risk factors of talaromycosis. MALDI-TOF MS is useful for rapid identification. Highly diverse T. marneffei sequence types were observed.

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.

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.

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.

Dirofilaria hongkongensis infection presenting as recurrent shoulder mass.

In 2012, a novel canine Dirofilaria species, D. hongkongensis was identified in Hong Kong that caused human diseases and subsequently reported in an Austrian traveller returning from the Indian subcontinent. Here we present a case of human infection by D. hongkongensis manifested as recurrent shoulder mass. Diagnosis was achieved by cox1 gene sequencing of the excised specimen. The case illustrated that parasitic infection represents an important differential diagnosis for musculoskeletal lesions.

Immunogenicity of intradermal hepatitis B vaccination in renal transplant recipients.

We investigated intradermal hepatitis B (HBV) vaccination in 24 renal transplant recipients who failed to develop hepatitis B surface antibody (anti-HBs) with intramuscular (i.m.) vaccination. All patients received recombinant HBV vaccine 5 microg intradermally every 2 weeks for 8 doses. Nine patients developed protective levels of anti-HBs (> 10 miu/mL) and two patients developed low levels of anti-HBs (4-6 miu/mL), giving an overall initial response rate of 45.8%. A booster of 40 microg was administered intramuscularly after 1 year. All initial responders developed an anti-HBs response (322.6 +/- 92.0 miu/mL). In addition, four patients who did not respond initially to the intradermal vaccination seroconverted after the booster. Responders (62.5%) and nonresponders had comparable age, gender, immunosuppressive medications, and duration of transplant. In conclusion, renal transplant patients who fail to respond to intramuscular HBV vaccination may benefit from intradermal vaccination followed by an intramuscular booster.