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Background: When individuals infected with human immunodeficiency virus (HIV) experience pulmonary infections, they often exhibit severe symptoms and face a grim prognosis. Consequently, early, rapid, and accurate pathogen diagnosis is vital for informing effective treatment strategies. This study aimed to use metagenomic next-generation sequencing (mNGS) and targeted mNGS (tNGS) to elucidate the characteristics of pulmonary infections in HIV and non-HIV individuals. Methods: This study enrolled 90 patients with pulmonary infection at the Department of Infectious Diseases of The First Hospital of Jilin University from June 2022 to May 2023, and they were divided into HIV (n=46) and non-HIV (n=44) infection groups. Their bronchoalveolar lavage fluid (BALF) was collected for mNGS analysis to evaluate the differences in pulmonary infection pathogens, and tNGS detection was performed on BALF samples from 15 HIV-infected patients. Results: A total of 37 pathogens were identified in this study, including 21 bacteria, 5 fungi, 5 viruses, 5 mycobacteria, and 1 mycoplasma. The sensitivity of mNGS was 78.9% (71/90), which is significantly higher than that of conventional methods (CTM) (39/90, P=1.5E-8). The combination of mNGS with CTM can greatly enhance the sensitivity of pathogen detection. The prevalence of Pneumocystis jirovecii (82.6% vs. 9.1%), cytomegalovirus (CMV) (58.7% vs. 0%), and Epstein-Barr virus (EBV) (17.4% vs. 2.3%) was significantly higher in the HIV infection group than in the non-HIV infection group (P<0.05). Although no statistically significant difference was observed, the detection rate of Mycobacteria was higher in HIV-infected patients (17.4%) than in the non-HIV group (6.8%). Furthermore, the tNGS results of BALF from 15 HIV-infected patients were not entirely consistent with the mNGS results., and the concordance rate of tNGS for the detection of main pathogens reached 86.7% (13/15). Conclusion: Next-generation sequencing (NGS) can accurately detect pathogens in the BALF of patients with pulmonary infection. The sensitivity of tNGS is comparable to that of mNGS. Therefore, this technique should be promoted in the clinic for better patient outcomes.

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The diagnosis of Bartonella is challenging due to its rarity and negative culture results. Once the diagnosis is delayed and proper treatment is not given, it can develop into infective endocarditis, which can be fatal. We reported a 60-year-old female patient who had recurrent fever for 5 months. After receiving ineffective treatment at the local hospital, she sought medical attention at our hospital. Laboratory blood indicators testing and imaging indicated infective endocarditis, and metagenomic Next Generation Sequencing (m-NGS) testing confirmed the diagnosis of Bartonella vinsonii infection. After surgical treatment and the combination of doxycycline and ceftriaxone sodium for anti-infective therapy, the patient recovered. Valuing the combination of multiple auxiliary diagnostic methods and improving the application of m-NGS in the detection of unknown pathogens can compensate for the current limitations in the diagnosis of Bartonella. Early diagnosis and treatment are extremely important for Bartonella endocarditis.

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Background: Although the emerging NGS-based assays, metagenomic next-generation sequencing (mNGS) and targeted next-generation sequencing (tNGS), have been extensively utilized for the identification of pathogens in pulmonary infections, there have been limited studies systematically evaluating differences in the efficacy of mNGS and multiplex PCR-based tNGS in bronchoalveolar lavage fluid (BALF) specimens. Methods: In this study, 85 suspected infectious BALF specimens were collected. Parallel mNGS and tNGS workflows to each sample were performed; then, we comparatively compared their consistency in detecting pathogens. The differential results for clinically key pathogens were confirmed using PCR. Results: The microbial detection rates of BALF specimens by the mNGS and tNGS workflows were 95.18% (79/83) and 92.77% (77/83), respectively, with no significant difference. mNGS identified 55 different microorganisms, whereas tNGS detected 49 pathogens. The comparative analysis of mNGS and tNGS revealed that 86.75% (72/83) of the specimens were complete or partial concordance. Particularly, mNGS and tNGS differed significantly in detection rates for some of the human herpesviruses only, including Human gammaherpesvirus 4 (P<0.001), Human betaherpesvirus 7 (P<0.001), Human betaherpesvirus 5 (P<0.05) and Human betaherpesvirus 6 (P<0.01), in which tNGS always had higher detection rates. Orthogonal testing of clinically critical pathogens showed a total coincidence rate of 50% for mNGS and PCR, as well as for tNGS and PCR. Conclusions: Overall, the performance of mNGS and multiplex PCR-based tNGS assays was similar for bacteria and fungi, and tNGS may be superior to mNGS for the detection of DNA viruses. No significant differences were seen between the two NGS assays compared to PCR.

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AIMS: This study aimed to evaluate the diagnostic performance of metagenomic next-generation sequencing (mNGS) with pancreatic fluid aspiration for the detection of infected pancreatic necrosis (IPN). METHODS: This retrospective observational study included 66 patients with suspected IPN. The participants simultaneously underwent pancreatic fluid aspiration mNGS, and microbial and blood culture. We compared the diagnostic performance of mNGS with that of culture in the detection of pathogens associated with IPN. RESULTS: Of the 66 patients, 45 (68.2 %) were confirmed to have IPN. Pancreatic fluid aspiration mNGS yielded positive results in 32 of these patients (71.1 %), significantly outperforming microbial culture results (25 patients, 55.6 %; P = 0.039); however, both methods exhibited similar specificity (95.2% vs. 100 %). The results of pancreatic fluid aspiration mNGS and microbial culture matched in 73.3 % (33/45) of patients with IPN. The turnaround time for the mNGS results was significantly shorter than that for the microbial culture method (P < 0.001). In addition, survival analysis demonstrated that a positive mNGS result was not associated with increased mortality (hazard ratio, 0.652; 95 % confidence interval 0.157-2.699, P = 0.555). CONCLUSIONS: Our study highlights the potential of mNGS for diagnosing IPN, with implications for improving patient care by facilitating early and accurate diagnosis, guiding appropriate interventions, and possibly improving patient outcomes.

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Next-generation sequencing (NGS) has revolutionized clinical microbiology, particularly in diagnosing respiratory infectious diseases and conducting epidemiological investigations. This narrative review summarizes conventional methods for routine respiratory infection diagnosis, including culture, smear microscopy, immunological assays, image techniques as well as polymerase chain reaction(PCR). In contrast to conventional methods, there is a new detection technology, sequencing technology, and here we mainly focus on the next-generation sequencing NGS, especially metagenomic NGS(mNGS). NGS offers significant advantages over traditional methods. Firstly, mNGS eliminates assumptions about pathogens, leading to faster and more accurate results, thus reducing diagnostic time. Secondly, it allows unbiased identification of known and novel pathogens, offering broad-spectrum coverage. Thirdly, mNGS not only identifies pathogens but also characterizes microbiomes, analyzes human host responses, and detects resistance genes and virulence factors. It can complement targeted sequencing for bacterial and fungal classification. Unlike traditional methods affected by antibiotics, mNGS is less influenced due to the extended survival of pathogen DNA in plasma, broadening its applicability. However, barriers to full integration into clinical practice persist, primarily due to cost constraints and limitations in sensitivity and turnaround time. Despite these challenges, ongoing advancements aim to improve cost-effectiveness and efficiency, making NGS a cornerstone technology for global respiratory infection diagnosis.

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Purpose: Metagenomic next-generation sequencing(mNGS) is a novel molecular diagnostic technique. For nucleic acid extraction methods, both whole-cell DNA (wcDNA) and cell-free DNA (cfDNA) are widely applied with the sample of bronchoalveolar lavage fluid (BALF). We aim to evaluate the clinical value of mNGS with cfDNA and mNGS with wcDNA for the detection of BALF pathogens in non-neutropenic pulmonary aspergillosis. Methods: mNGS with BALF-cfDNA, BALF-wcDNA and conventional microbiological tests (CMTs) were performed in suspected non-neutropenic pulmonary aspergillosis. The diagnostic value of different assays for pulmonary aspergillosis was compared. Results: BALF-mNGS (cfDNA, wcDNA) outperformed CMTs in terms of microorganisms detection. Receiver operating characteristic (ROC) analysis indicated BALF-mNGS (cfDNA, wcDNA) was superior to culture and BALF-GM. Combination diagnosis of either positive for BALF-mNGS (cfDNA, wcDNA) or CMTs is more sensitive than CMTs alone in the diagnosis of pulmonary aspergillosis (BALF-cfDNA+CMTs/BALF-wcDNA+CMTs vs. CMTs: ROC analysis: 0.813 vs.0.66, P=0.0142/0.796 vs.0.66, P=0.0244; Sensitivity: 89.47% vs. 47.37%, P=0.008/84.21% vs. 47.37%, P=0.016). BALF-cfDNA showed a significantly greater reads per million (RPM) than BALF-wcDNA. The area under the ROC curve (AUC) for RPM of Aspergillus detected by BALF-cfDNA, used to predict "True positive" pulmonary aspergillosis patients, was 0.779, with a cut-off value greater than 4.5. Conclusion: We propose that the incorporation of BALF-mNGS (cfDNA, wcDNA) with CMTs improves diagnostic precision in the identification of non-neutropenic pulmonary aspergillosis when compared to CMTs alone. BALF-cfDNA outperforms BALF-wcDNA in clinical value.

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The case presents a 47-year-old man with sudden abdominal pain and fever, but the cause was uncertain. Through metagenomic next-generation sequencing (mNGS) and detecting Q fever antibodies in serum, along with the patient's clinical and epidemiological history, a precise diagnosis was made, enabling timely and proper treatment.

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Q fever is a worldwide distribution disease caused by Coxiella burnetii（C. burnetii）, an obligate intracellular, Gram-negative acidophilic bacterium belonging to γ-proteobacterium. Most patients present with acute Q-fever accompanied by atypical flu-like symptoms, with only 1%-5% of cases may develop into persistent and focally infected foci, mainly manifest as endocarditis, osteomyelitis and prosthetic arthritis. In this case, the patient experienced an unexplained and uninterrupted fever up to 39.2 °C for a week, accompanied by chills and headaches, as well as abnormal liver function. The laboratory reported negative results for blood culture and respiratory-associated pathogens, however, the metagenomic next-generation sequencing (mNGS) reported that detection of 20 sequence reads of C. burnetii in the patient's peripheral blood. In addition, the patient had traveled to Sri Lanka, Iraq and Saudi Arabia before illness. In clinical, the treatment regimen was adjusted from empirically intravenous moxifloxacin 400 mg a day for 1 week to continuously oral minocyline 100 mg twice daily for 2 weeks. The patient was in good health without any adverse sequelae during outpatient visitation and the phone calls follow-up. In conclusion, the mNGS does provide an early and timely diagnostic basis for rare and difficult to culture pathogens, which contributes to the success of clinical anti-infection.

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INTRODUCTION: Lower respiratory tract infections(LRTIs) in adults are complicated by diverse pathogens that challenge traditional detection methods, which are often slow and insensitive. Metagenomic next-generation sequencing (mNGS) offers a comprehensive, high-throughput, and unbiased approach to pathogen identification. This retrospective study evaluates the diagnostic efficacy of mNGS compared to conventional microbiological testing (CMT) in LRTIs, aiming to enhance detection accuracy and enable early clinical prediction. METHODS: In our retrospective single-center analysis, 451 patients with suspected LRTIs underwent mNGS testing from July 2020 to July 2023. We assessed the pathogen spectrum and compared the diagnostic efficacy of mNGS to CMT, with clinical comprehensive diagnosis serving as the reference standard. The study analyzed mNGS performance in lung tissue biopsies and bronchoalveolar lavage fluid (BALF) from cases suspected of lung infection. Patients were stratified into two groups based on clinical outcomes (improvement or mortality), and we compared clinical data and conventional laboratory indices between groups. A predictive model and nomogram for the prognosis of LRTIs were constructed using univariate followed by multivariate logistic regression, with model predictive accuracy evaluated by the area under the ROC curve (AUC). RESULTS: (1) Comparative Analysis of mNGS versus CMT: In a comprehensive analysis of 510 specimens, where 59 cases were concurrently collected from lung tissue biopsies and BALF, the study highlights the diagnostic superiority of mNGS over CMT. Specifically, mNGS demonstrated significantly higher sensitivity and specificity in BALF samples (82.86% vs. 44.42% and 52.00% vs. 21.05%, respectively, p < 0.001) alongside greater positive and negative predictive values (96.71% vs. 79.55% and 15.12% vs. 5.19%, respectively, p < 0.01). Additionally, when comparing simultaneous testing of lung tissue biopsies and BALF, mNGS showed enhanced sensitivity in BALF (84.21% vs. 57.41%), whereas lung tissues offered higher specificity (80.00% vs. 50.00%). (2) Analysis of Infectious Species in Patients from This Study: The study also notes a concerning incidence of lung abscesses and identifies Epstein-Barr virus (EBV), Fusobacterium nucleatum, Mycoplasma pneumoniae, Chlamydia psittaci, and Haemophilus influenzae as the most common pathogens, with Klebsiella pneumoniae emerging as the predominant bacterial culprit. Among herpes viruses, EBV and herpes virus 7 (HHV-7) were most frequently detected, with HHV-7 more prevalent in immunocompromised individuals. (3) Risk Factors for Adverse Prognosis and a Mortality Risk Prediction Model in Patients with LRTIs: We identified key risk factors for poor prognosis in lower respiratory tract infection patients, with significant findings including delayed time to mNGS testing, low lymphocyte percentage, presence of chronic lung disease, multiple comorbidities, false-negative CMT results, and positive herpesvirus affecting patient outcomes. We also developed a nomogram model with good consistency and high accuracy (AUC of 0.825) for predicting mortality risk in these patients, offering a valuable clinical tool for assessing prognosis. CONCLUSION: The study underscores mNGS as a superior tool for lower respiratory tract infection diagnosis, exhibiting higher sensitivity and specificity than traditional methods.

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Background: Traditional microbiological detection methods used to detect pulmonary infections in people living with HIV (PLHIV) are usually time-consuming and have low sensitivity, leading to delayed treatment. We aimed to evaluate the diagnostic value of metagenomics next-generation sequencing (mNGS) for microbial diagnosis of suspected pulmonary infections in PLHIV. Methods: We retrospectively analyzed PLHIV who were hospitalized due to suspected pulmonary infections at the sixth people hospital of Zhengzhou from November 1, 2021 to June 30, 2022. Bronchoalveolar lavage fluid (BALF) samples of PLHIV were collected and subjected to routine microbiological examination and mNGS detection. The diagnostic performance of the two methods was compared to evaluate the diagnostic value of mNGS for unknown pathogens. Results: This study included a total of 36 PLHIV with suspected pulmonary infections, of which 31 were male. The reporting period of mNGS is significantly shorter than that of CMTs. The mNGS positive rate of BALF samples in PLHIV was 83.33%, which was significantly higher than that of smear and culture (44.4%, P<0.001). In addition, 11 patients showed consistent results between the two methods. Futhermore, mNGS showed excellent performance in identifying multi-infections in PLHIV, and 27 pathogens were detected in the BALF of 30 PLHIV by mNGS, among which 15 PLHIV were found to have multiple microbial infections (at least 3 pathogens). Pneumocystis jirovecii, human herpesvirus type 5, and human herpesvirus type 4 were the most common pathogen types. Conclusions: For PLHIV with suspected pulmonary infections, mNGS is capable of rapidly and accurately identifying the pathogen causing the pulmonary infection, which contributes to implement timely and accurate anti-infective treatment.

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Background: A rapid and precise etiological diagnosis is crucial for the effective treatment of bloodstream infection (BSI). In this study, the performance of probe capture-based targeted next-generation sequencing (tNGS) was compared to that of blood culture and metagenomic next-generation sequencing (mNGS) in detecting potential pathogens in patients with BSI. Methods: A total of 80 patients with suspected BSI were prospectively enrolled from 24 November 2023 to 30 December 2023 at Zhongshan Hospital, Shanghai, China. All 80 participants underwent simultaneous blood culture, blood mNGS, and blood tNGS after admission when febrile, and the results were compared. Results: Among the 80 participants, 11 were clinically diagnosed with noninfectious fever, and 69 were diagnosed with BSI. Blood tNGS had a higher sensitivity for the diagnosis of BSI than blood culture (91.3% vs. 23.2%, P<0.001) and blood mNGS (91.3% vs. 69.6%, P=0.001). There was no significant difference in specificity between blood mNGS and tNGS (81.8% vs. 100.0%, P=0.13). Blood tNGS demonstrated a faster turnaround time than blood culture and blood mNGS. In 22 (31.9%) patients with BSI, targeted adjustment of the anti-infectious therapy according to the blood tNGS results resulted in clinical improvement. Conclusions: Blood tNGS may be a promising tool for detecting potential pathogens in patients with BSI. The application of blood tNGS for BSI could guide anti-infectious treatment strategies and might improve clinical outcomes.

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Background: As a culture-independent method, metagenomic next-generation sequencing (mNGS) is widely used in microbiological diagnosis with advantages in identifying potential pathogens, guiding antibiotic therapy, and improving clinical prognosis, especially in culture-negative cases. Mycoplasma hominis (M. hominis) mediastinitis is a rare and severe disease for which etiological diagnosis is important but challenging. The application of mNGS in the etiological diagnosis of mediastinitis has seldom been studied. Methods: By searching the electronic medical history retrieval system with "Mycoplasma hominis" and "mediastinitis", seven patients diagnosed with M. hominis mediastinitis were reviewed in Zhongshan Hospital, Fudan University, Shanghai from 9 December 2020 to 14 February 2023. Microbiological cultures and mNGS were conducted for blood, abscess, and/or mediastinal fluid. Adjustment of the antibiotic therapy due to mNGS was assessed. A literature review was conducted in the PubMed database beginning in 1970 for M. hominis infection and mediastinitis. Results: For the seven patients, cultures of blood, abscess, and mediastinal fluid were negative whereas mNGS identified M. hominis in serum, abscess, and/or mediastinal fluid and was used to guide specific antibiotic therapy. The stringent mapped reads number of genera (SMRNG), stringent mapped reads number of species (SMRN), and coverage rate of M. hominis detection by mNGS were significantly higher in body fluid (abscess or mediastinal fluid) than in serum. All seven patients had underlying heart diseases and underwent previous cardiac surgery. The most common symptoms were fever and sternal pain. After detection of M. hominis, antibiotics were adjusted to quinolones or doxycycline except for one patient, whose diagnosis was clarified after death. Two patients died. Literature review since 1970 identified 30 cases of extra-genital infection caused by M. hominis. Including our seven new cases, 2 (5.4%) were neonates and 35 (94.6%) were adults. Thirty (81.1%) cases were postoperative infection and 15 (40.5%) had implanted devices. Five patients (13.5%) died. Conclusions: mNGS might be a promising technology in the detection of fastidious pathogens such as M. hominis. Accurate etiological diagnosis by mNGS could guide antibiotic therapy and facilitate clinical management.

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The metagenomic next-generation sequencing (mNGS) method is preferred for genotyping useful for the identification of organisms, illumination of metabolic pathways, and determination of microbiota. It can accurately obtain all the nucleic acid information in the test sample. Anthrax is one of the most important zoonotic diseases, infecting mainly herbivores and occasionally humans. The disease has four typical clinical forms, cutaneous, gastrointestinal, inhalation, and injection, all of which may result in sepsis or meningitis, with cutaneous being the most common form. Here, we report a case of cutaneous anthrax diagnosed by mNGS in a butcher. Histopathology of a skin biopsy revealed PAS-positive bacilli. Formalin-fixed paraffin-embedded (FFPE) tissue sample was confirmed the diagnosis of anthrax by mNGS. He was cured with intravenous penicillin. To our knowledge, this is the first case of cutaneous anthrax diagnosed by mNGS using FFPE tissue. mNGS is useful for identifying pathogens that are difficult to diagnose with conventional methods, and FFPE samples are simple to manage. Compared with traditional bacterial culture, which is difficult to cultivate and takes a long time, mNGS can quickly and accurately help us diagnose anthrax, so that anthrax can be controlled in a timely manner and prevent the outbreak of epidemic events.

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Leptospirosis is a zoonotic and neglected waterborne disease caused by the pathogenic helical spirochetes. Early diagnosis of leptospirosis remains challenging due to non-specific symptoms and the limited availability of rapid point-of-care diagnostic tests. Herein, we present a case where a patient suspected of having COVID-19 was diagnosed with leptospirosis using metagenomic next-generation sequencing (mNGS). This case highlights the potential of mNGS to diagnose leptospirosis in the context of the COVID-19 pandemic.

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OBJECTIVES: Early diagnosis of infectious diseases remains a challenge. This study assessed the diagnostic value of mNGS in infections and explored the effect of various factors on the accuracy of mNGS. METHODS: An electronic article search of PubMed, Cochrane Library, and Embase was performed. A total of 85 papers were eligible for inclusion and analysis. Stata 12.0 was used for statistical calculation to evaluate the efficacy of mNGS for the diagnosis of infectious diseases. RESULTS: The AUC of 85 studies was 0.88 (95%CI, 0.85-0.90). The AUC of the clinical comprehensive diagnosis and conventional test groups was 0.92 (95%CI, 0.89-0.94) and 0.82 (95%CI, 0.78-0.85), respectively. The results of subgroup analysis indicated that the PLR and NLR were 12.67 (95%CI, 6.01-26.70) and 0.05 (95%CI, 0.03-0.10), respectively, in arthrosis infections. The PLR was 24.41 (95%CI, 5.70-104.58) in central system infections and the NLR of immunocompromised patients was 0.08 (95%CI, 0.01-0.62). CONCLUSION: mNGS demonstrated satisfactory diagnostic performance for infections, especially for bone and joint infections and central system infections. Moreover, mNGS also has a high value in the exclusion of infection in immunocompromised patients.

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OBJECTIVES: Nocardia gipuzkoensis was first described as a novel and distinct species in 2020 by Imen Nouioui and pulmonary nocardiosis associated with N. gipuzkoensis was once reported in two bronchiectasis patients. Noteworthy, both reported N. gipuzkoensis cases showed sensitivity to trimethoprim/sulfamethoxazol (TMP-SMZ), which are usually recommended for empirical therapy. METHODS: We reported the third case of N. gipuzkoensis infection in a 16-year-old girl with chief complaints of cough and persistent chest and back pain. No underlying immuno-suppressive conditions and glucocorticoid use was revealed. Patchy lesions next to the spine and located in the posterior basal segment of the lower lobes of the left lung were seen in thorax computed tomography (CT), but no pathogenic bacteria were detected according to routine laboratory testings. RESULTS: Metagenomic next-generation sequencing (mNGS) combined with whole-genome sequencing (WGS) was used to classified our isolate from bronchoalveolar lavage fluid (BALF) as N. gipuzkoensis. It is worth mentioning that drug susceptibility testing of our isolate showed resistance to TMP-SMZ, which was never reported before. The patient improved remarkably both clinically and radiographically according to the treatment with imipenem-cilastatin infusion alone. CONCLUSION: mNGS and WGS showed excellent performance in identifying the Nocardia genus to the species level and improving the detection rate of N. gipuzkoensis ignored by traditional culture. Different from previously reported cases, the N. gipuzkoensis infection case showed resistance to TMP-SMZ, which is an unprecedented finding and a crucial addition to our understanding of the antibacterial spectrum of N. gipuzkoensis. The successful treatment with imipenem-cilastatin infusion alone in this case is a testament to the importance of precise identification and tailored antibiotic therapy.

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Background: Pulmonary trichomoniasis is considered a neglected disease due to failures in recognizing it, stemming from insensitive microbial methods and a lack of specific clinical features. This study aims to analyze the clinical implications of trichomonads detected in bronchoalveolar lavage fluid (BALF) by metagenomic next-generation sequencing (mNGS). Methods: This multicenter retrospective study included patients diagnosed with pneumonia, admitted to three tertiary hospitals in China from July 2018 to September 2022, with trichomonads detected in BALF through mNGS. The analysis covered demographics, comorbidities, symptoms, laboratory findings, mNGS results, clinical treatment, and outcomes of these patients. Results: A total of 17 patients were enrolled, comprising 14 males and 3 females. Trichomonas tenax and Trichomonas vaginalis were detected by mNGS in BALF samples of 15 and 2 patients, respectively. Patients were categorized into two groups based on the presence of risk factors for trichomonad infection, including immunocompromised conditions, uncontrolled diabetes mellitus, oral/periodontal diseases, and aspiration. Among 11 patients with risk factors (Case 1-11), 4 received nitromidazoles as part of comprehensive treatment, achieving a 100% treatment success rate. The remaining 7 patients, who did not receive nitromidazoles, had only one achieving relief after broad-spectrum antimicrobial therapy, resulting in a 14.3% treatment success rate. For the 6 patients without any risk factors for trichomonad infection (Case 12-17), none received nitromidazoles during hospitalization. However, 4 out of these 6 patients (66.7%) eventually recovered. Conclusion: mNGS proves to be an efficient tool for detecting trichomonads in BALF samples. Comprehensive analysis of clinical features and laboratory indicators is essential to distinguish between infection and colonization of trichomonads. Pulmonary trichomoniasis should not be overlooked when trichomonads are detected in BALF from patients with risk factors.

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Complicated case with fever or headache of unknown origin is currently one of the main challenges in clinical diagnosis. A retrospective analysis was conducted on a 27-year-old female patient hospitalized with headache and fever, and the pathogen species were ultimately determined by metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF). The culture results of CSF showed no bacterial or fungal growth. CSF cytology showed a significant increase in nucleated cells. Pathogenic index (corresponded to human gamma herpesvirus 4) of the microorganism after correcting for human background was 12846.77 with a host index (human resource) of 27822.48 by mNGS of CSF. The patient improved through antiviral treatment with ganciclovir. Epstein-Barr virus encephalitis is rare in immunocompetent adults, which can easily cause misdiagnosis and should be paid attention to. mNGS of CSF has significant advantages in the diagnosis of Epstein-Barr virus encephalitis.

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The positive detection rate of blood metagenomic next-generation sequencing (mNGS) was still too low to meet clinical needs, while pus from the site of primary infection may be advantageous for identification of pathogens. To assess the value of mNGS using pus in patients with sepsis, thirty-five samples were collected. Pathogen identification and mixed infection diagnosis obtained by use of mNGS or cultivation methods were compared. Fifty-three aerobic or facultative anaerobes, 59 obligate anaerobes and 7 fungi were identified by the two methods. mNGS increased the accuracy rate of diagnosing aerobic or facultative anaerobic infections from 44.4% to 94.4%; mNGS also increased the sensitivity of diagnosing obligate anaerobic infections from 52.9% to 100.0%; however, mNGS did not show any advantage in terms of fungal infections. Culture and mNGS identified 1 and 24 patients with mixed infection, respectively. For obligate anaerobes, source of microorganisms was analyzed. The odontogenic bacteria all caused empyema (n = 7) or skin and soft tissue infections (n = 5), whereas the gut-derived microbes all caused intra-abdominal infections (n = 7). We also compared the clinical characteristics of non-obligate anaerobic and obligate anaerobic infection groups. The SOFA score [9.0 (7.5, 14.3) vs. 5.0 (3.0, 8.0), P = 0.005], procalcitonin value [4.7 (1.8, 39.9) vs. 2.50 (0.7, 8.0), P = 0.035], the proportion of septic shock (66.7% vs. 35.3%, P = 0.044) and acute liver injury (66.7% vs. 23.5%, P = 0.018) in the non-obligate anaerobic infection group were significantly higher than those in the obligate anaerobic infection group. In patients with sepsis caused by purulent infection, mNGS using pus from the primary lesion may yield more valuable microbiological information.

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