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In the last few years, next-generation sequencing (NGS) has emerged as a technology for laboratory diagnosis of many culture-negative infections and slow-growing microorganisms. In this study, we describe the use of metagenomic NGS (mNGS) for rapid diagnosis of T. marneffei infection in a 37-year-old renal transplant recipient who presented with chronic pneumonia syndrome. Bronchoalveolar lavage for mNGS was positive for T. marneffei sequence reads. Prolonged incubation of the bronchoalveolar lavage revealed T. marneffei colonies after 6 days of incubation. Analysis of 23 cases of T. marneffei infections in renal transplant recipients from the literature revealed that the number of cases ranged from 1 to 4 cases per five years from 1990 to 2020; but increased rapidly to 9 cases from 2021 to 2023, with 7 of them diagnosed by NGS. Twenty of the 23 cases were from T. marneffei-endemic areas [southern part of mainland China (n = 9); Hong Kong (n = 4); northeastern India (n = 2); Indonesia (n = 1) and Taiwan (n = 4)]. For the 3 patients from non-T. marneffei-endemic areas [United Kingdom (n = 2) and Australia (n = 1)], they had travel histories to China and Vietnam respectively. The time taken for diagnosis by mNGS [median 1 (range 1 to 2) day] was significantly shorter than that for fungal culture [median 6 (range 3 to 15) days] (P = 0.002). mNGS is useful for picking up more cases of T. marneffei infections in renal transplant recipients as well as providing a rapid diagnosis. Talaromycosis is an emerging fungal infection in renal transplant recipients., (© 2024. The Author(s).)

Leishmania belongs to a genus of the protozoan parasites that causes leishmaniasis, and includes cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). In this case, Leishmania amastigotes were found on cytomorphology examination of the bone marrow specimen, followed by 1,076 Leishmania donovani reads using metagenomic next generation sequencing (mNGS). Since being definitely diagnosed with VL/HIV coinfection, the patient was treated with liposomal amphotericin B as the parasite-resistant therapy and was discharged after clinical cure. But nearly a year later, on the mNGS follow-up, L. donovani was detected in the patient's blood plasma specimen with 941 reads, suggesting that a relapse of leishmaniasis had occurred. These results indicate that leishmaniasis still exists in China and may represent a public health concern. This case could be helpful in the differential diagnosis of leishmaniasis, and for determining disease progression, prevention, and control of vectors and reservoir hosts. [ABSTRACT FROM AUTHOR]

• Metagenomic next-generation sequencing (mNGS) is superior to all previous etiological tests for tuberculosis (TB) in smear-negative extrapulmonary TB. • mNGS could identify all possible pathogens in extrapulmonary specimens at once, within 48 h. • Extrapulmonary specimens were not susceptible to contamination and effective mNGS results could be obtained. • The results did not significantly affect anti-TB therapy. The aim of this study was to assess the clinical utility of metagenomic next-generation sequencing (mNGS) on smear-negative extrapulmonary specimens collected in China. Specimens were tested by mNGS and other routine tests for tuberculosis (TB). The diagnostic accuracy of mNGS was calculated and compared with the final clinical diagnosis. The sensitivity of mNGS was found to be significantly higher than the sensitivities of the other routine TB tests. Receiver operating characteristic curve analysis showed that mNGS achieved the highest area under the curve (AUC) value of 0.79. The mNGS positive rate was highest for tuberculous meningitis. All non-tuberculous extrapulmonary pathogens were directly and simultaneously detected. mNGS appeared to be superior to all previous etiological tests for TB on smear-negative extrapulmonary specimens and could identify all possible pathogens at once within 48 h. [ABSTRACT FROM AUTHOR]

Objective: To determine the diagnostic accuracy of pneumococcal antigen detection in diagnosis of pneumococcal meningitis in children. Methods: Purulent meningitis was diagnosed according to European Society for Clinical Microbiology and Infectious Diseases (ESCMID) guideline between July 2014 and June 2016. Along with a cerebrospinal fluid (CSF) culture, pneumococcal antigen detection in cerebrospinal fluid (CSF) was performed, and further identification of pathogens was done with 16S rDNA-PCR and high-throughput sequencing. Results: CSF samples collected from 184 children (median age of 1.92 mo). CSF culture was used as the gold standard. 46 (25%) had positive results for culture and 10 (5.4%) were pneumococci; 34 (18.5%) were pneumococcal antigen positive. The sensitivity and specificity of pneumococcal antigen detection were 100% (95% CI: 89.4%–100%) and 86.2% (95% CI: 96.4%–99.9%), respectively. 92.3% (12/13) were confirmed by nucleic acid detection to be pneumococci. Conclusions: Pneumococcal antigen detection in CSF has adequate sensitivity and specificity in diagnosing pneumococcal meningitis. [ABSTRACT FROM AUTHOR]

Phytophthora pini is a globally distributed plant pathogen of the Phytophthora genus. This fungus-like organism was recently associated with foliage blight and shoot dieback disease of Rhododendron pulchrum Sweet, which threaten the health of natural ecosystems and horticultural crops in China. Early and accurate detection of P. pini is critical for effective disease prevention and management. In this study, a new target gene Ppinihp1, which is a hypothetical protein, identified from the whole-genome sequence of P. pini was used to establish a conventional polymerase chain reaction (PCR) assay for the detection of this pathogen. PCR results were proved to be positive for P. pini , but negative for other Phytophthora , Pythium , fungal isolates and Bursaphelenchus species. The test was sensitive enough to detect 100 pg of P. pini purified genomic DNA in a 50 μL reaction mixture. P. pini was also detected in artificially inoculated R. pulchrum samples. Finally, this novel Ppinihp1- PCR-assay was able to detect P. pini in 15 of 40 soil samples collected from R. pulchrum fields in Jiangsu, China. This method established the specificity, sensitivity, accuracy and convenience for P. pini detection, offering a dependable tool for the rapid screening of Phytophthora root and stem rot in R. pulchrum. • A new target Ppinihp1 was identified from the whole-genome sequence for PCR detection of P.pini. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggest that patients with advanced non–small-cell lung cancer (NSCLC) and specific genomic alterations including epidermal growth factor receptor and microtubule-associated protein-like 4 anaplastic lymphoma kinase could significantly benefit from molecular-targeted therapies compared with chemotherapy. Recently, immunotherapy based on programmed cell death 1 (PD-1) and its ligand (PD-L1) blockade prolong survival in patients with advanced NSCLC, especially in those patients with positive expression of PD-L1 and when used in the first-line setting. Therefore, the diagnosis, clinical staging and molecular genotyping must be quick and efficient so that we can make a timely and precise decision for treatment strategy. In our department, it takes a median 4 working days (range 3–6) for a new patient from initial respiratory consultation to treatment decision, whereas in many countries, 14 workdays is considered a reasonable timeline. In this article, we will provide detailed information on the diagnostic pathway for a new patient suspected of having lung cancer to the final treatment decisions in our department. [ABSTRACT FROM AUTHOR]

Highlights: [•] The reverse transcription-loop-mediated isothermal amplification assay was developed to detect avian influenza A (H7N9) virus epidemic in China. [•] The developed assays were evaluated using in vitro transcribed control RNA, clinical specimens, and clinical isolates. [•] The assays were shown to have high sensitivity and high specificity. [ABSTRACT FROM AUTHOR]

Abstract: Background: The quality of clinical specimens is a crucial determinant for virological diagnosis. Objectives: We compared the viral diagnostic yield for influenza A and respiratory syncytial virus (RSV) from the recently developed nasopharyngeal flocked swabs (NPFS) with nasopharyngeal aspirates (NPA) collected in parallel from 196 hospitalized children with acute respiratory infection during the peak period of influenza A and RSV activity in Hong Kong. Specimens were tested by RT-PCR for influenza A and RSV and viral load determined. They were also tested by direct immunofluorescence (DIF) for influenza A and B, RSV, parainfluenza types 1–3 and adenovirus. Results: Both NPA and NPFS had excellent sensitivity (100%) for detecting influenza A by RT-PCR but NPA was slightly more sensitive than NPFS for detecting RSV by both RT-PCR (100% vs. 92.3%) and DIF (87.2% vs. 84.6%) and for detecting influenza A by DIF (90.2% vs. 82.9%). Viral load for influenza A in NPA and NPFS was not significantly different but that for RSV was higher in NPA. Conclusion: NPA remains the optimal specimen for diagnosis of respiratory infections by RT-PCR and DIF. However, collection of NPFS is easier to perform in an out-patient setting, was more acceptable to parents and less likely to generate aerosols than NPA engendering potentially less infection control hazard. [Copyright &y& Elsevier]

Background: Cytological detection of early esophageal squamous cell carcinoma (ESCC) remains challenging. Therefore, we introduced a rapid cytological screening method and evaluated its efficacy as a pre-endoscopy screening for early ESCC and precursor lesions. Methods: This method consisted of a sponge sample retrieval, automatic liquid-based cytological treatment and slides preparation, computer-assisted screening and manual diagnosis. Efficacy for detection of early ESCC and precursor lesions was evaluated. Also, diagnostic efficiency was compared with manual diagnosis. Results: Eighty-three patients with early ESCC and precursor lesions and 2,090 asymptomatic participants with high risks of ESCC were enrolled. Whole procedure was accomplished within two working days. Abnormal cells were detected in all 83 patients, and in 272 (13.01%) subjects among 2,090 asymptomatic participants. Early ESCC, high-grade intraepithelial neoplasia, low-grade intraepithelial neoplasia and reflux esophagitis and normal endoscopic findings were detected in 8, 13, 11, 187 and 53 participants with abnormal cells, respectively. The calculated sensitivity, specificity, positive predictive value and negative predictive value for detection of early ESCC and precursor lesions were 100%, 88.34%, 11.76%, and 100%, respectively. Compared with manual diagnosis, this method was accomplished in a shorter time duration (5.4 ± 0.45 min vs 320.2 ± 132.4 min, p  < 0.001), a higher diagnostic accuracy (96.7% vs74.4%, p  = 0.015) and a better inter-observer agreement (93.3% vs66.7%, K = 0.286, p  < 0.001). Conclusions: Our study provides a promising methodology as pre-endoscopy screening for early ESCC and precursor lesions.

The ongoing global pandemic (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a huge public health issue. Hence, we devised a multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (mRT-LAMP-LFB) for diagnosing COVID-19. Using two LAMP primer sets, the ORF1ab (opening reading frame 1a/b) and N (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified in a single-tube reaction, and detected with the diagnosis results easily interpreted by LFB. In presence of FITC (fluorescein)-/digoxin- and biotin-labeled primers, mRT-LAMP produced numerous FITC-/digoxin- and biotin-attached duplex amplicons, which were determined by LFB through immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the test line of LFB) and biotin/treptavidin interaction (biotin on the duplex and strptavidin on the polymerase nanoparticle). The accumulation of nanoparticles leaded a characteristic crimson band, enabling multiplex analysis of ORF1ab and N gene without instrumentation. The limit of detection (LoD) of COVID-19 mRT-LAMP-LFB was 12 copies (for each detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. The analytical sensitivity of SARS-CoV-2 was 100% (33/33 oropharynx swab samples collected from COVID-19 patients), and the assay's specificity was also 100% (96/96 oropharynx swab samples collected from non-COVID-19 patients). The total diagnostic test can be completed within 1 h from sample collection to result interpretation. In sum, the COVID-19 mRT-LAMP-LFB assay is a promising tool for diagnosing SARS-CoV-2 infections in frontline public health field and clinical laboratories, especially from resource-poor regions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Talaromyces marneffei is a thermally dimorphic fungus endemic in south-east Asia. It predominantly occurs in both immunocompromised and immunosuppressed patients and can be fatal if diagnosis and treatment are delayed. The clinical manifestations of T. marneffei infection are nonspecific and rapid diagnosis of T. marneffei infection remains challenging., Case Presentation: A 24-year-old man came to our outpatient department with the sign of common skin lesions. The lesions were cuticolor follicular papules with or without central umbilication, nodules and acne-like lesions, which are common in syringoma, steatocystoma multiplex and trichoepithelioma. A dermatoscopy examination was performed to differentiate these skin lesions. The dermatoscopic images revealed circular or quasi-circular whitish amorphous structure with a central brownish keratin plug, providing the diagnostic clues of T. marneffei infection. Therefore, a skin scrapings culture, skin biopsy and serological detection for human immunodeficiency virus (HIV) were performed. The final diagnosis of this patient was T. marneffei and HIV co-infection., Conclusion: Rapid diagnosis of T. marneffei infection is clinically challenging since presenting clinical manifestations are nonspecific with significant overlap with other common conditions. This case highlights that dermatoscopy is a promising tool for the rapid diagnosis of T. marneffei infection in patients with nonspecific skin lesions, assisting clinicians to avoid delayed diagnosis or misdiagnosis.

Fusarium temperatum is an emerging maize pathogen that causes maize ear and stalk rot diseases and produces various mycotoxins including moniliformin, beauvericin, enniatins and fumonisin B1, which poses a potential risk to the human food or animal feed supply chains. Early detection of F. temperatum is crucial to prevent its derived mycotoxins from entering the food chain, and is also a useful tool in disease management practices. Here, we describe a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of F. temperatum. The 28S ribosomal DNA sequences (28S rDNA) of F. temperatum were used to design a set of six primers. The reaction conditions were optimized for developing a fast assay with high specificity and sensitivity, and were able to detect the presence of less than 10 pg of target DNA per reaction within 60 min. Furthermore, the resulting amplicons were visualized by adding SYBR Green I to the reaction tubes. Suspected F. temperatum infected maize stalk samples collected from Yunnan province, China were identified using the developed LAMP assay. In conclusion, the method not only provides a rapid and specific screening for the existence of F. temperatum in a bulk of maize samples without using sophisticated equipment, but also is potentially useful for other agriculturally important toxigenic fungi., (Copyright © 2018 Elsevier B.V. All rights reserved.)