Utility of clinical metagenomics in diagnosing malignancies in a cohort of patients with Epstein-Barr virus positivity.

Backgrounds: Differentiation between benign and malignant diseases in EBV-positive patients poses a significant challenge due to the lack of efficient diagnostic tools. Metagenomic Next-Generation Sequencing (mNGS) is commonly used to identify pathogens of patients with fevers of unknown-origin (FUO). Recent studies have extended the application of Next-Generation Sequencing (NGS) in identifying tumors in body fluids and cerebrospinal fluids. In light of these, we conducted this study to develop and apply metagenomic methods to validate their role in identifying EBV-associated malignant disease. Methods: We enrolled 29 patients with positive EBV results in the cohort of FUO in the Department of Infectious Diseases of Huashan Hospital affiliated with Fudan University from 2018 to 2019. Upon enrollment, these patients were grouped for benign diseases, CAEBV, and malignant diseases according to their final diagnosis, and CNV analysis was retrospectively performed in 2022 using samples from 2018 to 2019. Results: Among the 29 patients. 16 of them were diagnosed with benign diseases, 3 patients were diagnosed with CAEBV and 10 patients were with malignant diseases. 29 blood samples from 29 patients were tested for mNGS. Among all 10 patients with malignant diagnosis, CNV analysis suggested neoplasms in 9 patients. Of all 19 patients with benign or CAEBV diagnosis, 2 patients showed abnormal CNV results. The sensitivity and specificity of CNV analysis for the identification for tumors were 90% and 89.5%, separately. Conclusions: The application of mNGS could assist in the identification of microbial infection and malignancies in EBV-related diseases. Our results demonstrate that CNV detection through mNGS is faster compared to conventional oncology tests. Moreover, the convenient collection of peripheral blood samples adds to the advantages of this approach.

Clinical Diagnostic Performance of Droplet Digital PCR for Suspected Bloodstream Infections.

Accurate and timely etiological diagnosis is crucial for bloodstream infections (BSIs) due to their high disability and mortality. We conducted a single-center prospective cohort study to compare the digital droplet PCR (ddPCR) assay with traditional blood culture. A total of 169 blood samples from 122 patients with suspected BSIs were collected, mostly from the department of infectious diseases, the emergency department, and the intensive care units, and the clinical data were also recorded. Nucleic acid was extracted from the blood samples, and a 5-fluorescent-channel droplet digital PCR assay was performed and then fed back with the pathogen and its copies. In BSI patients, ddPCR reported an overall 85.71% (12/14) (95% confidence interval [CI], 56.15 to 97.48%) sensitivity, 100% (7/7) (95% CI, 56.09 to 100.00%) and 71.43% (5/7) (95% CI, 30.26 to 94.89%) sensitivity in patients without empirical treatment and in empirically treated patients, respectively. Compared to traditional blood culture, the overall detection rate of ddPCR was significantly higher, 11.27% (16/142) (95% CI, 6.78 to 17.93%) versus 30.28% (43/142) (95% CI, 23.01 to 38.64%), and the extra detection rate of ddPCR was 19.01% (27/142) (95% CI, 13.11 to 26.63%). Of the ddPCR-positive culture-negative cases, 74.19% (23/31) (95% CI, 55.07 to 87.46%) were consistent with the final clinical diagnosis, including 10 bacteria and fungi. The detection rate of ddPCR was significantly higher in patients with white blood cell (WBC) counts of >10 · 109/L, C-reactive protein (CRP) of >70 mg/L, or procalcitonin (PCT) of >0.9 ng/L. Pathogen loads detected by ddPCR are correlated with WBC, CRP, and especially, PCT levels, precisely and rapidly reflecting clinical disease progression. ddPCR has an important guiding value for the clinical use of antibiotics to achieve the best pathogen coverage and the antibacterial effect. Collectively, ddPCR showed a great diagnostic performance in BSIs and had an overall higher detection rate than blood culture. In addition, ddPCR could be used to dynamically monitor the disease progression and provide medication guidance on antibiotic use. IMPORTANCE ddPCR is a promising method to address the current challenges of BSI diagnosis and precise treatment, as it is highly efficient in DNA detection. It shortens the identification of BSI-related pathogens from several days of traditional bacterial culture to 4 to 5 h. It is extremely sensitive and more tolerant to PCR inhibitors, which may facilitate the amplification and enable the detection of a meager amount of DNA fragments in detecting BSI-related pathogens and drug-resistant genes. It can identify almost 20 pathogens in one reaction, which reduces the usage of clinical blood samples to no more than 2 mL. Additionally, dynamic monitoring, assessment of pathogens, and antibiotic resistance genes in patients could be used to guide timely and precise adjustment of antimicrobial prescription. The short turnaround time of ddPCR may have the potential to guide antimicrobial treatment in the very early stage of sepsis and reduce the mortality and disability rate of sepsis.

Comprehensive Surveillance and Sampling Reveal Carbapenem-Resistant Organism Spreading in Tertiary Hospitals in China.

Purpose: Carbapenem-resistant organisms (CROs) have posed a great threat to antibiotic use and induce multi-drug resistance. Contamination of the hospital environment and infection of healthcare workers (HCWs) are reported as sources of nosocomial infections. Here, we performed a comprehensive environment sampling and timely epidemiological investigation during outbreaks to investigate the role of the environment and HCWs in CRO transmission. Patients and Methods: We enrolled carbapenem-resistant organism outbreaks in ICU-1 of Huashan Hospital from January 2019 to March 2019, and ICU-2 located at west branch of Huashan Hospital from October 2019 to November 2019. Carbapenem-resistant Klebsiella pneumoniae (CRKP) and carbapenem-resistant Acinetobacter baumannii (CRAB) isolates were collected from the patients. We performed a real-time comprehensive environmental and HCW sampling in the two ICUs. Isolated strains from patients and the positive colonies from the screening were sent for whole-genome sequencing. Finally, phylogenetic trees were constructed. Results: CRAB and CRKP outbreaks simultaneously occurred in ICU-1; the outbreak involved 13 patients. Meanwhile, the CRKP outbreak in ICU-2 included 11 patients. Twelve out of 146 environment and HCWs samples in ICU-1 were carbapenem-resistant bacteria, including six CRKP and six CRAB strains. For ICU-2, hospital surfaces and HCWs were negative for CRKP. Phylogenetic analyses showed that CRKP strains in ICU-1 were classified into two clades: Clade 1 and Clade 2, sharing a high similarity of isolates from the environment and HCWs. The same phenomenon was observed in CRAB. Conclusion: A timely comprehensive sampling combined with genome-based investigation may aid in tracking the transmission route of and controlling the infections. The environment and HCWs could be contaminated during CRO transmission, which calls for strengthened prevention and control measures.

A case of fatal amoebic encephalitis caused by Balamuthia mandrillaris, China.

We reported a case of B.mandrillaris amoebic encephalitis in mainland China. Metagenomics next-generation sequencing helped initial diagnosis and then polymerase chain reaction of the B.mandrillaris in the infected nasal skin tissues reported positive and amoeba cysts were found in the tissue under microscopic observation.

Clinical application of Epstein-Barr virus DNA loads in Epstein-Barr virus-associated diseases: A cohort study.

OBJECTIVES: This study evaluated the diagnostic value of Epstein-Barr virus (EBV) DNA load in blood samples of patients with EBV-associated diseases, and proposed a strategy for the interpretation of positive EBV DNA results. METHODS: Derivation and validation cohorts were established to evaluate the clinical significance of EBV DNA loads in the peripheral blood mononuclear cells (PBMCs) and plasma from EBV-infected patients. EBV DNA loads were compared and receiver operating characteristic curves were employed to assess the optimal cutoff values of EBV DNA for identification of EBV-associated diseases. RESULTS: The derivation and validation cohorts comprised 135 and 71 subjects, respectively. EBV DNA loads in the PBMCs of the EBV-associated diseases group was significantly higher than that of the EBV non-associated diseases group (5.8 × 104 vs 7.8 × 103 copies/106 cells, P<0.0001). The diagnostic cut-off value of viral load in PBMCs for EBV-associated diseases was determined to be 1.6 × 104 copies/106 cells. The combined EBV DNA load cutoff in PBMCs and positive EBV DNA qualitative detection in plasma (>500 copies/mL) allowed for the differentiation of EBV-associated and non-associated diseases; the sensitivity and specificity were 80.6 and 96.8%, respectively. CONCLUSIONS: The strategy of combining EBV DNA loads in PBMCs and plasma will potentially help identify EBV-associated diseases.

Rapid detection of respiratory organisms with FilmArray respiratory panel and its impact on clinical decisions in Shanghai, China, 2016-2018.

BACKGROUND: In this study, we evaluated the diagnostic potential and clinical impact of an automated multiplex PCR platform (the FilmArray Respiratory Panel; FA-RP), specially designed for pathogen detection in respiratory tract infections in adults with unexplained pneumonia (UP). METHODS: A total of 112 UP patients in Shanghai, China, were enrolled prospectively and assessed using the FA-RP from October 2016 to March 2018. We examined the test results and their influence on clinical decisions. Furthermore, as a control group, we retrospectively obtained the clinical data of 70 UP patients between October 2014 and March 2016 (before the FA-RP was available). The two patient groups were compared with respect to factors, including general antimicrobial use and defined daily dose (DDD) numbers. RESULTS: Between October 2016 and March 2018, the positive rate obtained using FA-RP for UP was 76.8%. The primary pathogens in adults with UP were Influenza A/B (47.3%, 53/112). Compared with the patients before FA-RP was available, patients who underwent FA-RP testing had higher rates of antiviral drug use and antibiotic de-escalation during clinical treatment. FA-RP significantly decreased the total DDDs of antibiotic or antifungal drugs DDDs by 7 days after admission (10.6 ± 2.5 vs 14.1 ± 8.8, P < .01). CONCLUSIONS: The FA-RP is a rapid and sensitive nucleic acid amplification test method for UP diagnosis in adults. The application of FA-RP may lead to a more accurately targeted antimicrobial treatment and reduced use of antibiotic/antifungal drugs.

Tracking Carbapenem-Producing Klebsiella pneumoniae Outbreak in an Intensive Care Unit by Whole Genome Sequencing.

The presence of carbapenem-producing Klebsiella pneumoniae (CP-Kp) is a serious threat to the control of nosocomial infections. Plasmid-mediated horizontal transfer of the resistance gene makes it difficult to control hospital-acquired CP- Kp infections. Nine CP- Kp strains were isolated during an outbreak in the intensive care unit of Shanghai Huashan hospital in east China. We conducted a retrospective study to identify the origin and route of transmission of this CP-Kp outbreak. Whole-genome sequencing (WGS) analysis was performed on 9 clinical isolates obtained from 8 patients, and the results were compared to clinical and epidemiological records. All isolates were ST11 CP-Kp. Single-nucleotide polymorphisms and the presence and structure of plasmids indicated that this CP-Kp outbreak had different origins. These 9 isolates were partitioned into two clades according to genetic distance. Four plasmids, CP002474.1, CP006799.1, CP018455.1, and CP025459.1, were detected among the 9 isolates. The plasmid phylogeny and antibiotic resistance (AR) gene profile results were consistent with the sequencing results. We found that two clades of CP-Kp were responsible for this nosocomial outbreak and demonstrated the transmission route from two index patients. Plasmid carriage and phylogeny are a useful tool for identifying clades involved in disease transmission.

Isolation of Mycobacterium arupense from pleural effusion: culprit or not?

BACKGROUND: Mycobacterium arupense, first identified in 2006, is a slow-growing nontuberculous mycobacterium (NTM) and an emerging cause of tenosynovitis, potentially associated with immunosuppression. However, unlike the diagnostic value of its isolation from osteoarticular specimens, the significance of detecting M. arupense in respiratory specimens is not yet clear. CASE PRESENTATION: To our knowledge, we, for the first time, described the identification of M. arupense from the pleural effusion of an immunocompetent patient, who presented with fever and chylothorax. The symptoms resolved with doxycycline treatment for 45 days and a low-fat, high-protein diet. Follow-up at 14 months showed no relapse. CONCLUSIONS: Because the patient fully recovered without combined anti-NTM treatment, we did not consider M. arupense the etiological cause in this case. This indicates that M. arupense detected in pleural effusion is not necessarily a causative agent and careful interpretation is needed in terms of its clinical relevance.

Medical treatment of an unusual cerebral hydatid disease.

BACKGROUND: Hydatid disease is a worldwide zoonosis produced by the larval stage of cestodes of the Echinococcus genus. Hydatid disease primarily involves the liver and lungs. The brain is involved in less than 2% of cases. Surgery has long been the only choice for the treatment, but chemotherapy has been successfully replaced surgery in some special cases. CASE PRESENTATION: We report a rare hydatid disease case which presented with multiple lesions in right frontal lobe, an uncommon site, and in the liver and lungs. A 28-year-old woman presented with 6 months history of recurrent convulsion. Cranial magnetic resonance imaging found multiple lesions in right frontal lobe, so she was hospitalized for surgical treatment and received sodium valproate by oral for controlling epilepsy. Before the operation, other lesions were found in the liver and lungs by computerized tomography scan. There were multiple pulmonary nodules near the pleura and large cyst in the liver. The pathology of liver showed that it may be a hydatid disease. Then, positive serum antibodies for echinococcus antigen further confirmed our diagnosis. Since her central nerve system was involved, she received four pills (800 mg, about 17 mg/kg/day) albendazole treatment for 18 months without operation. Her symptoms abated and a follow-up magnetic resonance imaging showed that the lesion had obviously diminished after treatment. She was recurrence free 2 years after we stopped albendazole treatment. CONCLUSIONS: This case reveals an uncommon pattern of intracranial hydatid disease. Albendazole can be beneficial for some inoperable cerebral hydatid disease patients.

Hierarchy Low CD4+/CD8+ T-Cell Counts and IFN-γ Responses in HIV-1+ Individuals Correlate with Active TB and/or M.tb Co-Infection.

OBJECTIVE: Detailed studies of correlation between HIV-M.tb co-infection and hierarchy declines of CD8+/CD4+ T-cell counts and IFN-γ responses have not been done. We conducted case-control studies to address this issue. METHODS: 164 HIV-1-infected individuals comprised of HIV-1+ATB, HIV-1+LTB and HIV-1+TB- groups were evaluated. Immune phenotyping and complete blood count (CBC) were employed to measure CD4+ and CD8+ T-cell counts; T.SPOT.TB and intracellular cytokine staining (ICS) were utilized to detect ESAT6, CFP10 or PPD-specific IFN-γ responses. RESULTS: There were significant differences in median CD4+ T-cell counts between HIV-1+ATB (164/µL), HIV-1+LTB (447/µL) and HIV-1+TB- (329/µL) groups. Hierarchy low CD4+ T-cell counts (<200/µL, 200-500/µL, >500/µL) were correlated significantly with active TB but not M.tb co-infection. Interestingly, hierarchy low CD8+ T-cell counts were not only associated significantly with active TB but also with M.tb co-infection (P<0.001). Immunologically, HIV-1+ATB group showed significantly lower numbers of ESAT-6-/CFP-10-specific IFN-γ+ T cells than HIV-1+LTB group. Consistently, PPD-specific IFN-γ+CD4+/CD8+ T effector cells in HIV-1+ATB group were significantly lower than those in HIV-1+LTB group (P<0.001). CONCLUSIONS: Hierarchy low CD8+ T-cell counts and effector function in HIV-1-infected individuals are correlated with both M.tb co-infection and active TB. Hierarchy low CD4+ T-cell counts and Th1 effector function in HIV-1+ individuals are associated with increased frequencies of active TB, but not M.tb co-infection.

Moxifloxacin and gatifloxacin for initial therapy of tuberculosis: a meta-analysis of randomized clinical trials.

Moxifloxacin (MOX) and gatifloxacin (GAT) have exhibited promising mycobactericidal activity, and a number of clinical trials have been conducted in recent decades to compare the treatment efficacy of MOX-containing and/or GAT-containing regimens with the standard regimen. The aim of this meta-analysis for clinical trials of MOX- or GAT-containing regimens was to evaluate their treatment efficacy and safety in initial therapy for drug-sensitive tuberculosis (TB). Databases were searched for randomized controlled trials, and nine studies with 6980 patients were included. We found that fluoroquinolone substitution for isoniazid or ethambutol in short-course regimens might result in more frequent unfavorable treatment outcomes compared with the standard regimen-in particular, an increased incidence of relapse. In a per-protocol analysis, MOX-containing regimens had slightly higher rates of sputum culture conversion at two months than the standard regimen (RR 1.08, 95% CI 1.04-1.11, P <0.001); there was no significant difference in the rate of sputum conversion between the GAT-containing regimens and the standard regimen (RR 1.13, 95% CI 0.96-1.33, P = 0.13). There were no significant differences in the incidence of death from any cause, including TB, nor were there serious adverse events between the MOX- or GAT-containing regimens and the standard regimen. In conclusion, MOX or GAT might not have the ability to shorten treatment duration in the initial therapy for tuberculosis despite the non-inferiority or even slightly better efficacy in the early phase of treatment compared with the standard regimen. Furthermore, it is safe to include MOX or GAT in initial TB treatment.

Diagnosis of Tuberculous Meningitis Using a Combination of Peripheral Blood T-SPOT.TB and Cerebrospinal Fluid Interferon-γ Detection Methods.

OBJECTIVE: To evaluate the effectiveness of combined peripheral blood T-SPOT.TB and cerebrospinal fluid interferon-γ (cIFN-γ) detection methods in the diagnosis of tuberculous meningitis (TBM). METHODS: A total of 30 individuals with TBM and 39 control individuals without TBM participated in this study. IFN-γ-secreting T cells were detected by enzyme-linked immunospot (ELISPOT), and cIFN-γ was detected by enzyme-linked immunosorbent assay (ELISA). We collected cerebrospinal fluid from 10 patients in the TBM group on initial visit and at 4 weeks, to observe changes. RESULTS: The sensitivity and specificity of peripheral-blood T-SPOT.TB testing in the diagnosis of TBM were 70% and 87%, respectively. The area under the ROC curve of cIFN-γ for TBM diagnosis was 0.819, and the corresponding sensitivity and specificity were 83% and 85%, respectively. When T-SPOT.TB and cIFN-γ results were positive, the specificity and positive predictive value of TBM diagnosis reached 100%. CONCLUSIONS: Combined use of T-SPOT.TB and cIFN-γ could improve the diagnosis efficiency of TBM. Dynamic observation of cIFN-γ is also important in monitoring TBM because the level of this analyte significantly decreases after treatment.

The incidence and risk factors of meningitis after major craniotomy in China: a retrospective cohort study.

BACKGROUND: Meningitis after neurosurgery can result in severe morbidity and high mortality. Incidence varies among regions and limited data are focused on meningitis after major craniotomy. AIM: This retrospective cohort study aimed to determine the incidence, risk factors and microbiological spectrum of postcraniotomy meningitis in a large clinical center of Neurosurgery in China. METHODS: Patients who underwent neurosurgeries at the Department of Neurosurgery in Huashan Hospital, the largest neurosurgery center in Asia and the Pacific, between 1st January and 31st December, 2008 were selected. Individuals with only shunts, burr holes, stereotactic surgery, transsphenoidal or spinal surgery were excluded. The complete medical records of each case were reviewed, and data on risk factors were extracted and evaluated for meningitis. RESULTS: A total of 65 meningitides were identified among 755 cases in the study, with an incidence of 8.60%. The risk of meningitis was increased by the presence of diabetes mellitus (odds ratio [OR], 6.27; P = 0.009), the use of external ventricular drainage (OR, 4.30; P = 0.003) and the use of lumbar drainage (OR, 17.23; P<0.001). The isolated microorganisms included Acinetobacter baumannii, Enterococcus sp, Streptococcus intermedius and Klebsiella pneumonia. CONCLUSIONS: Meningitis remains an important source of morbidity and mortality after major craniotomy. Diabetic patients or those with cerebral spinal fluid shunts carry significant high risk of infection. Thus, identification of the risk factors as soon as possible will help physicians to improve patient care.

Retrospective study of 61 patients with adult-onset Still's disease admitted with fever of unknown origin in China.

Adult-onset Still's disease (AOSD), as a category of connective tissue diseases, has about 5∼9% of fever of unknown origin (FUO) cases. Diagnosis of AOSD was challenging because of its nonspecific characteristics. The present study analyzed clinical manifestations and laboratory findings in a series of patients with AOSD from eastern China. Medical records of 61 patients admitted with FUO and with a discharge diagnosis of AOSD were retrospectively evaluated and analyzed with special focus on clinical manifestations and laboratory findings. Compared with previous reports, most features of our patients had a similar incidence rate. Rash (79%), arthralgia (80%), and sore throat (84%) were the most frequent clinical manifestations in our series. Leukocytosis (80%), elevated ESR (98%) and CRP (100%), negative ANA (90%) and RF (93%), and high ferritin level (94%) were the most sensitive laboratory findings in our patients. AOSD was not a rare reason of FUO in eastern China. Fever, arthralgia, rash, sore throat, leukocytosis, neutrophilia, elevated ESR and CRP, negative ANA and RF, and high ferritin level were the most common clinical features in our series. The lack of highly specific characteristic makes the diagnosis of AOSD difficult compared with other diseases in FUO.

Spectroscopic properties and energy transfer analysis of Tm3+-doped BaF2-Ga2O3-GeO2-La2O3 glass.

This paper reports on the spectroscopic properties and energy transfer analysis of Tm(3+)-doped BaF(2)-Ga(2)O(3)-GeO(2)-La(2)O(3) glasses with different Tm(2)O(3) doping concentrations (0.2, 0.5, 2.0, 2.5, 3.0, 3.5, 3.5, 4.0 wt%). Mid-IR fluorescence intensities in the range of 1,300 nm-2,200 nm have been measured when excited under an 808 nm LD for all the samples with the same pump power. Energy level structure and Judd-Ofelt parameters have been calculated based on the absorption spectra of Tm(3+), cross-relaxation rates and multi-phonon relaxation rates have been estimated with different Tm(2)O(3) doping concentrations. The maximum fluorescence intensity at around 1.8 mum has been obtained in Tm(2)O(3)-3 wt% sample and the maximum value of calculated stimulated emission cross-section of Tm(3+) in this sample is about 0.48 x 10(-20) cm(2) at 1,793 nm, and there is not any crystallization peak in the DSC curve of this sample, which indicate the potential utility of Tm(3+)-doped BaF(2)-Ga(2)O(3)-GeO(2)- La(2)O(3) glass for 2.0-microm optical fiber laser.