Identification of Talaromyces marneffei Infection in an HIV-Negative Patient by ITS Sequencing.

Disseminated ankle mycosis is a life-threatening systemic infection caused by the emerging opportunistic and lethal fungal pathogen Talaromyces marneffei which is more common in HIV-positive patients. However, an increasing number of infections are occurring in HIV-negative patients. Here, we report a case of Talaromyces marneffei infection in HIV-negative patient. A 50s HIV-negative male patient with fever, cough, bloody sputum expectoration, pulmonary sarcoidosis and body rashes was hospitalized at Zhejiang Provincial People's Hospital. CT scanning showed pulmonary multiple nodules with apical bronchial occlusion, patchy infiltration and pathological biopsy demonstrated bronchiolitis obliterans with organized pneumonia and chronic active inflammation of lung tissue with infiltration of numerous lymphocytes, plasma cells, phagocytes and neutrophils. Laboratory tests revealed significantly increased white blood cells count 18.3 ×109/L, neutrophil count 15.34 ×109/L, monocyte count 0.66 ×109/L, platelet count 517 ×109/L, C-reactive protein 116 mg/L, erythrocyte sedimentation rate 112mm/h. The ß-D-glucan test was negative (33.06 pg/mL) while fungal culture of broncho alveolar lavage fluid revealed colonies with temperature-dependent dimorphic growth character and Talaromyces marneffei was confirmed by ITS sequencing of the colonies. The patient exhibited radiological improvement and clinical recuperation after intravenously guttae of voriconazole. Talaromycosis in immunocompetent and HIV-negative individuals is relatively rare and is characterized by an insidious onset, various clinical manifestations, and is clinically challenging. Fungal culture and ITS sequencing are warranted for diagnosis Talaromyces marneffei infection. This is the first report on identification of Talaromyces marneffei infection in an HIV-negative patient with skin involvement by ITS sequencing in Zhejiang.

Pacemaker Associated Aspergillus fumigatus Endocarditis: A Case Report.

Aspergillus endocarditis (AE) is a highly fatal infection that can occur in heart valve replacement, pacemaker implantation and other heart surgeries, and early recognition and sufficient diagnosis are challenging. Here, we report the case of a 68-year-old male with a history of dilated cardiomyopathy and pacemaker implantation who had a repeated fever with failed antibacterial treatment and sterile blood culture. He developed endocarditis, and the culture and biopsy of vegetation tissue showed the abundant presence of septate hyphae, which was subsequently identified as Aspergillus fumigatus by internal transcribed spacer (ITS) sequencing. Although the patient had serious side effects from voriconazole, he had a good prognosis following surgery and prolonged caspofungin antifungal therapy of 42 consecutive days. We discuss the diagnosis and treatment strategy of AE, and recommend galactomannan assays and next-generation sequencing for a timely diagnosis. Early surgical intervention combined with prompt antifungal therapy appears significant for survival.

A Rare Case of a Subcutaneous Abscess Caused by Nocardia cyriacigeorgica in an Immunocompetent Patient.

Background: Nocardia cyriacigeorgica, which mainly causes pleuropulmonary and disseminated nocardiosis, has been proved to be one of the most common opportunistic pathogens in patients with immunodeficiency, but the cases that cause subcutaneous abscesses in normal individuals are rare and should be paid attention to. Methods: The clinical data of a patient with cutaneous nocardiosis caused by Nocardia cyriacigeorgica in Zhejiang Provincial People's Hospital were retrospectively analyzed, including clinical manifestations, laboratory examinations, imaging examinations, medication and prognosis. Results: Magnetic resonance imaging (MRI) showed that there was a 26 mm × 73 mm abscess under the skin. The pus in the abscess was green. Gram staining showed positive branched rod-shaped and undivided hyphae. After culture, small wrinkle dry white small colonies were observed, and it was identified as Nocardia cyriacigeorgica by MALDI-TOF MS. Conclusion: We report the first case of a subcutaneous abscess caused by Nocardia cyriacigeorgica in an immunocompetent patient. Compared with cutaneous nocardiosis of which approximately 80% caused by Nocardia brasiliensis invasion, infection of Nocardia cyriacigeorgica is more insidious and latent, the features of the lesions are also unique. For this Nocardia cyriacigeorgica clinical isolate, the tested antibacterial drugs are generally sensitive and have an ideal prognosis after treatment with linezolid and timely debridement.

Serum Interleukin-26 is a Potential Biomarker for the Differential Diagnosis of Neurosyphilis and Syphilis at Other Stages.

Background: Interleukin-26 (IL-26) is an atypical proinflammatory cytokine due to its binding to circulating double-stranded DNA and direct antibacterial activity. Although IL-26 has been confirmed to be involved in the pathophysiology of cancer, chronic inflammatory diseases and infections, the diagnostic and prognostic values of IL-26 levels in syphilis patients are not clear. This study aimed to investigate IL-26 levels in different stages of syphilis progression. Methods: A total of 30 healthy controls and 166 patients with syphilis at different stages of disease progression were enrolled. Serum IL-26 levels were quantified in accordance with the protocols of RayBio® Human Interleukin-26 Enzyme Linked Immunosorbent Assay (ELISA) kits. Clinical laboratory diagnostic parameters and blood analysis data were detected and collected according to clinical medical laboratory standards. Results: The levels of serum IL-26 were significantly higher in neurosyphilis patients than in healthy subjects (6.87 (4.36, 12.14) and 1.67 (0.09, 4.89) pg/µL, respectively; ****p < 0.0001), latent syphilis (1.48 (0.40, 2.05) pg/µL, ****p < 0.0001), seroresistant syphilis (0.81 (0.20, 2.91) pg/µL, ****p < 0.0001) and secondary syphilis (1.66 (0.41, 4.25) pg/µL, ****p < 0.0001) with data presented as the median with interquartile range. The concentration of serum IL-26 was most sensitive to serum low-density lipoprotein concentration (r = -0.438, **p = 0.004) in latent syphilis, urine epithelial cells (r = 0.459, **p = 0.003) in seroresistant syphilis, and serum creatinine levels (r = 0.463, **p = 0.004) and urea creatinine ratio levels (r = 0.500, **p = 0.008) in secondary syphilis patients. There was no significant correlation with the concentration of IL-26 and toluidine red unheated serum test (TRUST) titers in each type of syphilis patient. Conclusion: Circulating IL-26 in serum displays diagnostic potential in the progression of neurosyphilis and warrants further evaluation in clinical trials.

Identification of sacrococcygeal and pelvic abscesses infected with invasive Mycoplasma hominis by MALDI-TOF MS.

BACKGROUND: Mycoplasma hominis is the smallest prokaryotic microorganism with no cell wall, high pleomorphism, and slower reproduction than bacteria. It is difficult for clinical technicians to find M. hominis through the negative Gram staining of specimens. Therefore, it is likely to miss detection in routine clinical smear etiological examination. M. hominis is generally considered to be a common colonizing bacterium in urogenital tract with low pathogenicity, and it is usually difficult to invade submucosal tissue and enter the bloodstream. METHODS: The abscesses of the patient were examined histopathologically, and the pus in the abscesses was extracted for etiological examination. MALDI-TOF MS was used to identify and confirmed the pathogens in the specimens. The commercial Mycoplasma isolation, culture, and drug sensitivity kit was used to determine antibiotic susceptibility. RESULTS: No pathogens were found after pathological and smear microscopic examination of the puncture fluid from the sacrococcygeal and pelvic abscesses. Until 48 h later, small, translucent, and gray-white colonies were observed in the blood plate culture results. The laboratory physician ultimately determined that the pathogen was M. hominis by MALDI-TOF MS. CONCLUSION: We report a case of extra-urogenital cystic abscesses infected by M. hominis, in order to improve clinicians' comprehensive understanding of the pathogenicity of Mycoplasma. In addition, the clinical laboratory technician should pay attention to the role of Wright-Giemsa staining of puncture fluid smear in the preliminary detection and the application of MALDI-TOF MS in identification of uncommon pathogenic microorganisms.

A rare case of pulmonary nocardiosis comorbid with Sjogren's syndrome.

BACKGROUND: Nocardia is an opportunistic pathogen, which occurs in patients with autoimmune diseases and immune dysfunction, and can cause bacteremia and other life-threatening complications. The clinical manifestations of Nocardia pneumonia are similar to tuberculous and other clinical common bacterial pneumonia, but its antibacterial treatments are different and detection methods are unique, which may lead patients to suffer for many years due to clinical misdiagnosis and missed diagnosis. METHODS: Imaging and laboratory examinations were performed for preliminary diagnosis, and next-generation sequencing was used to identify the exact species type of Nocardia in the bronchoalveolar lavage fluid (BALF) of the patient. RESULTS: Imaging and laboratory parameters preliminarily implied that the patient was infected with Nocardia with Sjogren's syndrome (SS), and NGS showed that the strain was N. terpenica. CONCLUSIONS: Accurate etiological diagnosis and corresponding antibiotics are key to improve the prognosis of pulmonary nocardiosis in this case. Nocardia pneumonia is rare in clinical practice; it is of great medical significance to improve the understanding of pulmonary nocardiosis.

A Comparison of Candida Detection in Sputum by the Conventional Culture and Fluorescent Polymerase Chain Reaction Methods.

BACKGROUND Candida is a pathogenic fungus. In recent years, the increase in immunosuppressive diseases has led to an increase in Candida infections, with the lungs being the most common site. Therefore, the aim of this study was to compare the positive detection rates of Candida in sputum samples by Candida culture and fluorescent polymerase chain reaction (PCR), and to explore a new method for rapid, accurate, and effective detection of Candida in sputum, providing swift evidence of clinical fungal infection. MATERIAL AND METHODS From October 2016 to March 2017, 300 sputum samples were collected and detected by the conventional culture method and fluorescent PCR method. The positive rate of Candida detection was compared between the 2 methods. RESULTS In the 300 sputum samples, the positive detection rate of Candida was 50% by the culture method and 65.67% by the fluorescent PCR method (P<0.001). Therefore, the positive detection rate of Candida was higher by the fluorescent PCR method. CONCLUSIONS The conventional culture method for Candida needs a longer duration (24 h to 48 h) and the positive detection rate is low. However, it takes only 3 h to detect Candida in sputum by the fluorescent PCR method, the positive detection rate is high, and can be used as a screening method for Candida in sputum samples. Additional large-scale clinical trials need to be completed to assess the correlation between fluorescent PCR and pulmonary Candida infection.

Prevalence, risk factors and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae in patients from Zhejiang, China, 2008-2018.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is emerging as a worldwide public health concern; however, the long-term molecular epidemiological surveillance of clinical CRKP in China is limited. We conducted a retrospective observational study (2008-2018) to assess the prevalence, susceptibility, risk factors and molecular epidemiology of clinical CRKP isolates. We found the prevalence of CRKP increased from 2.5%, 2008 to 15.8%, 2018. CRKP were significantly more frequent among hospitalized patients from ICU, and it was significantly more likely to be isolated from the capital city (Hangzhou) and the patients aged ≥60 years. Additionally, seasons and specimen types were associated with CRKP infections. The main CRKP sequence type (ST) was ST11, and bla KPC-2 was the most prevalent gene variant. Together these data reveal an increasing incidence and resistance trends among CRKP, especially the ST11-bla KPC-2-CRKP, in Zhejiang, during 2008-2018. Our findings are important for hospitals to limit its dissemination and optimize antibiotic administration.

Whole genome sequence of an Escherichia coli ST131 strain isolated from a patient with bloodstream infection in China co-harbouring blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6')-Ib-cr genes.

OBJECTIVES: Escherichia coli sequence type 131 (ST131) is an international multiresistant high-risk clone associated with a large number of clinical infections. Here we report the draft genome sequence of a ST131 clinical isolate (EC538) obtained from a patient with bloodstream infection (BSI) in China co-harbouring the blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6')-Ib-cr genes. METHODS: Antimicrobial susceptibility testing of E. coli EC538 was performed. DNA of E. coli EC538 was extracted and was sequenced using an Illumina HiSeqTM X Ten platform. Generated sequence reads were assembled using CLC Genomics Workbench. Contigs were annotated using Rapid Annotation using Subsystem Technology (RAST), and further bioinformatics analyses were performed. RESULTS: The total number of assembled bases was 5 420 040bp, with 5611 protein-coding sequences. Escherichia coli EC538 belongs to ST131 by multilocus sequence typing (MLST). The presence of blaKPC-2, blaCTX-M-3 and blaCTX-M-14 genes was detected in addition to other antimicrobial resistance genes conferring resistance to fluoroquinolones, aminoglycosides, trimethoprim, sulfonamides, tetracyclines, macrolides and rifampicin. CONCLUSION: To our knowledge, this is the first report of an E. coli ST131 strain from a BSI co-harbouring blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6')-Ib-cr genes in China. The presented genome sequence of a carbapenemase-producing E. coli ST131 strain could provide further insight into the genomic diversity of this highly virulent, multiresistant and successfully pandemic bacterial pathogen.

Risk Factors for Carbapenem-Resistant Pseudomonas aeruginosa, Zhejiang Province, China.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a public health concern worldwide, but comprehensive analysis of risk factors for CRPA remains limited in China. We conducted a retrospective observational study of carbapenem resistance in 71,880 P. aeruginosa isolates collected in Zhejiang Province during 2015-2017. We analyzed risk factors for CRPA, including the type of clinical specimen; the year, season, and region in which it was collected; patient information, including age, whether they were an outpatient or inpatient, and whether inpatients were in the intensive care unit or general ward; and the level of hospital submitting isolates. We found CRPA was more prevalent among isolates from patients >60 years of age and in inpatients, especially in intensive care units. In addition, specimen types and seasons in which they were collected were associated with higher rates of CRPA. Our findings can help hospitals reduce the spread of P. aeruginosa and optimize antimicrobial drug use.

Epidemiology and risk factors of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci infections in Zhejiang China from 2015 to 2017.

Background: Gram-positive bacteria are dangerous and challenging agents of infection due to their increasing resistance to antibiotics. We aim to analyse the epidemiology and risk factors of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) in Zhejiang China. Methods: Gram-positive bacteria (including S. aureus, Enterococcus faecalis and Enterococcus faecium) were collected from eighty-six hospitals of eleven cities in Zhejiang China from 2015 to 2017. The detection rates of MRSA and VRE infection were calculated for the non-duplicated isolate according to year, region, hospital level, patient age, specimen type and patient category. Meanwhile, the detected resistances of MRSA, E. faecalis and E. faecium to different antibiotics from 2015 to 2017 were compared. The risk factors and the differences in MRSA and VRE detection rates were compared using odds ratio (OR) with 95% confidence interval (95% CI) and Chi-square test respectively. Results: From 2015 to 2017, the detection rates of MRSA and VRE decreased gradually. The cities with the highest MRSA and VRE detection rates tended to be adjacent; for example, the neighbouring cities Hangzhou and Quzhou had simultaneously high rates of MRSA and VRE infection. Patients from IIIA hospital who were older than 75 years and in the intensive care unit (ICU) were most at risk. No vancomycin-resistant isolate was found in MRSA. Resistance of E. faecalis and E. faecium to vancomycin and linezolid decreased slightly and then maintained a low level. Conclusions: The detection rates of MRSA and VRE stayed at moderate and low levels during the three year period of this study, while local dissemination was found in MRSA and VRE isolates. Sustained surveillance is necessary to prevent the spread or clonal dissemination of drug-resistant strains in Zhejiang China.

Increasing prevalence of Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone and resistance to azithromycin in Hangzhou, China (2015-17).

Objectives: Development of resistance in Neisseria gonorrhoeae to ceftriaxone monotherapy or ceftriaxone plus azithromycin dual therapy is a global public health concern. The aim of this study was to analyse the trend in antimicrobial resistance in Hangzhou, China, over the period 2015-17. Methods: In total, 379 clinical isolates were collected from seven hospitals and antimicrobial susceptibility was determined using the agar dilution method. Isolates showing resistance to ceftriaxone, azithromycin or cefixime were analysed for the presence of resistance determinants. STs were determined with the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method and phylogenetic analysis and strain clustering was determined using porB and tbpB sequences. Results: Ceftriaxone resistance, decreased susceptibility to ceftriaxone and azithromycin resistance were observed in 3%, 17% and 21% of the isolates, respectively. This resulted in 5% of the isolates showing both decreased susceptibility to ceftriaxone and azithromycin resistance. Importantly, resistance levels to ceftriaxone and azithromycin increased over the study period, resulting in 5% ceftriaxone resistance, 27% decreased susceptibility to ceftriaxone and 35% azithromycin resistance in 2017 and 11% of the isolates showing both decreased susceptibility to ceftriaxone and azithromycin resistance. Phylogenetic and cluster analysis showed the emergence and expansion in 2017 of a clonally related cluster containing strains with high abundance of decreased susceptibility to ceftriaxone and/or cefixime, which was related to the presence of the mosaic penA allele X. Co-resistance to azithromycin was also observed in this cluster. Conclusions: Our findings have major implications for the future reliability of ceftriaxone monotherapy and ceftriaxone plus azithromycin dual therapy in China.

Safety of Staphylococcus aureus four-antigen and three-antigen vaccines in healthy adults: A meta-analysis of randomized controlled trials.

PURPOSE: Two new Staphylococcus aureus vaccines, S. aureus four-antigen (SA4Ag) and three-antigen (SA3Ag) vaccines, have good immunogenicity and tolerance. However, the safety of these vaccines is worth exploring. Here, we performed a meta-analysis to investigate the safety of SA3Ag and SA4Ag by evaluating systemic and local adverse events. METHODS: The Medline, EMBASE, and Cochrane databases were searched for randomized clinical trials confirming the safety of SA4Ag and SA3Ag. Two investigators independently selected suitable trials, assessed trial quality, and extracted data. RESULTS: Three studies comprising a total of 1,148 participants were included in this review. The two S. aureus vaccines did not increase systemic adverse events (relative ratio 1.1 [95% confidence interval 0.98, 1.24]), but increased the incidence of local adverse events (2.89 [2.15, 3.90]). However, the incidence of severe local adverse events (4.06 [0.78, 21.24]) did not rise significantly. CONCLUSIONS: SA4Ag and SA3Ag have acceptable safety in adults.

Amplification of electrochemical signal by a whole-cell redox reactivation module for ultrasensitive detection of pyocyanin.

A bioelectrochemical sensing system based on a novel whole-cell redox reactivation/cycling module for ultrasensitive detection of pyocyanin (the biomarker of Pseudomonas aeruginosa infections) was developed. The electroactive bacteria mediated redox reactivation module was constructed using Shewanella oneidensis MR-1 cells as the bioelectro-catalyst and lactate as the electron donor. It could regenerate reductive pyocyanin from its oxidative state, which enabled pyocyanin molecule repeatedly registered by the electrode. Uniquely, with this redox reactivation module, the electrochemical response of pyocyanin was amplified about 405 times (1.3 µA/nM vs. 3.2nA/nM). Thus, an ultrasensitive bioelectrochemical sensing system for pyocyanin quantification was developed by integrating the pyocyanin reactivation module with conventional electrochemical detection system. Remarkably, with this developed biosensing system, an extremely low LOD of 47±1pM was reached. Additionally, this biosensing system showed excellent resistance to interferences from human fluids or bacterial contamination. This work provided a simple, ultrasensitive and robust tool for pyocyanin detection, and more importantly, demonstrated a new dimension for electrochemical signal amplification in biosensing.

Characterization of aminoglycoside resistance and virulence genes among Enterococcus spp. isolated from a hospital in China.

This study investigated the aminoglycoside resistance phenotypes and genotypes, as well as the prevalence of virulence genes, in Enterococcus species isolated from clinical patients in China. A total of 160 enterococcal isolates from various clinical samples collected from September 2013 to July 2014 were identified to the species level using the VITEK-2 COMPACT system. The antimicrobial susceptibilities of the identified Enterococcus strains were determined by the Kirby-Bauer (K-B) disc diffusion method. PCR-based assays were used to detect the aminoglycoside resistance and virulence genes in all enterococcal isolates. Of 160 Enterococcus isolates, 105 were identified as E. faecium, 35 as E. faecalis, and 20 isolates were classified as "other" Enterococcus species. High-level aminoglycoside resistance (HLAR) for gentamicin, streptomycin, and both antibiotics was identified in 58.8, 50, and 34.4% of strains, respectively. The most common virulence gene (50.6% of isolates) was efaA, followed by asa1 (28.8%). The most prevalent aminoglycoside resistance genes were aac(6')-Ie-aph(2''), aph(2')-Id, aph(3')-IIIa, and ant(6')-Ia, present in 49.4%, 1.3%, 48.8% and 31.3% of strains, respectively. Overall, E. faecium and E. faecalis were most frequently associated with hospital-acquired enterococcal infections in Zhejiang Province. All aminoglycoside resistance genes, except aph(2'')-Id, were significantly more prevalent in HLAR strains than amongst high level aminoglycoside susceptible (HLAS) strains, while there was no significant difference between HLAR and HLAS strains in regard to the prevalence of virulence genes, apart from esp, therefore, measures should be taken to manage infections caused by multi-drug resistant Enterococcus species.

X-linked agammaglobulinemia combined with juvenile idiopathic arthritis and invasive Klebsiella pneumoniae polyarticular septic arthritis.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA can also present in combination with juvenile idiopathic arthritis (JIA), the major chronic rheumatologic disease in children. We report herein the first known case of a juvenile patient diagnosed with XLA combined with JIA that later developed into invasive Klebsiella pneumoniae polyarticular septic polyarthritis. An additional comprehensive review of XLA combined with JIA and invasive K. pneumoniae septic arthritis is also presented. XLA was identified by the detection of BTK mutations while the diagnosis of JIA was established by clinical and laboratory assessments. Septic arthritis caused by invasive K. pneumoniae was confirmed by culturing of the synovia and gene detection of the isolates. Invasive K. pneumoniae infections can not only result in liver abscesses but also septic arthritis, although this is rare. XLA combined with JIA may contribute to invasive K. pneumoniae infection.

Expression and purification of SenX3 from the Mycobacterium tuberculosis strain H37Rv in Escherichia coli.

Infection with the bacterium Mycobacterium tuberculosis (MTB) causes tuberculosis, a pulmonary infection that may be fatal if left untreated. Misuse or mismanagement of tuberculosis drugs may lead to drug-resistant pathogen forms that are difficult to treat and contribute to a global health problem. The MTB SenX3/RegX3 signal transduction system allows bacteria to externally sense the environment and mediate an appropriate internal response; SenX3 is also associated with MTB virulence, suggesting that this protein may provide a potential therapeutic target. To investigate the role of SenX3 and MTB drug resistance, SenX3 was cloned, expressed and purified in Escherichia coli. SenX3 was cloned from the genome of the MTB strain H37Rv by polymerase chain reaction and an internal NcoI restriction site was destroyed by site-directed mutagenesis to allow cloning into the pET-28b prokaryotic expression vector. SenX3 expression from the resulting pET­28b-mSenX3 plasmid was induced with isopropyl ß-D-thiogalactoside and the protein was purified using Ni-NTA agarose affinity chromatography. A pure protein of the expected size was identified. The examination of purified SenX3 protein is considered to enable the in­depth investigation of SenX3-mediated drug resistance.