Microbiomes detected by cerebrospinal fluid metagenomic next-generation sequencing among patients with and without HIV with suspected central nervous system infection.

BACKGROUND: Opportunistic infections in the central nervous system (CNS) can be a serious threat to people living with HIV. Early aetiological diagnosis and targeted treatment are crucial but difficult. Metagenomic next-generation sequencing (mNGS) has significant advantages over traditional detection methods. However, differences in the cerebrospinal fluid (CSF) microbiome profiles of patients living with and without HIV with suspected CNS infections using mNGS and conventional testing methods have not yet been adequately evaluated. METHODS: We conducted a retrospective cohort study in the first hospital of Changsha between January 2019 and June 2022 to investigate the microbiomes detected using mNGS of the CSF of patients living with and without HIV with suspected CNS infections. The pathogens causing CNS infections were concurrently identified using both mNGS and traditional detection methods. The spectrum of pathogens identified was compared between the two groups. RESULTS: Overall, 173 patients (140 with and 33 without HIV) with suspected CNS infection were enrolled in our study. In total, 106 (75.7%) patients with and 16 (48.5%) patients without HIV tested positive with mNGS (p = 0.002). Among the enrolled patients, 71 (50.7%) with HIV and five (15.2%) without HIV tested positive for two or more pathogens (p < 0.001). Patients with HIV had significantly higher proportions of fungus (20.7% vs. 3.0%, p = 0.016) and DNA virus (59.3% vs. 21.2%, p < 0.001) than those without HIV. Epstein-Barr virus (33.6%) was the most commonly identified potential pathogen in the CSF of patients living with HIV using mNGS, followed by cytomegalovirus (20.7%) and torque teno virus (13.8%). The top three causative pathogens identified in patients without HIV were Streptococcus (18.2%), Epstein-Barr virus (12.1%), and Mycobacterium tuberculosis (9.1%). In total, 113 patients living with HIV were diagnosed as having CNS infections. The rate of pathogen detection in people living with HIV with a CNS infection was significantly higher with mNGS than with conventional methods (93.8% vs. 15.0%, p < 0.001). CONCLUSION: CSF microbiome profiles differ between patients living with and without HIV with suspected CNS infection. mNGS is a powerful tool for the diagnosis of CNS infection among people living with HIV, especially in those with mixed infections.

Safety of different amphotericin B formulations among AIDS patients with invasive fungal disease: a retrospective observational study.

We conducted a retrospective, observational study among acquired immune deficiency syndrome (AIDS) patients with cryptococcal meningitis or talaromycosis to assess AmB formulations-related adverse events (AEs). Total 205 eligible patients were enrolled. Of them, 139 received AmB therapy, 51 received liposomal AmB (L-AmB) therapy, and 15 received AmB cholesteryl sulfate complex (ABCD) therapy. The incidences of total AEs between the AmB, L-AmB and ABCD group had no significant differences. The ABCD group had significantly higher incidences of hepatotoxicity and hematological toxicity than the AmB and L-AmB groups. The incidence of grade 3-4 hematological toxicity in the ABCD group was significantly higher than that in the AmB and L-AmB groups. Multinomial logistic regression models showed that compared with AmB, ABCD had a higher risk for the occurrence of grade 3-4 hematological toxicity (aOR = 43.924, 95%CI 6.296-306.418; p < 0.001). We demonstrated that ABCD was more prone to hepatotoxicity and hematological toxicity than AmB and L-AmB among AIDS patients, which is worth noting.

Clinical Characteristics of Refractory Coronavirus Disease 2019 in Wuhan, China.

BACKGROUND: Since December 2019, coronavirus disease 2019 (COVID-19), caused by severe adult respiratory syndrome coronavirus 2, occurred in Wuhan, and rapidly spread throughout China. This study aimed to clarify the characteristics of patients with refractory COVID-19. METHODS: In this retrospective single-center study, we included 155 consecutive patients with confirmed COVID-19 in Zhongnan Hospital of Wuhan University from 1 January to 5 February. The cases were divided into general and refractory COVID-19 groups according to the clinical efficacy of treatment after hospitalization, and the differences between groups were compared. RESULTS: Compared with patients with general COVID-19 (45.2%), those with refractory disease were older, were more likely to be male, and had more underlying comorbid conditions, a lower incidence of fever, higher maximum temperatures among patients with fever, higher incidences of shortness of breath and anorexia, more severe disease assessment at admission, higher neutrophil, aspartate aminotransferase, lactate dehydrogenase, and C-reactive protein levels, lower platelet counts and albumin levels, and higher incidences of bilateral pneumonia and pleural effusion (P < .05). Patients with refractory COVID-19 were more likely to receive oxygen, mechanical ventilation, expectorant, and adjunctive treatment, including corticosteroids, antiviral drugs, and immune enhancers (P < .05). Considering the factors of disease severity at admission, mechanical ventilation, and intensive care unit transfer, patients with refractory COVID-19 were also more likely to be male, have manifestations of anorexia on admission, and receive oxygen, expectorant, and adjunctive agents (P < .05). CONCLUSION: In nearly 50% of patients with COVID-19 obvious clinical and radiological remission was not achieved within 10 days after hospitalization. Male, anorexia, and no fever at admission was predictive of poor treatment efficacy.

Characteristics of Peripheral Lymphocyte Subset Alteration in COVID-19 Pneumonia.

BACKGROUND: In December 2019, novel coronavirus (SARS-CoV-2) pneumonia (COVID-19) was reported in Wuhan and has since rapidly spread throughout China. We aimed to clarify the characteristics and clinical significance of peripheral lymphocyte subset alteration in COVID-19. METHODS: The levels of peripheral lymphocyte subsets were measured by flow cytometry in 60 hospitalized COVID-19 patients before and after treatment, and their association with clinical characteristics and treatment efficacy was analyzed. RESULTS: Total lymphocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells decreased in COVID-19 patients, and severe cases had a lower level than mild cases. The subsets showed a significant association with inflammatory status in COVID-19, especially CD8+ T cells and CD4+/CD8+ ratio. After treatment, 37 patients (67%) showed clinical response, with an increase in CD8+ T cells and B cells. No significant change in any subset was detected in nonresponsive cases. In multivariate analysis, posttreatment decrease in CD8+ T cells and B cells and increase in CD4+/CD8+ ratio were indicated as independent predictors of poor efficacy. CONCLUSIONS: Peripheral lymphocyte subset alteration was associated with clinical characteristics and treatment efficacy of COVID-19. CD8+ T cells tended to be an independent predictor for COVID-19 severity and treatment efficacy.

Risk factors for delayed negative conversion of SARS-CoV-2 in patients with COVID-19 pneumonia: a retrospective cohort study.

The epidemic of coronavirus disease 2019 (COVID-19) began in China and had spread rapidly to many other countries. This study aimed to identify risk factors associated with delayed negative conversion of SARS-CoV-2 in COVID-19 patients. In this retrospective single-centre study, we included 169 consecutive patients with confirmed COVID-19 in Zhongnan Hospital of Wuhan University from 15th January to 2nd March. The cases were divided into two groups according to the median time of SARS-CoV-2 negative conversion. The differences between groups were compared. In total, 169 patients had a median virus negative conversion time of 18 days (interquartile range: 11-25) from symptom onset. Compared with the patients with short-term negative conversion, those with long-term conversion had an older age, higher incidence of comorbidities, chief complaints of cough and chest distress/breath shortness and severer illness on admission, higher level of leucocytes, neutrophils, aspartate aminotransferase, creatine kinase and erythrocyte sedimentation rate (ESR), lower level of CD3+CD4+ lymphocytes and albumin and more likely to receive mechanical ventilation. In multivariate analysis, cough, leucocytes, neutrophils and ESR were positively correlated with delayed virus negative conversion, and CD3+CD4+ lymphocytes were negatively correlated. The integrated indicator of leucocytes, neutrophils and CD3+CD4+ lymphocytes showed a good performance in predicting the negative conversion within 2 weeks (area under ROC curve (AUC) = 0.815), 3 weeks (AUC = 0.804), 4 weeks (AUC = 0.812) and 5 weeks (AUC = 0.786). In conclusion, longer quarantine periods might be more justified for COVID-19 patients with cough, higher levels of leucocytes, neutrophils and ESR and lower levels of CD3+CD4+ lymphocytes.

Life-threatening gastrointestinal bleeding caused by cytomegalovirus-induced duodenal ulcer in a patient with AIDS: A case report.

Background: The reasons for gastrointestinal bleeding among patients with acquired immune deficiency syndrome (AIDS) were complex. Here we present an unusual case of life-threatening gastrointestinal bleeding caused by a cytomegalovirus-induced duodenal ulcer in an AIDS patient. Case presentation: A 31-year-old male with AIDS was admitted on July 18, 2023, complaining of abdominal pain for 38 days and intermittent hematochezia for 12 days. During his hospitalization, gastrointestinal endoscopy attributed gastrointestinal bleeding to a giant duodenal ulcer. Furthermore, cytomegalovirus(CMV) infection was confirmed as the reason for the ulcer through metagenomic next-generation sequencing (mNGs), hematoxylin-eosin(HE) staining, and immunohistochemistry (IHC) staining for the biopsy tissue. The patient's gastrointestinal bleeding was stopped by interventional embolization. Following a 4-week course of anti-CMV treatment, the giant duodenal ulcer was cured. Conclusions: For AIDS patients with gastrointestinal bleeding, the CMV-induced gastrointestinal ulcer should be considered. Comprehensive mothods (mNGs, HE staining and IHC staining for biopsy tissue) were benefit for confirmed diagnosis. Beside anti-CMV treatment, the interventional embolization is a choice for hemostasis.

Case Report: Giant Oral Ulcers Attributed to Cytomegalovirus Infection in a Patient with AIDS.

Oral ulcers are often neglected in patients with AIDS. However, giant oral ulcers are uncommon and are usually suspected to be malignant lesions. Our study presents a case of giant ulcers in an AIDS patient that were initially suspected to be oral cancer. To assist with diagnosis, conventional microbiological tests, metagenomic next-generation sequencing, and a pathological examination were conducted on oral lesion biopsy specimens. The case was finally confirmed via hematoxylin-eosin staining and immunohistochemical staining to be a cytomegalovirus infection.

Metagenomic next-generation sequencing versus traditional laboratory methods for the diagnosis of central nervous system opportunistic infections in HIV-infected Chinese adults.

To evaluate clinical value of metagenomic next-generation sequencing (mNGS) in people living with HIV/AIDS (PLWHA) who had CNS disorders. Cerebrospinal fluid (CSF) samples from 48 PLWHA presenting with CNS disorders were sequenced using mNGS and compared with clinical conventional diagnostic methods. In total, 36/48 ss(75%) patients were diagnosed with pathogen(s) infection by mNGS, and the positive detection proportion by mNGS was higher than that by clinical conventional diagnostic methods (75% vs 52.1%, X2 = 5.441, P = 0.020). Thirteen out of 48 patients (27.1%) were detected with 3-7 pathogens by mNGS. Moreover, 77 pathogen strains were detected, of which 94.8% (73/77) by mNGS and 37.0% (30/77) by clinical conventional methods (X2 = 54.206, P < 0.001). The sensitivity and specificity of pathogens detection by mNGS were 63.9% (23/36) and 66.7% (8/12), respectively, which were superior to that by clinical conventional methods (23/36 vs 9/25, X2 = 4.601, P = 0.032; 8/12 vs 5/23, X2 = 5.029, P = 0.009). The application of mNGS was superior for its ability to detect a variety of unknown pathogens and multiple pathogens infection, and relatively higher sensitivity and specificity in diagnosis of CNS disorders in PLWHA.

Characterization of peripheral cytokine-secreting cells responses in HIV/TB co-infection.

Background: Currently the responses of peripheral cytokine-secreting cells in the natural course of human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection haven't been fully elucidated. Methods: The function of peripheral proinflammatory, regulatory and cytotoxic cytokine-secreting cells were investigated by direct intracellular cytokine staining (ICS) and flow cytometry, additionally, the absolute numbers of different cytokine-secreting cells were measured among patients with HIV/TB co-infection (HT group), and compared them with the healthy controls (HC group), patients with TB (TB group) and patients with HIV infection (HIV group). After one week's anti-TB treatment, the changes of the percentages of cytokine-secreting cells were further evaluated in TB and HT groups. Results: Totally 26 individuals in the HC group, 51 in the TB group, 26 in the HIV group and 29 in the HT group were enrolled. The HT. HT group exhibited significantly lower absolute numbers of IFN-γ+CD4+, IFN-γ+CD8+, TNF-α+CD4+, IL17A+CD4+ T cells and TNF-α+CD14+ monocytes than the TB and HIV groups. Compared with the TB group, the percentages of CD8+ T cells secreting IFN-γ and perforin (p=0.010; p=0.043) were significantly lower among the HT group. Compared with the HIV group, the percentages of CD4+, CD8+ T cells and CD14+ monocytes secreting TNF-α (p=0.013; p=0.001; p<0.001) were significantly decreased, and the percentage of CD8+ T cells secreting IL-17A (p=0.015) was significantly increased among the HT group. Both the percentages of CD4+ T cells secreting TGF-ß (p<0.001; p=0.001), and CD4+ and CD8+ T cells secreting granzyme A (all p<0.001), were significantly higher among the HT group than among the TB group and HIV group. After one week's anti-TB treatment, an increased percentage of CD4+ T cells secreting TNF-α (p=0.003) was found in the TB group, and an increased percentage of CD8+ T cells secreting TNF-α (p=0.029) was found in the HT group. Conclusion: Significantly different functional profiles of peripheral proinflammatory, regulatory, and cytotoxic cytokine-secreting cells were observed in the natural course of HIV/TB co-infection compared to TB and HIV infection alone, even though the absolute numbers of those cells were significantly lower in HIV/TB co-infection. TNF-α-secreting CD8+ T cells may be a more sensitive marker for early evaluation of anti-TB treatment efficacy in patients with HIV/TB co-infection.

A longitudinal sampling study of transcriptomic and epigenetic profiles in patients with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is a novel tick-borne infectious disease caused by a new type of SFTS virus (SFTSV). Here, a longitudinal sampling study is conducted to explore the differences in transcript levels after SFTSV infection, and to characterize the transcriptomic and epigenetic profiles of hospitalized patients. The results reveal significant changes in the mRNA expression of certain genes from onset to recovery. Moreover, m6A-seq reveals that certain genes related with immune regulation may be regulated by m6A. Besides the routine tests such as platelet counts, serum ALT and AST levels testing, distinct changes in myocardial enzymes, coagulation function, and inflammation are well correlated with the clinical data and sequencing data, suggesting that clinical practitioners should monitor the above indicators to track disease progression and guide personalized treatment. In this study, the transcript changes and RNA modification may lend a fresh perspective to our understanding of the SFTSV and play a significant role in the discovery of drugs for effective treatment of this disease.

Family cluster of three recovered cases of pneumonia due to severe acute respiratory syndrome coronavirus 2 infection.

The coronavirus disease (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, in late 2019 and has affected more than 1 270 000 people worldwide. The numbers of reported cases continue to rise and threaten global health. Transmissions among family members are frequently observed, although the route of transmission is partially known. Here we report three cases of SARS-CoV-2 infection within one family. Sequencing of the S gene of the viral genome showed 100% identity among samples, suggesting that the same strain caused the infection. Following treatment with oseltamivir and short-term methylprednisolone combined with symptomatic management, all three patients recovered within 3 weeks, as evidenced by the disappearance of their symptoms, clearance of pulmonary infiltrates and consecutive negative molecular diagnostic test findings. Our observations suggest the importance of preventing family transmission and the efficacy of current integrated treatment for mild/moderate pneumonia in COVID-19 cases.

Clinical Characteristics and Outcomes of Older Patients with Coronavirus Disease 2019 (COVID-19) in Wuhan, China: A Single-Centered, Retrospective Study.

BACKGROUND: In December 2019, the coronavirus disease 2019 (COVID-19) emerged in Wuhan city and spread rapidly throughout China and the world. In this study, we aimed to describe the clinical course and outcomes of older patients with COVID-19. METHODS: This is a retrospective investigation of hospitalized older patients with confirmed COVID-19 at Zhongnan Hospital of Wuhan University from January 1, 2020, to February 10, 2020. RESULTS: In total, 203 patients were diagnosed with COVID-19, with a median age of 54 years (interquartile range, 41-68; range, 20-91 years). Men accounted for 108 (53.2%) of the cases, and 55 patients (27.1%) were more than 65 years of age. Among patients who were 65 years and older, the mortality rate was 34.5% (19/55), which was significantly higher than that of the younger patients at 4.7% (7/148). Common symptoms of older patients with COVID-19 included fever (94.5%; n = 52), dry cough (69.1%; n = 38), and chest distress (63.6%; n = 35). Compared with young patients, older patients had more laboratory abnormalities and comorbidities. Through a multivariate analysis of the causes of death in older patients, we found that males, comorbidities, time from disease onset to hospitalization, abnormal kidney function, and elevated procalcitonin levels were all significantly associated with death. CONCLUSIONS: In the recent outbreak of COVID-19, our local hospital in Wuhan found that patients aged 65 and older had greater initial comorbidities, more severe symptoms, and were more likely to experience multiorgan involvement and death, as compared to younger patients.

Clinical characteristics of four cancer patients with SARS-CoV-2 infection in Wuhan, China.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the outbreak of pneumonia in Wuhan. The virus is highly infectious. Patients with cancer might be susceptible to the viral infection because of the immunosuppressive state cause by therapies on tumors. CASE PRESENTATION: We present the clinical features of four cancer patients who were infected with SARS-CoV-2 in late January of 2020 in our hospital. Cases 1 and 3 were diagnosed as mild and common type of coronavirus disease 2019 (COVID-2019) and survived from the viral infection. They acquired SARS-CoV-2 infection during their staying in hospital under radiotherapy and surgery of the tumors. Cases 2 and 4 suffered from severe type of COVID-19, and Case 2 was dead owning to the advanced age, uncontrolled chronic B cell lymphocytic leukemia and many other underlying diseases. The immunosuppressive state induced by liver transplantation and anti-rejection therapy might contribute to the severity of COVID-19 in Case 4, who suffered from hepatitis B related hepatocellular carcinoma. However, Case 4 was recovered from COVID-19 after a combination therapy against virus, bacteria and fungi, and also respiratory support. Nearly all patients showed a decrease in lymphocytes including total CD3+ T cells, B cells, and natural killer cells after infection of the virus. CONCLUSIONS: The severity of COVID-19 might be influenced by immune system state and underlying diseases in cancer patients. And the treatment of SARS-CoV-2 infection in cancer patients is challenged by the immunosuppressive state of these patients under chemotherapy or surgery.

Transport and numerical simulation of Cu2+ in saturated porous medium in the presence of magnetic nanoparticles.

Fe3O4 magnetic nanoparticles (MNPs) can control and remove heavy metal pollution from wastewater. This approach has gained broad attention due to its excellent surface properties. However, there have been limited studies for Cu2+ retention and transfer regulation in saturated porous media in the presence of MNPs. The objectives of this study were to analyze the migration and deposition mechanism of Cu2+ under different conditions through static adsorption and numerical models. The results indicated that the MNPs-quartz sand had better adsorption capacity for Cu2+ (59.1 mg/kg) than quartz sand only (26.84 mg/kg), and thus it inhibited the migration of Cu2+; the effect improved with increasing MNP content. Furthermore, high ion strength (IS) and flow velocity were beneficial to the migration of Cu2+, and a high pH inhibited the migration of Cu2+. The numerical simulation results showed that the two-site model (TSM) nicely fitted the migration of Cu2+ in quartz sand and MNPs-sand. The migration of Cu2+ in both media was affected by chemical nonequilibrium. We also found that the presence of MNPs had little impact on the dispersion of porous media by observing the fitting parameters D (dispersion coefficient) 0.202 for both media. Our results can evaluate the risk of heavy metal migration and retention in saturated porous media in the presence of nanoparticles; this can prevent aquifer pollution.

The effect of HBx gene on the apoptosis of hepatic cells.

To study the effect of HBx gene on the apoptosis of the cell lines (L02, HepG2) and the interaction between HBx and X-linked inhibitor of apoptosis protein (XIAP), the apoptosis of pcDNA3.1-HBx transiently transfected cell lines (L02, HepG2) was detected by flow cytometry and the mRNA expression of XIAP was assayed by real-time RT-PCR. Our study showed (1) the morphology of L02/pcDNA3.1-HBx was changed and the appearance of the cells mimicked that of HepG2 cells; (2) HBx gene could be detected in L02/pcDNA3.1-HBx and HepG2/ pcDNA3.1-HBx; (3) the apoptosis rate of L02/pcDNA 3.1-HBx was higher than that of L02 cells (P<0.01) and the apoptosis rate of HepG2/pcDNA3.1-HBx was lower than that of HepG2 cells (P<0.05); (4) the XIAP expression in L02 was about 3 times that in L02/pcDNA3.1-HBx cells (P<0.01), and the expression of XIAP in HepG2/pcDNA3.1-HBx was about 4 times that in HepG2 (P<0.01). It is concluded that HBx gene may promote the apoptosis of normal hepatocytes and inhibit the apoptosis of cells of hepatic carcinoma by regulating the expression of XIAP.

[Feasibility and its clinical significance of detection of LUNX mRNA expression in diagnosis of micrometastasis for non-small cell lung cancer].

BACKGROUND: There is important significance of micrometastasis for the individual treatment and prognosis of non-small cell lung cancer (NSCLC). LUNX is a lung-specific gene found recently. The aim of this study is to detect LUNX mRNA expression in NSCLC patients in order to discuss the possibility of indicating lung cancer micrometastasis by LUNX. METHODS: Fluorescence quantitative reverse transcription-polymerase chain reaction (FQ-RT-PCR) and ordinary RT-PCR were used to detect LUNX mRNA in cancer tissues, bone marrow and peripheral blood from 62 patients with NSCLC. Lung tissue, bone marrow and peripheral blood from 10 patients with pulmonary benign diseases and peripheral blood from 10 healthy volunteers were served as controls. RESULTS: LUNX mRNA was expressed in all the lung tissues, either malignant or benign. No bone marrow and peripheral blood sample was positive for LUNX mRNA in controls. The positive detection rate of LUNX mRNA for NSCLC was 38.7% (24/62) in bone marrow, 29.0% (18/62) in peripheral blood, and 45.2% (28/62) in either. The positive rate of LUNX mRNA for NSCLC in bone marrow increased according to the stage of disease and there was a statistical significance (P=0.02), aod there was a correlation between bone marrow and peripheral blood expression in NSCLC (P < 0.001). CONCLUSIONS: LUNX mRNA is an efficient indicating factor on sensitivity and specificity to detect early haematogenous dissemination of cancer cells for patients with NSCLC. This method may lead to an earlier diagnosis of metastasis for lung cancer and help to evaluate the cancer more correctly and make the best treatment plan.

The construction of a supramolecular polymeric rotaxane from bipyridine-ruthenium and cyclodextrin.

A luminescent supramolecular link is constructed by a very simple method using bipyridine-ruthenium and cyclodextrin, which displays not only a quasi-linear structure, but also a satisfactory fluorescence emission in both solution and the solid state.

Co-infection of visceral leishmaniasis and HIV-1: a surviving case in China and review of treatment strategies.

Co-infection of visceral leishmaniasis (VL) and human immunodeficiency virus type 1 (HIV-1) is known to have higher rates of initial treatment failure, relapse and mortality than in those without HIV-1 infection. Co-infection of VL and HIV-1 usually results in death by the end of treatment in previously reported cases in China. Here we report on a patient with VL and HIV-1 co-infection who received a high dose and an extended course of sodium stibogluconate treatment in addition to antiretroviral therapy (ART). This treatment regimen resulted in good control of VL and HIV-1 infection, while the conventional protocol of sodium stibogluconate treatment was not able to prevent multiple VL relapses. To the best of our knowledge, this is the first surviving case of VL and HIV-1 co-infection with this particular treatment regimen in China.

Promoter methylation and expression of TIMP3 gene in gastric cancer.

BACKGROUND: Gastric carcinoma development is a multi-stage process that involves more than one gene. Aberrant changes in DNA methylation are considered as the third mechanism that leads to anti-oncogene inactivation, which plays an essential role in tumor development. In this study, we assessed the relationship among the aberrant methylation of the promoter CpG islands of tissue inhibitor of metalloproteinase 3 (TIMP3) gene, its protein expression, and the clinicopathological features of gastric adenocarcinoma. METHODS: The methylation status of the promoter CpG islands and the protein expression of TIMP3 gene in tumors and adjacent normal mucosal tissues of 78 patients with gastric adenocarcinoma were detected by methylation-specific PCR (MSP) and immunohistochemistry. RESULTS: The CpG island methylation of TIMP3 was detected in tumor tissues, cancer-adjacent tissues, and lymph nodes with metastasis. In increasing order, the hypermethylation frequency of these tissues were 35.9% (28 of 78 non-neoplastic tissues), 85% (17 of 20 early-stage cases), 89.7% (52 of 58 progressive-stage cases), and 100% (78 of 78 metastatic lymph node). A marked difference was found between tumors and non-neoplastic tissues (P<0.05), but no difference existed among the subgroups of tumors (P>0.05). Immunohistochemistry analysis confirmed TIMP3 down-regulation in tumor tissues. The rate of TIMP3 gene expression was 100% in non-neoplastic tissues but apparently decreased to various extents at different stages, i.e., decreased to 30% (6/20) at the early stage, to 3.4% (2/58) at the progressive stage, and to 0% (0/78) in metastatic lymph nodes. Among the 70 tumor tissues with negative TIMP3 expression, 64 (91.4%) were hypermethylated and 6 were unmethylated (8.6%), indicating a significant association between hypermethylation and reduced or negative TIMP3 expression (P<0.01). CONCLUSION: The hypermethylation of the promoter region in CpG islands is the main mechanism of TIMP3 gene expression and may provide evidence for the molecular diagnosis and stage evaluation of gastric cancer. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1756134016954958.