Sexually transmitted coinfections among at-risk HIV-positive MSM: implications for optimal preemptive treatment.

Background: Concurrent sexually transmitted infections (STIs) are common in sexually active populations. We aimed to estimate the prevalence and coinfection rates of bacterial STIs among sexually active, HIV-positive men who have sex with men (MSM), and to assess the potential benefits of different combination treatment regimens in managing concurrent bacterial STIs. Methods: From September 2021 to September 2023, HIV-positive MSM underwent STI testing when they had symptoms suggestive of STIs or recently acquired hepatitis C virus (HCV) infection or early syphilis. The oral rinse, rectal swab, and urethral swab specimens were tested for Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma spp., Ureaplasma spp., and Trichomonas vaginalis with the use of multiplex real-time polymerase-chain-reaction assays. The estimated coinfection rates were used to evaluate the benefits of different combination treatment regimens for managing coinfections. Results: During the study period, 535 participants (median age, 37 years; and CD4 count, 615 cells/mm3) were enrolled. On their first visits, at least one bacterial pathogen was detected in 57.9% and concomitant bacterial infections were found in 32.9% of the participants. The most commonly identified pathogen was U. urealyticum (36.3%), followed by C. trachomatis (22.8%), and N. gonorrhoeae (19.8%). The factors associated with any bacterial STIs included older age (per 1-year increase, adjusted odds ratio [AOR], 0.97; 95% confidence interval [CI], 0.95-1.00), early syphilis (AOR, 1.87; 95% CI, 1.22-2.84), and having more than 5 sex partners in the preceding 3 months (AOR, 2.08, 95% CI, 1.07-4.06). A combination therapy of benzathine penicillin G with a 7-day course of doxycycline could simultaneously treat 27.1% of C. trachomatis coinfections in participants with early syphilis, while a combination therapy of ceftriaxone with doxycycline could simultaneously treat 40.6% of chlamydial coinfections in participants with gonorrhea. Conclusion: Bacterial STIs were prevalent and concomitant infections were not uncommon among sexually active, HIV-positive MSM, supporting regular screening for bacterial STIs. The effectiveness of preemptive use of doxycycline as combination therapy for concurrent STIs warrants more investigations.

Optimal Frequency of Hepatitis C Virus (HCV) RNA Testing for Detection of Acute HCV Infection Among At-risk People With Human Immunodeficiency Virus: A Multicenter Study.

Using 3-stage pooled-plasma hepatitis C virus (HCV) RNA testing performed quarterly among at-risk people with human immunodeficiency virus (PWH), we found that if testing had been performed every 6 or 12 months, 58.6%-91.7% of PWH who recently acquired HCV would be delayed for diagnosis and might contribute to onward HCV transmission with longer durations.

Risky sexual practices and hepatitis C viremia among HIV-positive men who have sex with men in Taiwan.

BACKGROUND: Understanding the risk behaviors associated with sexually-transmitted hepatitis C virus (HCV) infection among men who have sex with men (MSM) may inform the public health policies and interventions aiming to achieve HCV microelimination. METHODS: HIV-positive MSM who had one of the following conditions were enrolled to undergo face-to-face questionnaire interviews to collect information on their sexual practices in the past 12 months: (1) elevation of aminotransferases in the past 6 months; (2) acquisition of sexually transmitted infections in the past 6 months; and (3) previous HCV infections. Plasma HCV RNA were tested at enrolment and every 3 months during follow-up. Baseline characteristics and risky behaviors were compared to identify factors associated with HCV viremia between HCV-viremic MSM and HCV-aviremic MSM in multivariate analysis. RESULTS: Among 781 MSM with a median age of 36 years, 57 (7.3%) had HCV viremia and 724 (92.7%) no HCV viremia during follow-up. A high proportion (38.9%) of the participants reported having used recreational drugs in the past 12 months, with 34.4% of them having slamming, but only 4.8% reported having shared their injection equipment. In multivariate analysis, use of recreational drugs (adjusted odds ratio [aOR], 2.14; 95% CI, 1.16-3.96), having participated in group sex (aOR, 2.35; 95% CI 1.24-4.40) and having had condomless receptive anal intercourse (aOR, 1.97; 95% CI 1.07-3.62) were significantly associated with HCV viremia. CONCLUSION: Among high-risk HIV-positive MSM, use of recreational drugs and risky sexual contacts were associated with HCV viremia, suggesting the mucosal contacts as the major route of HCV transmission.

Three-Stage Pooled Plasma Hepatitis C Virus RNA Testing for the Identification of Acute HCV Infections in At-Risk Populations.

Timely diagnosis and treatment of hepatitis C virus (HCV) infection may prevent its transmission. We evaluated the performance and cost reductions of the pooled plasma HCV RNA testing strategy to identify acute HCV infections among people living with HIV (PLWH). PLWH with sexually transmitted infections, elevated aminotransferases within the past 6 months or past HCV infections (high-risk) and those without (low-risk) were enrolled prospectively. Participants underwent three-stage pooled plasma HCV RNA testing every 12 to 24 weeks until detection of HCV RNA or completion of a 48-week follow-up. The three-stage strategy combined 20 individual specimens into a stage 1 pool, 5 individual specimens from the stage 1 pool that tested positive for HCV RNA in the stage 2 mini-pool, followed by testing of individual specimens of the stage 2 mini-pool tested positive for HCV RNA. A simulation was constructed to investigate the cost reductions and pooled sensitivity and specificity under different combinations of HCV prevalence and pool/mini-pool sizes. Between June 25, 2019 and March 31, 2021, 32 cases of incident HCV viremia were identified in 760 high-risk PLWH that were enrolled 834 times, giving an incidence rate of 56.6 per 1000 person-years of follow-up (PYFU). No cases of HCV viremia were identified in 557 low-risk PLWH during a total of 269.2 PYFU. Simulation analysis suggested that this strategy could reduce HCV RNA testing cost by 50% to 86% with HCV viremia prevalence of 1% to 5% and various pooled sizes despite compromised pooled sensitivity. This pooled plasma HCV RNA testing strategy is cost-saving to identify acute HCV infections in high-risk populations with HCV viremia prevalence of 1% to 5%. IMPORTANCE Our three-stage pooled plasma HCV RNA testing successfully identified HCV viremia in high-risk PLWH with a testing cost reduction of 84.5%. Simulation analysis offered detailed information regarding the selection of pool and mini-pool sizes in settings of different HCV epidemiology and the performance of HCV RNA testing to optimize the cost reduction.

Hepatitis C microelimination among people living with HIV in Taiwan.

To reach the WHO target of hepatitis C virus (HCV) elimination by 2025, Taiwan started to implement free-of-charge direct-acting antiviral (DAA) treatment programme in 2017. Evaluating the progress of HCV microelimination among people living with HIV (PLWH) is a critical step to identify the barriers to HCV elimination. PLWH seeking care at a major hospital designated for HIV care in Taiwan between January 2011 and December 2021 were retrospectively included. For PLWH with HCV-seropositive or HCV seroconversion during the study period, serial HCV RNA testing was performed using archived samples to confirm the presence of HCV viremia and estimate the prevalence and incidence of HCV viremia. Overall, 4199 PLWH contributed to a total of 27,258.75 person-years of follow-up (PYFU). With the reimbursement of DAAs and improvement of access to treatments, the prevalence of HCV viremia has declined from its peak of 6.21% (95% CI, 5.39-7.12%) in 2018 to 2.09% (95% CI, 1.60-2.77%) in 2021 (decline by 66.4% [95% CI, 55.4-74.7%]); the incidence has declined from 25.94 per 1000 PYFU (95% CI, 20.44-32.47) in 2019 to 12.15% per 1000 PYFU (95% CI, 8.14-17.44) (decline by 53.2% [95% CI, 27.3-70.6%]). However, the proportion of HCV reinfections continued to increase and accounted for 82.8% of incident HCV infections in 2021. We observed significant declines of HCV viremia among PLWH with the expansion of the DAA treatment programme in Taiwan. Further improvement of the access to DAA retreatments is warranted to achieve the goal of HCV microelimination.

Performance of Hepatitis C Virus (HCV) Core Antigen Assay in the Diagnosis of Recently Acquired HCV Infection among High-Risk Populations.

How the hepatitis C virus (HCV) core antigen (HCVcAg) assay performs in detecting recently acquired HCV infection among people living with HIV (PLWH) and HIV-negative men who have sex with men (MSM) is rarely assessed in the Asia-Pacific region. High-risk participants, including PLWH with sexually transmitted infections (STIs), HCV clearance by antivirals or spontaneously, or elevated aminotransferases, HIV-negative MSM with STIs or on HIV preexposure prophylaxis, and low-risk PLWH were enrolled. Blood samples were subjected to 3-stage pooled-plasma HCV RNA testing every 3 to 6 months until detection of HCV viremia or completion of the 1-year follow-up. The samples at enrollment and all of the archived samples preceding the detection of HCV RNA during follow-up were tested for HCVcAg. During June 2019 and February 2021, 1,639 blood samples from 744 high-risk and 727 low-risk PLWH and 86 HIV-negative participants were tested for both HCV RNA and HCVcAg. Of 62 samples positive for HCV RNA, 54 (87.1%) were positive for HCVcAg. Of 1,577 samples negative for HCV RNA, 1,568 (99.4%) were negative for HCVcAg. The mean HCV RNA load of the 8 individual samples positive for HCV RNA but negative for HCVcAg was 3.2 (range, 2.5 to 3.9) log10 IU/mL, and that of the remaining 54 samples with concordant results was 6.2 (range, 1.3 to 8.5) log10 IU/mL. The positive predictive value (PPV) and negative predictive value (NPV) of HCVcAg were 85.7% and 99.5%, respectively. In at-risk populations, HCVcAg has a high specificity and NPV but lower sensitivity and PPV, particularly in individuals with low HCV RNA loads. IMPORTANCE The HCV core antigen assay has a high specificity of 99.4% and negative predictive value of 99.5% but a lower sensitivity of 87.1% and positive predictive value of 85.7% in the diagnosis of recently acquired HCV infection in high-risk populations. Our findings are informative for many countries confronted with limited resources to timely identify acute HCV infections and provide effective direct-acting antivirals to halt onward transmission.

The role of urease in the acid stress response and fimbriae expression in Klebsiella pneumoniae CG43.

BACKGROUND/PURPOSE: Two urease operons were identified in Klebsiella pneumoniae CG43, ure-1 and ure-2. This study investigates whether a differential regulation of the expression of ure-1 and ure-2 exists and how urease activity influences the acid stress response and expression of type 1 and type 3 fimbriae. METHODS: The ureA1 and ureA2 gene specific deletion mutants were constructed. Promoter activity was assessed using a LacZ reporter system. The sensitivity to acid stress was determined by assessing the survival after pH 2.5 treatment. The influence on type 1 and type 3 fimbriae expression was assessed using western blotting and mannose-sensitive yeast agglutination and biofilm formation assay, respectively. RESULTS: Bacterial growth analysis in mM9-U or modified Stuart broth revealed that ure-1 was the principal urease system, and ure-2 had a negative effect on ure-1 activity. Deletion of the fur or nac gene had no apparent effect on the activity of Pure1, Pure2-1, and Pure2-2. The Pure2-2 activity was enhanced by deletion of the hns gene. ureA1 deletion increased acid stress sensitivity, whereas the deleting effect of ureA2 was notable without hns. Deletion of ureA1 or ureA2 significantly induced the expression of type 1 fimbriae but decreased MrkA production and biofilm formation. CONCLUSION: ure-1 is the primary expression system in K. pneumoniae CG43, while ure-2 is active in the absence of hns. Impairment of urease activity increases the sensitivity to acid stress, and the accumulation of urea induces the expression of type 1 fimbriae but represses type 3 fimbriae expression.

Sexually-transmitted hepatitis C virus reinfections among people living with HIV in Taiwan: the emerging role of genotype 6.

Hepatitis C virus (HCV) reinfections after successful treatment with direct-acting antivirals (DAAs) pose a significant challenge to HCV elimination, especially among high-risk people living with HIV (PLWH). In this study, PLWH who had achieved HCV viral clearance with DAAs were included between January 2018 and June 2021. PLWH having acquired HCV infections after 2017 were classified as "recent-infection group," and those before 2017 as "remote-infection group," and the incidences of HCV reinfection were compared between two groups. Clinical and behavioural characteristics were evaluated to identify associated factors with HCV reinfection. A total of 284 PLWH were included: 179 in the recent-infection group and 105 in the remote-infection group. After a median follow-up of 2.32 years (interquartile range [IQR], 0.13-3.94), the overall incidence of HCV reinfection was 5.8 per 100 person-years of follow-up (PYFU). The incidence in the recent-infection group was significantly higher than that in the remote-infection group (9.8 vs. 0.4 per 100 PYFU, p < 0.001). The leading HCV genotypes before DAA treatment were genotypes 2 (31.0%), 1b (26.8%), and 6 (21.8%); however, genotype 6 (58.8%) became predominant upon reinfection. Younger age (adjusted odds ratio [aOR] per 1-year increase, 0.95; 95% CI, 0.90-0.99), condomless receptive anal sex (aOR, 14.5; 95% CI, 2.37-88.8), rimming (aOR, 3.87; 95% CI, 1.14-13.1), and recent syphilis (aOR, 2.73; 95% CI, 1.26-5.91) were linked to HCV reinfections. In conclusion, PLWH acquiring HCV after 2017 had a significantly higher risk for sexually-transmitted HCV reinfections. The predominance of HCV genotype 6 reinfections suggests possible on-going clustered HCV infections among at-risk PLWH.

Evolution of Hepatitis B Virus (HBV) Serologic Markers Among Antiretroviral-Naive Young People Living With Human Immunodeficiency Virus Who Had Undergone Neonatal HBV Vaccination and Initiated Antiretroviral Therapy.

BACKGROUND: With initiation of antiretroviral therapy (ART) containing nucleos(t)ide reverse-transcriptase inhibitors (NRTIs) with anti-hepatitis B virus (HBV) activity, the evolution of HBV serologic markers among people living with human immunodeficiency virus (PLWH) who were born in the era of nationwide neonatal HBV vaccination is rarely investigated. METHODS: This retrospective cohort study evaluated the changes of HBV serologic markers (hepatitis B surface antigen [HBsAg], antibody to hepatitis B surface antigen [anti-HBs], and antibody to hepatitis B core antigen [anti-HBc]) of PLWH who had undergone neonatal HBV vaccination. Clinical characteristics were analyzed and the incidences of evolution of HBV serologic markers were estimated. RESULTS: Between 2004 and 2020, 608 PLWH (mean age, 24 years) were included and 62.0% initiated tenofovir-containing ART: 13 (2.1%) were HBsAg-positive, 312 (51.3%) tested triple-negative, 209 (34.4%) had vaccine-induced seroprotection against HBV, and 74 (12.2%) tested positive for anti-HBc with or without anti-HBs. Among 492 PLWH who received a median follow-up of 2.8 years, 4 cases of incident HBV infection occurred (0.59 per 100 person-years of follow-up [PYFU]) in PLWH testing triple-negative at baseline despite ART containing NRTIs with anti-HBV activity. Of PLWH with seroprotection against HBV at baseline, 38 subsequently lost anti-HBs (4.46 per 100 PYFU) and 4 cases of incident HBV infection occurred (0.47 per 100 PYFU). PLWH with an anti-HBs antibody titer ≥100 mIU/mL at baseline (adjusted hazard ratio [aHR], 0.10 [95% confidence interval {CI}: .02-.42]) and CD4 ≥500 cells/µL during follow-up (aHR, 0.51 [95% CI: .30-1.00]) were less likely to lose HBV seroprotection. CONCLUSIONS: Among young PLWH who had undergone neonatal HBV vaccination, evolution of HBV serologic markers and incident infections occurred despite ART containing NRTIs with anti-HBV activity.

Recently acquired hepatitis C virus infection among people living with human immunodeficiency virus at a university hospital in Taiwan.

BACKGROUND: Little is known about the engagement in hepatitis C virus (HCV) care and completion of HCV treatment in people living with human immunodeficiency virus (HIV) (PLWH) who have HCV coinfection in the Asia-Pacific region. Examining the HCV care cascade can identify barriers to the completion of HCV treatment and facilitate achievement of HCV micro-elimination in PLWH. AIM: To investigate the care cascade of incident HCV infections among PLWH in Taiwan. METHODS: PLWH with incident HCV infections, defined as HCV seroconversion, were retrospectively identified by sequential anti-HCV testing of all archived blood samples at National Taiwan University Hospital between 2011 and 2018. All PLWH with incident HCV infections were followed until December 31, 2019. The care cascade of HCV examined included all incident HCV-infected patients, the percentages of anti-HCV antibodies detected by HIV-treating physicians in clinical care, plasma HCV RNA load tested, HCV RNA positivity diagnosed, referral to treatment assessment made, anti-HCV treatment initiated, and sustained virologic response achieved. Those who had HCV seroconversion during the interferon (IFN) era (2011-2016) and the direct-acting antiviral (DAA) era (2017-2018) were analyzed separately. The duration of HCV viremia-from the date of seroconversion to viral clearance by treatments or until the end of observation-and the incidence of sexually transmitted infections (STIs) during the HCV viremic period were estimated. RESULTS: During the study period, 287 of 3495 (8.2%) PLWH (92.3% being men who have sex with men) who were HCV-seronegative at baseline developed HCV seroconversion by retrospective testing of all archived blood samples. Of the 287 incident HCV infections, 277 (96.5%) had anti-HCV antibodies detected by HIV-treating physicians, 270 (94.1%) had plasma HCV RNA determined and 251 (87.5%) tested positive for HCV RNA. Of those with HCV viremia, 226 (78.7%) were referred to treatment assessment, 215 (74.9%) initiated anti-HCV treatment, and 202 (70.4%) achieved viral clearance. Compared with that in the IFN era, the median interval from HCV seroconversion by retrospective testing to detection of HCV seropositivity by HIV-treating physicians was significantly shorter in the DAA era {179 d [interquartile range (IQR) 87-434] vs 92 d (IQR 57-173); P < 0.001}. The incidence rate of STIs in the DAA vs the IFN era was 50.5 per 100 person-years of follow-up (PYFU) and 38.5 per 100 PYFU, respectively, with an incidence rate ratio of 1.31 (95% confidence interval 0.96-1.77), while the duration of HCV viremia was 380 d (IQR 274-554) and 735 d (IQR 391-1447) (P < 0.001), respectively. CONCLUSION: While anti-HCV therapies are effective in achieving viral clearance, our study suggests more efforts are needed to expedite the linkage of PLWH diagnosed with incident HCV infections to HCV treatment.

A myeloid leukemia factor homolog involved in encystation-induced protein metabolism in Giardia lamblia.

BACKGROUND: Giardia lamblia differentiates into resistant cysts as an established model for dormancy. Myeloid leukemia factor (MLF) proteins are important regulators of cell differentiation. Giardia possesses a MLF homolog which was up-regulated during encystation and localized to unknown cytosolic vesicles named MLF vesicles (MLFVs). METHODS: We used double staining for visualization of potential factors with role in protein metabolism pathway and a strategy that employed a deletion mutant, CDK2m3, to test the protein degradation pathway. We also explored whether autophagy or proteasomal degradation are regulators of Giardia encystation by treatment with MG132, rapamycin, or chloroquine. RESULTS: Double staining of MLF and ISCU or CWP1 revealed no overlap between their vesicles. The aberrant CDK2m3 colocalized with MLFVs and formed complexes with MLF. MG132 increased the number of CDK2m3-localized vesicles and its protein level. We further found that MLF colocalized and interacted with a FYVE protein and an ATG8-like (ATG8L) protein, which were up-regulated during encystation and their expression induced Giardia encystation. The addition of MG132, rapamycin, or chloroquine, increased their levels and the number of their vesicles, and inhibited the cyst formation. MLF and FYVE were detected in exosomes released from culture. CONCLUSIONS: The MLFVs are not mitosomes or encystation-specific vesicles, but are related with degradative pathway for CDK2m3. MLF, FYVE, and ATG8L play a positive role in encystation and function in protein clearance pathway, which is important for encystation and coordinated with Exosomes. GENERAL SIGNIFICANCE: MLF, FYVE, and ATG8L may be involved an encystation-induced protein metabolism during Giardia differentiation.

A Novel Multiprotein Bridging Factor 1-Like Protein Induces Cyst Wall Protein Gene Expression and Cyst Differentiation in Giardia lamblia.

The capacity to synthesize a protective cyst wall is critical for infectivity of Giardia lamblia. It is of interest to know the mechanism of coordinated synthesis of three cyst wall proteins (CWPs) during encystation, a differentiation process. Multiprotein bridging factor 1 (MBF1) gene family is a group of transcription coactivators that bridge various transcription factors. They are involved in cell growth and differentiation in yeast and animals, or in stress response in fungi and plants. We asked whether Giardia has MBF1-like genes and whether their products influence gene expression. BLAST searches of the Giardia genome database identified one gene encoding a putative MBF1 protein with a helix-turn-helix domain. We found that it can specifically bind to the AT-rich initiator promoters of the encystation-induced cwp1-3 and myb2 genes. MBF1 localized to cell nuclei and cytoplasm with higher expression during encystation. In addition, overexpression of MBF1 induced cwp1-3 and myb2 gene expression and cyst generation. Mutation of the helixes in the helix-turn-helix domain reduced cwp1-3 and myb2 gene expression and cyst generation. Chromatin immunoprecipitation assays confirmed the binding of MBF1 to the promoters with its binding sites in vivo. We also found that MBF1 can interact with E2F1, Pax2, WRKY, and Myb2 transcription factors that coordinately up-regulate the cwp genes during encystation. Using a CRISPR/Cas9 system for targeted disruption of mbf1 gene, we found a downregulation of cwp1-3 and myb2 genes and decrease of cyst generation. Our results suggest that MBF1 is functionally conserved and positively regulates Giardia cyst differentiation.

Seroepidemiology of the human herpesvirus 8 infection among people living with HIV in Taiwan, 2014-2018.

BACKGROUND: Human herpesvirus type 8 (HHV-8) can be transmitted through unprotected sex as HIV. We aimed to investigate the seroincidence of HHV-8 and associated factors among people living with HIV (PLWH). METHODS: From 2014 to 2018, blood samples of PLWH on the first date of HIV care were determined for antibodies against HHV-8. Individuals testing HHV-8-seronegative at baseline were followed for at least four months to estimate the annual seroconversion rate. To identify the factors associated with HHV-8 seroconversion, we compared the clinical characteristics between seroconverters and non-seroconverters who were matched for observation duration. RESULTS: The HHV-8 seroprevalence increased from 8.1% in 2014 to 20.0% in 2018. HHV-8 seroconversion occurred in 154 (14.7%) PLWH after a total of 2652.16 person-years of follow-up (PYFU), resulting in an overall incidence rate of 5.62 per 100 PYFU, which increased from 3.20 to 6.84 per 100 PYFU during the study period. HHV-8 seroconverters were less likely to have chronic hepatitis B virus (HBV) infection (1.9% vs 10.6%) and more likely to be antiretroviral-naïve on entry into care (87.7% vs 75.4%) (both p < 0.05). In multivariate logistic analysis, men who have sex with men (MSM) (adjusted odds ratio [aOR], 2.22; 95% CI, 1.01-4.86), being antiretroviral-naïve (aOR, 2.91; 95% CI, 1.27-6.67), and chronic HBV infection (aOR, 0.13; 95% CI, 0.03-0.61) at baseline were associated with HHV-8 seroconversion. CONCLUSIONS: An increasing trend of HHV-8 infection was observed among PLWH in Taiwan between 2014 and 2018. MSM and being antiretroviral-naïve were associated with higher risk for HHV-8 seroconversion.

Trends of recent hepatitis C virus infection among HIV-positive men who have sex with men in Taiwan, 2011-2018.

BACKGROUND: Increasing trends of HCV infection have been reported among HIV-positive men who have sex with men (MSM) in Europe, Australia and North America. The trends of recently acquired HCV infection among HIV-positive MSM are less clear in Asia-pacific region. METHODS: All HIV-positive patients seeking care at a university hospital in Taiwan tested for anti-HCV IgG at least once annually to estimate the incidence of HCV seroconversion during 2011-2018. HCV genotyping and sequencing were performed and multivariate logistic regression analysis was conducted to identify the factors associated with HCV seroconversion among MSM. FINDINGS: During the study period, 3495 HCV-seronegative patients (86·4% MSM) were included and 294 (8·4%) with recent HCV infection were identified, in whom 281 (95·6%) were MSM, during a total of 16,361·86 person-years of follow-up (PYFU), giving an overall incidence rate of 17·97 per 1000 PYFU, which increased from 14·28 per 1000 PYFU in 2011 to 25·38 per 1000 PYFU in 2018 (p<0·001). HCV seroconversion among MSM was associated with aspartate aminotransferase ≥37 U/L (adjusted odds ratio [AOR] 7·50, 95% CI 4·17-13·50), alanine aminotransferase ≥41 U/L (AOR 7·47, 95% CI 4·11-13·58), and syphilis acquisition (AOR 2·88, 95% CI 1·67-4·97). Among the 277 (94·2%) with HCV viremia, genotype 2a (n = 116) was the leading genotype, followed by 1b (n = 85), 6a (n = 34), and 1a (n = 21). Genotypes 3a and 6a increased from 0% and 5·2%, respectively, in 2011-2014 to 4·1% and 17·1% in 2015-2018. Phylogenetic analysis revealed increased clusters in genotypes 2a, 3a and 6a from 2011-2014 to 2015-2018. INTERPRETATION: An expanding HCV epidemic among HIV-positive MSM is occurring in Taiwan. Improving access to HCV testing and early linkage to treatment are needed to curb the expanding HCV epidemic. FUNDING: This research was supported by a grant from National Taiwan University Hospital, Taipei, Taiwan (NTUH.106-003347 to Hsin-Yun Sun).

DNA topoisomerase IIIß promotes cyst generation by inducing cyst wall protein gene expression in Giardia lamblia.

Giardia lamblia causes waterborne diarrhoea by transmission of infective cysts. Three cyst wall proteins are highly expressed in a concerted manner during encystation of trophozoites into cysts. However, their gene regulatory mechanism is still largely unknown. DNA topoisomerases control topological homeostasis of genomic DNA during replication, transcription and chromosome segregation. They are involved in a variety of cellular processes including cell cycle, cell proliferation and differentiation, so they may be valuable drug targets. Giardia lamblia possesses a type IA DNA topoisomerase (TOP3ß) with similarity to the mammalian topoisomerase IIIß. We found that TOP3ß was upregulated during encystation and it possessed DNA-binding and cleavage activity. TOP3ß can bind to the cwp promoters in vivo using norfloxacin-mediated topoisomerase immunoprecipitation assays. We also found TOP3ß can interact with MYB2, a transcription factor involved in the coordinate expression of cwp1-3 genes during encystation. Interestingly, overexpression of TOP3ß increased expression of cwp1-3 and myb2 genes and cyst formation. Microarray analysis confirmed upregulation of cwp1-3 and myb2 genes by TOP3ß. Mutation of the catalytically important Tyr residue, deletion of C-terminal zinc ribbon domain or further deletion of partial catalytic core domain reduced the levels of cleavage activity, cwp1-3 and myb2 gene expression, and cyst formation. Interestingly, some of these mutant proteins were mis-localized to cytoplasm. Using a CRISPR/Cas9 system for targeted disruption of top3ß gene, we found a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that TOP3ß may be functionally conserved, and involved in inducing Giardia cyst formation.

HCV reinfections after viral clearance among HIV-positive patients with recent HCV infection in Taiwan.

BACKGROUND: Higher rates of hepatitis C virus (HCV) reinfection after viral clearance have been well described among HIV-positive men who have sex with men (MSM) in Europe. The epidemiology of HCV reinfection, however, has rarely been investigated among HIV-positive patients in Asia-Pacific region. METHODS: We retrospectively identified HIV-positive patients with recent HCV infection who had cleared their primary infection, either spontaneously or via treatment, between January 2011 and May 2018. All included patients were observed until 31 March 2019. HCV reinfection was defined as recurrent HCV viraemia after achieving viral clearance with anti-HCV treatment or after spontaneous clearance. RESULTS: During the study period, 219 HIV-positive patients (90.4% MSM) were diagnosed with recent HCV infection. Viral clearance with successful treatment was achieved in 108 patients (49.3%) and spontaneous clearance occurred in 20 (9.1%); of them, 18 (14.1%) acquired HCV reinfections, resulting in an incidence rate of 8.2 per 100 person-years of follow-up (95% CI 5.2-13.1). With the adjusted Cox proportional hazards model, we found a higher reinfection risk in patients with syphilis (adjusted hazard ratio 10.3, 95% CI 1.4-77.8, P = .023) compared to those without syphilis. HCV RNA testing, if performed only following syphilis and elevated aminotransferases, might miss 44.4% and 33.3% of HCV reinfections, respectively. CONCLUSIONS: Similar to the findings in Europe, we observed a high incidence of HCV reinfection among HIV-positive Taiwanese with recent HCV infection, which was significantly associated with syphilis. To identify HCV reinfections, annual HCV RNA testing should be instituted instead of testing driven by symptoms, syphilis or elevated aminotransferases.

Hepatitis E Virus Infections Among Human Immunodeficiency Virus-Positive Individuals During an Outbreak of Acute Hepatitis A in Taiwan.

Hepatitis A virus (HAV) and hepatitis E virus (HEV) share the similar fecal-oral transmission route. During an outbreak of sexually transmitted acute hepatitis A among men who have sex with men (MSM) worldwide between 2015 and 2017, we investigated the possibility of sexual transmission and related morbidity of HEV infection among human immunodeficiency virus (HIV)-positive patients. From March 1, 2015 to August 31, 2017, anti-HEV immunoglobulin G was retrospectively determined among 3,293 HIV-positive patients, who were mainly MSM (87.6%) with a median CD4 count of 575 cells/µL. Prevalence and incidence of HEV infection were 3.7% (123 of 3,293) and 4.35 per 1,000 person-years of follow-up (PYFU), respectively, which were significantly lower compared with those of HAV infection (31.1% [996 of 3,204] and 12.61 per 1,000 PYFU, respectively). The number of patients with HEV infection did not increase with the hepatitis A epidemic. The factor associated with prevalent HEV infection was older age (per 1-year increase, adjusted odds ratio, 1.07; 95% confidence interval, 1.05-1.09), but neither sexual orientation nor acquisition of sexually transmitted infections was related to prevalent or incident HEV infection. Among 23 patients with incident HEV infection, 22 patients had viremia caused by HEV genotype 4. No patients had prolonged HEV viremia or clinical symptoms, and only a mild elevation of serum aminotransferase, ranging from 34 to 77 IU/L, was noted. Although 4 patients had hepatitis for a prolonged duration of between 8 and 17 months, no abdominal imaging revealed liver fibrosis or cirrhosis. Conclusion: HEV endemicity remained low among HIV-positive patients in Taiwan during the outbreak of acute hepatitis A. Our data suggest that sexual transmission of HEV with significant morbidity of HEV infection, if any, is rare in this population.

Development of CRISPR/Cas9-mediated gene disruption systems in Giardia lamblia.

Giardia lamblia becomes dormant by differentiation into a water-resistant cyst that can infect a new host. Synthesis of three cyst wall proteins (CWPs) is the fundamental feature of this differentiation. Myeloid leukemia factor (MLF) proteins are involved in cell differentiation, and tumorigenesis in mammals, but little is known about its role in protozoan parasites. We developed a CRISPR/Cas9 system to understand the role of MLF in Giardia. Due to the tetraploid genome in two nuclei of Giardia, it could be hard to disrupt a gene completely in Giardia. We only generated knockdown but not knockout mutants. We found that knockdown of the mlf gene resulted in a significant decrease of cwp gene expression and cyst formation, suggesting a positive role of MLF in encystation. We further used mlf as a model gene to improve the system. The addition of an inhibitor for NHEJ, Scr7, or combining all cassettes for gRNA and Cas9 expression into one plasmid resulted in improved gene disruption efficiencies and a significant decrease in cwp gene expression. Our results provide insights into a positive role of MLF in inducing Giardia differentiation and a useful tool for studies in Giardia.

Molecular epidemiology of acute HCV infection in HIV-positive patients from Hong Kong, Taipei, Tokyo.

BACKGROUND: Acute hepatitis C virus (HCV) infections have been increasingly reported among human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) in the Asia-Pacific region. It remains unknown whether international network of HCV transmission has occurred in this region. METHODS: HIV-positive patients with acute HCV infection, defined as HCV seroconversion within a year or documented acute hepatitis with seroconversion, diagnosed in Hong Kong, Taipei and Tokyo during 2010-2016 were included in this molecular epidemiology study. The NS5B region of the HCV genome (365 bp) was amplified using nested polymerase chain reaction and sequenced. RESULTS: Of 234 HIV-positive patients with acute HCV infection, all were male with 94% being MSM. At the diagnosis of acute HCV infection, 73.5% had concurrent sexually transmitted diseases and 88.0% were receiving combination antiretroviral therapy. The most prevalent HCV genotype was 3a, 2a and 1b in Hong Kong, Taipei and Tokyo respectively. Nine independent clusters belonging to five genotypes (1b, 2a, 2c, 3a and 6a) were identified, each of which occurred in one city without overlapping except for one 3a sequence from Taipei that was closely related genetically to the Hong Kong cluster. CONCLUSIONS: No international network of HCV transmission was identified among HIV-positive patients in the three Asia-Pacific cities. The transmission dynamics of sexually acquired HCV differed by city, but the risk of intercity clustering should not be ignored.

Patterns of emergent resistance-associated mutations after initiation of non-nucleoside reverse-transcriptase inhibitor-containing antiretroviral regimens in Taiwan: a multicenter cohort study.

BACKGROUND: Increasing trends of resistance-associated mutations (RAMs) to non-nucleoside reverse-transcriptase inhibitors (nNRTIs) have raised concerns about the effectiveness of the regimens in the national HIV treatment programs in resource-limited countries. We aimed to retrospectively investigate the incidence and patterns of emergent RAMs of HIV-1 in HIV-positive adults experiencing virological failure to first-line nNRTI-containing combination antiretroviral therapy (cART) in Taiwan. PATIENTS AND METHODS: Between June 2012 and March 2016, 1138 antiretroviral-naïve HIV-positive adults without baseline RAMs who initiated nNRTI-containing regimens were included for analysis. Virological failure was defined as plasma viral load (PVL) ≥200 copies/mL after 6 months of cART or confirmed PVL ≥200 copies/mL after achieving PVL <50 copies/mL. Population sequencing was retrospectively performed to detect baseline and emergent RAMs. RAMs were interpreted using the International AIDS Society-USA 2016 mutations list. RESULTS: Seventy-one patients (6.2%) developed virological failure, which occurred in 14.8% (43/291), 3.9% (26/675), and 1.2% (2/172) of patients receiving 2 nucleoside reverse-transcriptase inhibitors (NRTIs) plus nevirapine, efavirenz, and rilpivirine, respectively. Among those, 53 (74.6%) had emergent RAMs identified, which included 43 (81.1%), 53 (100.0%), and 1 (1.9%) with RAMs to NRTIs, nNRTIs, and protease inhibitors, respectively; and 43 (81.1%) had multi-drug resistance. The most common emergent RAMs to NRTIs were M184V/I (42.3%) and K65R (28.2%), and those to nNRTIs were Y181C (42.3%), K103N (15.5%), G190A/E/Q (12.7%), V179D/E (12.7%), and V108I (9.9%). CONCLUSION: While the rates of virological failure varied with the nNRTI used, the rate of emergent RAMs of HIV-1 to NRTIs and nNRTIs among the antiretroviral-naïve patients who failed nNRTI-containing cART remained low.