[Analysis of HIV-1 genetic subtype and pretreatment drug resistance among men who have sex with men infected with HIV-1 from 19 cities of 6 provinces in China].

Objective: To investigate the distribution of HIV-1 genetic subtypes and pretreatment drug resistance (PDR) among men who have sex with men (MSM) from 19 cities of 6 provinces in China. Methods: From April to November 2019, 574 plasma samples of ART-naive HIV-1 infected MSM were collected from 19 cities in Hebei, Shandong, Jiangsu, Zhejiang, Fujian, and Guangdong provinces, total ribonucleic acid (RNA) was extracted and amplified the HIV-1 pol gene region by nested polymerase chain reaction (PCR) after reverse transcription. Then sequences were used to construct a phylogenetic tree to determine genetic subtypes and submitted to the Stanford drug resistance database for drug resistance analysis. Results: A total of 479 samples were successfully amplified by PCR. The HIV-1 genetic subtypes included CRF01_AE, CRF07_BC, B, CRF55_01B, CRF59_01B, CRF65_cpx, CRF103_01B, CRF67_01B, CRF68_01B and unrecognized subtype, which accounted for 43.4%, 36.3%, 6.3%, 5.9%, 0.8%, 0.8%, 0.4%, 0.4%, 0.2% and 5.5%, respectively. The distribution of genetic subtypes among provinces is statistically different (χ2=44.141, P<0.001). The overall PDR rate was 4.6% (22/479), the drug resistance rate of non-nucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors, and protease inhibitors were 3.5% (17/479), 0.8% (4/479) and 0.2% (1/479), respectively. The PDR rate of recent infections was significantly higher than that of long-term infections (χ2=4.634, P=0.031). Conclusions: The HIV-1 genetic subtypes among MSM infected with HIV-1 from 19 cities of 6 provinces in China are diverse, and the distribution of subtypes is different among provinces. The overall PDR rate is low, while the PDR rate of recent infections was significantly higher than that of long-term infections, suggesting the surveillance of PDR in recent infections should be strengthened.

[Application of limiting antigen avidity enzyme immunoassay for estimating HIV-1 incidence in men who have sex with men].

Objective: To estimate the incidence of HIV-1 infection in men who have sex with men (MSM) in key areas of China through HIV-1 limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA), analyze the deviation from the actual results and identify influencing factors, and provided reference for improving the accuracy of estimation results. Methods: Based on the principle of the cohort randomized study design, 20 cities were selected in China based on population size and the number of HIV-positive MSM. The sample size was estimated to be 700 according to the HIV-1 infection rate in MSM. MSM mobile phone app. was used to establish a detection appointment and questionnaire system, and the baseline cross-sectional survey was conducted from April to November 2019. LAg-Avidity EIA was used to identify the recent infected samples. The incidence of HIV-1 infection was calculated and then adjusted based on the estimation formula designed by WHO. The influencing factors were identified by analyzing the sample collection and detection processes. Results: Among the 10 650 blood samples from the participants, 799 were HIV-positive in initial screening, in which 198 samples (24.78%) missed during confirmation test. Only 621 samples were received by the laboratory. After excluding misreported samples, 520 samples were qualified for testing. A total of 155 samples were eventually determined as recent infection through LAg-Avidity EIA; Based on the estimation formula , the incidence of HIV-1 infection in MSM in 20 cities was 4.06% (95%CI:3.27%-4.85%), it increased to 5.53% (95%CI: 4.45%-6.60%)after the adjusting for sample missing rate. When the sample missing rate and misreporting rate were both adjusted, the incidence of HIV-1 infection in the MSM increased to 5.66% (95%CI:4.67%-6.65%). The actual incidence of HIV-1 infection in MSM in the 20 cities might be between 4.06% and 5.66%. Conclusions: Sample missing and misreporting might cause the deviation of the estimation of HIV-1 infection incidence. It is important to ensure the sample source and the quality of sample collection and detection to reduce the deviation in the estimation of HIV-1 infection incidence.

[Homozygous ectonucleotide pyrophosphatase/phosphodiesterase 1 variants in a girl with hypophosphatemic rickets and literature review].

Objective: To investigate the clinical features and genetic characteristics of patients with ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene variants. Method: The clinical data of a patient with ENPP1 homozygous variants from Capital Institute of Pediatrics was collected, the related literature was searched from China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform, National Center from Biotechnology Information and PubMed by using search term "ENPP1" , "hypophosphatemic rickets" . The literature retrieval was confined from 1980 to February 2017. The clinical manifestations, bone metabolism examinations, X-RAY and genotypes were reviewed. Result: Our patient was an 11 years old girl, with 7 years history of lower limb malformation. She showed significant valgus deformity of the knee (genu valgum). Metabolic examination revealed reduced level of plasma phosphate (0.86 mmol/L), a normal level of plasma calcium (2.30 mmol/L) and an elevated alkaline phosphatase level of 688 IU/L. The calcium-phosphorus product was 25.9. A homozygous nonsense variants of ENPP1 gene, c.783C>G (p.Tyr261X) in exon 7 was identified in the patient. Both parents were heterozygous carriers. Literature review identified 3 Chinese patients from one publication and 17 cases from twenty one publications around the world. None of the patients was found PHEX variants which is the most common variants among hypophosphatemic rickets patients. The disease onset age was 11 months to 10 years. Eight patients had short stature, five patients had the history of generalized arterial calcification of infancy. Four suffered from deafness, three showed localized calcifications of arteries, three patients manifested pseudoxanthoma elasticum and two suffered from ossification of posterior longitudinal ligament. Nine missense variants, six splicing variants and 4 nonsense variants were reported among these twenty patients. c.783C>G was found in two Chinese patients. Conclusion: ENPP1 gene mutation was a cause of patient with hypophosphatemic rickets. Comorbid features included generalized arterial calcification of infancy, early onset hearing loss, pseudoxanthoma and ossification of posterior longitudinal ligament. ENPP1 gene testing should be performed on hypophosphatemic rickets patients without PHEX gene variants. Long-term follow up is recommended. The most common types of ENPP1 gene variants were nonsense/splicing variants. The gene c.783C>G was the most common variants in Chinese patients.

[Comparative study on the HIV epidemic between the Chinese and Burmese drug users in drug rehabilitation places in Dehong Prefecture, Yunnan Province].

Objective: To compare the prevalence of HIV infection and its risk factors among Chinese and Burmese drug users living in Dehong Prefecture, Yunnan Province. Methods: We obtained plasma specimens and gathered demographic data from 7 867 drug users with Chinese or Burmese nationality attending rehabilitation clinics in Dehong Prefecture from October 2014 to September 2015. Of these, 7 756 individuals who gave valid questionnaire responses, including 5 389 Chinese and 2 367 Burmese, were enrolled in the study after giving informed consent. We used the Chi-squared test to compare the demographic characteristics and HIV prevalence between the Chinese and Burmese drug users. Logistic regression was then used to identify risk factors for HIV infection. Results: The HIV infection rate of 7 756 subjects (aged (35.45 ± 10.91) years old) was 7.18%, in which Burmese with higher HIV infection rate (9.38%, 222/2 367) than Chinese (6.22%, 335/5 389) (χ2=24.21, P<0.001). In chinese drug users, OR (95%CI) of HIV infection of those aged 25-34 years old, 35-44 years old and ≥45 years old were 2.88 (1.46-5.69), 5.72 (2.87-11.40) and 3.48 (1.66-7.27),compared with those aged below 25 years;Compared with married participants, OR (95% CI) of those unmarried and divorced were 1.44 (1.08-1.93) and 1.56 (1.09-2.24); Jingpo drug users were 1.47 (1.07-2.04) times to get HIV infection,compared with Han ethnicity; OR (95%CI) of HIV infection for IDUs was 11.48 (9.73-16.01) compared with NIDUs.In Burmese drug users, OR (95% CI) of HIV infection for females was 0.50 (0.26-0.93) compared with men;Compared with those aged below 25 years,those aged 25-34,35-44 and ≥45 years had OR (95% CI) of 1.82 (1.18-2.77), 2.90 (1.82-4.62) and 2.31 (1.24-4.30), respectively; OR (95% CI) of Jingpo participants was 2.22 (1.44-3.41) compared with Han nationality; OR (95%CI) of HIV infection for IDUs was 10.61 (7.68-14.64) compared with NIDUs. Conclusion: The HIV infection rate of Burmese drug users was higher than that of Chinese drug users. Measures of HIV prevention and control should be mainly allocated in those 25 years above, non-married, Jingpo ethnicity and IDU in Chinese drug users, while in Burmese drug users,those females, 25 years above, Jingpo ethnicity and IDU should be higlighted in HIV prevention and control.

[Comparison of genotypes of hepatitis C virus between Chinese and Burmese drug users living in Dehong Prefecture, Yunnan Province].

Objective: To analyze the distribution of hepatitis C virus (HCV) genotypes among Chinese and Burmese drug users in Dehong Prefecture, Yunnan Province. Methods: Plasma specimens and relevant epidemiological data were collected from 7 545 drug users attending rehabilitation centers and methadone clinics in Dehong Prefecture from January to September in 2015, of which 752 were positive for HCV antibodies. HCV RNA was then extracted from 139 random specimens (64 Burmese and 75 Chinese) and the CE1 and NS5B regions were amplified by nested PCR. The sequences of these HCV genes were aligned, a phylogenetic tree was constructed using MEGA 6.0.6 and the average genetic discrete rate of each subtype group was calculated. We used Fisher's exact test to compare distribution of HCV subtypes from different people, using analysis of variance to compare the discrete rates of different CE1 and NS5B subtypes. Results: While we successfully amplified RNA from 43 specimens provided by Burmese patients (67%), including 31 with CE1 region and 38 with NS5B region, 52 from Chinese patients (69%) were successfully amplified including 43 with CE1 region and 45 with NS5B region. We found that 3b and 6n were the predominant subtypes and were found in 27% (n=14) and 37% (n=19) of Chinese and 28% (n=12) and 33%(n=14) of Burmese specimens. Subtypes 6u, 3a, 1a and 1b were present in 14%(n=7), 19%(n=10), 2% (n=1) and 2%(n=1) of Chinese specimens and 16%(n=7), 5%(n=2), 16%(n=7) and 2%(n=1) of Burmese specimens respectively. While the prevalence of subtype 1a was higher among samples from Burmese patients than Chinese patients (P=0.015), the presence of subtype 3a was higher among the latter (P=0.031). The discrete rates of CE1 region subtypes 1a, 1b, 3a, 3b, 6n and 6u were 0.048±0.007, 0.091±0.013, 0.074± 0.008, 0.061 ± 0.006, 0.136 ± 0.009 and 0.031 ± 0.005 (F=516.26, P<0.001). The discrete rates for NS5B region subtypes 1a, 1b, 3a, 3b, 6n and 6u, meanwhile, were 0.032±0.006, 0.065±0.012, 0.058±0.008, 0.041± 0.005, 0.059 ± 0.008, 0.045 ± 0.006 (F=45.11, P<0.001). Conclusion: Six HCV subtypes were identified among drug users in Dehong Prefecture, of which 6n and 3b were predominant. There were statistically significant differences in the distribution of subtypes 1a and 3b, which have been circulating for a long time in this population, between samples from Chinese and Burmese participants.

[Tracing hepatitis C virus transmission by using high-throughput sequencing technique].

OBJECTIVE: An approach for analysis of hepatitis C virus (HCV) quasispecies using Hiseq high-throughput sequencing (hereinafter referred to as Hiseq sequencing) technique was developed and then applied to investigate a possible case of HCV needle sharing transmission. METHODS: One case of HCV antibody seroconversion (P1) was found in a methadone clinic on January 15, 2015. Four HCV antibody positive injecting drug users (IDUs), P2 to P5, suspected to be involved in needle sharing transmission with P1 during the period (after March 24, 2014) that P1 may be infected with HCV were investigated, and another 28 HCV antibody positive IDUs were selected as controls (C1 to C28). These controls came from the same methadone clinic or lived in the same town with P1. The RNAs were extracted from the plasma specimens and then reverse-transcribed into cDNA. After HCV subtyping, Hiseq sequencing was performed to detect and sequence the HCV quasispecies (263 bp) in the specimens with the same subtype as P1. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated. RESULTS: The HCV subtype of specimen P1 was 3b. All the other specimens with the same subtype were P2, C7, C12, C14, C15, C16, C19, C20 and C28. Hiseq sequencing was successfully performed in 9 out of these 10 specimens, and 249 753 to 1 086 333 (average 869 608) cleaned sequences representing 3 to 172 (average 48) unique HCV quasispecies were obtained. The medians (P50) of intrapersonal genetic diversities from the 9 specimens were 0.4% to 12.3%. The P50 (P25, P75) of genetic diversities between P1 and the other 8 specimens were 19.0% (18.4%, 19.8%), 10.4% (2.8%, 18.3%), 19.6% (17.8%, 21.4%),24.9% (23.8%, 26.1%), 19.8% (18.7%, 20.7%), 20.1% (18.9%, 21.2%), 20.6% (20.0%, 21.1%), 23.6% (22.4%, 24.8%). There were no significant difference between the genetic diversities of P1 and P2 and those of P1 and other 7 specimens (H=9.40, P=0.100). The genetic diversities between few HCV quasispecies from P1 and few ones from C7 were 0. Phylogenetic tree analysis indicated that there was no HCV transmission relationship between P1 and P2, but there was HCV transmission relationship between P1 and C7. CONCLUSION: With the feature of high-throughput, easier operation and lower cost, Hiseq sequencing technique has high practical value in tracing HCV transmission at the quasispecies level.