Changes of Viral Load,Drug Resistance,and CD4 Cell Count in HIV-1–Infected Patients Receiving Antiretroviral Therapy in Hebei Province,China,2003

Abstract

In this study, a total of 462 samples of whole blood were collected from 36 patients enrolled. During the entire course of antiretroviral therapy (ART) from 2003 to 2019, both the CD4 cell count and viral load (VL) of study patients were examined annually, and an HIV-1 genotypic drug resistance (DR) assay was carried out using an in-house method when the HIV-1 VL was >1,000 copies/mL. The results indicated that 13 (36.1%) experienced treatment failure and 23 (63.9%) experienced treatment success among 36 patients. The proportion of patients with effective treatment after the adjustment of ART regimens was significantly higher than before adjustment (χ² = 33.796, p < .001). Furthermore, the frequencies of HIV-1 DR mutations before adjustment were higher than those after adjustment (t = 3.345, p = .002). In particular, among 23 patients with effective treatment after adjustment, the means ± standard deviations of the VLs and CD4 cell counts before adjustment were 3.85 ± 0.65 log RNA copies/mL and 226.83 ± 106.06 cells/mm³, respectively, compared with 2.19 ± 0.58 log RNA copies/mL and 367.68 ± 174.62 cells/mm³, respectively, after adjustment. Obviously, there were statistically significant differences in the changes in VL (t = 8.728, p < .001) and CD4 cell count (t = −4.476, p < .001) before and after adjustment. Therefore, patients who received updated ART regimens containing LPV/r and TDF after adjustment achieved better therapeutic effects compared with patients who received initial ART regimens harboring D4T/AZT or NVP. This suggests that future research is needed to initiate the surveillance of DR, VLs, and CD4 cell counts immediately after HIV diagnosis, and dynamic changes in these indicators so as to optimize ART effects.

Introduction

In 2019, it was estimated that there were 38.0 million people infected with HIV worldwide.¹ Since the global use of highly active antiretroviral therapy (ART) was initiated in 1996, 26.0 million HIV-infected people had received ART as of June 2020.¹ Over more than three decades, the extensive global use of ART has significantly reduced HIV-associated morbidity, mortality, disease transmission, and the incidence of new infections, thereby effectively prolonging survival among patients and reducing HIV transmission.^2

–5 Despite the success of ART, 1.7 million people were newly infected with HIV and 690,000 HIV-infected individuals died from diseases related to AIDS in 2019.¹

Hebei province has undertaken China's National Free Antiretroviral Treatment Program (NFATP), which has been largely successful.^2

–5 With changes to the NFATP guidelines,⁶ treatment criteria and regimens have been appropriately adjusted in Hebei: at the beginning of 2003, HIV-1–infected people with a CD4 cell count <200 cells/mm³ received ART, compared with a CD4 cell count <350 cells/mm³ in 2012, and a CD4 cell count <500 cells/mm³ in 2014, with therapy being initiated regardless of CD4 cell count in 2016. A total of 15,178 HIV/AIDS patients, of whom 14,245 were receiving ART, remained in the whole province by the end of October 2020.⁷ Hebei represents an area that has undergone a large increase in treatment coverage, from 9.9% in 2003 to 90.7% in 2020, and the viral suppression rate among those receiving ART achieved an obvious increase from 11.6% to 98.1% over this time period.

The aim of this study was to evaluate the wide scale outcome of ART through an observational cohort study. We examined the CD4 cell count, viral load (VL), and drug resistance (DR) among HIV/AIDS patients, observed their clinical symptoms over the previous 3 months, and analyzed, in detail, clinical changes throughout the entire course of ART from 2003 to 2019.

Materials and Methods

Participants

We selected participants experiencing long-term ART according to the following criteria: (1) age ≥18 years, (2) duration of therapy ≥9 years, (3) blood samples could be collected from them annually during the ART, and (4) the follow-up data were complete. A total of 36 patients were recruited who were diagnosed as anti-HIV-1–positive by western blot analysis between 1995 and 2009. At that time, the national criterion for receiving free ART was a CD4 cell count <200 cells/mm³, and ART was initiated for these patients between 2003 and 2010, when their CD4 cell counts were at the required level. All participants signed written informed consent statements. The study was approved by the local Ethics Committee at Hebei Provincial Centers for Disease Control and Prevention. The protocol number was IRB(S)2020-002.

During the entire ART period from 2003 to 2019, the initial therapeutic regimen for each patient was only updated once between 2009 and 2015. All patients had undergone ART for 9–16 years. A total of 462 samples of whole blood (10 mL) were collected from these 36 patients from 2003 to 2019. Their clinical symptoms over the past 3 months were also recorded via face-to-face interviews. HIV-1 subtypes were preliminarily analyzed using the online HIV BLAST (https://www.hiv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html) and identified using the online REGA HIV-1 Subtyping Tool 3.0 (http://dbpartners.stanford. edu:8080/RegaSubtyping/stanford-hiv/typingtool/).

ART regimens

In this study, initial ART regimens comprised first-line antiretroviral drugs, which included 3TC+D4T+NVP (58.3%, 21/36), 3TC+AZT+NVP (13.9%, 5/36), DDI+AZT+NVP (11.1%, 4/36), DDI+D4T+EFV (8.3%, 3/36), DDI+D4T+NVP (2.8%, 1/36), 3TC+AZT+EFV (2.8%, 1/36), and 3TC+D4T+EFV (2.8%, 1/36). Among these, the NVP-based regimen was the most frequent, accounting for 86.1% (31/36), followed by the 3TC-based regimen (77.8%, 28/36), the D4T-based regimen (72.2%, 26/36), the AZT-based regimen (27.8%, 10/36), the DDI-based regimen (22.2%, 8/36), and the EFV-based regimen (13.9%, 5/36).

After the adjustment of ART regimens, updated ART regimens included LPV/r+3TC+TDF (63.9%, 23/36), LPV/r+3TC+AZT (16.7%, 6/36), 3TC+AZT+NVP (8.3%, 3/36), LPV/r+AZT+TDF (2.8%, 1/36), 3TC+TDF+NVP (2.8%, 1/36), 3TC+D4T+NVP (2.8%, 1/36), and 3TC+TDF+EFV (2.8%, 1/36). Updated ART regimens included both the second-line regimen of LPV/r (83.3%, 30/36) and first-line regimens (3TC+AZT/TDF/D4T+NVP/EFV: 16.7%, 6/36). Furthermore, AZT (two cases: Hb03 and Hb25), D4T (three cases: Hb30, Hb31, and Hb36), and DDI (one case: Hb33) in the initial ART regimens were replaced by TDF or D4T, AZT or TDF or AZT, and 3TC in the first-line regimens after adjustment, respectively. Among the updated regimens, the proportions of regimens containing 3TC and TDF were 97.2% (35/36) and 69.4% (25/36), respectively. However, updated regimens containing NVP, which was the most widely used drug in initial ART regimens, only accounted for 14.0% (5/36).

Laboratory tests

Annually, the CD4 cell count was determined from 50 μL of each whole blood sample (FACS Count; Becton–Dickinson, Franklin Lakes, NJ). The remaining whole blood samples were centrifuged at 3,000 rpm to produce blood plasma, and the resulting blood plasma samples were frozen at −80°C before testing for the HIV-1 VL and DR. The HIV-1 VL was quantified using an Amplicor HIV-1 monitor test (COBAS TaqMan 48; Roche, Switzerland). According to the WHO HIV DR surveillance guidelines, a VL ≥1,000 copies/mL was classified as virological failure. In this study, the VL values were converted into log RNA copies/mL. The VL and CD4 cell count of each patient enrolled in this study were examined once per year during the entire course of ART. In samples that showed virological failure on ART, an HIV-1 DR genotypic assay was implemented using an in-house method, as previously described.⁸

HIV-1 DR gene mutation analysis was performed based on a fragment of the HIV-1 pol gene (1.3 kb, HXB2:2147–3462) using the Stanford University HIV DR Database online sequence analysis tool (http://hivdb.stanford.edu/). The Stanford HIVdb algorithm classifies the degree of DR into low-, intermediate-, or high-level resistance.

Results

Demographic and epidemiological information about the study patients

As given in Table 1, the ratio of females to males was 2:1 among the 36 patients. Regarding age, 88.9% (32/36) of patients were >25 years old, and four patients infected with HIV-1 through mother-to-child transmission (MTCT) were younger than 24 years. Married, unmarried, and divorced or widowed patients accounted for 69.4% (25/36), 16.7% (6/36), and 13.9% (5/36), respectively. The majority of patients (97.2%, 35/36) were of Han ethnicity. Blood transmission was the predominant infection route and accounted for 63.9% (23/36), including paid plasma donor (41.7%, 15/36) and blood transfusion (22.2%, 8/36), followed by transmission via heterosexual sexual contact (25.0%, 9/36), and MTCT (11.1%, 4/36). Of note, 94.4% (34/36) of patients had attained junior middle school or less, reflecting a poor education level; furthermore, 86.1% (31/36) were farmers. The subtypes identified by this study included subtype B (97.2%, 35/36) and subtype C (2.8%, 1/36). Of the study patients, 94.4% had no sexually transmitted disease (STD) history and 5.6% were infected with an STD through nonmarital sexual contact.

Table 1.

Demographic and Epidemiological Information of Study Patients

Characteristics	Patients
Characteristics	Cases	%
Gender
Male	12	33.3
Female	24	66.7
Age, years
19–24	4	11.1
25–49	11	30.6
50–78	21	58.3
Marital status
Married	25	69.4
Unmarried	6	16.7
Divorced/widowed	5	13.9
Ethnicity
Han	35	97.2
Man	1	2.8
Sample source
HIV-infected individual's child	4	11.1
VCT	4	11.1
Test before-operation	7	19.4
HIV-infected individual's spouse	5	13.9
Paid plasma donor	14	38.9
STD diagnostic testing	2	5.6
Transmission routes
Paid plasma donor	15	41.7
Heterosexual	9	25.0
Blood transfusion	8	22.2
MTCT	4	11.1
Education level
Junior middle school or less	34	94.4
Other^a	2	5.6
Subtype
B	35	97.2
C	1	2.8
Occupation
Farmer	31	86.1
Student	3	8.3
Other	2	5.6
Contact history
Sexual contact^b	9	25
Single collection of plasma	15	41.7
Blood transfusion	8	22.2
Mother to child	4	11.1
Initial therapeutic regimen
3TC+NVP based regimen	26	72.2
DDI based regimen	8	22.2
3TC+EFV-based regimen	2	5.6
Updated therapeutic regimen
LPV/r+3TC based regimen	29	80.5
3TC+NVP based regimen	5	13.9
Other^c	2	5.6
STD history
No	34	94.4
Yes	2	5.6
Total	36	100.0

Including college and above (one case) and senior middle school (one case).

Including nonmarital sexual contact (two cases) and couple sexual transmission (seven case).

Including LPV/r+AZT+TDF and 3TC+EFV+TDF.

MTCT, mother-to-child transmission; STD, sexually transmitted disease.

As given in Table 1, only the free first-line antiretroviral drugs were included in initial ART regimens, and therapeutic regimens containing 3TC and NVP accounted for 72.2% of patients (26/36). In the updated ART regimens, regimens containing the second-line drugs LPV/r and TDF accounted for 80.5% (29/36) of patients, and 13.9% (5/36) of patients received the first-line regimens harboring 3TC and NVP.

HIV-1 DR mutations detected throughout the period of ART implementation

As given in Supplementary Tables S1–S3, DR mutations were detected in 88.9% (32/36) of patient samples. Throughout the period of ART implementation, 13.9% (5/36) of samples had a single non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation, 75.0% (27/36) harbored NNRTI and nucleoside reverse transcriptase inhibitor (NRTI) dual mutations, and no protease inhibitor (PI) and single NRTI mutations were observed. Furthermore, Supplementary Table S4 shows that 14.3% (8/36) of patients harbored thymidine analogue mutations (TAMs; six cases) or Q151M complex mutations (two cases), accounting for 25.0% (8/32) of patients exhibiting DR. The occurrence of DR mutations (83.3%, 30/36) in patients undergoing initial ART before adjustment was significantly higher than that (41.7%, 15/36) in patients after adjustment (χ² = 10.356, p = .001).

Of interest, Hb35 (Supplementary Table S3), Hb33 (Supplementary Table S3), Hb36 (Supplementary Table S2), and Hb34 (Supplementary Table S3) had undergone ART for 13, 14, 14, and 16 years, respectively; however, no resistance-associated mutations were detected in these four patients during the entire ART. Before adjustment for ART regimens, 83.3% (30/36) of patients receiving initial ART regimens exhibited greater than two consecutive DR mutations. After adjustment, 41.7% (15/36) of patients exhibited at least one DR mutation during the updated ART.

In 36 patients who had undergone ART for 9–16 years, a total of 213 blood plasma samples showed virological failure (VL ≥1,000 copies/mL). An HIV-1 genotypic DR assay was performed using these plasma samples and 151 HIV-1 pol gene sequences (1.3 kb) were also obtained from these samples. Detailed data on the HIV-1 DR mutations are provided in Table 2. The most frequent DR mutation was M184V (49.7%, 75/151), followed by K103N/S (35.1%, 53/151). In NRTI coding regions, T215Y/N/F was the second most frequent mutation, accounting for 27.8% (42/151), followed by M41L (18.5%, 28/151), K70R (10.6%, 16/151), K219E/N/Q (10.6%, 16/151), L210W (9.9%, 15/151), D67N (9.3%, 14/151), V75M/I (7.3%, 11/151), T69D/S (4.6%, 7/151), and all other mutations occurred at a frequency of ≦4.0%.

Table 2.

HIV-1 DR Mutations Among Patients Receiving Antiretroviral Therapy Between 2003 and 2019

DR mutations	Patients related to mutation site (%)	Occurrence number of mutation site (n = 151) ^a	Frequency (%)	Initial ART before drug adjustment	Updated ART after drug adjustment
Nucleoside reverse transcriptase inhibitors
M184V	26 (72.2)	75	49.7	52	23
T215Y/N/F	13 (36.1)	42	27.8	29	13
M41L	10 (27.8)	28	18.5	17	11
K70R	7 (19.4)	16	10.6	11	5
K219E/N/Q	7 (19.4)	16	10.6	10	6
L210W/LW	5 (13.9)	15	9.9	7	8
D67N	6 (16.7)	14	9.3	8	6
V75M/I	3 (8.3)	11	7.3	3	8
T69D/S	2 (5.6)	7	4.6	3	4
Q151M	2 (5.6)	6	4.0	5	1
K65R	3 (83.)	6	4.0	5	1
A62V/AV	3 (8.3)	5	3.3	3	2
L74V/I	3 (8.3)	5	3.3	3	2
F116Y	1 (2.8)	3	2.0	3	0
F77L	1 (2.8)	2	1.3	1	1
E44D	1 (2.8)	1	0.7	0	1
Non-nucleoside reverse transcriptase inhibitors
K103N/KN/S	19 (52.8)	53	35.1	43	10
Y181C	14 (38.9)	38	25.2	26	12
V106A/M/I	9 (25)	19	12.6	13	6
G190A/S	5 (13.9)	16	10.6	12	4
H221Y	4 (11.1)	15	9.9	10	5
Y188L	4 (11.1)	12	8.0	10	2
V108I	4 (11.1)	11	7.3	6	5
K101E/H/P	5 (13.9)	11	7.3	9	2
K238T	6 (16.7)	9	6.0	6	3
F227L	3 (8.3)	8	5.3	1	7
V179E/D	3 (8.3)	8	5.3	7	1
N348I	2 (5.6)	2	1.3	2	0
M230L/I	2 (5.6)	2	1.3	0	2
P225H	2 (5.6)	2	1.3	1	1
E138Q	1 (2.8)	2	1.3	2	0

A viral load ≥1,000 was classified as virological failure based on WHO HIV DR surveillance guideline; During ART, there were a total of 213 blood plasma samples with virological failure, and HIV DR genotyping was performed using these plasma samples; 151 HIV-1 pol gene sequences (1.3 kb) were obtained from 213 samples with viral load ≥1,000.

ART, antiretroviral therapy; DR, drug resistance.

In the NNRTI coding regions, Y181C was the second most frequent mutation, accounting for 25.2% (38/151), followed by V106A/M/I (12.6%, 19/151), G190A/S (10.6%, 16/151), H221Y (9.9%, 15/151), Y188L (8.0%, 12/151), V108I (7.3%, 11/151), K101E/H/P (7.3%, 11/151), K238T (6.0%, 9/151), FZZ7L (5.3%, 8/151), V179E/D (5.3%, 8/151), and all other mutations occurred at a frequency of <2.0%. Significantly, patients harboring M184V and T215Y/N/F mutations accounted for 96.3% (26/27) and 48.1% (13/27) of those resistant to NRTIs, respectively, and K103N/S and Y181C mutations accounted for 59.4% (19/32) and 43.8% (14/32) of those resistant to NNRTIs, respectively. The frequencies of HIV-1 mutations (Table 2) before adjustment for ART regimens were higher than those after adjustment (t = 3.345, p = .002). In contrast to the DR mutations distributed among the 32 patients after the initial ART, HIV-1 DR mutations after adjustment were mainly distributed among 13 patients experiencing treatment failure (76.5%, 13/17).

As described previously,⁹ the mutations found in this study could cause low-, intermediate-, or high-level resistance to first-line antiretroviral drugs at different stages of ART. Figure 1 shows that the frequencies of DR to NVP, EFV, 3TC, FTC, ABC, RPV, DOR, AZT, D4T, DDI, ETR, and TDF were 70.2% (106/151), 70.2% (106/151), 54.3% (82/151), 54.3% (82/151), 53.0% (80/151), 45.0% (68/151), 41.1% (60/151), 37.1% (56/151), 35.1% (54/151), 33.8% (51/151), 30.5% (46/151), and 49.8% (45/151), respectively. The details of the resistance levels to 12 antiretroviral drugs caused by mutations are given in Figure 1. There was a high level of resistance to each drug. The four drugs with the highest levels of resistance were NVP (99.1%; 105/106), 3TC (95.1%; 78/82), FTC (95.1%; 78/82), and EFV (78.3%; 83/106).

FIG. 1.

HIV-1 drug resistance to first-line antiretroviral drugs at different stages of ART. ART, antiretroviral therapy.

Treatment outcomes in HIV-1 patients receiving ART from 2003 to 2019

Among the 36 patients, 36.1% (13/36) experienced treatment failure and 63.9% (23/36) experienced effective treatment. The proportions of patients with effective treatment after adjustment (63.9%, 23/36) were significantly higher than those before adjustment (0.0%, 0/36) (χ² = 33.796, p < .001).

Virological outcomes, CD4 cell counts, and DR in HIV-1 patients receiving first-line ART before adjustment

Table 3 provides data relating to the study patients for whom first-line ART was initiated between 2003 and 2010. The ART regimens were adjusted for these patients between 2009 and 2015. As given in Supplementary Tables S1–S3, during the initial ART regimen, only 2 (Hb22 and Hb36) of the 36 patients achieved immunological reconstruction, accounting for 5.6% (2/36), and CD4 cell counts for patients Hb22 and Hb36 reached 504 and 657 cells/mm³, respectively, in the final year of initial ART. The CD4 cell counts of nine patients (25.0%, 9/36) were >200 cells/mm³, but <500 cells/mm³. During the course of initial ART, the CD4 cell counts in 25 patients (69.4%, 25/36) showed a general decline, falling <200 cells/mm³ in the later stage of initial treatment. This included 10 patients with CD4 cell counts <100 cells/mm³ and 15 patients with CD4 cell counts between 100 and 200 cells/mm³.

Table 3.

Diagnosis Date, Therapeutic Regimens and Clinical Symptoms of 36 Study Patients

Patient ID	Date of diagnosis	Initial ART regimen	Initial date of therapy	Updated ART regimen	Updated date of ART	Clinical symptoms in the past 3 months
Hb01	November 9, 2009	3TC+D4T+NVP	June 6, 2010	LPV/r+3TC+TDF	June 30, 2014	Lymphadenectasis, herpes zoster, cough, expectoration
Hb02	November 26, 2007	3TC+AZT+NVP	June 1, 2009	LPV/r+AZT+TDF	August 19, 2014	Headache, dyspnea, blurred vision, anemia. This patient died from diseases related to AIDS on September 1, 2015
Hb03	November 29, 2007	3TC+AZT+NVP	June 1, 2009	3TC+TDF+NVP	August 12, 2012	Fever, cough, blurred vision. This patient died from diseases related to AIDS on June 25, 2014
Hb04	January 29, 2008	3TC+AZT+NVP	September 12, 2008	LPV/r+3TC+TDF	June 7, 2011	—
Hb05	March 26, 2004	3TC+D4T+NVP	May 5, 2004	LPV/r+3TC+TDF	August 1, 2011	Anemia, sick, vomiting
Hb06	March 6, 1995	DDI+D4T+EFV	October 24, 2003	LPV/r+3TC+TDF	October 20, 2011	—
Hb07	January 22, 2010	3TC+AZT+EFV	May 7, 2010	LPV/r+3TC+TDF	November 24, 2013	—
Hb08	December 17, 2004	DDI+AZT+NVP	July 22, 2005	LPV/r+3TC+TDF	July 20, 2012	—
Hb09	November 18, 2004	3TC+D4T+NVP	November 27, 2006	LPV/r+3TC+TDF	November 19, 2012	Fever, cough, dyspnea, projectile vomiting, persistent or intermittent fever (>38°C, >1 month)
Hb10	November 24, 2004	DDI+AZT+NVP	December 2, 2004	LPV/r+3TC+TDF	March 11, 2014	—
Hb11	April 1, 2003	3TC+D4T+NVP	October 25, 2003	LPV/r+3TC+TDF	April 29, 2011	Cough, sleep hyperhidrosis, erythra, persistent or intermittent fever (>38°C, >1 month)
Hb12	March 26, 2004	3TC+D4T+NVP	May 1, 2004	LPV/r+3TC+TDF	April 1, 2011	—
Hb13	November 3, 2006	3TC+D4T+NVP	November 9, 2006	LPV/r+3TC+AZT	September 10, 2014	Fever, vomiting, persistent or intermittent fever (>38°C, >1 month), herpes zoster
Hb14	December 14, 2001	3TC+D4T+NVP	March 31, 2006	LPV/r+3TC+TDF	June 30, 2014	Cough, projectile vomiting, blurred vision, erythra, persistent or intermittent fever (>38°C, >1 month), herpes zoster
Hb15	March 14, 1995	3TC+D4T+NVP	March 31, 2004	LPV/r+3TC+TDF	June 30, 2010	Sleehyperhidrosis, sick
Hb16	March 6, 1995	DDI+D4T+EFV	May 12, 2004	LPV/r+3TC+TDF	May 26, 2009	Cough, dyspnea, chest pain, blurred vision, sleep hyperhidrosis, sick, headache, erythra, persistent diarrhea (>1 month)
Hb17	November 30, 2007	3TC+D4T+NVP	January 26, 2008	LPV/r+3TC+AZT	December 25, 2012	Fever, erythra, herpes zoster, sick, vomiting, headache
Hb18	March 6, 1995	3TC+D4T+NVP	December 29, 2006	LPV/r+3TC+AZT	May 20, 2013	Herpes zoster
Hb19	March 1, 1995	DDI+AZT+NVP	October 25, 2004	LPV/r+3TC+TDF	January 15 2010	Herpes zoster, erythra, blurred vision, dyspnea. This patient died from PCP on November 24, 2016
Hb20	December 30, 2005	3TC+D4T+NVP	December 10, 2008	LPV/r+3TC+TDF	May 24, 2011	Fever, cough
Hb21	June 9, 2009	3TC+D4T+NVP	June 16, 2009	LPV/r+3TC+TDF	May 20, 2013	Influenza
Hb22	April 12, 2002	DDI+D4T+NVP	October 3, 2004	LPV/r+3TC+TDF	January 5, 2015	—
Hb23	June 9, 2005	3TC+D4T+NVP	December 31, 2006	LPV/r+3TC+TDF	August 1, 2011	Fever, cough, expectoration, projectile vomiting, headache, erythra, herpes zoster
Hb24	April 1, 2010	3TC+D4T+NVP	April 20, 2010	LPV/r+3TC+TDF	April 24, 2015	Persistent or intermittent fever (>38°C, >1 month), anti-HCV positive; This patient died from PCP on December 21, 2015
Hb25	December 29, 2007	3TC+AZT+NVP	April 15, 2008	3TC+D4T+NVP	June 20, 2014	—
Hb26	February 23, 2006	3TC+D4T+NVP	July 26, 2006	LPV/r+3TC+AZT	September 3, 2012	—
Hb27	August 10, 2009	3TC+D4T+EFV	June 2010/06/18	LPV/r+3TC+TDF	May 25, 2015	Vomiting
Hb28	March 6, 1995	3TC+D4T+NVP	May 24, 008	LPV/r+3TC+AZT	July 20, 2015	Fever, cough, expectoration, projectile vomiting, headache, erythra, Persistent or intermittent fever (>38°C, >1 month), herpes zoster
Hb29	August 19, 2004	3TC+D4T+NVP	May 24, 2008	LPV/r+3TC+AZT	July 20, 2015	Sleep hyperhidrosis, headache, blurred vision, erythra, lymphadenectasis, herpes zoster; This patient died from diseases related to AIDS on February 18, 2016
Hb30	March 31, 2006	3TC+D4T+NVP	April 1, 2007	3TC+AZT+NVP	June 30, 2014	Herpes zoster, erythra
Hb31	October 17, 1999	3TC+D4T+NVP	June 24, 2010	3TC+TDF+EFV	June 25, 2015	Herpes zoster, erythra, persistent or intermittent fever (>38°C, >1 month)
Hb32	July 24, 2006	3TC+AZT+NVP	April 20, 2008	LPV/r+3TC+TDF	July 8, 2011	—
Hb33	March 17, 2005	DDI+AZT+NVP	April 6, 2005	3TC+AZT+NVP	March 22, 2012	—
Hb34	March 6, 1995	DDI+D4T+EFV	October 24, 2003	LPV/r+3TC+TDF	May 11, 2009	—
Hb35	June 1, 2006	3TC+D4T+NVP	June 22, 2006	LPV/r+3TC+TDF	October 18, 2011	Fever, influenza, dyspnea
Hb36	May 10, 2005	3TC+D4T+NVP	June 10, 2005	3TC+AZT+NVP	November 22, 2012	Sick, vomiting, diarrhea

PCP, Pneumocystis carinii pneumonia.

Therefore, these 25 patients had undergone immunological failure. Furthermore, each of the patients experienced virological failure between one and seven times. The mean value for virological failure per patient was 3.8 (138/36) times. Particularly in the later stage of initial treatment, counting backwards from the final year of initial treatment to the beginning of initial treatment, the proportions of patients who showed 1-, 2-, 3-, 4-, 5-, and 6 years of consecutive virological failure were 8.3% (3/36), 25.0% (9/36), 25.0% (9/36), 22.2% (8/36), 13.9% (5/36), and 5.6% (2/36), respectively; furthermore, the proportions of patients who showed 1-, 2-, 3-, 4-, and 5 years of consecutive DR to reverse transcriptase (RT) inhibitors were 16.7% (6/36), 36.1% (13/36), 16.7% (6/36), 2.8% (1/36), and 2.8% (1/36), respectively, and the remaining nine patients showed no DR within the final 3 years of initial treatment.

Virological outcomes, CD4 cell counts, and DR in HIV-1 patients receiving updated ART after adjustment

The ART regimens of the study patients were adjusted because of the poor effects observed with the initial ART regimens, as mentioned previously. After adjustment, second-line ART regimens and first-line ART regimens accounted for 83.3% (30/36) and 16.7% (6/36) of updated ART regimens, respectively. According to changes in the annual VL, CD4 cell count, and DR, as well as clinical symptoms reported over the past 3 months, 13 (36.1%) of 36 patients were diagnosed with treatment failure (Supplementary Table S1). All 13 patients with treatment failure presented different clinical symptoms over the past 3 months (Table 3), which included Pneumocystis carinii pneumonia (PCP), dyspnea, blurred vision, anemia, sleep hyperhidrosis, erythra, persistent or intermittent fever (>38°C, >1 month), and herpes zoster.

Furthermore, Hb02, Hb03, Hb19, Hb24, and Hb29 died from diseases related to AIDS, such as PCP, in months 12, 22, 82, 8, and 7 after updated ART initiation, respectively. These 13 patients with treatment failure had experienced serious virological failure, immunological failure, and DR to RT inhibitors from the start of the initial ART regimen to the end of the updated ART regimen. Moreover, throughout the observation of clinical symptoms over the past 3 months, we found that all 13 patients experienced a range of clinical symptoms (Table 3), such as PCP, dyspnea, blurred vision, anemia, and persistent diarrhea (>1 month). Compared with the annual means ± standard deviations of the VLs of 4.41 ± 0.46 log RNA copies/mL and CD4 cell counts of 141.8 ± 65.7 cells/mm³ before adjustment, the annual means ± standard deviations of the VLs and CD4 cell counts after adjustment showed a small change at 3.72 ± 1.26 log RNA copies/mL and 160.72 ± 65.7 cells/mm³, respectively.

Specifically, the annual mean values for VLs and CD4 cell counts for five of the patients who died were 4.51 log RNA copies/mL and 141.8 cells/mm³, 4.45 log RNA copies/mL and 173.2 cells/mm³, 4.57 log RNA copies/mL and 38.5 cells/mm³, 5.0 log RNA copies/mL and 84.5 cells/mm³, and 4.15 log RNA copies/mL and 180.6 cells/mm³, respectively. Supplementary Table S1 provides data indicating the changes in VLs and CD4 cell counts for the 13 patients.

The remaining 23 patients (63.9%, 23/36) underwent effective treatment after adjustment. The means ± standard deviations of the VLs and CD4 cell counts before adjustment for ART regimens were 3.85 ± 0.65 log RNA copies/mL and 226.83 ± 106.06 cells/mm³, respectively; however, the means ± standard deviations of the VLs and CD4 cell counts after adjustment were 2.19 ± 0.58 log RNA copies/mL and 367.68 ± 174.62 cells/mm³, respectively. Of note, paired-sample t-tests indicated that there were statistically significant differences in the changes in VLs (t = 8.728, p < .001) and CD4 cell counts (t = −4.476, p < .001) before and after adjustment. Table 3 shows that 12 patients had no symptoms, and 11 patients experienced some symptoms, such as a fever, cough, influenza, and vomiting, over the past 3 months, but their symptoms were relatively mild and lasted only a short duration, suggesting that these symptoms may have been caused by adverse effects from antiretroviral drugs.

In general, after the initiation of updated ART regimens, the VLs of these 23 patients showed an obvious decreasing trend year by year. However, their CD4 cell counts showed an increasing trend year by year (Supplementary Tables S2 and S3). In the later stage of the updated ART regimens (counting backwards from the final year of updated ART to the start of the updated ART), the VLs of all 23 patients had fallen to <1,000 copies/mL with the exception of 6 patients for whom viral rebounds occurred in the second or third year. In this study, in the final year of the updated ART regimen, the VLs of almost all patients had fallen to below the minimum detectable level (<20 copies/mL), with the exception of Hb26 with 2.09 log RNA copies/mL, Hb01 with 2.82 log RNA copies/mL, and Hb18 with 2.09 log RNA copies/mL.

With the use of updated ART regimens year by year, a recovery of immunologic function was observed in 23 patients. For 10 patients, the CD4 cell counts rose rapidly year by year, and the means of their CD4 cell counts reached 660.5 (543–801) cells/mm³ in the final year of the updated ART regimens, obtaining immunologic reconstruction. For the other 13 patients, the CD4 cell counts did not reach 500 cells/mm³, but in the final year of the updated ART regimen, their CD4 cell counts (mean: 340.00 ± 81.00 cells/mm³) rose significantly (t = 6.741, p < .001) compared with their CD4 cell counts before adjustment (mean: 142.92 ± 71.42 cells/mm³). No DR mutations were found in these patients.

Discussion

According to China's National Free Antiretroviral Treatment Guidelines (Fourth Edition),⁶ the VL, CD4 cell count, and clinical symptoms are three vital indicators that can be used to estimate disease progress. Our results indicated that most patients who received updated ART regimens containing LPV/r and TDF after adjustment achieved better therapeutic effects compared with those receiving initial ART regimens harboring D4T/AZT or NVP. In the first-line regimens after adjustment, the replacement of DDI (Hb33) and D4T (Hb36) with 3TC and AZT, respectively, improved the treatment effects. However, our study results were not in line with those of a previous study that showed that LPV/r had poor therapeutic effects as an initial treatment drug.⁹ This suggests that the effect of treatment was improved significantly by adjusting treatment regimens in which LPV/r and TDF were included when the first-line initial ART regimens were unsuccessful.

Compared with the levels before ART, the CD4 cell counts increased by 30% or 100 cells/mm³ following ART, and immunological reconstitution was achieved when CD4 cell counts were >500 cells/mm³.^10,11 In our study, by analyzing the changes in CD4 cell count, VL, and DR in HIV/AIDS patients receiving 9- to 16-year ART and their clinical symptoms over the past 3 months, we found that 23 of 36 (63.9%) patients who experienced first-line initial ART failure experienced effective ART after adjustment of ART regimens. The VLs of these 23 patients showed an obvious decreasing trend year by year after the updated ART regimen was initiated and an increasing trend in their CD4 cell counts.

However, the remaining 13 patients experienced serious virological failure, immunological failure, and DR to RT inhibitors from the start of the initial ART regimen to the end of the updated ART regimen, and experienced a range of WHO-defined stage II, III, or IV clinical symptoms, such as PCP, dyspnea, and so on. Five of these 13 patients died from diseases related to AIDS, such as PCP, soon after the start of updated ART regimens. Furthermore, the frequencies of HIV-1 mutations before adjustment for ART regimens were higher than those after adjustment (t = 3.345, p = .002).

We deduced that the reasons associated with treatment failure were as follows: (1) poor adherence: possibly owing to the following: (i) 94.4% of patients had a poor level of education (junior middle school or less); (ii) 6 of 13 patients with treatment failure experienced blurred vision, influencing their cognitive ability; and (iii) 66.7% of study patients experienced clinical symptoms over the past 3 months; (iv) the adverse effects of drugs. For example, DDI and D4T cause clinical diseases. The above factors can induce discomfort and lead to missed doses or incomplete continuation of treatment. (2) A lower baseline CD4 cell count: in 15%–20% of patients it was difficult to achieve normal CD4 cell counts because of their lower CD4 cell count (<200 cells/mm³) before ART, even if their VLs were completely suppressed.⁶ In our study, initial ART was started in patients between 2003 and 2010, when their CD4 cell counts before ART were <200 cells/mm³, and in some cases even as low as 5 cells/mm³. (3) DR: with the extensive use of ART worldwide, HIV-1 DR has become widespread.

In this study, the patients for whom treatment failure occurred had experienced resistance to RT inhibitors from the beginning of initial ART regimens to the end of updated ART regimens, and HIV-1–resistant strains had become the dominant strains. Even after undergoing a prolonged period of ART, six patients presented TAMs and two patients presented Q151M mutation, leading to high-level resistance to AZT and TDF, respectively.

In conclusion, patients who received updated ART regimens containing LPV/r and TDF after adjustment achieved better therapeutic effects compared with those who received initial ART regimens harboring D4T/AZT or NVP. Future research is needed to initiate the surveillance of DR, VL, and CD4 cell counts immediately after HIV diagnosis, and dynamic changes in these indicators.

Footnotes

Acknowledgments

The authors thank all staff involved in this study. The authors thank Liwen Bianji (Edanz) for editing the English text of a draft of this article.

Availability of Data and Materials

The authors confirm data and material used in this study are included in the article and Supplementary Materials. Patients` names will not be shared according to the principle of confidentiality in China`s AIDS prevention and control regulations. Study sequences have been submitted to GenBank with accession numbers: OP277803–OP277917.

Authors' Contributions

Q.L. and X.L. designed the study. M.L. and X.L. acquired the gene sequences. Y.L., Y.W., N.A., and M.L. carried out the experiments. J.X. and X.L. analyzed and interpreted the data. L.M. carried out the investigation of epidemiology. X.L. wrote the article. All authors read and agreed to the published version of the article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Natural Science Fund of Hebei Province (Grant No. H2016303006) and the Key Research Items of Hebei Province (Grant No. 192777107D), China. There was no additional external funding received for this study.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

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Supplementary Material

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Changes of Viral Load,Drug Resistance,and CD4 Cell Count in HIV-1–Infected Patients Receiving Antiretroviral Therapy in Hebei Province,China,2003–2019

Abstract

Introduction

Materials and Methods

Participants

ART regimens

Laboratory tests

Results

Demographic and epidemiological information about the study patients

HIV-1 DR mutations detected throughout the period of ART implementation

Treatment outcomes in HIV-1 patients receiving ART from 2003 to 2019

Virological outcomes, CD4 cell counts, and DR in HIV-1 patients receiving first-line ART before adjustment

Virological outcomes, CD4 cell counts, and DR in HIV-1 patients receiving updated ART after adjustment

Discussion

Footnotes

Acknowledgments

Availability of Data and Materials

Authors' Contributions

Author Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material