MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH
NATIONAL INSTITUTE OF HYGIENE AND EPIDEMIOLOGY
*






DO THI NHAN









RESEARCH OF HIV DRUG RESISTANCE AMONG
PATIENTS ON FIRST LINE ARV THERAPY
IN SELECTED PROVINCES






Specialization: Public Health
Code: 62 72 03 01

HANOI – 2014


The dissertation was completed at the
National Institute Of Hygiene And
Epidemiology

Supervisor: 1 Nguyen Thanh Long, Ass.Prof. PhD.
2 .Nguyen Van Trang, PhD


Oppenent 1: Prof. Dao Van Dung, the Central Propaganda Department
Oppenent 2: Ass.Prof. Nguyen Duc Hien, National Hospital of Tropical Disease
Oppenent 3: Ass.Prof. Nguyen Vu Trung, Hanoi Medical University of Medical


The dissertation will be defended before thesis committee at
National Institute Of

Hygiene And Epidemiology, at hour day month year 2014

The thesis information can be looked at following libraries:
- National Library
- Library of the National Institute of Hygiene and Epidemiology

i



MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH
NATIONAL INSTITUTE OF HYGIENE AND EPIDEMIOLOGY
*






DO THI NHAN









RESEARCH OF HIV DRUG RESISTANCE AMONG

PATIENTS ON FIRST LINE ARV THERAPY
IN SELECTED PROVINCES






Specialization: Public Health
Code: 62 72 03 01













HANOI – 2014
ii



LIST OF ABBREVIATIONS


Abbreviations English words Vietnamese meaning
HIV
Human immunodeficiency
virus

Virus gây suy giảm miễn dịch ở người
AIDS
Acquired Immunodeficiency
Syndrome
Hội chứng suy giảm miễn dịch mắc phải
HAART High active antiretroviral

therapy
Điều trị kháng retrovirus hoạt tính cao
ARV Antiretroviral Kháng retrovirus
NNRTI
Nonnucleoside reverse
transcriptase inhibitors

Nhóm thuốc ức chế men sao chép ngược
không ph
ả
i nucleoside

NRTI
Nucleoside reverse
transcriptase inhibitors

Nhóm thuốc ức chế men sao chép ngược
nucleoside


PI Protease inhibitors Nhóm thuốc ức chế protease
3TC Lamivudine
ABC Abacavir
AZT Zidovudine
EFV Efavirenz
NVP Nevirapine
TDF Tenofovir
BN Bệnh nhân
KTC confidence interval Khoảng tin cậy
RT Reverse transcriptase Gen sao chép ngược
TAM Thymidine analogue mutations

Các đột biến tương tự thymidine
WHO World Health Organization Tổ chức Y tế Thế giới
TTYT Trung tâm y tế
PKNT Phòng khám ngoại trú
OR Odd Ratio Tỷ suất chênh
PCR Polymerase Chain Reaction Phản ứng chuỗi trùng hợp
TLVR Tải lượng vi rút
T1 Thời điểm bắt đầu điều trị
T2 Thời điểm kết thúc nghiên cứu
TTPC HIV/AIDS Trung tâm phòng chống HIV/AIDS
1


INTRODUCTION

The expansion of antiretroviral therapyn (ART) can lead to the emergence and
spread of HIV drug-resistant due to poor adherence to treatment or supply ARV

discontinuously, or monitoring of HIV drug resistance is not fulfilled enough lead to limit
the effectiveness of ART.
HIV Drug Resistance (HIVDR) testing is neither routinely available nor
recommentded for individual patient manegement in most resource-limited countries,
including Vietnam. Genotyping is expensive and complex. However, the monitoring of
patient and clinic fators, early warning indicators for HIV drug resistance, associated with
the emergence of preventalble HIVDR is comparatively inexpensive and can be used to
identify these factors and reduce their harmful consequences. It also reflects the
implementation of HIV drug-resistant prevention programs at both the clinics and
program. Besides, the current situation of HIV drug resistance status in patients on ART
will provide the evidence for interventions to be implemented at all levels of the OPC and
program to maximize treatment effectiveness of ART. In Vietnam there still have not any
studies yet which reflect the status of early warning factors for HIV drug resistance as
well as no prospective research about HIV drug resistance status on patients from the time
they start using ARV treatment.
Therefore, we conducted a "Research of HIV drug-resistant among patients on first
line ART in selected provinces" with two objectives:
1. Describe the current condition of Early Warning Indicators of HIV drug
resistance at out patient clinic (OPC) in selected provinces from 2010 to 2012.
2. Identify HIV drug resistance outcomes and associated factors among patients on
first line ARV therapy.

New findings of the Thesis:
1. Provide the real situation of early warning indicators and their relationship with
HIV Drug resistance emergence in clinic sites and program level.
2. Provide the data of HIV drug-resistance among the patients on ART, include the
HIVDR prevalecne at iniating of ART, outcomes of HIVDR and HIVDR mutations after
12 months of ART initiating.
These are scientific evidences in providing and deceloping policy related to
expansion the ART effectively program, minimize the HIV drug-resistant situation in

Vietnam.

The layout of the thesis:
The dissertation consists of 142 pages (excluding appendices), 4 chapters include
Introduction: 2 pages; Chapter 1- Overview: 29 pages; Chapter 2 - Subjects and methods
Study: 24 pages; Chapter 3 - Results: 35 pages; Chapter 4-Discussion: 32 pages;
Conclusion: 2 pages; Recommendations: 1; 41 tables, 13 charts, 3 photos, 3 diagrams and
142 references, of which 22 Vietnamese and 120 documents in English.


2



CHAPTER 1
OVERVIEW

1.1 Early warning indicators for HIV drug resistance (EWI)
The early warning indicators of HIV drug resistance reflects the practice of ART at
the HIV/AIDS OPC treatment which include the appropriate prescripbing, procurement
and supply management of ARV drug, the relevant factors adherence to the treatment of
patients, viral load suppression and retention on first line ART. These factors are
associated with the HIV drug resistance emergence during ARV treatment.
1.2. HIV drug resistance situation of patients on ART
1.2.1 HIV drug resistance before ART initiation
There is an increase in the prevalence of HIV drug resistance mutations before the
ARV treatment, ranging from 4.8% - 6.8% from 2007 to 2010. Mainly is the surge with
NNRTI group. This involves the expanded use of Nevirapine for prevention of HIV
transmission from mother to child.
1.2.2. Drug-resistant HIV of patients on ART

According to WHO HIV Drug Resistance report in 2012, the HIV drug resistance
prevention after 12 months of ART initiating was 76.1%, HIV drug resistance was 5.1%
and possible HIV drug resistance was 18.8%.
In Vietnam, there have been some studies of HIV drug resistance, but they are the
cross-sectional studies, the researchers monitored populations of patients on ARV
treatment. There is not any study on the status of early warning indicators or cohort
monitoring of emergence of HIV drug resistance since initiating of antiretroviral therapy.


Chapter 2
METHOD OF STUDY

Methodology is designed according to the method which recommended by World
Health Organization for research/survey on HIV drug resistance.
2.1. Describe the current condition of Early Warning Indicators of HIV drug
resistance at out patient clinic (OPC) in selected provinces from 2010 to 2012
2.1.1. Object, venue and time line
2.1.1.1. Study participants
Participant inclusion criteria:
- HIV/AIDS patients with first line ART;
- Patient initiating of first line ART appropriate with requirement at the time of
data collection for each early warning indicators for HIV drug resistance.
Exclusion criteria:
- Patients not on ART or on the second line ART.
- Patients with time point of initiating of ART not consistent with the time
requirements of each EWI in the collection of samples.
3




2.1.1.2. Venue and timeline of study
Venue: 42 outpatient clinics (OPCs) treatment for HIV/AIDS in 23 provinces were
selected according to the following criteria:
- Providing ART for at least one year
- Representative of central, provincial and district level
- Representative of North, Central and South geographic regions
Time for collecting data: May 2010 – December 2012.
2.1.2. Method of the Research
2.1.2.1. Design of the Research: Cross-sectional descriptive methods
2.1.2.2. Research index: 5 indexs are selected as follow:
EWI 1: The proportion of initial ART prescribtion congruent with national
guideline.
EWI 2: the proportion of % patients lost to follow up at 12 months.
EWI 3: The proportion of % patients still retention on first line ART at 12 months
EWI 4: The proportion of patienst on-time clinical appointment keeping
EWI 5: Percentage of months in a designated year in which there were no ARV
drug stock-outs.
2.1.2.3. Time frame for collecting sample:
For EWI 1: The time for collecting sample was selected for each year of data
collecting, as follows:
Year 2010: Patients initiated ARV treatment from January to December 2009.
Year 2011: Patients initiated ARV treatment from January to December 2010
Year 2012: Patients initiated ARV treatment from January to December 2011.
For EWI 2 and EWI 3: The time for collecting sample was selected for each year
of data collecting, as follows:
Year 2010: Patients initiated ARV treatment from January to December 2008.
Year 2011: Patients initiated ARV treatment from January to December 2009
Year 2012: Patients initiated ARV treatment from January to December 2010.
For EWI 4: All patients on ARV treatment come as per dating in the 4th quarter of
the year preceding the year of data collection.

For EWI 5: Reports on coming in and out status of ARV of 12 months of the year
preceding the year of data collection.
2.1.2.4. Sample size
The formula for calculating sample sizes of each performance index as per
recommended by the WHO:
N = n
0
/ (1 + (n
0
-1)/n); n
0
= Z
2
*p*(1-p) / e
2

In which:
Z = 1,96; p = 0,5 (estimated prevalence of each index); e = Accury = 0,07 (based
on 95% with ± 7% deviation); n = number of patient with ARV treatment at OPC.
However, based on actual data, the research team decided to collect all the cases
eligible for the research.




4





Table 2.1. The number of medical reports and medication inventory situation was
collected for each index per year.

Indicators Number of medical reports/medication
inventory collected per collecting data year
2010 2011 2012
EWI 1 The proportion of initial ART
prescribtion congruent with
national guideline 5122 4542 4678
EWI 2 The proportion of % patients
lost to follow up at 12 months
5.631 4.778 4.727
EWI 3 The proportion of % patients
still retention on first line ART
at 12 months 5631 4778 4727
EWI 4 The proportion of patienst on-
time clinical appointment
keeping
4.365 5.134 5.536
EWI 5 Percentage of months in a
designated year in which there
were no ARV drug stock-outs
12 12 12
2.1.3. Data processing
Data was collected, entried and managed by using Excel software.
2.2 Identify HIV Drug Reristance and relevant factors among patienst on
ARV therapy first line
2.2.1. Target population, venues, and time line
2.2.1.1. Target pupulation
- Participant inclusion criteria

Patients attending the four selected OPCs who meet the following criteria:
- > 16 years old, and
- Individuals who consent following the written informed consent process, and
- Who are eligible to initiate, and do initiate, an adult first-line ART regimen at a
participating site. This includes individuals who have had previous ART elsewhere
(including in the private sector).
- Participant exclusion criteria
Patients who meet any of the following criteria are excluded from the survey:
1. Individuals enrolled in a clinical trial or clinical research study.
2. Individuals who are part of an observational cohort for whom more follow-up
efforts are made than for other ART patients treated at the site.
3. Individuals restarting ART, who have previously started and stopped ART at the
sentinel survey site.
4. Individuals who on ART transferring in from another ART site.
2.2.1.2. Study sites and timeline
5



Study sites: four adult OPCs have been chosen: Hai Phong out patient clinic (OPC)
in Viet Tiep hospital, Hai Duong OPC is located in Hai Duong Provincial AIDS Center
(PAC), District 1 OPC in Ho Chi Minh City and District 10 OPC in Ho Chi Minh City .
Time line:: 12/2009 to 12/2011.
2.2.2. Study Method
2.2.2.1. Method : Cohort study.
2.2.2.2. Sample size at each clinic
Survey Sample size calculation
According WHO’s guidelines, sample size at endpoint required (n) will be
obtained folloings fomular:
N * (p * (1 - p))

n =
(N - 1) * (e
2
/ z
2
) + (p * (1 - p))

n : sample size at endpoint;
N: total number of patients selected sites; p: estimated prevalence of HIV Drug
Resistance; e: unknown proportion within 0.1 (i.e. 10%) of the
true population proportio; Where z is the α / 2 point of the Normal distribution and
ignoring the finite population correction this formula simplifies to:
z
2
* (p * (1 - p))
n =
e
2

n = 96

The estimated sample size required at each OPC to ensure at least 96 patients will
be classifiable at the end of the 12 month study period, 96 individuals initially starting
ART at the site, plus an additional number calculated for that site, reflecting the numbers
who transferred out and those who died. Actual samplle size in each OPC as folloings:
- OPC in Việt – Tiệp Hospital in Hai Phong City: 127 patients
- OPC in Hai Duong PAC: 118 patients
- OPC in District 1 in Ho Chi Minh City: 134 patients
- OPC in District 10 in Ho Chi Minh City: 122 patients
Total patient have recruited in 4 selected OPCs: 501 patients

2.2.2.4. Diagram of research:
6






Diagram 2.1: Diagram of research
2.2.2.3. Main result s of HIV Drug Resistance (HIVDR) in study
- HIV Drug Resistance before initiating of ARV Treatment:
- HIV drug resistance classification at defined endpoints:
HIV drug resistance prevention: defined as HIV RNA suppression to < 1,000
copies/ml as measured using a quality assured viral load assay in patients still on first-line
ART at 12 months.
Possible HIVDR: defined as a detectable viral load with no detectable HIVDR
mutations. Specimens from patients still on a first-line ART regimen with a viral load
≥1,000 copies/ml and no evidence of HIVDR on genotype testing are classified as having
possible HIVDR. Patients who have been lost to follow-up or who stopped ART within
the first 12 months of ART, and from whom no specimens are available for classification,
are also classified as having possible HIVDR. Other patients for whom no endpoint viral
load or HIVDR result is available, and who did not transfer out or die during their first 12
months of HIVDR, are also classified as having possible HIVDR.
HIV Drug Resistance (HIVDR): defined as the presence of one or more mutations
leading to low, intermediate, or high-level resistance to one or more relevant antiretroviral
7



drugs detected by standard HIVDR genotyping at the endpoint on therapy at 12 months or

at time of switch to second line regimens.
2.2.2.5 Rresearch procedures
Recruiment of patients at base line (T1)
At the the time of commencement of first-line ART, in the last of counselling
session, all patients with whom the decision has been made to initiate ART at the sentinel
site on or after the identified survey start date will be enrolled consecutively until the
effective survey sample size is reached. If patient don’t agree to attend research, they will
continue to receive ARV treatment as routine procedure.
At the time of enrolment (baseline), basic patient information was abstracted from
the patient records, including socio-demographic information, previous ARV exposure
(including PMTCT, previous ART), regimen prescribed and date of start of ART, CD4
count and WHO clinical staging. Plasma was collected at enrolment for viral load and
genotypic HIV drug resistance testing if viral load ≥ 1000 copies/ml. All plasma
specimens at enrolment was collected on the day ART is prescribed for the patient but
before the patient has consumed any dose of ART. The information extracted includes the
dates of scheduled and actual appointments, the dates of scheduled and actual pharmacy
pick-ups, changes in regimens, adherence as measured by the 30-day before endpoint of
research. At the endpoints of switch and on first-line ART at 12 months, plasma was
collected, at the same time as routine testing, for viral load quantification and HIVDR
testing was performed on specimens with detectable viral loads.
Specimens sent to the Pasteur Institute (PI) for VL and genotyping. This is a lab
accredited by WHO for HIVDR testing. Tests were done with two plasma cryotubes (1
for VL, 1 for HIVDR), the remaining cryotubes will be kept frozen at the HIVDR
laboratory as reserves. Specimens with VL ≥ 1,000 copies/ml will be selected to perform
genotypic sequencing. The gennotypic sequencing results were analysed by Seqman –
DNASTAR Version 9.0. In order to evaluate the drug resistance level, data entried to
online HIVDR genotyping – Stanford version 5/2013. Resistant level to each drug was
evaluated base on the total point of all mutations in 5 levels: sensitivity, low capacity to
resit, low level of resistance, middle level of resistance and high level of resistance.
2.2.2.6 Data management and analysic

The data entried to EpiInfor 6.0 then was exported to data analysis STATA 11
software.
2.2.3. Ethical consideration
This research received approcal of IRB Committee in Ministry of Health and
Hanoi Public Healthe School for implementing.
8




Chapter 3
RESULTS

3.1. Describe of HIV Drug Resistance Early Warning Indicators (EWI) in 42
HIV/AIDS out patient clinics
3.1.1 General of results
Table 3.1. Number of patient records and ARV drug reports collected in 3 years
(2010 – 2012)
EWIs
Number of patient
records collected 2010 2011 2012
EWI 1
% of initial ART
prescribtion
congruent with
national guideline
Target: 100%
Number of patients
initiating ART at the site
on the designated EWI

sample start date 5122 4542 4678
Number of patients
initiating ART at the site
who are prescribed an
appropriate first-line
national ART regimen
5116
(99,9%)
4542
(100%)
4678
(100%)
EWI 2
% patients lost to
follow up at 12
months
Target: <20%
Number of patients
initiating ART at the site
on the designated EWI
sample start date 5631 4778 4727
Number of patients
initiating ART at the site
who, during the first 12
months after ART
initiation, did not attend
a clinical consultation
and did not pick up ARV
drugs within 90 days
(≤90 days) after the date

of their last missed
appointment, or within
90 days (≤90 days) after
the last ART run-out
date
304
(5,4%)
277
(5,8%)
280
(5,9%)
EWI 3
% Retention on
first line ART at
12 months
Target: ≥70%
Number of patients
initiating ART at the site
on the designated EWI
sample start date 5631 4778 4727
Number of adult patients
initiating ART at the site
who are on an
4645
(82,5%)
3921
(82,1%)
3950
(83,6%)
9




appropriate first-line
ART regimen 12
months after ART
initiation
EWI 4
% on-time clinical
appointment
keeping
Target: >80%
Number of patients who
attended a clinical
consultation on the
quarter 4 of year 4365 5134 5536
Number of patients who
attended two consecutive
scheduled after a
baseline consultation in
the quarter 4 of year
3938
(90,2%)
4454
(86,8%)
4918
(88,8%)
EWI 5
Percentage of
months in a

designated year in
which there were
no ARV drug
stock-outs (cross-
sectional)
Target: 100%
12 months 12 12 12
Number of months in
which there were no
stock-out days of any
ARV drug routinely used
at the site
12 12 12

Comments: 100% of OPCs having no ARV stock-outs over a 12 month period in 3
years. In general, all EWIs for 42 sites met WHO’s target.
3.1.2 Results of each EWIs:
EWI 1 (Prescribing of ART congruent with national gudeline): overall, 92,9% of
42 OPCs monitored met the target of 100% of patients receiving prescription for ART
congruent with national guidelines with 92,9%, 100% and 100% in 2010, 2011 and 2012
respectively.
EWI 2 (Lost to follow-up at 12 months): Overall, the indicator about loss to follow
up at 12 month of ART initiation (EWI 2) was low with results in 2010, 2011 and 2012
with 5.4% (range from 0% - 28.3%), 5.8% (range from 0% - 24.5%) and 5.9% (range
from 0% - 17.1%) respectively. The proportion of OPCs met the WHO target of ≤20%
patients lost to follow up at 12 months were 97.6%, 90.5% và 100% in 2010, 2011 and
2012 respectively.
EWI 3 (Retention on first line ART at 12 months): Results of EWI 3 were 82,5%
(range from 56.5% - 100%), 82.1% (range from 55.1%- 97.4%), and 83.6% (range from
47.1% - 100%) in 2010, 2011 and 2012 respectively. The proportion of OPCs met the

WHO target of ≥70 patients retention on first line after 12 months were 83.3%, 88.1% và
88.1% in 2010, 2011 and 2012 respectively.
EWI 4 (on-time clinical appointment keeping): Results of EWI 4 were 90.2%
(range from 16.7% - 100%), 86.8% (range from 42.3% - 100%), and 89% (range 43.4% -
100%) in 2010, 2011 and 2012 respectively. The proportion of OPCs met the WHO target
of ≥ 80% patients keeping 100% of clinical appoitments on time were 83.3%, 76.2% and
81% in 2010, 2011 and 2012 respectively.
10



EWI 5 (ARV supply continuity): 100% of OPCs met WHO’s target of 100% ARV
supply continuity.
3.1.3. EWIs in 4 OPCs implementing of monitoring of HIV Drug resistance among
patients on first line ARV:
These OPCs were OPC in Hai Duong Provincial AIDS Center, OPC in Viet Tiep
Hospital in Hai Phong city, 2 OPCs in Health Center in District 1 and in District 10 in Ho
Chi Minh City. EWI 2, EWI 3 and EWI 5 met WHO’s target. With EWI 1, the proportion
of patients got prescribing of ART congruent with national guideline were 98.9%, 100%
and 100% in 2010, 2011, and 2012 respectively. OPC in Hai Duong PAC and OPC in
District 10 in Ho Chi Minh Cityt did not meet WHO’s target in 2011. In 2012, all EWIs in
4 OPCs met the WHO’s target.
3.2. Idenfity the HIVDR characteristic on patients on ART
3.2.1 Charateristic of HIVDR at T1
3.2.1.1. Demographic characteristics
Among 504 patients who meet the criterials for 1
st
line ART, two patients refused
to participate the study, one patient without speciment. The ratio of male patient was
higher than female patient (63.3% vs 36.7%). The average age of patients was 32.5 years

old. The transportation rates through sexsual intercourse and IDUs were 57.7% and
39.2%, respectively. One patient was infected through MSM while 13 patients (2.8%)
were not indentified the transportation route.
Clinical and Immunological characteristic when starting ART
At T1, there were 32 patients (6.4%) exposed to ARV previously, 147 patients
infected and treated TB (29.3%).
The distribution of CD4 results when starting the study is showed in table 3.10
Table 3.10. CD4 results distribution when starting the treatment
Characteristic
District 1-
HCMC
(n = 134)
District 10-
HCMC
(n = 122)
Hai Duong

(n= 127)
Hai Phong

(n = 118)
Total
(501)
CD4 results (cell/ml)
Mean 144 142,3 97,1 123,1 126,8
Median 142 167 56 87,5 106
Variation 1-725 2–408 14-603 4-387 1-725
CD4 results, percentage (%)
≤50 39 (29,1) 31 (25,4) 62 (48,8) 45 (38,1) 177 (35,3)


51–100 10 (7,5) 18 (14,8) 23 (18,1) 18 (15,3) 69 (13,8)
101–250 72 (53,7) 66 (54,1) 34 (26,8) 39 (33,1) 211 (42,1)

251–350 8 (6,0) 5 (4,1) 4 (3,2) 15 (12,7) 32 (6,4)
>350 5 (3,7) 2 (1,6) 4 (3,2) 1 (0,9) 12 (2,4)
Comments: Median of CD4 number when starting treatment is 106 cell/mm
3
, in
which the lowest CD4 median is Hai Duong (56 cell/mm
3
) and highest median is District
10 HCMC with 167 cell/mm
3
.
.

VL level when starting the treatment is showed in Table 3.14
11



Table 3.14. HIV VL when starting the treatment
HIV VL
(copy/ml)
Dist 1-
HCMC
(N = 134)
Dist 10-
HCMC
(N = 122)

Hai Duong

(N= 127)
Hai Phong

(118)
Total
(501)
Mean
316.228 436.516 537.032 239.883 371.535
<250 4 (3,0) 1 (0,8) 0 (0,0) 2 (1,7) 7 (1,4)
250- <1000 0 (0,0) 0 (0,0) 1 (0,8) 2 (1,7) 3 (0,6)
≥ 1000 – 5000 3 (2,2) 1 (0,8) 0 (0,0) 2 (1,7) 6 (1,2)
>5000 127 (94,8) 120 (98,4) 126 (99,2) 112 (94,9) 485 (96,8)
Comments: the mean of HIV VL at starting point of the treatment is 371,535
copies/ml. Almost of HIV VL level are more than 5.000 copies/ml (96.8%) when starting
ART.

3.2.1.3. HIVDR mutation and resistant level with ARV on patients with HIVDR
mutation at T1
Among 501 patients participated in the study, 491 samples with HIV VL ≥ 1000
copies/ml were genotyping sequenced for HIVDR. There were 17 patients with HIV Drug
Resistance mutation (3.5%). Those were 6 patients from the OPC of district 1 HCMC, 2
patients from District 10, HCMC, 4 patients from PAC Hai Duong and 5 patients from
Viet Tiep hospital, Hai Phong. The results of HIVDR are showed in the Figure 3.7



Figure 3.7. HIVDR mutations distribution at T1


Comments: M184V, V75M and K219K are NRTI mutation which has higher
frequency than the other NRTI mutations. One patien has TAM mutation. For NNRTI,
Y181C mutation has highest frequency.
12



3.2.2. Clinical and immunological characteristic at the ending point of the study
– 12 months after ART 1
st
line (T2)
After 12 months of 1
st
line ART, 415 patients maintained the 1
st
line ART and no
patient was officially transferred to 2
nd
line ART. After removing patients with illegal 2
nd

line ART, dead and reffered patients, results of 447 patients were used to analyze the data.
The characteristic of CD4 number at T2 is showed in table 3.16

Table 3.16. Characteristic of CD4 number at T2
Characteristic
District 1-
HCMC
(n = 124)
District

10-
HCMC
(n= 110)
Hai
Duong
(n= 121)
Hai Phong

(n=102)
Total
(n= 447)
CD4 (number,%)

≤100 7 (5,7) 2 (1,9) 9 (9,1) 10 (10,1) 28 (6,6)
101 – 350 75 (61,5) 65 (61,9) 71 (72,5) 62 (62,7) 273 (64,4)
>350 40 (32,8) 38 (36,2) 18 (18,4) 27 (27,2) 123 (29,0)
Comments: The number of CD4 at T2 is significantly increased. The ratio of
patients with CD4 less than 100 cells/ml is reduced from 39.1% to 6.6%.

3.2.2.4. Treatment adherence
The level of treatment adherence is showed in table 3.19.
Table 3.19. Treatment adherence of patients
Characteristic
(number,%)
District 1-
HCMC
Distrcit 10-
HCMC
Hai
Duong Hai Phong Total

(n = 124) (n = 110) (n= 111) (n= 102) (n= 447)
Rate of on time re-examination
≥ 80% 123 (99,2) 110 (100) 95 (85,6) 97 (95,1) 425 (95,1)
<80% 1 (0,8) 0 (0,0) 16 (14,4) 5 (4,9) 22 (4,9)
Rate of distributed drug was taken (data from 418 patients)
<80% 3 1 2 4 10
80 – <90% 0 2 0 3 5
≥90% 113 (97,4) 95 (96,9) 102 (98,1) 93 (93) 403 (96,4)
Comment: 95.1% patient has on time re-examination rate ≥ 80%. 96.4% of
patients reported that more than 90% of distributed drugs were taken.
13



3.2.2.6. HIV VL level at T2
Among 415 patients maintained in 1
st
line ART at T2, 401 cases were tested with
HIV VL. The results are showed in Figure 3.6



Figure 3.6. HIV VL distribution at T2
Comment: Majority of the patients (94.5%) has VL at depressed level (< 1.000
copies/ml). 22 patients have VL ≥ 1000 copies/ml. All patients at OPC of district 10
have VL at depressed level.
3.2.3. Characteristic of HIVDR at T2
3.2.3.1. Indicator of HIVDR at T2
Out of 401 patients received HIV VL testing at T2, 379 patients have HIV VL less
than 1000 copies/ml (94.5%) and 22 patients has HIV VL ≥1000 copies/ml. 55 patients

has possible HIVDR including lost-track patients (29), stopped treatment patients (03), 9
patients has HIV VL ≥1.000 copies/ml after 12 months of treatment but cannot identify
the HIVDR mutation, 14 patients on 1
st
line ART without HIV VL testing. Among 22
patients have HIV VL ≥1.000 copies/ml, 20 patients had successfully sequenced the
genotype. 13 out of 20 patients have HIVDR mutation.
Table 3.23. Summary of patients who received evaluation on HIVDR at T2
Detected HIVDR Possible HIVDR HIVDR prevention
OPC Việt Tiệp 1 8 93
OPC Hải Dương 4 20 87
District 1 Health
Center in Ho Chi
Minh City 8 13 103
District 10 Health
Center in Ho Chi
Minh City 0 14 96
Total 13 55 379
14



Comments: OPC in District 1 was the highest of patients with HIVDR. No cases
identified with HIVDR in OPC 10. OPC in Hai Duong PAC was the highest of patient
with possible HIVDR.
The results of outcomes of HIVDR in T2 are showed in Figure 3.8.



Figure 3.8: The results of outcomes of HIVDR in T2.

Comment:The rate of HIVDR prevention is at high level for 4 OPCs with the
highest is the OPC at Viet-Tiep Hospital in Hai Phong and lowest is the OPC in Hai
Duong. The general HIVDR rate for 4 OPCs is low (2.9%) and the highest is the OPC at
District 1 in HCMC (6.5%). The rate of potential HIVDR for 4 OPCs is 12.3% with the
highest at OPC Hai Duong (18%) and lowest at Viet Tiep OPC (7.8%).
3.2.3.2. Progress of HIVDR cases at the ART initiaion ( T1)
Among 17 patients with HIVDR mutation at T1, of which there are 11 patients
achieved HIVDR prevention at T2 (64.7%), 2 patients continued to emerge new HIVDR
mutation, 2 patient’s loss-to-follow up and 2 patients died.
3.2.3.3. Distribution of HIVDR mutation by ARV classification at T2
Distribution of patients with HIVDR mutation by ARV classification at T2 has been
presented in Figure 3.10

15




Figure 3.10 Distribution of HIVDR mutation by ARV classification among 13
patients with HIVDR at T2
Comment: M184V is the mutation of NRTI with the most prequency (38.5%). Four
TAM mutations that have been observed include D67N, M41L, K70R and K219E.
Y181C is the mutation of NNRTI which occured with highest frequency (46.2%). One
patient developed 3 mutations at T2 (D67N+K70R+K219E).

3.2.4. Factors associated with HIVDR mutation at T2
3.2.4.1. Factors associated with HIVDR emerging at T1
Bivariate and multivariate analysis showed that age is the factor associated with
the emerging of HIVDR at T1. Patients with one year older, the risk of HIVDR increased
by 1.07 time (OR 1,07 (KTC95% 1,01 – 1,13).

3.2.4.2. Factors associated with new HIVDR emerging and accumulative HIVDR
at T2
Factors with the emerging of new HIVDR muation and accumulative HIVDR at
T2 are young ages from 18-25, the increase of CD4 cells, HIV viral load at the initiation
of treatment and the emerging of HIVDR mutation at the beginning of treatment. Those
factors affect the occurrence of HIVDR T2.
For accumulative HIVDR mutation at T2, possibility of patients aged 18-25 with
accumulative HIVDR mutation is 11.3 times higher than in patients aged> 35; an increase
of 50 CD4 cells in the treatment course will reduce the risk of accumulative HIVDR at
T2 by 44% (nearly half); each increase of log copy/ml at the start of treatment reduces the
risk of accumulative HIVDR mutation at T2 by 3.8 times; patients with HIVDR mutation
at the initiation of ARV treatment face the risk of accumulative HIVDR mutation 23.3
times higher.
For new HIVDR mutations at T2, patients aged 18-25 are at risk of HIVDR 13.58
times hgher compared to the over 35; an increase per 50 CD4 cells in the treatment course
will reduce the risk of accumulative HIVDR at T2 by 39%; if HIV viral load at the
initiation of treatment ≥450,000 copies/ml, the risk of new HIVDR mutation at T2
increases by 6.53 times.

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Chapter 4
DISCUSSSION

4.1. Description of HIV drug resistance early warning indicators in HIV/AIDS
clinics
Results of 5 HIV drug resistance early warning indicators from 42 HIV/AIDS
clinics in 2010, 2011 and 2012 showed HIV/AIDS prevention activities in Vietnam were

generally well performed. Generally, HIV drug resistance early warning indicators during
3 years of follow up met WHO targets.
However, the results also showed that in some clinics, EWI 2 (percentage of
patients who are lost to follow-up), EWI 3 (retention on appropriate 1st line) and EWI 4
(percentage of on-time ARV pick-up) did not meet WHO targets over 3 years. Those were
indicators of loss to follow up, retention on 1st line and on-time ARV pick up. Some
studies suggested that if patients who on NNRTI based ARV regimens stop taking drugs
more than 48 hours, the risk of virological failure and drug resistance will increase. In
Vietnam, re-examination appointment is scheduled as same as ARV picking up time.
Therefore if a patient misses the appointment, the risk of treatment interruption will
increase and in turn increase the virological failure and HIV drug resistance.
While the indicators of lost to follow-up was improved (all clinics met WHO target
in 2012), the indicators of retention on appropriate 1st line and on-time ARV pick-up still
be most difficult to be improved in clinics. In 3 consecutive years, there were5 clinics had
indicators of retention on appropriate 1st line and on-time clinics appointment lower than
WHO’s targets. The retention on first line ART regimens depended on lost to follow up,
stop to treatment, death or switching to second line ART. In fact, the second line
switching in clinics was very low. Especially, in Khanh Hoa provincial HIV/AIDS
prevention center, the percentage of retention on 1st line regiment was 61,7% in 2010,
55,1% in 2011 and 66,1% in 2012. This data suggested that patients could die within 12
months after ART initiation. The understanding of cause of death need to be focused to
propose the solutions that helps to reduce morbidity and mortality.
On-time clinic appointment for re-examination is an indicators that cannot be met
WHO targets by many clinics. There was one clinics that had 43%-45% patients who had
on-time appointment. In Vietnam during study period, the pick-up time was scheduled as
same as re-examination appointment. This fact showed that there were a significant
number of patients who did not take drugs sufficiently as needed. After each time of data
collection and analysis, the results were shared and discussed with HIV/AIDS prevention
center and outpatient clinics regarding these issues. Many solutions were raised, including
consultation on patient medication adherence that was applied. However, the result

showed that the situation of on-time re-examination appointment did not improve. This
suggested the needs of ARV drugs supply models that make sure the continuity of ARVs
drug supply, minimizing the virological failure that facilitating drug resistance.
Results from data of HIV DR EWIs in In 3 consecutive years disclosed issues that
need to be addressed. Many clinics had 2-3 indicators that did not met WHO targets,
including indicators of patients who lost to follow up, retention on first line ART and low
rate of on-time re-examination appointments. Some clinics had indicators that did not
17



met WHO target over years. This showed that clinics need to be strengthened on
consultation on patient medication adherence, to reduce the lost to follow up and to avoid
missed appointment. At programme level, it is need to improve early diagnosis and ART
for HIV infection to improve the early detection and treatment. Early ART will assist the
retention on first line ART. Additionally, while expanding ART to grassroots level
(district and commune levels), it is important to ensure continued ARVs drug supply for
patients and to prioritize distribution to prevent drugs from being out of stock.
4.2. Identify HIV drug resistances among patient receiving first line ART
4.2.1 HIV drug resistance at ART initiation
4.2.1.1 Pre-ART HIV drug resistance mutation rate
The study results showed that the general prevalence of pre-ART HIV drug resistance
at ART initiation for 4 outpatient clinics was as low as 3.5% (17/490), with lowest
prevalence at district 1 clinic and highest at district 10 clinic. The HIV drug resistance at
ART initiation could reflex the prior infection of drug resistance HIV or past ART. This
result was in accordance with low transmitted HIV drug resistance in Vietnam.
4.2.1.2 ARV drug resistance and resistance categorization of HIV drug
resistance mutation at T1
HIV drug resistances which were detected at T1 and T2 were similar as other studies.
There were 3 patients with TAMs which are cross mutations to most of NRTIs, including

TDF. These mutations caused cross mutation to other drugs of NNRTI class, often seen
when taking nevirapine and caused highly resistance to this drug but low resistance to
efaviren. The occurrence of these mutations may reflex a previous infection with drug
resistance HIV strains or history of previous ART exposure. There were major mutations
with high resistance to some current first line ART drugs (including K103N, Y181C,
Y188C, and G190A – are NNRTI-associated mutations; and V75M and M184V – are
NRTI-associated mutations). These mutations can cause virological failure among
patients with mutations and can increase the risk of HIV drug resistance transmission to
community.
4.2.2. HIV drug resistance at 12 months after ART initiation
4.2.2.1 HIV drug resistance prevention and HIV drug resistance at T2
All 4 clinics met WHO target of HIV drug resistance prevention. The prevalence of
HIV drug resistance prevention for 4 clinics was as high as 84.8%. The prevalence of HIV
drug resistance at 12 months of ART was as low as 2,9%, but the prevalence of possible
HIV drug resistance was as high as 12.3%.
The prevalence of HIV drug resistance prevention in our study (84,8%) was higher
than other studies. HIV drug resistance rate at 12 months of first line ART (2.9%) was
lower than other reports.
4.2.2.2 Results of possible HIV drug resistance at T2
Although prevalence of HIV drug resistance was low, possible HIV drug resistance
rate was as high as 12.3% that was associated with discontinuation of antiretroviral
therapy (ART). The interruption of NNRTI based regimens 48 hours and more was
strongly associated with selection of NNRTI resistance and increase the risk of virological
failure. The lost to follow up was seen at all 4 clinics. This suggested that HIV resistance
prevalence may higher than in our study and the district 10 clinics may have patients with
HIV drug resistance.
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4.2.2.4. Treatment failure and HIV drug resistance
In our study, there were 9/22 patients (40.9%) who have treatment failure (HIV viral
load >1000 copies at 12 months of first line ART) but no HIV drug resistance mutation
was detected. That means treatment failure is not always associate with ARV drug
resistance.
4.2.3. Factors associated with the occurrence of HIV drug resistance mutation at
T2
Firstly, it was the age at ART initiation: the prevalence of HIV drug resistance tends to
be higher in younger age. This trend is only significant in the age group of 18-25. Among
young people, it may be more difficult to accept a chronic disease and adherence to
treatment than older groups. This result was similar with other studies.
Secondly, viral load at ARV initiation was associated with the occurrence of HIV drug
resistance mutation at 12 months. Regarding the risk of HIV drug resistance at T2, the
occurrence of HIV drug resistance mutations increase by 3.8 times for every increase in
log10 (OR 3.8, CI95%: 1.5 – 9.4). For patients with viral load at ART initiation ≥ 450.000
copies/ml, the risk of occurrence of HIV drug resistance was 6,53 times higher (OR 6.53,
CI95% 1,23-34,61). This result was accordance with other studies.
Thirdly, HIV drug resistance at ART initiation was also increase the risk of
acquirement of additional HIV drug resistance mutation. Compare to non pre-ART
mutations, patients with pre-ART HIV drug resistance mutation have 23.3 (OR 23.3,
CI95%: 3,3-164,5) times higher risks of HIV drug resistance occurrence at 12 months of
ART. This result was accordance with other studies
Fourthly, for every 50 cells/ml increment of baseline CD4, the risk of HIV drug
resistance occurrence decrease by 44%, range of 41% - 76%, or in other words, the risk of
HIV drug resistance was halved.
4.2.4. Limitations of the Study
There were some limitations in our study. The selection of 4 outpatient clinics based
on their capacities and convenience of specimen shipping for molecular testing was not
representative for the country. Additionally, the issues related to history of ARV exposure
and self reported may not reflex the real facts.

19



CONCLUSION

1. Description of HIV drug resistance early warning indicators in HIV/AIDS
clinics:
1.1. HIV drug resistance early warning indicators for 42 outpatient clinics during 3
years met WHO targets as following:
- Percentage of patients with standard ART prescriptions: 99.9%, 100%, 100%.
- Percentage of patients who lost to follow up within 12 months of ART: 5,4%,
5,8% and 5,9%.
- Percentage of patients retained on first line ART at 12 months: 82,5%, 82,1% and
83,6%.
- Percentage of patients who kept on time clinic appointment: 90,2%, 86,8% and
88,8%
- ARV drug supply continuity indicators: 100% over years. The ARV drugs were
supplied continuously without stock out.
1.2. However, there were 15/42 (35,7%) outpatients clinics with at least one out of
3 indicators of ART discontinuation, retain on first line ART and on time clinic
appointment did not meet WHO target. One clinic with one of 3 above indications did not
meet WHO target for 2 consecutive years. This presented a potential risk of increase in
HIV drug resistance among patients on ART.
2. Identify HIV drug resistance among patients receiving first line ART in 4
outpatient clinics:
2.1. Percentage of patients archiving viral load suppression (< 1000 copies/ml) at 4
OPC was higher than WHO target: 94.5% of patients at 12 months of ART with HIV viral
load < 1000 copies/ml.
2.2. Percentage of HIV drug resistance prevention at 12 months of ART was high:

84,8% patients with HIV drug resistance prevention at 12 months of first line ART.
2.3. Percentage of patients with HIV drug resistance who receiving first line ART
in 4 OPC at base line and at 12 months of ART was low.
Specify:
- 3.5% patients with at least one HIV drug resistance mutation at baseline.
- 2.9% patients with at least one HIV drug resistance mutation at 12 months of
ART.
2.4. However, percentage of possible HIV drug resistance at 12 months of ART
was quite high in 4 OPC: 12.3% patients with possible HIV drug resistance mutation.
Therefore the prevalence of HIV drug resistance may be higher in real life than the results
was observed.
2.5. Virological failure did not mean occurrence of HIV drug resistance: 40.9%
patients with virological failure (≥1000 copies/ml) but not HIV drug resistance mutation
was detected.
2.6. HIV drug resistance at baseline was observed in 3 drug classes of NRTI,
NNRTI and PI. At 12 months of ART, only mutation to NNRTI and NNRTI were
observed, no mutation to PI was detected. There were 3 patients with TAM, one of 3
patients had 3 TAMs. This is a mutation that cause cross resistance to NRTI drugs.
20



3. Factor impacted into the occurrence of HIV drug resistance mutations at 12
months of ART including high HIV viral load at baseline, pre-ART HIV drug resistance
mutations, age group of 18-25 years old and every 50 cells/ml increment of baseline CD4.


RECOMMENDATIONS



From the study results and to strengthen the effectiveness of treatment and
minimize HIV drug resistance, the study team proposes some following
recommendations:
1. Collect, analyze and use the results of HIV drug resistance EWIs survey in
monitoring ART in HIV/AIDS clinics.
2. Implement survey about factors associated to adherence of patients, and carry out
the adherence suport interventions for cases on ART to reduce the lacks of adherence and
lost to follow up.
3. Monitor HIV drug resistance among patients on ART by time of ART initiation
to identify the trend on HIV drug resistance and HIV drug resistance mutations in
population of HIV patients receiving ART.
4. Perform HIV viral load testing routinely in patients on ART to early detect
virological failure manifestation.
5. HIV drug resistance genotyping before switching to second line ART should be
performed to reduce the unnecessary switching.





21



LIST OF PUBLISHED ARTICLES

1. Do Thi Nhan, Nguyen Thanh Long, Bui Duc Duong, Doan Thi Thuy Linh,
Nguyen Van Trang, Nguyen Van Kinh, Nguyen Thi Hoai Dung, Nguyen Thi Minh Thu,
(2012), Antiretroviral therapy and monitoring of HIV early warning indicators of drug
resistance to asses antiretroviral therapy in some HIV/AIDS clinics, Journal of Preventive

Medicine, Volume XXII, No 6 (133), pages 99-106.
2. Nguyen Thanh Long, Do Thi Nhan, Nguyen Vu Thuong, Truong Thi Xuan
Lien, (2013), HIV Drug resistance among adult patients on first line antiretroviral (ARV)
at HIV/AIDS clinics, Journal of Vietnam Medical Health, Volume 406, May 2013, No 2,
Pages 42-46.