Can HIV e e se ansc ip ase ac i i y
assay be a low-cos al e na i e o i al
load moni o ing in esou ce-limi ed
se ings?
Soham Gup a,
1
Riya Palchaudhu i,
1
Ujjwal Neogi,
2
Hi esa e S ini asa,
1
Pe Asho n,
3
Ayesha De Cos a,
4
Clas Källande ,
5
Ani a She
6
To ci e: Gup a S,
Palchaudhu i R, Neogi U,
e al. Can HIV e e se
ansc ip ase ac i i y assay be
a low-cos al e na i e o i al
load moni o ing in esou ce-
limi ed se ings?. BMJ Open
2016;6:e008795.
doi:10.1136/bmjopen-2015-
008795
▸P epublica ion his o y o
his pape is a ailable online.
To iew hese iles please
isi he jou nal online
(h p://dx.doi.o g/10.1136/
bmjopen-2015-008795).
SG and UN a e co-
co esponding au ho s.
Recei ed 28 May 2015
Re ised 16 No embe 2015
Accep ed 27 No embe 2015
Fo numbe ed a ilia ions see
end o a icle.
Co espondence o
Soham Gup a;
[email p o ec ed] and
Ujjwal Neogi; ujjwal.neogi@
ki.se
ABSTRACT
Objec i e: To e alua e he pe o mance and cos o
an HIV e e se ansc ip ase-enzyme ac i i y (HIV-RT)
assay in compa ison o an HIV-1 RNA assay o
ou ine i al load moni o ing in esou ce limi ed
se ings.
Design: A coho -based longi udinal s udy.
Se ing: Two an i e o i al he apy (ART) cen es in
Ka na aka s a e, Sou h India, p o iding ea men unde
he Indian AIDS con ol p og amme.
Pa icipan s: A coho o 327 HIV-1-in ec ed Indian
adul pa ien s ini ia ing i s -line ART.
Ou come measu es: Pe o mance and cos o an
HIV-RT assay (ExaVi Load V3) in compa ison o a
gold s anda d HIV-1 RNA assay (Abbo m2000 ) in a
coho o 327 Indian pa ien s be o e (WK00) and
4 weeks (WK04) a e ini ia ion o i s -line he apy.
Resul s: Plasma i al load was de e mined by an HIV-
1 RNA assay and an HIV-RT assay in 629 samples
(302 pai ed samples and 25 single ime poin samples
a WK00) ob ained om 327 pa ien s. O e all, a s ong
co ela ion o =0.96 was obse ed, wi h good
co ela ion a WK00 ( =0.84) and a WK04 ( =0.77).
Bland-Al man analysis o all samples showed a good
le el o ag eemen wi h a mean di e ence (bias) o
0.22 log
10
copies/mL. The pe o mance o ExaVi Load
V3 was no nega i ely a ec ed by a ne i apine/e a i enz
based an i e o i al egimen. The pe es cos o
measu ing plasma i al load by he Abbo m2000
and ExaVi Load V3 assays in a basic lab se ing was
$36.4 and $16.8, espec i ely.
Conclusions: The s ong co ela ion be ween he HIV-
RT and HIV-1 RNA assays sugges s ha he HIV-RT
assay can be an a o dable al e na i e op ion o
moni o ing pa ien s on an i e o i al he apy in
esou ce-limi ed se ings.
T ial egis a ion numbe : ISRCTN79261738.
INTRODUCTION
The p incipal aim o an i e o i al he apy
(ART) is du able supp ession o eplica ing
plasma i us o unde ec able le els, he eby
delaying disease p og ession and p olonging
su i al.
12
Expanding access o ART in
esou ce-limi ed se ings along wi h close
moni o ing is needed o success ul ea -
men ou comes. In high income se ings, his
is achie ed by pe o ming quan i a i e i al
load moni o ing e e y 3–6 mon hs,
3
as i al
load moni o ing de ec s ea ly ea men
ailu e. Howe e , in esou ce-limi ed se ings,
he apeu ic ou come is e alua ed ei he on
he basis o a CD4 T cell coun o clinical
findings,
4
nei he o which accu a ely p e-
dic s i al supp ession.
5
Ea ly de ec ion o
i al ailu e by moni o ing he i al load also
p o ides he oppo uni y o in ensi y adhe -
ence counselling o imp o e adhe ence o
ART, po en ially leading o esupp ession o
i al load be o e he e olu ion o
d ug- esis an i us can ake place.
6
The cu en ly used i al load assays a e
based on he amplifica ion o HIV-1 i ion
S eng hs and limi a ions o his s udy
▪To he bes o ou knowledge, he cu en s udy
is he mos ho oughly e alua ed s udy o ExaVi
Load V3 HIV e e se ansc ip ase-enzyme ac i -
i y (HIV-RT assay) om India o da e.
▪This s udy was pe o med in a la ge numbe o
pa ien s in a longi udinal manne looking in o
he e ec s o he non-nucleoside e e se an-
sc ip ase inhibi o s-based he apy and d ug
esis ance mu a ions on he pe o mance o he
HIV RT-enzyme ac i i y assay.
▪Though he assay has been alida ed be o e in
o he non-C sub ype se ings, in his s udy he
HIV-RT assay was alida ed on a la ge scale in
a sub ype C p edominan se ing and sub ype C
is he mos p edominan sub ype globally, mo e
so a ec ing he esou ce-limi ed se ings.
▪This s udy was limi ed o pa ien s hailing om
Sou he n India and a pe o mance e alua ion
and cos -e ec i eness o he HIV-RT assay
needs o be accessed on a na ional le el.
Gup a S, e al.BMJ Open 2016;6:e008795. doi:10.1136/bmjopen-2015-008795 1
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RNA, which is conside ed imp ac ical o wide-scale use
in esou ce-limi ed se ings, as i equi es in as uc u e,
acili ies o molecula diagnos ics, expensi e equipmen
and skilled echnicians, which a e o en una ailable.
7
Simple , less expensi e i al load assays would be e y
use ul in he esou ce-limi ed en i onmen s ha a e
mos impac ed by his epidemic.
An al e na i e o measu e he HIV-1 RNA is o
measu e he ac i i y o he i al e e se ansc ip ase
(RT) enzyme. The ExaVi Load assay (Ca idi, AB,
Sweden), a low-cos and echnically less demanding
assay using an ELISA-based me hod o measu e RT
enzyme ac i i y, has shown p omising esul s.
8–19
Al hough p e ious e sions o he es ha e been e alu-
a ed agains se e al PCR-based HIV es s ha measu e
i al RNA, he e a e ew compa a i e s udies be ween
ExaVi Load V3 and such molecula eal- ime
assays.
14 16 18
The mos ecen V3 o he ExaVi Load
assay has an enhanced sensi i i y (lowe de ec ion limi
200 copies/mL) and educed u na ound ime com-
pa ed o V2.
14 18
HIV-1 sub ype C (HIV-1C) is he dominan s ain in
mos low and middle income coun ies like India, Sou h
A ica and E hiopia,
20
and he need o a simple low
cos i al load moni o ing ool is a p io i y in hese se -
ings. Da a a ailable om coun ies domina ed by he
HIV-1C epidemic a e limi ed. Whe e a ailable, e alua-
ions ha e been mainly pe o med wi h ea lie e sion
assays (ExaVi Load V1 and V2), which had lowe de ec-
ion limi s o 400 copies/mL.
7112122
S udies e alua ing
Exa i Load V3 ha e been mainly pe o med in non-C
domina ed coun ies.
14 16 18 19
Thus, we aimed o e alua e he pe o mance o he
HIV RT-enzyme ac i i y (HIV RT) assay (ExaVi Load
V3) in compa ison o a ‘gold s anda d’HIV RNA load
(Abbo m2000 eal- ime PCR) assay in a coho o
pa ien s be o e and a e ini ia ion o fi s -line ART in
Indian se ings. We also compa ed i al load measu e-
men s om bo h assays in a subse o pa ien s wi h
d ug- esis an mu a ions a baseline. Fu he , we s udied
he di e ence in cos s o he wo i al load assays in he
con ex o ou labo a o y se ing om a p o ide
pe spec i e.
MATERIALS AND METHODS
S udy pa icipan s and samples
Be ween Ap il 2010 and Sep embe 2011, EDTA plasma
samples we e collec ed om HIV-1-in ec ed adul
pa ien s a ending he In ec ious Disease Clinic,
S . John’s Medical College and Hospi al, Bangalo e
(main si e) and ART cen e, K ishna Rajend a Hospi al,
Myso e (pe iphe al si e) en olled in he HIVIND ando-
mised con olled ial (T ial egis a ion:
ISRCTN79261738).
23
All he pa ien s included in he
s udy ini ia ed ART wi h e e se ansc ip ase inhibi o
(RTI) d ugs, ha is, wo nucleoside e e se ansc ip ase
inhibi o s (NRTI), zido udine (AZT) o s a udine (d4T)
wi h lami udine (3TC) + one non-nucleoside e e se
ansc ip ase inhibi o (NNRTI), ei he ne i apine
(NVP) o e a i enz (EFV) as pe he s anda d na ional
AIDS p og amme guidelines.
24
Plasma samples we e collec ed a wo ime poin s; (1)
p io o ART ini ia ion (no longe han 3 mon hs p io )
and (2) 4 weeks a e ART ini ia ion. The plasma
samples we e sepa a ed wi hin 6 h o EDTA whole blood
collec ion, aliquo ed and s o ed a −80°C in he main
si e. Plasma samples aliquo ed in he pe iphe al si e
we e s o ed a −20°C and anspo ed o he main si e
on d y ice (e e y 2 weeks) and hen s o ed a −80°C
p io o es ing.
E hical s a emen
E hical app o als o he conduc o he ial we e
ob ained om he Ins i u ional E hical Re iew Boa d o
S John’s Medical College Hospi al, Bangalo e (IERB 1/
369/08-92/2008) and K ishna Rajend a Hospi al,
Myso e (NO/PS/173/2010). All pa ien s pa icipa ing in
he HIVIND s udy ha e gi en hei w i en consen .
Plasma HIV-1 RNA assay
Plasma HIV-1 RNA load was measu ed in he pa ien
coho using he Abbo Real-Time PCR, m2000 sys em
wi h a manual RNA ex ac ion p ocedu e on an
m2000sp sample p epa a ion sys em as pe he manu ac-
u e ’s ins uc ions. The assay was pe o med using an
ini ial olume o 0.2 mL plasma, which p o ides limi s
o quan ifica ion be ween 150 copies/mL (lowe limi o
de ec ion) and 10 000 000 copies/mL (uppe limi o
de ec ion). In e e y un, a nega i e con ol, a low posi-
i e con ol and a high posi i e con ol supplied in he
Abbo Real ime HIV-1 con ol ki we e included. This
measu e using Real-Time PCR was conside ed as he
gold s anda d. This p o ocol was alida ed by an ex e nal
quali y con ol p og amme by he Quali y Con ol o
Molecula Diagnos ics, Glasgow, Sco land (QCMD,
h p://www.qcmd.go ) on a 2010 panel (Consis ing o
ou HIV-1B samples, wo HIV-1C samples, one HIV-1A/
G sample and one HIV-1 nega i e sample) and ob ained
a highly sa is ac o y sco e.
Plasma HIV RT enzyme ac i i y assay
The i al RT enzyme ac i i y was quan ified using Ca idi
ExaVi Load V3 as pe he ins uc ions o he manu ac-
u e .
14
In an ELISA-based o ma , he RT ac i i y o he
RT enzyme eco e ed om 1 mL o pa ien plasma was
de e mined and compa ed o a s anda d cu e based on
known amoun s o an HIV 1 ecombinan RT
cons uc ed om he BH10 isola e. The RT ac i i y in
he sample was exp essed as em og am HIV-1 RT
ac i i y/mL ( g/mL) plasma ha was ansla ed in o
RNA copies/mL equi alen by he ExaVi Load analyse
so wa e. The lowe de ec ion limi o he assay was
≥1 g/mL ha is, ≥200 copies/mL. In e e y un, a single
posi i e con ol and a nega i e con ol we e used o
moni o he pe o mance o he RT ex ac ion p ocess
2Gup a S, e al.BMJ Open 2016;6:e008795. doi:10.1136/bmjopen-2015-008795
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and o access be ween un a ia ions. The posi i e
con ol was p epa ed by pooling he EDTA plasma om
a high HIV-1 i al load sample and HIV-1 nega i e
plasma; 1.2 mL was aliquo ed and s o ed in a −80°C
eeze . The labo a o y pe sonnel unning he ExaVi
Load V3 assay we e blind o he plasma HIV-1 RNA
alues.
HIV-1 sub yping and baseline d ug esis ance
Geno ypic esis ance es ing (GRT) was pe o med on
he baseline plasma samples. B iefly, he e e se an-
sc ip ase (RT) egion o he HIV-1 pol gene was ampli-
fied and sequenced using he p ime s desc ibed by us
p e iously.
25
HIV-1 sub yping was de e mined on he
basis o he pol gene as well as he en gene (whe e e
sequence da a we e a ailable).
26–28
using he maximum
likelihood (ML) phylogene ic ee based on e e ence
sequences downloaded om he Los Alamos Da abase
(h p://www.hi .lanl.go ). P ima y d ug esis ance ana-
lysis was e alua ed using he WHO lis o mu a ions om
2009 (WHO_SDRM 2009).
29
Compa a i e cos analysis
We did an analysis compa ing he cos s be ween Abbo
m2000 and ExaVi Load V3 om a p o ide (labo a-
o y se ice p o ide ) pe spec i e. We used cos s om
ou lab o his pu pose. Cos s conside ed included
annui ised capi al cos s o he wo di e en ins umen s
including ope a o -supplied ins umen s. These a e
ins umen s which a e necessa y in case o a new labo a-
o y. In case o Abbo , he ope a o -supplied ins u-
men s included single-channel mic opipe es, wo d y
ba hs and a o ex. Fo ExaVi Load V3, hey included
mic opipe es (bo h single-channel and mul ichannel),
an ELISA pla e eade , an incuba o , a ocke and a
o ex.
Cos s o s a -up ki s, human esou ce cos s (includ-
ing ime o aining), annual main enance, eagen s
and o he consumables we e also conside ed. We
assumed he wo king li e o Abbo m2000 and
ExaVi Load V3 o be 5 yea s, and a discoun a e o 5%
was applied.
Assump ions
Numbe o pa ien s: The e a e a p esen 1500
ART-expe ienced pa ien s in ou ART cen e. Assuming
ha i al load moni o ing o hese pa ien s will equi e
o be pe o med e e y 6 mon hs, he e will be 3000
samples a yea .
Main enance cos s: In he case o Abbo m2000 he
cos s o se icing, main enance o ins umen and cali-
b a ion o lase head. In he case o Abbo m2000 he
annual main enance cos included he cos s o se -
icing, main enance o ins umen and callib a ion o
lase heads.
Human esou ce skills and aining equi emen s:
F om ou expe ience, 1 mon h was equi ed o ain a
echnician on Abbo m2000 . One week was equi ed
o aining on ExaVi load V3. We also conside ed ha
he echnician handling Abbo m2000 would equi e
o be mo e senio and expe ienced (sala y $300 pe
mon h) compa ed o he echnician wo king wi h
ExaVi Load V3 (sala y $200 pe mon h).
Time o each me hod o es ing: Fo Abbo m2000 ,
a ba ch o 24 eac ions which comp ise 21 samples and
3 con ols will in ol e a o al ime o 8 h ( om he
beginning o RNA ex ac ion up un il ob aining esul s),
o which 5 h in ol e he echnician’s ime. Fo Exa i
Load V3, he assay is pe o med in ba ches o 30
samples. Though he u na ound ime is 48 h, i in ol es
5 h o ac ual hands-on ime pe ba ch. Cos s o ime o
he echnicians we e calcula ed agains he sala ies men-
ioned abo e.
In he cos compa ison analysis, we did no conside
cos s associa ed wi h he sample collec ion, s o age and
anspo a ion as hese a e common o bo h es s.
S a is ical analysis
All s a is ical analyses we e pe o med a e he HIV RT
and HIV-1 RNA le el alues we e log
10
ans o med. Fo
analysis, he lowe limi o de ec ion o he HIV RT assay
(≤200 copies/mL) was conside ed; samples showing
<200 copies/mL by any o he assays we e assigned a
alue o 199 copies/mL. Wi h Exa i Load V3, we
achie ed a a ying uppe de ec ion limi anging om
>360 000 o >770 000 in di e en uns. Thus, samples
wi h a i al load o >360 000 ( he lowes ange o he
uppe de ec ion limi ob ained o ExaVi Load V3) by
any o he assays we e assigned a i al load o 360 000
copies/mL. The diagnos ic ag eemen be ween he HIV
RT assay and he HIV-1 RNA assay a di e en i al load
cu -o s was de e mined om he κs a is ic. Pea son’s
co ela ion coe ficien ( ) was calcula ed o s udy he
co ela ion be ween log
10
HIV RT ac i i y (copies/mL
equi alen s) and log
10
HIV RNA (copies/mL). Howe e ,
since his coe ficien does no ake in o accoun he pos-
sibili y ha one measu e may di e consis en ly om he
o he , we u he assessed he le el o ag eemen using
pai wise Bland-Al man plo s. This plo compa es he
measu es be ween he wo es s by plo ing he di e -
ence in he wo VL measu es agains he a e age o he
wo measu es.
RESULTS
Sample cha ac e is ics
Plasma i al load was ob ained om 629 samples col-
lec ed om 327 HIV-1-in ec ed adul pa ien s, o which
302 we e pai ed (be o e ART and 4 weeks a e ART ini-
ia ion). HIV-1C was he p edominan sub ype obse ed
in 98.1% (313/319) o he pa ien s. Six ou o 319 geno-
yped pa ien s (1.9%) showed he p esence o non-C
sub ype s ains, namely BC ecombinan (1), BD ecom-
binan (1), A1C ecombinan (2) and HIV-1A1 (2).
Ele en pa ien s (3.4%) showed he p esence o single
p ima y d ug- esis an mu a ions, wi h six samples
Gup a S, e al.BMJ Open 2016;6:e008795. doi:10.1136/bmjopen-2015-008795 3
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ha bou ing NRTI-associa ed d ug- esis an mu a ions
(DRMs) and fi e samples wi h NNRTI-associa ed mu a-
ions ( able 1).
Compa ison be ween HIV RT ac i i y and HIV-1 RNA load
assay
The e we e 54 samples (8.5%) ha we e quan ifiable by
he HIV-1 RNA assay bu we e below he de ec ion limi
o he HIV RT assay ( able 2). A a lowe limi o quan i-
fica ion o 200 copies/mL, 90.7% o he samples showed
a quan ifiable i us by he HIV RT assay. The pe cen age
o he samples wi h a quan ifiable i al load by he HIV
RT assay inc eased wi h highe i al load cu -o s by he
HIV-1 RNA assay as shown in able 2. O e all, he e was
accep able ag eemen obse ed be ween he HIV RT
and HIV-1 RNA assays, wi h excellen ag eemen
obse ed a highe alues o plasma i al load ≥3.0 log
10-
copies/mL (κ=0.76). O all he samples, 81.7% (514/
629) had i al load alues by he HIV RT assay, which di -
e ed by<0.5 log
10
uni s om he HIV-1 RNA alues,
while 99.2% (624/629) o he samples di e ed by <1.0
log
10
uni s.
A s ong posi i e co ela ion was obse ed be ween he
plasma i al load alues by he HIV RT and HIV-1 RNA
assays ( =0.96) in all he samples. A good co ela ion was
no ed in ART-naï e samples ( =0.84) as well as in
samples a week 04 o ART ( =0.77; figu e 1).
Bland-Al man plo s o all he samples showed good
le els o ag eemen wi h a mean di e ence (bias) o
0.22 log
10
copies/mL, wi h accep able limi s o ag ee-
men (−0.45 and +0.89 log
10
copies/mL). A good le el
o ag eemen was also obse ed sepa a ely a baseline
(mean di e ence bias o 0.25; ange o accep able limi
o ag eemen : −0.39 and +0.89 log
10
copies/mL) and a
WK04 (mean di e ence bias o 0.19; ange o accep able
limi o ag eemen : −0.52 and +0.89 log
10
copies/mL;
figu e 2).
In luence o cu en ART and d ug- esis an mu a ions on
RT-enzyme ac i i y
Table 1 shows ha he mean log
10
di e ence be ween
he HIV RT and HIV-1 RNA assays bo h be o e and a e
ini ia ion o ART was no significan ly di e en (<0.25
log
10
copies/mL) bu was well wi hin he clinically
accep ed limi o 0.5 log
10
copies/mL. Thus, he pe -
o mance o he HIV RT assay is no a ec ed by he p es-
ence o he NNRTI (ne i apine/e a i enz)-based ART
egimen. Also, he p esence o ei he NRTI-associa ed
DRMs (n=6; M41L: 1, D67N: 1, T69D: 1, M184I: 1 and
T215S: 2) and NNRTI-associa ed DRMs (n=5; Y181C: 1,
K101E: 1 and K103N: 2) showed an accep able change
Table 1 Compa ison o HIV-1 plasma VL le els measu ed by he HIV RT and HIV-1 RNA assays by ART s a us, HIV-1
sub ypes and RT-d ug esis an mu a ions
Sample ype Numbe o samples
Mean i al load±SD in
log
10
copies/mL
Mean log
10
i al load
di e ence±SD in
log
10
copies/mLExaVi Load V3 Abbo m2000
All samples 629 3.98±1.3 4.19±1.3 0.22±0.3
ART S a us
Naï e (baseline a WK00) 327 5.07±0.6 5.33±0.5 0.25±0.3
Expe ienced (WK04) 302 2.79±0.5 2.97±0.6 0.19±0.4
Sub ype a WK00 (n=319)*
C 313 5.08±0.6 5.33±0.4 0.25±0.3
Non-C 6 5.05±0.5 5.3±0.4 0.25±0.3
DRMs a WK00 (n=319)*
Wild ype (no DRM) 308 5.07±0.6 5.32±0.5 0.25±0.3
NRTI mu a ions 6 5.41±0.3 5.55±0.01 0.15±0.3
NNRTI mu a ions 5 5.26±0.2 5.62±0.2 0.36+0.2
*Geno yping pe o med only in baseline samples.
ART, an i e o i al he apy; DRM, d ug- esis an mu a ions, NNRTI, non-nucleoside e e se ansc ip ase inhibi o s; NRTI, nucleoside e e se
ansc ip ase inhibi o s; RT, e e se ansc ip ase; VL, i al load.
Table 2 Ag eemen be ween he HIV RT assay and HIV-1 RNA assay a di e en PVL le els
PVL by HIV-1 RNA
In copies/mL (log
10
copies/mL) Ag eemen κValue
Numbe o samples
de ec ed by Abbo m2000
Pe cen age o Samples
de ec ed by ExaVi Load V3
≥200 (2.3) 89.1 0.46 580 90.7
≥400 (2.6) 88.1 0.57 550 93.5
≥1000 (3.0) 89.7 0.76 458 97.6
≥5000 (3.7) 94.4 0.89 344 99.7
≥10 000 (4.0) 96.8 0.94 324 100
PVL, plasma i al load; RT, e e se ansc ip ase.
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(<0.4 log
10
copies/mL) in mean log
10
di e ence
om he co esponding alue among wild ypes.
Al hough he samples wi h mu a ions a e small, i indi-
ca es ha he p esence o NRTI and NNRTI DRMs did
no nega i ely impac he es pe o mance.
Cos compa ison o he assays
The labo a o y cos o i al load moni o ing o
HIV-in ec ed pa ien s analysed in ou coho by bo h
Abbo m2000 (HIV-1 RNA) and ExaVi Load V3
(HIV RT) is shown in able 3. The pe es cos o he
plasma i al load measu ed by Abbo m2000 and
ExaVi Load V3 was $36.4 and $16.8, espec i ely. Thus,
by using ExaVi Load V3, $19.6 pe es can be sa ed. In
a labo a o y wi h a p e-exis ing basic se -up o
ELISA-based assays, ExaVi Load V3 can be pe o med
a $16.1, sa ing $20.2 pe es . Mos o he expense sa ed
by using ExaVi Load V3 was due o (1) lowe capi al
cos s (ins umen s $37 750 agains $2000) (2) he lowe
cos o he assay eagen s ($15/ es agains $31/ es ;
able 3).
Figu e 1 Co ela ion be ween he Abbo m2000 and
ExaVi Load V3 assays o (A) all 629 samples showing
=0.96 (B) 327 baseline (WK00) samples showing =0.84 and
(C) 302 4-weeks pos -ART (WK04) samples showing =0.77.
ART, an i e o i al he apy.
Figu e 2 Bland-Al man plo wi h 95% CI o limi s o
ag eemen be ween HIV-1 i al loads measu ed wi h he
Abbo Real-Time m2000 assay and he ExaVi Load V3
assay o (A) all 629 samples showing a mean bias o 0.22
wi h 95% limi s o ag eemen anging om −0.45 o 0.89 (B)
327 baseline (WK00) samples showing a mean bias o 0.25
wi h 95% limi s o ag eemen anging om −0.39 o 0.89 (C)
302 4 weeks pos -ART (WK04) samples showing a mean bias
o 0.19 wi h 95% limi s o ag eemen anging om −0.52 o
0.89. ART, an i e o i al he apy.
Gup a S, e al.BMJ Open 2016;6:e008795. doi:10.1136/bmjopen-2015-008795 5
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DISCUSSION
A good co ela ion be ween he HIV RT and HIV-1 RNA
assays was obse ed in he cu en HIV-1C p edominan
se ing in India. The ag eemen be ween he es s was
no significan ly a ec ed by he NRTI/NNRTI-based
an i e o i al egimen used. Ea lie s udies pe o med
on panels o di e en sub ypes and ecombinan s ha e
sugges ed ha he HIV RT assay de ec s all HIV-1 and
HIV-2 sub ypes wi h simila e ficiency.
14 15 30
This assay,
ExaVi Load V3, can he e o e be an a ac i e op ion
o i al load moni o ing in Indian se ings.
This s udy compa ed he ExaVi Load V3 assay wi h he
Abbo m2000 HIV-1 RNA assay and obse ed an excel-
len co ela ion ( =0.96). An ea lie s udy om London
compa ing he same es s obse ed a simila co ela ion
( =0.94).
14
S ong co ela ions be ween he ExaVi Load
V3 and Roche HIV-1 RNA-based assays ha e also been
obse ed by wo o he s udies, by G eeng ass e al
18
om
Aus alia (Roche Cobas Amplico ; =0.85) and Huang
e al
16
om China (Roche Cobas TaqMan 48; =0.95).
Nei he o hese s udies was pe o med in HIV-1C domi-
na ed se ings. The HIV RT assay showed a good ag ee-
men wi h he HIV-1 RNA assay a he clinically impo an
i al load h eshold o 1000 copies/mL, which is used by
he WHO o define i al ailu e o fi s -line he apy and is
also mos o en used as he cu -o o d ug esis ance
geno yping.
31
The pe o mance o he Exa i Load
below 1000 copies/mL is mode a e.
In gene al, we obse ed an unde es ima ion o i al
load o 0.22 log
10
RNA copies by he HIV RT assay,
which is simila o wha has been obse ed in o he
s udies.
14 16 32
These wo su oga e assays use e y di e -
en me hods o quan i ying he plasma i al load. The
HIV-1 RNA assays quan i y he amoun o i al RNA i e-
spec i e o RNA unc ionali y, while he HIV RT assay
quan ifies he amoun o ac i e RT enzyme. The
calib a ion cons an used o ansla e RT ac i i y in o
RNA copies was es ima ed om a s udy o an Aus alian
coho
18
and is no comple ely accu a e o all combina-
ions o HIV RNA assays and coho s wi h a ying
sub ype composi ions. The a ia ion obse ed is,
howe e , well wi hin he accep able limi o <0.5 log
10
copies.
NNRTI d ugs bind o he RT enzyme, inhibi i s ac i -
i y and p e en i al eplica ion. Se e al a icles ha e dis-
cussed he possibili y ha enzyma ically inac i e RT d ug
complexes could esul in unde quan ifica ion o RT in
ela ion o RNA.
29 30 33
These s udies we e, howe e ,
c oss-sec ional and ne e ound any e idence o
educed RT ac i i y du ing NNRTI he apy. In con as
o p e ious coho s, he longi udinal sampling in ou
s udy p o ides op imal ma e ial o e alua ion o he
e ec s o NNRTI-based d ug egimens. When compa -
ing HIV i al load da a om he same pa ien coho
be o e and a e onse o ART, we ound a mean log
10
di e ence be ween ExaVi Load V3 and Abbo m2000
o 0.25 o naï e pa ien s and 0.19 o expe ienced
pa ien s ( able 1). Thus, he di e ence be ween he es s
did no inc ease a e onse o he apy. This suppo s e i-
dence ha he cu en NNRTI con aining he apy does
no ad e sely influence he eco e y o RT enzyme
ac i i y.
On a small numbe o samples, we assessed i he p es-
ence o d ug- esis an mu a ions dec eased he RT
fi ness so as o influence he pe o mance o he HIV
RT assay. We had six samples wi h single NRTI mu a ions
and fi e samples wi h single NNRTI mu a ions and
obse ed no e idence ha hei p esence caused any sig-
nifican di e ence in he associa ion be ween RT
enzyme ac i i y and HIV-1 RNA load. These esul s we e
no unexpec ed and suppo e idence om p e ious
s udies by Nap a nik e al and Rooijen e al indica ing
ha he p esence o NRTI o NNRTI mu a ions do no
a ec he ela ionship be ween RT enzyme ac i i y and
HIV-1 RNA load.
34 35
Resis ance o NRTIs is media ed by
a p imi i e DNA edi ing unc ion ha is in oduced in o
he HIV RT by ce ain mu a ions. An ene gy dependen
base excision eac ion emo es he las base in he
g owing DNA chain. This equi es an ene gy dona o ,
usually ATP o GTP, and migh dec ease RT eac ion el-
oci y. This happens eadily in i o, bu he eac ion con-
di ions in he cu en RT assay do no suppo his
eac ion.
36
To he bes o ou knowledge, his is he mos ho -
oughly e alua ed s udy o ExaVi Load V3 om India o
da e. Thus a , he e ha e been h ee compa a i e
s udies om India ha ha e been epo ed om he
s a es o Andh a P adesh (Anan pu ),
37
Tamil Nadu
(Chennai)
7
and New Delhi.
32
Iqbal e al
7
om Chennai
c oss-sec ionally e alua ed he ExaVi Load assay V1 and
Roche Amplico Moni o assay. They ound a good
ag eemen be ween he wo es s and a significan
in e se co ela ion be ween ExaVi Load and CD4+
T-cell coun . Al a ez-U ia e al
37
om Anan pu
Table 3 Cos compa ison be ween he HIV RT assay and
HIV-1 RNA assay o a labo a o y doing 3000 es s/yea
Cos i ems ($)
Abbo
m2000 Cos
($)
ExaVi Load
V3 Cos ($)
Annui ised cos s o
capi al ins umen s
8719 462
Annui ised cos o
ope a o supplied
ins umen s
293 1540
Annual main enance cos 1126 666
Cos s o ki s pe yea 93 000 45 000
Consumables pe yea 4616 1846
T aining ime o lab s a
o un he es
300 50
Sala y cos s (3000 es s
pe yea )
1200 1000
To al ($) 109 634 50 534
Cos / es ($) 36.4 16.8
US$1=60 INR.
RT, e e se ansc ip ase.
6Gup a S, e al.BMJ Open 2016;6:e008795. doi:10.1136/bmjopen-2015-008795
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compa ed he accu acy o ExaVi V3 wi h Roche Cobas
TaqMan HIV-1 es and Roche Amplico HIV-1 DNA
assay o ea ly in an diagnosis. ExaVi pe o med well
showing 100% sensi i i y and 99% specifici y, bu no
quan i a i e co ela ions we e e alua ed. A mo e ecen
s udy by Kokkayil e al
32
compa ed ExaVi Load V3 wi h
Roche Cobas TaqMan among 75 ART naï e pa ien s and
epo ed no s a is ically significan di e ence be ween
he wo assays.
The e a e a ew d awbacks o he HIV RT assay. The
long u na ound ime o 48 h makes i appea labou
in ensi e, hough he ac ual hands-on ime is app oxi-
ma ely 5 h. The p olonged incuba ion ime is c i ical o
achie e assay sensi i i y. Fo s anda d pe o mance, he
assay equi es 1 mL o plasma, which is high in compa i-
son o he equi emen o HIV-1 RNA assays, hus limi -
ing i s possible use ulness in paedia ic popula ions.
Howe e , a ecen s udy by G eeng ass e al
19
obse ed
ha sample olumes down o 0.25 mL wi h VL >800
copies/mL can be u ilised o paedia ic moni o ing.
The ExaVi Load assay does no p o ide a s anda d posi-
i e and nega i e con ol, hus equi ing he lab o
p epa e i s own con ols, which may comp omise he
quali y assu ance o he assay. Addi ionally, we no ed ha
he quali y o he wa e used o washing is impo an as
impu i ies and bac e ial con aminan s p esen in wa e
may con ain polyme ases which can c ea e backg ound
noise and inc ease he le el o he lowe de ec ion limi .
In spi e o hese ca ea s, he HIV RT assay has ad an-
ages o e he HIV-1 RNA assays in esou ce-limi ed se -
ings because i is an ELISA-based assay and can be
pe o med in any ou ine lab a a lowe cos . The ExaVi
Load assay equi es a cheape and main enance ee
s a -up ki as compa ed o eal- ime assays. We obse ed
ha pe o ming he HIV RT assay ou inely in ou
cen e would sa e us $20.2 pe sample as compa ed o
an HIV-1 RNA assay. A mo e basic labo a o y, which
equi es ins alling basic ELISA equipmen , would sa e
$19.6 pe sample ( able 3).
The use o CD4 cell coun as a p ognos ic ma ke has
been deba ed; i is a gued ha his coun may no eflec
he ac ual i al load s a us o he pa ien .
38
The cos asso-
cia ed wi h i al load moni o ing using HIV-1 RNA assays,
despi e being lowe han PCR assays, is a majo limi ing
ac o o i s implemen a ion. Cu en ly, in India, i al load
es ing has been phased in o suppo pa ien s ailing fi s -
line ART. In he yea 2012, abou 4157 i al load es s we e
pe o med unde he Na ional AIDS Con ol O ganisa ion
(NACO).
39
Conside ing ha $19.6 could ha e been sa ed
pe sample by pe o ming an HIV RT assay, he cos sa ing
o hese 4157 i al load es s could ha e amoun ed o $
81 477 i an HIV RT assay had been used.
In ou cos -compa ison analysis, we ha e used a p o-
ide pe spec i e (lab se ice). We acknowledge ha his
is a na owe pe spec i e han a socie al one, which
would include pa ien cos s, oppo uni y cos s among
o he cos s. Howe e , he pu pose o ou analysis was o
p o ide in o ma ion o labo a o ies in esou ce
cons ained se ings, o en aced wi h decisions in he
ace o igh budge s, and hus a socie al pe spec i e was
no conside ed necessa y. A labo a o y manage aced
wi h a limi ed budge would concen a e en i ely on
cos s ha ha e an immedia e impac on he /his own
budge ; his is he pe spec i e adop ed in his s udy.
NACO is now conside ing aking up he moni o ing
app oach ecommended by he WHO o diagnose and
confi m ART ailu e. Conside ing ha he e a e cu -
en ly 604 987 HIV-1-in ec ed indi iduals ecei ing fi s -
line ART a 380 cen es sp ead ac oss he coun y, he
cos educ ion o u ilising HIV RT compa ed o HIV
RNA plasma load can be subs an ial.
40
Scaling up ART equi es he c i ical suppo o HIV-1
i al load moni o ing. E idence om he compa a i e
pe o mance o he HIV RT assay wi h HIV-1 RNA assays
om ou s and o he s udies om India indica es ha
he ExaVi Load assay could se e as an a o dable al e -
na i e o moni o pa ien s on ART.
Au ho a ilia ions
1
Di ision o Clinical Vi ology, Depa men o Mic obiology, S . John’s Medical
College, Bangalo e, India
2
Di ision o Clinical Mic obiology, Depa men o Labo a o y Medicine,
Ka olinska Ins i u e , S ockholm, Sweden
3
Depa men o In e na ional Heal h, Uni e si y o Tampe e School o
Medicine, Tampe e, Finland
4
Depa men o Public Heal h Sciences, Ka olinska Ins i u e, S ockholm,
Sweden
5
Ca idi AB, Uppsala, Sweden
6
Depa men o Pedia ics, S John’s Medical College Hospi al, Bangalo e,
India
Acknowledgemen s The au ho s would like o hank he HIVIND s udy eam
o hei ou s anding e o s in ec ui ing and ollowing he s udy pa icipan s
and collec ion o samples. They would also like o hank P a a Nalini Sahoo
and Shwe ha D Rao, S . John’s Resea ch Ins i u e, o pe o ming labo a o y
expe imen s pe aining o HIV-1 d ug esis ance and HIV-1 RNA load. They
a e e y much hank ul o S a an Sjödahl, Ca idi AB o his echnical help
and suppo wi h he ExaVi Load V3 assay. Ujjwal Neogi acknowledges he
suppo ecei ed om Ka olinska Ins i u e Resea ch Founda ion G an s
(2014 obi41250). The au ho s also acknowledge he s a a he ART Cen e,
S . John’s Medical College Hospi al and KR Hospi al, Myso e o hei
excellen eamwo k and pa ien ca e and NACO and Ka na aka S a e AIDS
P e en ion Socie y (KSAPS) o hei suppo . Mos impo an ly, hey a e e e
hank ul o he pa ien s o hei pa icipa ion in he s udy.
Con ibu o s UN, ADC, CK, PA and AS concei ed he s udy. SG, UN, HS,
ADC, CK and AS designed he expe imen s. SG and RP pe o med he
expe imen s. PA was esponsible o ex e nal lab moni o ing and in e im
quali y assessmen . SG, UN, ADC, CK and AS analysed he da a. CK and AS
con ibu ed eagen s/ma e ials/analysis ools. SG, RP, UN, HS, PA, ADC, CK
and AS con ibu ed o he w i ing o he manusc ip .
Funding The s udy was inancially suppo ed by a g an unde he Eu opean
Union F amewo k P og amme 7 (p ojec no: 222946).
Compe ing in e es s CK is an employee o Ca idi AB, Uppsala, Sweden. All
he o he au ho s ha e decla ed ha no compe ing in e es s exis .
Pa ien consen Ob ained.
E hics app o al E hical app o als o he conduc o he ial we e ob ained
om he Ins i u ional E hical Re iew Boa d o S John’s Medical College
Hospi al, Bangalo e (IERB 1/369/08-92/2008) and K ishna Rajend a Hospi al,
Myso e (NO/PS/173/2010). All pa ien s pa icipa ing in he HIVIND s udy ha e
gi en hei w i en consen .
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Da a sha ing s a emen No addi ional da a a e a ailable.
Open Access This is an Open Access a icle dis ibu ed in acco dance wi h
he C ea i e Commons A ibu ion Non Comme cial (CC BY-NC 4.0) license,
which pe mi s o he s o dis ibu e, emix, adap , build upon his wo k non-
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he o iginal wo k is p ope ly ci ed and he use is non-comme cial. See: h p://
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8Gup a S, e al.BMJ Open 2016;6:e008795. doi:10.1136/bmjopen-2015-008795
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moni o ing in esou ce-limi ed se ings?
be a low-cos al e na i e o i al load
Can HIV e e se ansc ip ase ac i i y assay
Asho n, Ayesha De Cos a, Clas Källande and Ani a She
Soham Gup a, Riya Palchaudhu i, Ujjwal Neogi, Hi esa e S ini asa, Pe
doi: 10.1136/bmjopen-2015-008795
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