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Biologicals 38 (2010) 59–64

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Biologicals
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Nucleic acid testing (NAT) in high prevalence–low resource settings


Magdy El Ekiaby a, *, Nico Lelie b, Jean-Pierre Allain c
a
Shabrawishi Hospital Blood Transfusion Center, Finni Square, Dokki, Giza, Egypt
b
Chiron, Novartis Vaccines & Diagnostics SAS, 10 Rue Chevreul, Suresnes, France
c
Department of Haematology, University of Cambridge, Cambridge, UK

a r t i c l e i n f o a b s t r a c t

Article history: Blood screening by NAT for major transfusion transmitted viral infections (TTIs) was originally intended
Received 23 October 2009 to complement serology for detection of infected donations. Reports from developed countries showed
Accepted 23 October 2009 limited marginal value to NAT blood screening in improving blood safety. Reports on NAT results from
Europe indicated yield of 1:0.6 million donations for HBV, <1:M for HCV and HIV-1-related to low
Keywords: prevalence of TTI. In contrast, prevalence of TTI in resource-limited countries is almost always high. As
Nucleic acid testing
a result, more incident cases can be expected among first-time blood donors. Most reports of NAT blood
NAT
donation screening in these countries showed NAT confirmed yield as high as 1/2800 for HBV and 1/3100
HIV
HBV blood donations for HCV as reported from Thailand and Egypt, respectively. The issues for low resource
HCV countries are mostly the high cost of NAT but also the requirements of staff qualification, adequate
Developing countries facilities, reagent procurement and maintenance of delicate equipment. Alternatives to commercial NAT
are the use of combos antigen-antibody for HIV and HCV, anti-HBc for HBV and in-house NAT. Most of
these alternatives have been reported but very few comparisons are available. Once yield data is avail-
able, models for estimation of feasibility and cost-effectiveness are proposed to help decision-making.
Ó 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

1. Introduction period at the time of donation is generally very low. Only in


countries with a high prevalence and incidence of infection are
NAT blood screening for key TTIs was originally intended to likely to yield a significant number of window period donations.
complement serological screening for detection of donations Thus although the risk of transfusing a blood unit collected during
infectious for those viruses. The main advantage of NAT screening is the window period may be decreased using NAT, the actual benefit
interdiction of new incident cases during the window period in most populations should be determined and balanced against
infections. An additional benefit, which was identified as a result of the complexity and high cost of setting-up, performing and
accumulated experience of the use of the NAT was the identifica- sustaining NAT [2–4].
tion of occult hepatitis B carrier status, which can potentially be The implementation of NAT blood screening at the national or
infectious. Most populations in resource-limited regions suffer blood establishment level requires an appropriate infrastructure,
from high prevalence rate of these TTIs, and are expected to have defined test algorithm, existing quality assurance and quality
more frequent incident cases, as well as more occult carriers. control, confirmatory assays for reactive results, management
Consequently NAT screening of TTIs in these populations would be procedures for donors with confirmed positive test results. In
expected to identify more yield cases as compared to the developed addition, an evaluation of cost-effectiveness should be performed
world and thus to be more cost-effective. before considering such a screening program. Several developing
The introduction of NAT should be considered only when an countries have recently implemented NAT screening and have
efficient and effective program of serological testing is in place [1] published their experience and presented their conclusions on the
and there is clear evidence for additional benefits of NAT. Although value of this strategy according to locally or nationally defined
NAT reduces the window period of infection, in many cases the criteria. This review summarizes these experiences and their
incremental gain is minimal as the actual reduction in the window conclusions.
period may be very small and the number of donors in the window
1.1. Blood screening program for TTIs

* Corresponding author. Tel.: þ20 2 338 4684; fax: þ20 2 3338 4679. A screening program may be developed to implement the
E-mail address: elekiaby@tedata.net.eg (M. El Ekiaby). national policy on blood screening to identify and prevent the

1045-1056/$36.00 Ó 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.biologicals.2009.10.015
60 M. El Ekiaby et al. / Biologicals 38 (2010) 59–64

release of any donations reactive for specific TTIs using the most 3. Collecting epidemiological evidence
reliable and cost-effective approach [5]. As a general philosophy, it
is understood that for any country, particularly those with scarce Epidemiological evidence should be collected on a relatively
resources, the most important priorities should be considered. First large, representative, cross-section of the donor population
is the adequacy of the blood supply for patient requirements. There including first-time volunteer and replacement donors, as well as
is no definitive number establishing the extent of such need but in repeat donors. In many areas, only screening data is available but
developing countries where most blood demand is for emergency this may not provide accurate information as only confirmed
indications of massive blood loss or acute anemia, a supply ranging positive results are informative. For antibody or antigen assays,
between 10 and 20 units/1000 inhabitants is generally cited. reactivity with two separate assays is the minimum approach for
Second, this blood should be as safe as possible within the limits of confirmation, particularly where prevalence is high. When
affordability. Depending on resources, blood testing might be only combo assays are used, the presence of antigen only (separate
for anti-HIV, or anti-HIV and HBsAg or for all three main markers antibody assay negative) indicates window period infections,
(HIV, HBV, anti-HCV). The use of combo antibody/antigen to which can be used to determine the incidence of infections. The
improve HIV or HCV safety depending on local epidemiology can be next step would be to collect NAT data, within or prior to
considered at relatively modest increase in costs over antibody the pilot stage to determine the potential added efficacy of the
testing alone. Envisaging the introduction of NAT requires a number method. NAT data collected with a Triplex system provides data
of preliminary steps described below. These include: (1) defining on HIV, HCV and HBV in a single study. For HBV safety, identi-
local or regional epidemiology; (2) performance of pilot studies to fying window period infection is important but it is also impor-
generate data as well as to establish operational and financial tant to detect HBV DNA in HBsAg negative, anti-HBc positive
parameters; and (3) determination of strategy taking into consid- donations. The alternative strategy for additional HBV safety is
eration not only technical and operational issues but most impor- screening for anti-HBc. This approach may lead to discarding
tantly cost-effectiveness. These preliminary steps usually imply the large proportions of donations unless anti-HBc positive samples
existence of a national blood policy that should address national are also tested for anti-HBs and only donations without anti-HBs
requirements for the screening of whole blood and apheresis or with anti-HBs titer <100 IU/L are discarded. In such circum-
donations prior to their release for clinical or manufacturing use. stances, the cost of discarding collected units and of recruiting
Such a policy should define mandatory screening for specific new donors to compensate for such loss of supply as well as the
infections markers and screening for other TTIs, based on national percentage of repeat donors have to be taken into consideration
epidemiological data on blood-borne pathogens. It should also in the costing of the strategy [6–8]. For instance, a unit of blood
outline the measures taken to ensure that all screening is per- may cost $20 in Sub-Saharan Africa when collected mostly from
formed in the context of effective, quality-managed blood trans- replacement donors, $50 in India or in African centralized
fusion services and require the most efficient use of available volunteer only systems, making these estimations widely
resources. The need for, and the role of, confirmatory testing should different. Preliminary calculations indicate that at a $20 blood
also be clearly defined. cost, NAT becomes cost-effective above 30% anti-HBc positive
units but at 15% discard rate when blood cost reaches $50 or
2. National blood collection and screening strategy above (van Hulst, personal communication).

Blood safety depends on donor selection and donor/blood


testing. In areas of high prevalence of viral infection, donor- 4. Pilot phase
dependent blood safety may rely more upon assuring repeat
donation than on the classical distinction between volunteer and The pilot phase is critical as it provides not only the addi-
replacement donors. Laboratory screening of blood donations is the tional epidemiologic data needed but also an evaluation of the
second step that determines whether or not a donor or a donation technology(ies) available, exposure of staff to equipment and
are non-reactive for specific markers of infection and blood is methods, assessment of algorithms for screening and confir-
therefore safe to release for clinical or manufacturing use. In most mation, and defines the changes in facilities and operations
cases, laboratory screening is performed after the blood donor created by the new method. In some cases such as in Thailand,
selection and the donation process. The expectation is that the Hong Kong or Taiwan, both commercial triplex assays were
majority of the donations collected would be from healthy, unin- successively or concomitantly evaluated [9–11]. In these three
fected, individuals and would be non-reactive. However, some countries, centralized high throughput systems were already in
donations will be repeat reactive and procedures must be in place place and some funding was available for the evaluation. As
to distinguish between true and false reactivity by the process of HBV was the only high prevalence infection, screening in pools
confirmation. of six to eight or in individual unit samples revealed differ-
Consideration should also be given to the management of ences in the frequency of yield cases both window period and
donors who are deferred after their blood is reactive in screening occult HBV infection (OBI) as well as in the frequency of HBsAg
tests and deferred and then of those confirmed infected. Reliable confirmed positive cases negative for HBV DNA. The data were
confirmation of screening reactivity is essential, helping both in the then presented to the authorities for decision-making in which
management of donors and in the understanding of the epidemi- both testing efficacy and cost of screening were taken into
ological profile of blood-borne infections in the donor population. consideration. In other evaluations, only one assay was
In addition to the screening strategy, the blood screening program assessed, either commercial or in-house [12–15]. In situations
should define the confirmation algorithm for repeat reactive test where resources and expertise are limited, it may be appro-
results. Each country must decide on the TTIs to be tested as part of priate to utilize evaluation data from external sources to assess
the blood screening program and select a screening strategy potential assays and systems. In all cases, however, it is
appropriate to its specific situation. This will be influenced by the essential that an effective process is defined and put in place to
incidence and prevalence of infection, the costs of testing, ensure that new assays and systems are considered and
the capacity and infrastructure of the blood transfusion service and possibly introduced only following extensive investigation,
the available resources. evaluation and validation.
M. El Ekiaby et al. / Biologicals 38 (2010) 59–64 61

5. Laboratory quality systems of HBV outside of the window period. The implementation of new
systems for screening is best undertaken in a stepwise fashion with
Effective quality systems are essential for the overall effective- appropriate data collected, cost-effectiveness calculated and
ness of the blood screening program and to prevent the trans- resources allocated for establishing long-term operations and
mission of infection through transfusion. Quality systems should quality systems [16].
not be limited to laboratories only but should encompass all BTS
activities to ensure that all donations are screened correctly and 8. Procurement and supply of test kits and reagents
handled appropriately before and after laboratory testing. For
instance, in blood services where the number of blood samples Once the decision is made to implement a strategy including
collected is modest, screening is performed at several day intervals NAT, continuity in the supply test kits, reagents and consumables
during which part of the blood stock is quarantined. The distur- required for quality testing depends on reliable procurement and
bances of such quarantine might create for storage of blood and to supply systems is needed. Interruptions in the supply of test kits
the blood availability must be carefully evaluated. Meeting defined and reagents may result in the temporary inability of screening
quality standards will ensure the safety and clinical efficacy of facilities to screen for TTIs thus resulting in issuance of unscreened
blood and blood products as well as protecting the health and blood for transfusion. Similarly, maintenance of the delicate
safety of all staff that come into contact with them. equipment required to perform the assays needs to be assured.
Formal procurement processes, where appropriate using existing
6. Human resources national procurement systems are recommended to ensure the
sustainability of the screening program. A reliable procurement and
A sufficient number of qualified, trained and competent staff supply system not only benefits the BTS, but also ensures that each
should be available to perform the laboratory activities associated supplier is fully aware of the test kits and reagents required, the
with blood screening, including the implementation of quality usage rates and the quantities needed. Transportation and storage
systems. Such training prior to the pilot phase of the evaluation will of reagents (some of them need to be kept frozen), particularly
be provided either on site supervised by commercial company staff during airport transit in tropical conditions can be a challenging
or, probably more effectively, through in depth training in an problem. The manufacturer and the BTS should ensure that reliable
external laboratory for people already knowledgeable in molecular cold chain systems are in place to assure compliance at all times
biology would be very beneficial as it would provide the blood [17,18].
center with new skills that may be applied to a range of targets.
During the weeks required for such training, not only the screening 9. Testing approaches
method(s) but also methods for confirmation and in depth under-
standing of the methodologies involved is needed as most of the 9.1. Commercial and In-house NAT blood screening assays
time, staff will be without external support when problems occur.
Continued support by electronic communication for data inter- Since the late 1990s several European blood centers imple-
pretation or technical issues by the specialists at the training site mented NAT blood screening. These centers used either in-house or
are often psychologically and technically very helpful. Blood commercial assays. The technologies used were either direct and
transfusion services would benefit from working with national reverse transcription-mediated amplification by polymerase chain
health and education authorities to ensure that education and reaction (PCR) or transcription-mediated amplification of particular
training institutions provide suitable opportunities for staff quali- genome sequences. In-house assays must be carefully validated,
fication and training. In-service training programs established and should include positive, negative, and internal controls and should
reviewed at appropriate intervals and measures adopted to retain always be monitored by the use of external quality assurance
experienced staff will help to ensure that laboratories function panels [19]. In contrast to the situation in developed countries, in
effectively. many developing countries, no specific requirements are in place to
make commercial assays mandatory. In-house assays can be more
7. Financial resources easily implemented at a fraction of the cost offered by western
commercial companies. However, as mentioned above, assay
Experience has demonstrated that in terms of blood safety, quality and quality assurance rules must be equally applied all
either economic or political criteria are usually critical in the assays whether commercial or in-house.
decision-making process. It is therefore essential that detailed and
evidence-based information be given to the decision-making 9.2. NAT blood screening experience in resource-limited countries
bodies. Recently, several articles have been published examining
cost-effectiveness issues related to HIV and HBV blood safety since Several resource-limited countries have implemented NAT
they are the most critical in most developing countries. The case of blood screening either on a national or center level. For example,
Egypt is unique since HCV is the most frequent infection in blood the Thai National Blood Center collected 486,676 seronegative
donors. The Working Party on Infectious Diseases of the Interna- blood donations in 2007 tested by NAT using the Novartis TIGRIS/
tional Society of Blood Transfusion has developed a program which Procleix Ultrio test and the Roche Cobas s 201/Cobas TaqScreen
uses the input of basic operational and epidemiologic information multiplex (MPX) test (Table 1). The NAT yield rate for human
to determine the cost-efficiency of a given intervention. Prelimi- immunodeficiency virus Type 1 (HIV-1), hepatitis C virus (HCV),
nary results obtained with this program indicated that HIV NAT in and hepatitis B virus (HBV) was 1:97,000, 1:490,000, and 1:2800,
Ghana where the prevalence is around 4% in the general population respectively. A majority of occult HBV infections, detected by both
and 2% in blood donors was not cost efficient [16]. In contrast, it tests, were identified. HIV-1 and HCV window period cases were
demonstrated that an in-house Triplex NAT in pools of 10 samples detected with both tests [9]. In Taiwan (which is not a developing
following pre-donation screening for anti-HIV, anti-HCV and country but a country with high HBV infection rate) among 10,727
HBsAg performed with rapid tests was more cost efficient than seronegative donations, 12 HBV NAT yield cases (0.11%) and one
microtiter plate EIAs (Van Hulst, 2009, submitted). One of the main HCV NAT yield case (0.01%) were detected. Follow-up results for 1–
uncertainties in the assessment of cost-efficiency is the infectivity 8 months showed that the HCV yield case was a window case and
62 M. El Ekiaby et al. / Biologicals 38 (2010) 59–64

Table 1
Pilot studies of Triplex NAT in four blood centers in four developing countries.

Country Author Dominant Serology NAT Mode N samples Seroþ/ Seroþ/ Sero-/ WP infection
genotypes NATþ DNA DNAþ
South Africa Vermeulen [20] A1/D HBV Ultrio ID 732,250b 358 17 14 4
C HIV 483 2 4
5 HCV 24 3 0
Thailand Nantachit [14] B/C HBV Ultrio ID 5083 268 10 7 0
CRF-AE HIV 13 0 0
3 HCV 34 0 0
Thailand Phikulsod [9] B/C HBV Ultrio ID 486,676 126 94 24
CRF-AE HIV S201 MP6 5
3 HCV 2
Ghana Owusu-Ofori E HBV In- MP10 5109 13 13 0
[15] house
E HBV ID 709 17 0 12 0
CRF02_AG HIV 0
2 HCV 0
Egypt El Ekiaby [22,23] D HBV Ultrio ID 15,655 164 23 6 0
HIV 0 0
4 HCV 444 130 5

all HBV NAT yield cases were occult carriers. The authors concluded external control. Samples were analyzed in pools of six to 12
that Triplex NAT detected occult HBV and reduced the HCV window donations, each donation included in two pools, one horizontal and
period. The yield rate, especially occult HBV, was 10- to 100-fold one vertical, permitting the immediate identification of a reactive
higher than that in developed, HBV non-endemic, countries. donation, obviating the need for pool resolution. The whole process
Therefore, NAT implementation for routine donor screening in took 6–8 h and results were issued in parallel with serology. The
a more cost-effective manner should contribute to safer blood method was shown to detect all six HCV genotypes with a sensi-
transfusion in Taiwan [10]. tivity of 500 IU/mL. Until July 2005, 139,678 donations were tested
In South Africa, NAT and serology yield rates in first-time, and 315 (0.23%) were found reactive for HCV RNA. Except for five
lapsed, and repeat donors were analyzed (Table 1). The HIV, HBV, false–positives, all reactive samples were also antibody positive and
and HCV ID-NAT window phase yield rates in 732,250 blood no WP donations were identified (Table 2). Detection of WP
donations tested as single samples were 1:45,765, 1:11,810, and donation in the population studied would probably require testing
1:732,200, respectively [20]. In Ghana, an in-house Triplex NAT for of a larger number of donations [12]. This method detects all HIV-1
HIV-1, HCV and HBV in pools of 10 individual plasmas has been group M subtypes, plus group N and O, with a detection threshold
evaluated in a pilot mode in over 5109 samples seronegative after of 500 IU/mL. After validation, the method replaced p24 Ag testing
pre-donation serological screening with rapid tests (Table 1) [15]. In which had been in use for blood donation screening since 1996.
this case, NAT identified not only samples missed by the HBsAg and From July 2001 to February 2006, 102,469 donations were tested
anti-HCV rapid test initial screening but also 1:400 occult HBV and 41 (0.04%) were found HIV-RNA reactive (Table 2). One window
cases. This rate increased to 1:60 when tested in individual dona- period infection was observed giving a yield of 1:102,469 [13].
tions. Of note, Ghana is one of the countries in the world with the In Mexico an exploratory comparison of the performance of the
highest rate of HBV seropositivity (80%) and chronic HBV infection standard immunoassay-based screening tests for the HBV and HCV
(15%). Despite a 4% prevalence of HIV infection in the general and NAT, in blood donations was conducted [26]. From January
population (2% in blood donors), no HIV window period infections 1999 to March 2005, 94,806 blood donors were screened for anti-
were identified. HCV antibodies and for hepatitis B surface antigen (HBsAg).
In Brazil, an ‘‘in-house’’ RT-PCR method was developed that Molecular screening was performed on 100 consecutive blood
allows the simultaneous detection of the RNA of HCV and an donations to detect HBV DNA and HCV RNA by home-made PCR

Table 2
Pilot screening for single virus by NAT in blood donors from developing countries.

Country Author Dominant genotypes Serology NAT Mode N samples Sero-/NATþ NAT yield
Brazil Wendel [12] 1 HCV In-house MP6–12 139,678 0
Brazil Levi [13] B HIV In-house MP6–12 102,469 1
Mexico Chiquete [20] D/F HBV In-house ID 100 1
Mexico Chiquete [20] 1 HCV In-house ID 100 1
Mexico Garcia-Montalvo [26] HBV In-house ID 11,240 >13 <1:865
Lebanon El-Zaatari [27] D HBV In-house ID 5511 11 1:501
Lebanon Ramia [28] D HBV In-house ID 2505 7 1:358
Mongolia Tsatsralt-Od [29] D HBV In-house ID 403 5 1:81
China Ren B/C HBV S201 MP8 14,305 10 1:1430
Malaysia Lam B/C/D HBV Ultrio ID 43,393 12 1:3,616
India Makroo [7] D HBV Ultrio ID 12,224 6 1:2037
C HIV Ultrio ID 12,224 2 1:6112
1/3 HCV Ultrio ID 12,224 1
Chaudhuri [30] D HBV In-house ID 24,674 >40 <1:617
Iran Behzad-Behbahani [6] D HBV In-house ID 2000 16 1:125
Kuwait Al Radwan D HBV In-house 121,384 5 1:24,275
Pakistan Bhatti [31] D HBV In-house ID 966 5 1:193
Mozambique Cunha [32] A1 HBV In-house ID 1577 23 1:69
M. El Ekiaby et al. / Biologicals 38 (2010) 59–64 63

techniques without sample pooling. In the 75-month period of can be expected among first-time blood donors. Most of the reports
serologic screening, HBsAg was detected in 219 donors (0.23%; 95% of NAT screening in these countries showed NAT yield cases as high
CI, 0.20–0.26%) and anti-HCV antibodies in 922 (0.97%; 95% CI, as 1:60 blood donation [15] for HBV and 1/3100 blood donations for
0.90–1.03%). The annual trend for HBsAg prevalence had a signifi- HCV [23]. Also of importance in the consideration of NAT blood
cantly decreasing pattern over the years, whereas that for anti-HCV screening in resource-limited areas is the importance of assessing
did not. In the molecular screening of seronegative samples, HBV infectivity for HCV and HBV [22,23]. While these reports and their
DNA was detected in one donor (1%) and HCV RNA in another (1%). projections suggest to support recommending the use of the NAT
Conclusion was that NAT can detect cases of HBV and HCV infec- blood screening in resource-limited countries, there are many
tions that standard immunoassay techniques cannot, even in critical elements to carefully evaluate before any recommendation
a highly selected population such as blood donors. Large-scale can be made. The level of development of the blood services in
studies are warranted for NAT to be considered as a method for these countries and their ability to adapt this complicated and
routine screening of HBV and HCV in Mexican blood banks [21]. expensive technology as well as its integration in the general blood
As shown in Table 2, a relatively large number of preliminary service is very challenging. Most importantly, cost/effectiveness
studies of NAT applied to blood donors in developing and high and affordability of implementation are paramount in making such
prevalence countries have been published. Most studies targeted decision.
HBV because of its high prevalence in many areas. The frequency of
HBV DNA in HBsAg seronegative blood donations was as high as
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