Current concepts in molecular testing

Current concepts in molecular testingJ. E. LeviHospital Sírio Libanês Blood Bank, São Paulo, SP, Brazi

Introduction

In the last decade methods targeting nucleic acids (Nucleic

Acids Tests) of infectious agents became a routine in the

blood bank setting. The most intensive use is in the primary

screening of blood donations for the hepatitis B and C

viruses (HBV and HCV) and the human immunodeficiency

virus (HIV) [1]. NAT have also been instrumental in the

rapid prevention of transmission of emerging agents such

as the West Nile Virus in North America [2].

Methodological issues

There are three steps in common to all NAT methods:

(1) Nucleic acids extraction.

(2) Amplification of targeted genes.

(3) Detection of the amplified material.

Extraction has been the most difficult step to automate,

being the main obstacle in achieving 100% automated sys-

tems for molecular screening of blood donations. However,

in the last few years, at least two distinct fully automated

platforms became available (Roche Cobas s201, Novartis

Tigris, Emeryville, CA). Apparently, precise automated han-

dling of small volumes, commonly employed in molecular

biology techniques, is hard to incorporate into instruments.

It is still an unsolved inconsistency the fact that we use for

NAT screening no more than 200 ll of plasma ⁄ sera, but we

need to feed the systems with tubes containing at least

1Æ2 ml of plasma ⁄ sera. Nevertheless, these systems are

expected to improve in the future by taking benefit of the

growing science of nanotechnology [3]. Intensive research

is being made in the field of miniaturization of all three

basic steps, aiming to obtain devices that can be used as

point-of-care tests, with minimal requirements of instru-

ments, with the same sensitivity already displayed [4].

Step 1 is generic for all kinds of targets, aiming to

remove macromolecules bound to the nucleic acids and

other potential molecules that may inhibit the polymerases,

finally delivering a pure solution of DNA ⁄ RNA. In contrast,

step 2 relies on the appropriate choice of oligonucleotides

that are highly complementary to the genomes to be

detected, but also presenting particular biochemical proper-

ties necessary for the specific assays. Assays also differ on

the kind of nucleic acid to be amplified. The polymerase

chain reaction (PCR), is the most used and recognized DNA

amplification technology [5]. To allow RNA targets, such as

RNA viruses, but also mRNAs, to be targeted by PCR, a pre-

vious or concomitant reverse-transcription step is required

to generate cDNA. This has been conjugated into a single

step reaction by employing enzymes able to cover both

activities (reverse transcription + DNA polymerase [6].

Other similar methods such as the Ligase Chain Reaction

(LCR) [7] did not find, so far, widespread use in blood

screening. There are alternative methodologies that use

RNA, instead of DNA, as the template molecule to be ampli-

fied. The Nucleic Acid Sequence Based Amplification (NAS-

BA) [8] and Transcription Mediated Amplification (TMA)

[9] are examples of innovative technologies that use also

repetitive cycles to generate identical copies of an RNA

stretch. Being RNA an unstable molecule, these methods

are less prone to amplicon contamination, as RNA is

expected to be rapidly degraded in the environment.

Another advantage is the temperature requirements, RNA

secondary structure is fragile, and RNA synthesis may

occur at 37–41 �C, making these methods less demanding

for instruments such as thermocyclers. Coupled with pro-

cesses that magnify amplification signals, current NATs

display an analytical sensitivity and specificity that cannot

be matched by existing serological assays.

Contamination of lab instruments and reagents by previ-

ously amplified material (amplicons) has been the night-

mare of every molecular biologist in diagnostics. This

caveat of amplification methods has demanded segregation

of the three steps of the process in distinct rooms with

restricted flow of persons and materials in between them.

Fortunately, fully automated systems are now available,

making it possible to carry the whole process in the same

room.

NAT false-negatives/positives

Since infectious agents are constantly evolving and modi-

fying their genomes through random and selective muta-

tions and recombination, NAT are challenged by these new

nucleotide sequences, making molecular surveillance an

Correspondence: José Eduardo Levi, Centro de Imunologia e Imunogené-tica, Avenida Brigadeiro Luiz Antônio 2533 2� andar, CEP 01401-000, SãoPaulo, SP, BrazilE-mail: [email protected]

ISBT Science Series (2011) 6, 67–71

STATE OF THE ART 2A-E2.3 ª 2011 The Author(s).ISBT Science Series ª 2011 International Society of Blood Transfusion

67

essential tool in the constant improvement of NAT. This is

illustrated by the failure of existing NAT to detect rare HIV

mutants [10], specific HCV genotypes [11] and, more fre-

quently reported, viral load assays that sub and overquanti-

fy particular genotypes of HCV and HIV [12,13].

One case of HIV transmission by a window period (WP)

donation missed by NAT-testing, was attributed to muta-

tions in the primer binding site on the particular viral strain

[14]. Moreover, the odd finding of donors with confirmed

antibody reactivity for HIV and individual NAT non-reac-

tive, harbouring viraemias much above the limit of detec-

tion of the blood screening NAT assay, as determined by

commercial viral load methods, illustrates how mutations

may lead to NAT failure [15]. This phenomenon (emergence

of new mutants) that will certainly continue to occur,

endorses the complementary role of NAT and antibody

tests, providing maximum safety of transfusion.

In addition to the reduction of the WP, another extraor-

dinary feature of NAT is the very high specificity obtained

by well-designed assays. As an example, in a recent report

from the American Red Cross on the molecular screening of

approximately 3Æ7 million donations for HBV, HCV and

HIV, the specificity of the NAT employed (Procleix Ultrio,

Novartis Diagnostics, Emeryville, CA) assay was 99Æ995%.

In our own experience with the MPx assay on the cobas

s201 platform (Roche Molecular Systems, Pleasanton, CA),

after testing 120 000 donations, no false-positive result

was verified.

Cost-effectiveness

As shown in many developed countries, NAT screening for

HBV, HCV and HIV is not cost-effective, being the decision

to use them justified as a health priority for the society

[16,17]. Paradoxically, NAT screening may prove to be

financially reasonable in developing countries, where the

incidence of these agents in the blood donor population is

higher than in the countries that first introduced NAT

screening, providing a higher yield of WP donations

blocked by the test. As a comparison, while in the US, after

10 years of NAT testing 32 HIV and 244 HCV yield cases

were detected among 66 million donations [18] (1:2 mil-

lion and 1:270 000 respectively) in South Africa [19] 16

HIV window period donations were interdicted among

732 250 evaluated (1:45 765) and in Egypt, among 15 655

first-time blood donors, five HCV WP were identified,

depicting an yield of 1:3100 [20].

NAT · antigen detection

Use of antigenic tests, where NAT is not affordable, has

been advocated by some [21,22]. Tests for the HIV p24 cap-

sid antigen were used in the past with disappointing results

and tests for the core antigen of HCV are available and in

use for the same purpose of reducing the WP. In general,

antigenic tests are able to detect approximately 70% of the

NAT-yield cases [22]. Replacement of HBsAg testing by

HBV NAT in combination with anti-HBc is under debate

[23], and may be possible in the near future, when sensitiv-

ity for this agent improves and testing of single units

instead of pools becomes a standard [24]. If this algorithm

is to be adopted, it may require, in countries with a high

anti-HBc prevalence, additional testing of positive units by

a quantitative anti-HBs assay, in order to recover units

positive for anti-HBc but negative for HBV-DNA and con-

taining protective titres of anti-HBs, an approach currently

used by the Japanese Red Cross [25].

Contributions to the knowledge of thenatural history of viral infections

Large-scale adoption of NAT has greatly contributed to the

evolution of the knowledge on the natural history of HBV,

HCV and HIV infections. HCV NAT was firstly introduced

worldwide, confirming that 15–30% of the infected hosts

do clear the virus [26]. Moreover, it also provided solid evi-

dence that the chronic serosilent carriers exist indeed, but

in a very small prevalence [27]. Previous reports suggested

that approximately 10% of all community-acquired HCV

infections are serosilent [28], what would have led to a

much larger number of HCV-RNA only donors detected by

NAT screening. In 10 years, data from the American Red

Cross depicted only three such cases [18]. For HIV, NAT

screening of millions of donors reinforced the established

knowledge that spontaneous clearance of HIV-1 does not

occur in humans. Even so, the figure of the so called ‘elite

controller’ was observed in approximately 5% of all HIV

positive donors (antibody+). These are individuals con-

firmed reactive for HIV-1 (EIA and Western blot+) whose

RNA viral load is undetectable by ultrasensitive tests avail-

able (limit of detection = 20 IU ⁄ ml) [29]. Follow-up and

thorough investigation of genetic and immunological char-

acteristics of these patients may bring new insights into

HIV treatment and control. Finally, HBV continues to rep-

resent the most complex agent for proper evaluation of lab-

oratorial results. Countries that use a triple screening for

HBV (anti-HBc, HBsAg and HBV-DNA) have found all pos-

sible combinations of test results [30]. Each combination

may have a biological explanation, excluding test errors,

and demand investigation and further testing until a final

interpretation may be achieved. In countries where anti-

HBc is not adopted, donors negative for HBsAg and reactive

for HBV-DNA are named as ‘occult B infections (OBI)’ [31].

The most common mechanism behind OBIs are individuals

chronically infected, presenting HBsAg levels below the

limit of detection of ultrasensitive HBsAg assays, but

68 J. E. Levi

� 2011 The Author(s).ISBT Science Series � 2011 International Society of Blood Transfusion, ISBT Science Series (2011) 6, 67–71

maintaining low titres of HBV-DNA, in general below

200 IU ⁄ ml. They tend to bear concomitant anti-HBc and

anti-HBs and thus present a very low risk for HBV trans-

mission. WP HBV donations, on the other hand, are highly

infectious, and are the main source of the existing risk of

transfusion transmitted infections (TTI), principally where

NAT for HBV is not adopted [32]. A particular feature of

HBV, apart from being a DNA virus, in contrast to RNA

viruses as HCV and HIV, is its slow replication, leading to a

prolonged serological WP, and not allowing the adoption

of large pools on NAT screening, since individuals in the

WP may have viraemias close to the limit of detection,

going undetected if diluted by the pool factor. These facts

have pushed companies to enhance the sensitivity of their

NAT assays towards HBV. Although the gain in sensitivity

has been noticeable, the ultimate goal is to have universal

individual testing, which may be reasonable in the near

future.

NAT for non-viral microorganisms

Bacterial and fungi detection by molecular assays present

an extremely attractive idea that has been proving hard to

be accomplished. As all bacteria share common conserved

sequences, for example the 16S ribosomal gene, it is pos-

sible to have one single primer pair covering all known

and unknown species [33], which may also be found for

the 18S ribosomal gene of Fungi [34]. If available, these

methods would provide an alternative to the expensive

and cumbersome culture techniques, while offering a

higher throughput [35]. Nevertheless, the widespread con-

tamination of reagents, including water, with fragments of

bacterial DNA, has prevented the development of such

assays.

Limited research has been carried on the potential use

of NAT for blood screening for parasites. Until recently,

TT of protozoa like Trypanosoma cruzi (Chagas disease)

and Plasmodium sp. (malaria) was a matter of concern

only in endemic areas. The exponential growth of travel-

ling and migratory movements brought these agents to

the agenda of the blood bank community in developed

countries as well. In non-endemic areas, the main source

of risk stems from asymptomatic chronic carriers, mostly

unaware of their parasitized condition, eventually volun-

teering for blood donation. They are prevented from

donating either by questions in the predonation interview

that identify a link to endemic areas, and ⁄ or by targeted

serological testing [36]. For these chronic carriers, NAT

screening would be of limited contribution, since in gen-

eral they harbour very low levels of parasitaemia. Seed et

al. in Australia detected only two PCR positive individuals

among 2697 donors serologically reactive for Plasmodium

sp antibodies [37] while Leiby et al., using several blood

drawings of large volume, obtained PCR positive results

in 63% out of 52 donors seroreactive for T. cruzi antibod-

ies [38]. In contrast, in endemic areas, due to the high

prevalence, it would be unrealistic to adopt serological

tests for Plasmodium sp. In these areas NAT would have a

potential role in detecting both serosilent and WP donors.

Noticeably, two studies on donors from blood banks in

the Brazilian Amazon depicted 0Æ5–3% of PCR positive

donations found negative by the standard mandatory

malarial test [39,40]. Even though higher rates of NAT

positivity are observed in seropositive T. cruzi donors, use

of genomic technologies for blood screening are not upon

consideration since Chagas is a vanishing disease and

serological screening is currently adopted in endemic

areas [41].

Future perspectives

So far, NAT for infectious agents have not achieved the

desirable multiplexing capability depicted, for example, in

current array platforms for determination of blood groups

[42].

The holy grail of molecular testing would be a single

assay displaying a very high analytical and clinical sensi-

tivity, down to one or less genome-equivalents ⁄ ml, includ-

ing all potential agents transmissible by blood. Several

attempts are being pursued towards that [3,43]. However,

the decrease in sensitivity for each target, observed when

multiplexing dozens of primer pairs, still hampers the

application of these methods to screening of blood-trans-

missible agents, as the maximum sensitivity is an absolute

requirement. In contrast, microarrays for blood group

genotyping do not suffer from this limitation; as we all

have at least one copy of each gene per cell, minimal

amounts of whole blood offer enough DNA for routine

genotyping.

The putative bloodchip for transfusion transmissible

agents would be a technological response to the ever grow-

ing number of targets for which we would like to test dona-

tions for. On the other hand, the evolution of pathogen

reduction technologies may render blood units sterile and

testing unnecessary in the future [44].

Disclosures

None.

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Current concepts in molecular testing