DNA microarrays in the clinic:infectious diseasesVladimir Mikhailovich,1 Dmitry Gryadunov,1 Alexander Kolchinsky,2

Alexander A. Makarov,1* and Alexander Zasedatelev1*

SummaryWe argue that the most-promising area of clinicalapplication of microarrays in the foreseeable future isthe diagnostics and monitoring of infectious diseases.Microarrays for the detection and characterization ofhuman pathogens have already found their way intoclinical practice in some countries. After discussing thepersistent, yet often underestimated, importance ofinfectious diseases for public health, we consider thetechnologies that are best suited for the detection andclinical investigation of pathogens. Clinical application ofmicroarray technologies for the detection of mycobac-teria, Bacillus anthracis, HIV, hepatitis and influenzaviruses, and other major pathogens, as well as theanalysis of their drug-resistance patterns, illustrate ourmain thesis. BioEssays 30:673–682, 2008.� 2008 Wiley Periodicals, Inc.

Introduction

The FDA discriminates two types of diagnostics based on the

analysis of nucleic acids (nucleic acids testing, NAT): genetic,

which ‘‘determines the presence or absence of a particular,

targeted DNA sequence already known. . .to be related to a

health outcome; and genomic, which measures gene expres-

sion, usually by mRNA as surrogate.’’(1) In a recent review of

the clinical applications of microarrays, Jordan surveyed both

approaches for the detection of clinically relevant features in

the genes and gene expression patterns of the patients.(2) The

author concluded: ‘‘. . .(T)he adoption of DNA arrays is taking

place at a slower rate than expected. . .’’ This is not surprising,

however, when we take into account that most of the reviewed

applications target cancer, which remains the highest, yet at

the same time the most intractable, target of such translational

research.

Historically, cancer and cardiovascular diseases only

became the major causes of mortality relatively recently, first

in developed countries, then gradually in most of the

developing world. The radical increase of longevity between

1900 and 1950 was due to the introduction of new chemo-

therapies against infectious diseases and, in the following two

decades, to the reduction in infant mortality resulting from

better maternal care and a decrease in malnutrition.

Translational research and fundamentally new approaches

have not played significant roles in the overall improvement of

cancer treatment, although some relatively rare cancers

became curable. One striking example of the failure of

cancer-related discoveries to influence clinical progress is

the history of p53.(3) Since its discoveryalmost 30 years ago, at

least 4 billion dollars have been spent on p53-related

research,(4) because it has been assumed that ‘‘determining

the p53 status of cancers would rapidly acquire major clinical

significance’’. Yet, ‘‘(a) recent meta-analysis of. . .studies in

colon cancer has. . .confirmed anecdotal understanding that

this is not the case. . .(T)his can be seen as a massive failure of

translational research.’’(3)

Currently, the impact of infectious disease on mortality in

developed and some developing countries is low, about 1–3%

of all deaths. At the same time, in other developing countries, it

remains much higher. For instance, 20% of all deaths in India

are caused by infections while, in Zimbabwe, this figure is as

high as 74%, with 63% of all deaths caused by HIV/AIDS.(4)

Overall, in 1990, 27% of all deaths in the world were due to

infectious causes;(5) in 2002, the last year for which the data

are available, this number decreased to 20%.(5)

However, accurate death statistics collected by WHO, as

noted in many of its own documents, are not available. One of

the WHO bulletins on the subject reads: ‘‘There are only

23 countries classified as having high-quality (cause of death)

data. There are 28 countries classified as having low-quality

data; these include some high-income countries, such as

1Engelhardt Institute of Molecular Biology, Russian Academy of

Sciences, Moscow, Russia.2Health Front Line, Ltd., Champaign, IL.

Funding agencies: This work has been financially supported by the

Russian Academy of Sciences’ Program on Molecular and Cellular

Biology and by contracts #02.190.11.30, #02.512.11.2089,

#02.512.11.2184 and #02.527.11.9006 with the Federal Agency of

Sciences and Innovations of the Russian Federation.

*Correspondence to: Alexander Zasedatelev and Alexander A.

Makarov, Engelhardt Institute of Molecular Biology, Russian Academy

of Sciences, 32 Vavilov St, 119991 Moscow, V-334, Russia.

E-mail: zas@biochip.ru and aamakarov@eimb.ru

DOI 10.1002/bies.20781

Published online in Wiley InterScience (www.interscience.wiley.com).

BioEssays 30:673–682, � 2008 Wiley Periodicals, Inc. BioEssays 30.7 673

Abbreviations: NAT, nucleic acid testing; FDA, Food and Drug

Administration (USA); POCT, point-of-care testing; S/N, signal/noise

ratio; QC, quality control; HPV, human papilloma virus; HBV, hepatitis

B virus; HCV, hepatitis C virus.

What’s new?

Greece and Portugal.’’ Moreover, death statistics for the most

populous countries, India and China, are based on sampling,

not actual count.(6)

It is quite certain that the available vital statistics diminish

the apparent importance of infectious diseases. First,

infections disproportionately affect the younger part of the

population; therefore its share in the so-called ‘‘years of life

lost’’ is higher.(7) Second, the relatively low mortality caused

by infections in developed countries is not exclusively the

result of their low incidence. To some extent, it is the result of

successful management of infections using antibiotics and

antiviral drugs. Third, infections resulting from underlying

chronic conditions may not be reflected in the overall vital

statistics. For instance, the immediate cause of death of

patients with suppressed immunity may be an opportunistic

infection but, in the death certificate, the listed cause will be

their primary condition—HIV or cancer, if the infection

emerged as the result of chemotherapy treatment. Finally,

dangerous and unusual infections can emerge as the result

of bioterrorism.

In addition to the direct impact of infections on mortality,

many chronic diseases may be initially caused by pathogens.

Thus, human papillomavirus is the cause of most cervical

cancers,(8) hepatitis viruses cause liver cancers.(9,10) and the

bacteria Helicobacter pilori is often responsible for stomach

ulcers and can lead to cancer.(11) Moreover, any chronic

inflammation may be a strong risk factor for malignancy.(12)

Contrary to public perception, therefore, infectious dis-

eases remain at the forefront of modern health care, and the

principal problem is that pathogens are constantly moving

targets. All pathogens sooner or later develop resistance to

new drugs, while viruses rapidly evolve into new strains with

better immune tolerance and more efficient transmission.

Generally speaking, infectious diseases are understood

better than many other common conditions, including cancer.

There are large databases of sequences of pathogens; many

genomes are sequenced fully, including different variants and

strains; the genetic basis of drug resistance and immune

tolerance is often (though not always) known. To address this

field of research and clinical diagnostics, National Institute of

Allergies and Infectious Diseases (NIAID), a member of NIH,

created a network of ‘‘Bioinformatics resource centers for

biodefense and emerging or re-emerging infectious dis-

eases’’(13) and created and regularly updates ‘‘List of Emerg-

ing and Re-Emerging Infectious Diseases’’.(14)

This review will complement the aforementioned paper by

Jordan,(2) which did not mention the diagnostics of infectious

diseases. We outline selected microarray-based technologies

and their clinical applications in this field, emphasizing the

most-recently published work. This outline does not attempt to

provide a comprehensive review, and the works chosen for

citation only illustrate the potential of microarrays to detect and

characterize clinically important pathogens.

Microarray technologies suitable for diagnostics

The demands of clinical diagnostics enforced by national

regulatoryagencies are much stricter than the requirements of

experimental technologies encouraged solely by voluntary

compliance. The quality of data obtained using microarrays, as

well as their inter-laboratory compatibility, is an actively

discussed technical challenge (for review, see Ref. 15). This

is especially true in case of genomic testing, when the results

depend on the preparation, storage and processing of

samples and the synthesis of hybridization probes. The

problems are serious enough that the Microarray Quality

Control (MAQC) project created by FDA in the United

States(16) stopped short of recommending microarray meas-

urements of gene expression (genomic testing) for clinical

diagnostics. In contrast, the combination of genetic testing with

low-density arrays and limited sets of oligonucleotides, which

is most frequently used for the diagnostics of infectious

diseases, can give clear qualitative answers and has much

better standing.

The criteria for quality assessment can be categorized into

those applied to the whole arrays (background variance

across the array and within batches of manufactured arrays,

dynamic range of signal level, precision of the grid) and those

related to individual elements of the arrays (S/N ratio for

positive controls, variance in spot size, variance of signal

intensity across the sets of replicates). While a certain

percentage of ‘‘flagged’’ elements is considered acceptable

in experimental settings, QC procedures must reject arrays

intended for clinical diagnostics if they contain faulty elements.

A good example of careful evaluation of a diagnostic DNA

chip was published by Lee et al.(17) The authors tested DNA

arrays for the detection of human papilloma virus (HPV).

These arrays were approved by Korea Food and Drug

Administration (KFDA) and evaluated by the authors in

accordance with KFDA requirements. Interestingly, these

requirements include ‘‘Clinical effectiveness of the device’’,

in contrast, for instance, to CE (conformite europeenne)

certification adopted by EU and limited to safety of the device

and its manufacture process. In this respect, KFDA require-

ments are similar to FDA policy in the United States.

Technology platforms for clinical diagnostics

It appears that, so far, just a few of the multitude of microarray

technologies have proved suitable for clinical applications.

First, this is the technology of photolithographic synthesis

of probes on the surface of quartz wafers developed by

Affymetrix (GeneChip1

). The resulting two-dimensional micro-

arrays became the first to be approved by the FDA for genetic

testing — clinical diagnostics of mutations in two P450

proteins.

Affymetrix GeneChips carry up to 106 individual elements

(features) and are able to re-sequence by hybridization rather

long segments of DNA and identify large number of variations.

What’s new?

674 BioEssays 30.7

Many applications of Affymetrix GeneChips for identification

and typing of pathogens are reviewed at the company web

site(18) and include studies of Streptococcus pyogenes and

other respiratory pathogens, about 200 strains of Staph-

ylococcus, rifampicin resistance in M. tuberculosis, oxacillin

resistance in S. aureus, typing of Neisseria meningitides,

simultaneous detection of a large spectrum of bacterial and

viral pathogens, and a GeneChip with more than 60,000

probes able to identify about 9,000 species of bacteria using

polymorphisms in their 16S ribosomal RNA.

While these data may have outstanding importance for

clinical science, the relative complexity of this approach limits

its usage to advanced laboratories and makes it expensive. For

routine clinical diagnostics of infectious diseases, it is often

more convenient to have limited and reliable information

generated by the use of low-density arrays, in which individual

elements detect specific sequence variations.

Thus, the most-popular microarray technology in clinical

diagnostics today is low-density printing of pre-synthesized

oligonucleotides on chemically treated slides; they often

consist of 96 features to match standard plates. Such slides

are easily prepared, inexpensive and require only the most

basic equipment for printing, hybridization and scanning.

Contrary to the Affymetrix chips, low-density home-printed

arrays can be adapted to new probes or new overall design

without contacting the manufacturer. Such arrays can be

converted into affordable point-of-care testing (POCT) devi-

ces and even field systems.

A versatile and robust method based on the immobilization

of DNA probes in three-dimensional gel drops has been

developed in the laboratory of Andrei Mirzabekov at the

Institute of Molecular Biology in Moscow.(19) The three-

dimensional structure of individual elements and liquid

environment provide significant advantages for clinical diag-

nostics. Namely, it allows using relatively short oligonucleo-

tides for efficient identification of point mutations, not to

mention more extensive sequence variations. Reliable dis-

crimination between matching and mismatching samples,

which can be achieved using gel-based arrays, allows the

number of elements necessary to detect specific sequences to

be minimized. Decreasing the initial concentration of the

probes accelerates the process of discrimination between

perfect and imperfect duplexes upon hybridization, and

optimization of washing (dissociation) can further improve

the discrimination; this occurs in accordance with theoretical

predictions.(20) Direct comparison of hybridization on flat

microarrays and three-dimensional microchips shows that,

although hybridization on flat microarrays is slightly faster,

gel-based microchips discriminate between perfect and

imperfect duplexes more efficiently and generate 5- to 20-fold

stronger signals.(21)

Because of the high water content, high porosity and

complete isolation of gel pads from each other, they can

function as reaction tubes of nanoliter volume. In particular,

this concept has been used in the development of the so-called

‘‘on-chip PCR’’ procedure, which combines PCR amplification

both inside the pads and in overlay solution with hybridization

with immobilized probes (Fig. 1).(22) Pilot experiments were

designed to identify bacteria within the Bacillus cereus group,

which includes B. anthracis. The first unambiguous identifica-

tion of B. anthracis by hybridization of bacillus RNA on

microarrays was reported in 1999,(23) and then using on-chip

PCR in 2001.(24) The same approach was also used for the

detection of mutations in M. tuberculosis conferring drug

resistance.(25) The most-recent application of this technology

is real-time on-chip PCR for identification and quantification of

HIV, HBC and HCV in donor blood.(26)

Another promising technology employs electrochemical

detection of the hybridization events on multiplex microarray

using enzyme-linked assaysor immobilization of the probes on

the surface of silicon wafers.(27) For instance, electrochemical

detection of hybridization on microarrays was successfully

employed for the detection of toxin-encoding genes respon-

sible for pathogenicity in the Bacillus cereus group.(28) The

latter technology does not even require PCR amplification of

target sequences and is therefore very convenient for

conversion into a field-deployed test. In the future, this

microarray format can be further developed into a disposable

cartridge, thus diminishing the possibility of cross-contami-

nation of samples. A similar electrochemical detection of

hybridization eventswithin a disposable cartridge is used in the

eSensor1, a cystic fibrosis carrier detection kit, developed by

Clinical Micro Sensors, Inc. (Pasadena, CA) and approved by

FDA for marketing.

An interesting approach has been proposed by Aragon

et al.(29) During the PCR amplification step, biotinylated

nucleotideswere incorporated in the product. For the detection

of certain resistance mutations, the authors hybridized the

product with immobilized probes, washed the arrays, and

stained them with avidin–peroxidase conjugate and colored

peroxidase substrate. Hybridized sequences were visible as

dark spots without any additional labeling, and the results were

in reasonable concordance with more traditional methods.

Mycobacterium tuberculosis

Traditional evaluation of drug resistance in clinical samples of

M. tuberculosis complex takes at least four weeks, causing

either a delay in treatment, or administration of often

ineffective antimycobacterial drugs. The resistance to two

first-line antibiotics, isoniazid and rifampicin, may be caused

by several different genetic changes, which could be identified

by molecular methods. Besides, NAT allows detecting

resistance to other drugs and to perform strain typing for

epidemiological monitoring (for review, see Ref. 30).

Gryadunov et al. developed a biochip for the detection

of rifampicin-resistant and isoniazid-resistant strains of

What’s new?

BioEssays 30.7 675

M. tuberculosis.(31) The biochip identifies over 95% of

rifampicin-resistant and more than 80% of isoniazid-resistant

M. tuberculosis strains in sputum samples.

The biochip consists of 77 gel elements and detects the

27 most-common mutations in the rpoB gene responsible

for rifampicin resistance, 11 mutations in the katG gene, five

mutations in the promoter region of the inhA gene, and five

mutations in the intergenic regulatory region of the ahpC–

oxyR genes, all of which can cause resistance to isoniazid.

Fig. 2 shows these microarrays after hybridization and

illustrates the interpretation of the results.

The performance of gel-based biochips was successfully

tested by an independent laboratory in the USA.(32) The

system displayed a sensitivity of 80% and a specificity of 100%

relative to conventional testing for rifampicin resistance. All

discrepancies between the microarray test and culture test

were caused by either rare mutations not detectable by the

microarray probes, or by unknown mutations. Gel-based

biochips were also used for large-scale epidemiological

studies of drug resistance in Kyrgyz Republic.(33,34)

The newest generation of TB-biochips identifies mutations

responsible for the emerging resistance of M. tuberculosis to a

highly effective second-line fluoroquinolone antibiotics.(35)

The sensitivity and specificity of the method are 93% and

100%, respectively. Due to the discrimination power of gel-

based hybridization, just 20 individual elements sufficed for

the detection of the majority of known mutations conferring this

resistance. Both TB-biochips for the analysis of rifampicin/

isoniazid or fluoroquinolones susceptibility were among the

first in the world to be certified by a national regulatory agency,

Ministry of Public Health of Russian Federation, for clinical

application.

Staphylococcus aureus

There is a growing threat of staphylococcal infections because

of the rapid spread of drug resistance and the ability of some

strains to produce toxins. Similarly to M. tuberculosis,

molecular tests, in particular microarray-based, provide the

necessary diagnostic tool for fast and reliable characterization

of this pathogen.

Monecke and Ehricht designed a diagnostic microarray that

identifies the genus and species of the pathogen and detects

more than 20 genes of drug resistance and 10 genes encoding

various toxins simultaneously.(36) An efficient diagnostic

microarray has been developed and tested by Zhu et al.(37)

Their array differentiates S. aureus from other staphylococci

using mutations in the variable region of 16S rRNA and detects

genes encoding resistance to seven antibiotics. The method

was successfully tested on about 400 samples. Another, even

Figure 1. A: On-chip PCR and B: its allele-specific variant. The initial PCR stage (not shown) occurs in reaction solution outside the gel

pad. Reaction mixture contains forward and labeled reverse primers. During the next stage, the immobilized forward primers are involved

in amplification along with free reverse primers from surrounding solution (A1). The immobilized primers are extended enzymatically

(A2), forming new target chains attached to the gel via their 50 termini. These chains serve as targets for further amplification (A3) and

eventually form tethered double-stranded hybridization complexes that either contain a fluorescent label (A4), or acquire the ability to bind

non-specific fluorescent dyes (e.g. SYBR1 green used for on-chip real-time PCR). Since each gel pad contains primers specific to one

target only, the kinetics of increase of fluorescence in each gel pad reflects the accumulation of the corresponding amplification product.

Fluorescence signals in gel pads are measured at the end of each elongation step, when specific double-stranded hybridization complexes

remain stable due to their high melting temperature, in contrast to shorter non-specific products. An oligonucleotide specific to the middle

part of the target product could serve as an additional specificity control (A5). Primer extension occurs only when the terminal nucleotide is

complementary to the tested sequence (B6); otherwise, the allele-specific PCR is not possible (B7).

What’s new?

676 BioEssays 30.7

more general, approach to the detection of drug resistance is

based on a microarray that can identify almost 100 different

genes responsible for drug resistance found in many different

bacteria.(38)

In many clinical situations with limited access to even most-

basic clinical laboratories, there is a need for rapid identi-

fication of pathogens from a broad variety of microorganisms.

Zhu et al. developed a microarray for identification of five

species of bacteria most common in clinical settings, including

Staphylococcus.(39) The method does not require PCR,

making it especially suitable for field applications, and its

specificity is better than 98%. The authors employed the

so-called base stacking hybridization discovered and explored

earlier by Vasiliskov et al.(40)

Malaria

Malaria remains one of the most frequent causes of death

among infectious diseases. According to some estimates, up

to 500 million people in the world are infected, leading to high

morbidity and mortality. The environmental ban on DDT

resulted in dramatic increase in the prevalence of malaria,

while the pathogen acquired resistance to chloroquine,

quinine and other common treatments.

Molecular studies of Plasmodium falciparum began rela-

tively late. NAT of P. falciparum is hindered by the unusually

high AT content of its DNA. Direct testing of the pathogen for

drug resistance is difficult enough. Recent advances in

sequencing and molecular mapping of Plasmodium genome

yielded many SNPs linked to resistance genes or directly

responsible for the resistance. Microarray-based analysis of

the resistance genes already reached better than 94%

specificity, can be performed even on asymptomatic patients

and is highly cost effective.(41) In a recent review, molecular

approaches, in particular microarray-based methods, are

considered the most-promising direction in the diagnostics

and characterization of the malaria pathogen.(42)

Figure 2. Analysis of M. tuberculosis susceptibility to rifampin (RIF) and isoniazid (INH) by hybridization on gel-based biochip. A: The

diagnostic microarray (biochip). The microarray detects 27 most-common mutations in the rpoB gene responsible for RIF

resistance (the corresponding area is marked yellow) and 21 mutations in the katG, inhA, ahpC genes, which cause the resistance to

INH (blue). The biochip also contains probes complementary to the IS6110 element (green) present in most mycobacteria belonging to M.

tuberculosis complex. Gel elements marked ‘R’ serve as negative controls, whereas marker pads ‘M’ are necessary for automatic

processing of fluorescence images. The pads with immobilized oligonucleotides are clustered in 21 groups, which are depicted as boxes.

Each group corresponds to a single variable amino acid position in rpoB and katG genes or variable nucleotide in the promoter regions of

inhA and ahpC genes. One gel pad within each group contains oligonucleotides matching the wild-type sequence (grey circles). Other gel

pads contain immobilized oligonucleotides forming perfect duplexes with different mutant variants of the same codon (white circles). Thus,

only one oligonucleotide in each group could form a perfect duplex with the target DNA. After hybridization, the fluorescence signals are

analyzed within each group separately to decide whether mutation(s) are present in the amino acid position concerned.B:Hybridization of a

wild-type DNA sample on the biochip. The maximum fluorescence intensity within each group is observed in gel pads containing

oligonucleotides corresponding to the wild-type sequence. Positive signals in gel pads with IS6110 probes confirm that the tested strain

belongs to the M. tuberculosis complex. This particular pattern indicates that standard therapy with RIF and INH could be applied.

C: Fluorescence pattern resulting from hybridization of DNA sample containing mutations Ser 531>Leu, TCG>TTG in the rpoB gene and

Ser 315>Thr and AGC>ACC in the katG gene differs from the WT pattern (signal changes are marked by the dashed frames). This TB

strain is considered multidrug-resistant, and therapy with the second-line drugs must be used.

What’s new?

BioEssays 30.7 677

Viruses

The major challenge in the molecular diagnostics of many

viruses,especially influenza,HIVandhepatitisC, is their extreme

variability and mutability. Sequences of viruses of the same

species or even serotype may differ by more than 30%, and

selection of primer sequences for PCR amplification and target

segments for the subsequent hybridization requires extensive

analysis of available databases and phylogenetic trees.

HBV

Impressive results were achieved by Chen et al., who

developed a microarray for detection of resistance of hepatitis

B virus (HBV) to lamivudine, one of the major anti-retroviral

drugs.(43) Expected mutations in HBV gene encoding

DNA polymerase were detected using microarrays in all

388 samples found mutant by direct sequencing. The

sensitivity of the microarray to the presence of HBV was

tested on almost 1000 samples and reached 99.7%. Moreover,

the microarray was able to detect mutant variants against the

background of wild-type virus in patients with mixed infections.

HIV

HIV-infected patients are treated with a mixture of drugs, the

so-called highly active antiretroviral therapy (HAART), which

decreases both morbidity and mortality. Under the pressure of

the drugs, the virus mutates and develops resistance. The

situation is further complicated by pre-existing sequence

polymorphisms and by the complex nature of resistance often

resulting from a combination of mutations.

The complexity of genetic testing of drug resistance in HIV

was reviewed in 2001,(44) and the situation has not changed

much since then. In general, the level of resistance to specific

drugs cannot be easily deduced from the sequence of the

virus. The HIV GeneChip for sequencing of HIV isolates by

hybridization offered in the past by Affymetrix currently is not

even mentioned on its web site. HIV drug resistance is

determined for practical purposes mostly from clinical obser-

vations.

However, certain HIV polymorphisms, including those

associated with drug resistance, can be identified using

dedicated microarrays and employed in epidemiological

studies. For instance, Roudinskii et al.(45) used gel-based

biochips to examine the occurrence of the so-called secondary

protease mutation V77I. The detailed study followed the route

of transmission of this particular strain in Eastern Europe.

Precision screening of more than 100 samples was possible

because of the ability of gel-immobilized oligonucleotides to

discriminate single nucleotide mismatches.

Influenza

Influenza causes high morbidity and mortality throughout the

world. Its diagnosis based on clinical symptoms is unreliable,

but timely administration of antiviral drugs is considered highly

cost effective and curative. Massive implementation of

affordable molecular diagnostics of influenza will serve many

purposes: improving discrimination of influenza from other

respiratory diseases, facilitating prophylaxis and treatment,

monitoring the spread of specific types and subtypes of the

virus, and helping to prevent pandemic of new highly

pathogenic and contagious strains.

Influenza viruses are divided into groups A, B and C, and

each of them subdivided into several subtypes based on

serological characteristics of two proteins, hemagglutinin (H)

and neuraminidase (N). The C virus is mostly asymptomatic;

B virus usually causes minor illness, but can be dangerous in

the elderly; Aviruses are the most dangerous, and some of the

A subtypes can cause pandemics. High mutability of the

influenza viruses is the major challenge in the design of

diagnostic microarrays.

Microarrays for identification of influenza A recognizing all

16 forms of hemagglutinins and 9 neuraminidases were

designed and successfully tested.(46) These microarrays

(commercial name GreenChips) consist of a relatively high-

density set of 15,000 60-mers and require rather advanced

technology for the manufacture of the chips, preparation of

probes and analysis of the results, so the authors do not

consider their approach ‘‘a stand-alone diagnostic platform’’.

Dawson et al. developed another technology for identifica-

tion of both modern(47) and historical(48) samples of the Avirus

(commercial name MChips). MChips employs low-density

arrays and identify polymorphisms in the so-called M gene of

influenza virus, which strongly correlate with the subtypes of

the agglutinin and neuraminidase genes. MChip targets

primarily clinical applications. Its strength stems from novel

software used for the analysis of sequence databases,

selection of highly informative sets of diagnostic oligonucleo-

tides and sophisticated analysis of the relative intensities of

fluorescence after hybridization of the probes.(49) The MChip

was successfully used for identification of influenza B in both

historical and modern clinical samples(50) and for identification

of subtypes of B virus and subtypes of A virus currently

circulating in humans (A/H3N2 and A/H1N1) and birds (A/

H5N1).(51) When referenced to traditional cultural typing of the

viruses and to typing using RT-PCR, the performance of

MChip equaled or exceeded that of commercial immunoassay

QuickVue (Quidel Corp., San Diego, CA).

Fesenko et al.(52) developed a gel-based biochip, which

detects 15 subtypes of HA and two most significant subtypes of

NA in influenza virus. The microchip was validated using a panel

of 21 reference strains and successfully tested on 41 samples

isolated during the poultry epizootics in Novosibirsk (Russia,

2005). The sensitivity and specificity of the method were 76%

and 100%, respectively, compared with standard techniques of

virus isolation followed by immunoassay. The whole procedure

can be performed in 10 hours—significantly less than conven-

tional cultivation-based identification.

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678 BioEssays 30.7

Polio virus

The exceptional importance of microarrays in the epidemio-

logical studies of poliovirus can be illustrated by the work by

Cherkasova et al.(53) (Fig. 3), in which the divergence of natural

populations and genetic maps resulting from recombination

were rapidly devised. One aspect emphasized in this work is

the sensitivity of microarray-based tests to the presence

of minor sequence variants. These can easily occur in rapidly

diverging viral populations and are not detected by direct

sequencing without additional tedious cloning experiments.

Besides, microarray-based screening is far cheaper and

enables to process more samples.

In another work on poliovirus published by the same

group,(54) microarrays were used to detect spontaneous

mutations in polio vaccine resulting in dangerous new variants.

A comparable work performed by traditional cultural methods

would take 2–3 weeks. What is even more important, any work

with cultures of potentially virulent polioviruses requires

biosafety 3 level of containment, while amplification of viral

RNA and hybridization of the products with microarray do not

present any safety concerns.

Orthopoxviruses

The genus Orthopoxvirus includes many species causing

infections in different vertebrates. The most dangerous of

these viruses, Variola, which causes smallpox, was com-

pletely eradicated in human populations as declared by WHO

in 1980. The immunizations were discontinued and the

majority of today’s world population has no immunity against

the smallpox. Meanwhile, samples of Variola have been

preserved in two specialized collections in USA and former

USSR, currently the Russian Federation. One frightening

possibility is that Variola will be used by bioterrorists. In

addition, there are several species of Orthopoxviruses

circulating in natural reservoirs. Mutations in these viruses

may produce new strains contagious for humans. Thus,

infections with monkeypox virus were recorded in Congo in

Figure 3. Analysis of genetic variations during microevolution

of polioviruses. A: Microarray analysis of viral recombination

(MAVR) assay. Results of hybridization of Sabin oral polio

vaccine (OPV) and vaccine-derived poliovirus (VDPV) strains.

Microarray consisted of genotype-specific oligoprobes for

identification of Sabin oral polio vaccine (OPV) strains. Recent

outbreaks of poliomyelitis in different countries were caused by

VDPV strains that fully regained their virulence and ability to

circulate in human populations. MAVR analysis identified the

genomic recombination schemes of VDPV strains 14, 11264,

15763, 14829, 9457. B: Microarrays for resequencing and

sequence heterogeneity (MARSH) assay. Mutational profiles of

the VP1 coding region of Sabin 1 viral stocks were obtained at

different passage levels. The microarray contained oligonu-

cleotides overlapping at half-length covering the region coding

for VP1, the most-variable capsid protein of poliovirus. Two

microarrays were simultaneously hybridized with fluorescently

labeled RNA samples prepared from the reference Sabin strain

and from a test strain from different passage level. The intensity

of normalized fluorescent signal from each spot in the reference

microarray was divided by the intensity of the respective signal

in the test array. Mutations in the test sample resulted in

decreased binding to some oligoprobes and thus higher ratio.

The peak size varied, reflecting the nature of different mutations

and their number and location within oligoprobes. C: Raw

sequencing data of VP1 genomic regions of Sabin 1 passages

3, 4, 6 and 9. These data confirm the results of MARSH analysis

and reflect gradual accumulation of mutations. (Courtesy of

Professor Konstantin Chumakov, Center for biologics evalua-

tion and research, Food and drug Administration, USA).

What’s new?

BioEssays 30.7 679

1996–2001 and later in the United States, and cowpox

infections were reported in humans in Europe and Brazil (for

references, see Refs 55,56).

Traditional methods of detection and characterization of

Orthopoxviruses are slow and laborous, and cannot be

performed in the field. The advantages of microarray-based

technologies for this application are especially striking (for

more detailed discussion see Ref. 57). Besides Orthopoxvi-

ruses, the microarrays carry specific probes for several strains

of herpes viruses, which during the early phase of infection can

cause a clinical pattern similar to Orthopoxviruses. Successful

detection and identification of these viruses has been achiev-

ed most recently using both traditional two-dimensional

arrays(56,58) and gel-based three-dimensional biochips.(57)

Identification of pathogens in complex mixtures

The need to identify pathogens in complex environmental or

clinical samples arises in the course of counter-terrorism

measures and many other situations. Contrary to traditional

immunological methods, microarrays can carry a large

number of probes directed against many diverse viruses and

bacteria. Identification of bacteria is usually achieved by

hybridization of segments of ribosomal genes, which are

relatively easy to amplify with universal primers, with variable

internal probes. In some cases, when the variability of

the ribosomal genes is insufficient, the sequence of the gyr

gene is used as an additional source of polymorphisms. Other

probes may be placed on the array to detect genes encoding

toxins and antibiotic resistance. The bottleneck in the design of

microarray-based tests for broad spectrum of pathogens is

often the choice of sequences to be amplified and correspond-

ing primers for multiplex PCR.

One application of broad NAT designed to detect viruses is

monitoring of safety of donor blood supplies. Since antibodies

against viruses may not appear in circulation immediately after

the infection, detection of viral nucleic acids is a more-reliable

alternative. Microarrays were developed to detect the most-

important viruses—HIV and hepatitis B and C—in donor

blood, and the sensitivity of the pilot tests was sufficient for

further development of the method.(58) In particular, gel-based

biochips developed by Khodakov et al.(26) allow blood-borne

pathogens to be quantified.

A microarray for the detection of food-born pathogens was

designed and successfully tested by Wang et al.(59) The

authors amplified the variable region of the 16S RNA gene and

hybridized it to oligonucleotides corresponding to unique

sequences in 115 different bacteria; during pilot experiments,

113 of these were identified correctly. The sensitivity of the

array was 102 CFU. The same group developed a microarray

for identification of fungal pathogens carrying specific probes

for 122 species.(60)

One of the most challenging clinical tasks is the correct

diagnosis of respiratory infections. A microarray-based test with

electrochemical enzyme-linked registration was developed by

Lodes et al.(61) The array detects four major bacterial pathogens

and nine viruses. In a similar work, Lin et al. screened more than

400 nasal wash specimens from patients with febrile respiratory

disease.(62) The microarray detection showed better than 98%

agreement with other detection methods with the sensitivity

101–103 genomic copies. The scale of this study enabled

researchers to demonstrate the presence of multiple pathogens

in 13% of samples. Interestingly, the high sensitivity of micro-

array-based detection of pathogens causing generic respiratory

infections calls for a complete consideration of clinical pattern,

since such pathogens are known to reside in healthy individuals,

especially children(63) and elderly.(64)

Conclusion

So far, clinical applications of oligonucleotide microarrays for

detection, identification, characterization and monitoring of

pathogens proved to be more successful than microarrays

intended for diagnosis, prognosis and management of non-

infectious diseases. As far as the growth of commercial

applications of NAT is concerned, it is anticipated to increase

from 8% of all molecular diagnostics in 2005 to 14% in 2010

(the total number include all immunological testing).(65) It is

quite likely that a significant share of this growth will take place

in mass applications of the diagnostics of infectious diseases

displacing or verifying some immunological tests.

Judging by publications, microarray-based diagnostics is

being developed in many countries, but the status of its actual

clinical implementation is mostly unknown. Because of the

dominating size of American diagnostic market and trans-

parency of its regulatory environment, the approval of micro-

arrays for clinical use in the USA is usually immediately made

public. The situation in EU countries is less clear, because

there is no explicit requirement of clinical relevance for new

tests, and information about the rest of the world is almost

impossible to find.

Nevertheless, we believe that microarray technologies for

the detection of pathogens hold great promise. They are

affordable, require small capital investment and are easily

converted into POCT and field applications. Detection and

identification of pathogens usually employ low-density arrays,

which are designed for straightforward answers without the

need for sophisticated equipment and data processing. Such

arrays better satisfy regulatory requirements. Last, but not

least, a significant proportion of humankind is in dire need of

effective diagnostics and treatment of infectious diseases,

and, even with the highest standards of health care, we will

always face the threat of emerging infections.

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