Methods for the diagnosis of blood-borne parasitic infections have stagnated in the last 20–30 years. However, recently, there has been a tremendous effort to focus research on the development of newer diagnostic methods focusing on serological, molecular, and proteomic approaches. This article examines the various diagnostic tools that are being used in clinical laboratories, optimized in reference laboratories and employed in mass screening programmes.

by A. Ricciardi and Dr M. Ndao

Blood-borne protozoans are the causative agents of some of the world’s most devastating and prevalent parasitic infections. This group of pathogens includes members of the Trypanosoma, Leishmania, Plasmodium, Toxoplasma, and Babesia genera. Most of these infections, with the exception of toxoplasmosis and babesiosis, have always been described as being tropical or subtropical. However, the increase in international travel as well as the arrival of new immigrants has made some of these tropical diseases realities in developed countries as well. In addition, infection via contaminated blood (transfusions and organ transplants) has become a major problem. Clearly, the transmission of blood-borne protozoans is boundless and the actual number of cases is underestimated. Quick diagnosis has always been a priority in order to determine the appropriate treatment and prevent fatalities. In addition, now more than ever, advances in diagnostics can help prevent transmission and provide active surveillance. Currently, diagnostic and reference laboratories use an array of techniques including microscopy, serological assays, and molecular assays. Here, the advantages and disadvantages of the methods will be discussed.

Toxoplasmosis
Toxoplasmosis, caused by Toxoplasma gondii, has a worldwide distribution. In immunocompetent individuals, more than 80% of primary Toxoplasma infections are asymptomatic [1]. Toxoplasmosis becomes a problem when an individual is immunocompromised or during pregnancy. Diagnosis of toxoplasmosis varies according to the immune status of the patient.

Diagnosis of immunocompetent individuals relies on serology. Early antibody responses can be detected via methods such as the dye test, immunofluorescent assay, and agglutination test whereas later IgG titres are detected by enzyme-linked immunosorbent assay (ELISA). For many years, the Sabin-Feldman dye test was the gold standard diagnostic technique due to its sensitivity and specificity. In recent years, few laboratories have continued to use this method and rather focused on newer techniques such as indirect immunofluorescent antibody tests, hemagglutination tests, capture ELISAs, and immunosorbent agglutination assays (ISAGAs). Serological assays lack the capacity to differentiate between recent and older infections; IgM levels can persist for over two years [2]. In order to determine whether an infection is recent, avidity ELISA is performed. This assay verifies IgG avidity and is based on the concept that as the immune response progresses, an immunoglobulin’s affinity for a specific antigen will increase [3].

Diagnosis of Toxoplasma infection during pregnancy is crucial in order to prevent congenital toxoplasmosis. Prenatal diagnosis involves performing real-time polymerase chain reaction (PCR) using amniotic fluid. The PCRs used often target the B1 gene of the parasite [1]. Upon delivery, PCR is performed on either the placenta or the cord blood serum in order to detect parasites. ISAGAs are also often performed. If the tests are positive, cord blood samples at one week of life are sent to a reference laboratory [1]. Follow up serology is again performed at one month and then every two to three months. There have been recent advances in the field of toxoplasmosis post-natal diagnosis. An ELISA assay that measures interferon-gamma levels upon stimulation of whole blood cells with Toxoplasma crude antigens has been developed. This method has proven to be both sensitive and specific [4].

In the case of immunocompromised patients, a quick diagnosis is essential because the infection can be fatal. Diagnosis relies on detecting parasites either by PCR or microscopy. Microscopic examination of Giemsa-stained tissues or smears is the quickest and most inexpensive method for diagnosing toxoplasmosis. However, poor sensitivity is the major pitfall of this method. PCR can also be performed on blood or cerebral spinal fluid (CSF) samples in order to detect parasite DNA. However, the degree of sensitivity attained by the PCRs is questionable and requires further investigations [1].

Leishmaniasis
Protozoans of the Leishmania genus are transmitted to humans via sand fly bites. Visceral leishmaniasis (VL), which is a lethal infection if left untreated, can also be transmitted by blood transfusions, organ transplants, and sharing of needles among intravenous drug users.

Direct parasitological methods, such as microscopy and cultures, are the gold standard methods when diagnosing VL. These methods have high specificity, but varying sensitivity. Direct detection of parasites is performed by microscopic examination of aspirates from spleen, bone marrow, or lymph nodes [5]. Using spleen samples increases sensitivity, but the procedure to obtain the aspirates risks internal bleeding. Parasite culturing from aspirates is widely used by reference laboratories.

Extensive research on the development of Leishmania serological assays has uncovered a myriad of candidate diagnostic antigens. The most promising antigens were the kinesin-related proteins. From this group, rK39 was the most tested antigen [6–8]. The rK39 antigen has been used to develop an immunochromatographic strip test (ICT)-based rapid diagnostic test which is advantageous for mass screening in endemic areas. This test requires a drop of peripheral blood and can be completed in approximately fifteen minutes [7]. Although the rK39 ICT rapid test was quite successful in Asia, it was often unable to detect Leishmania infections in African patients [5]. Additionally, rapid diagnostic tests still need standardization in order to become a regular practice in clinical laboratories.

PCR is the main molecular tool for Leishmania diagnosis due to its high sensitivity and reliability. Different PCR target sequences that are commonly used include ribosomal RNA genes, kinetoplast DNA, mini-exon derived RNA, internal transcribed spacer regions, etc., [5]. Quantitative PCR is useful because it allows for the quantification of parasites as well as species typing. Furthermore, this technique can be used to monitor treatment efficiency. Unfortunately, equipment requirements as well as the high cost limit the use of PCR for mass screening purposes in the field. The introduction of loop-mediated isothermal amplification (LAMP) could facilitate the use of molecular techniques for diagnostics. LAMP is highly specific, carried out under isothermal conditions, quick, and requires less complicated equipment (5). Moreover, reagents can be kept at room temperature, and there are no post-PCR steps. Assessment of drug treatment can also be carried out through the use of nucleic acid sequence based amplification (NASBA) which amplifies RNA sequences under isothermal conditions. Coupled to oligochromatography, NASBA can be used to monitor the progression from active disease to cure [9].

Chagas Disease (American Trypanosomiasis)
Chagas disease is the result of an infection with the blood-borne protozoan Trypanosoma cruzi. The parasite is transmitted by the triatomine bug. The second most important mode of transmission is via contaminated blood. This includes blood transfusions, organ transplants, and congenital transmission.
During the acute stage of Chagas disease, parasites can be observed in the blood. For this reason, diagnosis is carried out by direct microscopic viewing of Giemsa-stained thin and thick blood smears [10]. Parasites may also be detected through the use of hemocultures. In Chagas endemic areas, xenodiagnosis may be performed. This method involves allowing the naïve triatomine bug to take a blood-meal from the patient, and then analysing the bug for the presence of trypanosomes. It is believed that with continued research, molecular methods will eventually replace indirect diagnostic techniques such as blood cultures and xenodiagnosis [10]. However, molecular tests need to be standardized for routine clinical practice.
During the chronic stage of Chagas disease, diagnosis relies on serology; however, these tests often yield results that are difficult to interpret [10]. Commonly used, standardized serological assays include indirect immunofluorescence (IIF), indirect hemagglutination (IHA), and ELISA. IIF and IHA are commonly used due to their good sensitivity; however, their results are operator-dependent, and there is a lack of studies which analyse their reproducibility [10]. Currently, the immunoblot and radioimmunoprecipitation assays are in the process of being standardized. Both tests showed promise in early studies. A great deal of work is also being focused on the development and standardization of molecular methods such as PCR, which could be useful in monitoring chronic phase, reactivation, and treatment response.

As previously mentioned, disease transmission can also occur from mother to child, leading to congenital Chagas. Screening of neonates can be performed via direct methods, such as microscopy, or PCR using venous or cord blood samples from the newborn. These tests have very high sensitivity when performed during the first month of life [10]. Serological analysis may also be performed.

Sleeping Sickness (African Trypanosomiasis)
Trypanosoma brucei is the causative agent of African trypanosomiasis, and it is transmitted via the bite of the tsetse fly. During the first stage of the disease, parasites can be found circulating in the peripheral blood. The second stage is marked by parasites crossing the blood-brain barrier and infecting the central nervous system (CNS). The parasitic subspecies dictates geographic distribution, prognosis, and diagnosis.
T. b. gambiense causes West African trypanosomiasis, which is a slow progressing disease and is characterized by low parasite loads [11]. Definite diagnosis is carried out by microscopic observation of blood, lymph node aspirate, or CSF for the presence of parasites. In the field, the card agglutination test for trypanosomiasis (CATT/T. b. gambiense) has been widely used since its development in 1978 (12). Whole blood is used, and the assay directly detects T. b. gambiense specific antibodies. CATT/T. b. gambiense is cheap, quick, and highly sensitive. However, the test can give rise to false positives in individuals who are co-infected with malaria [12]. Although CATT/T. b. gambiense is the most sensitive, similar tests such as micro-CATT and LATEX/ T. b. gambiense can also be used. If these assays generate positive results, they need to be confirmed by microscopy or other molecular methods.

T. b. rhodesiense causes East African trypanosomiasis, which progresses quickly and is characterized by high parasite loads (11). For this subspecies, there is no diagnostic equivalent to the CATT/T. b. gambiense. However, diagnosis by microscopic observations of thick and thin smears is simple due to the elevated parasite load associated with T. b. rhodesiense.

Microscopy is the most practical technique to be used in rural areas. However, microscopy requires adequately qualified personnel in order to prevent misdiagnosis. Molecular methods would substantially improve the diagnosis of African trypanosomiasis. PCR techniques have been developed to screen the CSF of patients. The discovery of the SRA gene in T. b. rhodesiense has proven to be a breakthrough for the promotion of PCR techniques. Reactions targeting this gene have the potential to identify a single trypanosome [11]. There has also been the introduction of fluorescence in-situ hybridization in combination with peptide nucleic acid probes aimed towards ribosomal RNA. However, these tools for diagnosis are new and require further optimization. Extensive research is being focused on standardizing molecular techniques and rendering them more accessible. The use of LAMP is a step forward in improving molecular
approaches [11].

Future research needs to focus on the improvement of molecular diagnostic techniques. Currently, second stage infections are diagnosed by microscopic observation of CSF. Research is being conducted to test various cytokines and antibodies as biomarkers for CNS infection [11].

Malaria
Malaria is the most important parasitic infection in the world due to its high mortality. The causative agents, parasites of the Plasmodium genus, are transmitted by Anopheles mosquitoes. Quick diagnosis is essential in order to determine the appropriate treatment as well as to prevent further transmission.

Microscopy is the gold standard for laboratory diagnosis. This method involves detecting parasites in Giemsa-stained thick and thin blood smears. However, microscopic results are operator-dependent, thereby causing the sensitivity to vary. A great deal of effort has been focused on developing rapid diagnostic tests (RDTs) which can be used in the field. These tests can supplement microscopy, but they cannot replace it yet. Current RDTs are serology based and use three different Plasmodium antigens: Plasmodium histidine-rich protein, Plasmodium lactate dehydrogenase, or Plasmodium aldolase [13]. These tests are quick, easy to perform, and require minimal patient samples. However, they are not specific for species such as P. malariae, P. ovale, and P. knowlesi. Furthermore, false positives may be observed due to cross-reactions in patients with Schistosoma mekongi or rheumatoid factor [14]. In addition, the tests inefficiently detect P. falciparum infections from South America, as this species does not produce the common histidine-rich proteins [15].

Currently, there are no commercially available molecular assays. Although some reference and government laboratories have developed their own molecular assays, their availability is limited. LAMP is currently in the spotlight. Poon et al. developed a LAMP test which detected the target sequence of P. falciparum 18S ribosomal RNA gene [16]. They stated that the price of this test was one tenth that of a conventional PCR. Recently, LAMP was further simplified in the form of a card test. It was used in combination with DNA filter paper and melting curve analysis. This system was shown to be highly specific and sensitive [17]. Improvement of the LAMP technique should be geared towards the development of rapid diagnostic tests which could potentially be used in the field.

Babesiosis
Babesiosis is caused by parasites belonging to the Babesia genus that are spread by certain ticks commonly found in North America. The parasites infect red blood cells (RBCs), and consequently cause hemolytic anemia. The disease can be fatal in splenectomy patients, immunocompromised individuals, and the elderly. Diagnosis is complicated by the symptoms’ resemblance to other tick-borne illnesses.

The gold standard of babesiosis diagnosis relies on detecting the parasites in the patients’ RBCs. This is achieved by microscopic observation of thick and thin blood smears. Babesia infections can be easily mistaken for P. falciparum infections [18]. Additionally, false negatives are common in immunocompetent individuals whose parasitemia can be lower than 1% [18]. Samples are often sent to reference laboratories in order to confirm ambiguous results. IFFs are used to detect anti-babesial IgM and IgG [18]. They are sensitive, specific, and reliable. ELISAs and immunoblots, although not standardized, can be performed to confirm the IFF results. However, compared to IFFs, Babesia detecting ELISAs require higher concentrations of antigen and have varying sensitivity [18]. Future research on babesiosis diagnosis is aimed at developing multiplex PCR assays that will be able to detect several tick-borne infections. PCR assays have the potential to yield positive results from 100µl blood samples containing as little as three parasites; demonstrating the incredible advantage that molecular techniques could contribute to diagnosis of this parasitic disease [18].

Proteomics
Dr Momar Ndao’s laboratory focuses on the improvement and advancement of diagnosis. Through our work, we hope to encourage the development of proteomic strategies for the diagnosis of parasitic infections. Mass-spectrometry platforms are the future of proteomics, and they can be used to identify biomarkers from biological fluids. Some techniques that can be used to analyse protein expression include matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MALDI-TOF MS), surface-enhanced laser desorption ionization time-of-flight mass-spectrometry (SELDI-TOF MS), liquid chromatography combined with mass-spectrometry, isotope-coded affinity tags, and isobaric tags for relative and absolute quantification [19]. When SELDI is used, samples are directly spotted onto chemically active ProteinChip Array surfaces which can be chosen based on specific chemical and biological properties. With MALDI, samples are mixed with the matrix component prior to loading on a chip. These proteomic platforms can be useful in identifying biomarkers that are indicative of a specific pathophysiological state. Currently, members of our laboratory are using both SELDI and MALDI techniques extensively to identify biomarkers of blood borne parasites.

Summary
Quick and correct diagnosis of parasitic infections is crucial to avoid deaths and further disease transmission. Diagnostic methods include parasitological techniques, such as microscopy and culturing, serological assays, and molecular tests [Table 1]. Although several serological and molecular diagnostic tools are being tested and used by certain reference laboratories, results are always confirmed by microscopy which remains the gold standard. Many newer assays have not been standardized yet, thus, forcing diagnosticians to rely on microscopic observations. Unfortunately, the evolution of diagnosis in the field of parasitology has been slow to progress. Fortunately, in recent years, several groups have focused their research on the improvement of diagnostics. Current research emphasizes the development and optimization of molecular techniques such as PCR and LAMP. Additional work must concentrate on rendering molecular diagnostics more accessible. Although relatively new at the moment, proteomic platforms seem to be the future of diagnosis. These new techniques can identify biomarkers which can categorize susceptible individuals, distinguish between the different stages of an infection, and monitor whether treatments lead to cure. Diagnostic research has made much progression, however, there is still a lot of work to be done and improvements to be made. In order to better the diagnosis of blood-borne parasitic infections, research plus communication is the answer.

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The authors
Alessandra Ricciardi, BSc
National Reference Centre for Parasitology, Research Institute of the McGill University Health Center, Montreal, Canada

Momar Ndao, DVM, MSc, PhD
National Reference Centre for Parasitology at the Montreal General Hospital, Montreal, Quebec, Canada
E-mail: momar.ndao@mcgill.ca

Noroviruses are the most common cause of viral gastroenteritis in humans. In recent years diagnostic methods for Noroviruses, especially real-time reverse transcription-polymerase chain reaction (RT-PCR) for the detection of Norovirus-RNA, have been improved and become more widely available.

by Dr Christoph Metzger-Boddien

Noroviruses are transmitted by fecally contaminated food or water, by person-to-person contact, and via aerosolization of the virus and subsequent contamination of surfaces. They are the most common cause of viral gastroenteritis in humans [15]. Symptoms include nausea, vomiting, diarrhea, and stomach cramping. Additional symptoms are fever, chills, headache, muscle aches and a general sense of tiredness. The onset of symptoms can begin quickly and an infected person may feel sick after a very short period of time. In most people, the illness lasts for about one or two days. People with Norovirus illness are contagious from onset of symptoms until at least three days after recovery. Some people may be contagious for even longer. Noroviruses are highly contagious. The estimated dose is as low as 18 viral particles. Approximately 5 billion infectious doses can be present in each gram of feces during peak shedding [16]. Infection can be more severe in young children and elderly people. Dehydration can occur rapidly and may require medical treatment or hospitalization [10].

Sporadic disease
In recent years diagnostic methods for Noroviruses, especially real-time reverse transcription-polymerase chain reaction (RT-PCR) for the detection of Norovirus-RNA, were improved and became more widely available. Subsequently, it became obvious that Noroviruses are the leading cause of sporadic gastroenteritis in all age groups. In Germany, since the implementation of the notification requirement according to §§6 and 7 of the infection protection act (Infektionsschutzgesetz, IfSG) a rise of reported cases can be observed with a seasonal accumulation during the winter months from October to March (2001: 9,223 cases, 2004: 64,973 cases, 2007: 201,242 and 2008: 212,769 cases, source: Robert-Koch-Institute, RKI, Berlin), but still a high estimated number of unreported cases remain.

Outbreaks
Noroviruses are the predominant cause of gastroenteritis outbreaks worldwide. Data from the United States and European countries show that Norovirus is responsible for approximately 50% of all reported gastroenteritis outbreaks (range: 36%–59%) [12]. Periodic increases in Norovirus outbreaks are associated with the emergence of new GII.4 strains. These emergent GII.4 strains are rapidly replacing existing strains predominating in circulation and sometimes cause seasons with high Norovirus activity, as in 2002–2003 and 2006—2007 [17, 20]. Genetic drift successfully promotes the re-emergence of GII-4 variants in the population [13]. Because the virus can be transmitted by food, water and contaminated environmental surfaces as well as directly from person to person, and because there is no long-lasting immunity to Noroviruses, outbreaks can occur in a variety of institutional settings (e.g. nursing homes, hospitals, and schools) and affect people of all ages. Multiple routes of transmission can occur within an outbreak; for example, point-source outbreaks from a food exposure often result in secondary person-to-person spread within an institution or community [4]. Of the 1,518 Norovirus outbreaks in the USA, during 2010 – 2011, laboratory confirmed by the CDC, 59% were from long-term care facilities (889 outbreaks); 8% were from restaurants (123 Outbreaks); 7% were from parties & events 7% (99 outbreaks); 4% were from hospitals (65 outbreaks); 4% were from schools (64 outbreaks); 4% were from cruise ships (55 outbreaks); and 14% were from other and unknown events (223 outbreaks) [10].

Foods that are commonly involved in outbreaks of Norovirus infection are e.g. leafy greens, fresh fruits, and shellfish. However, any food that is served raw or is being handled after cooking can get contaminated.

In Germany, according to data published by the RKI, the number of Norovirus outbreaks has increased by 20% between 2009 and 2010. Recently, the RKI published the final report of a huge outbreak of acute gastroenteritis in five Eastern German federal states. The source of the outbreak was a batch of deep-frozen strawberries. In total, over 11,000 cases of disease occurred. It was Germany’s largest foodborne outbreak of gastroenteritis, with several hundred institutions affected. In a considerable proportion of tested patients, Noroviruses were found [4].

Analysis of outbreak costs
In fact there is a huge socio-economic impact of Norovirus-associated diseases. A study of Johnston et al. 2007, showed the costs of an outbreak including the estimated loss of revenue because of unit closures, sick leave and cleaning expenses [7]. Because of the high contagiousness of Noroviruses early diagnosis in order to set up appropriate hygiene interventions is the most useful measure. In 2004, Lopman et al. showed, that diagnosis of the first case within three days instead of four reduces the duration of an outbreak by seven days [5, 8].

Diagnostic methods
The clinical specimens used for Norovirus diagnosis in most cases are stool and vomit samples. There is no cell culture method for the isolation of Noroviruses from clinical specimens available. Therefore, the majority of clinical virology laboratories perform RT-PCR assays for Norovirus detection. Additionally, for preliminary identification of Norovirus as the cause of gastroenteritis outbreaks, there are enzyme immunoassays (EIA) and rapid tests available. However, these kits are not recommended for individual diagnosis.

Real-time RT-PCR assays
The region between ORF1-ORF2 is the most conserved region of the Norovirus genome, with a high level of nucleotide sequence identity across strains within a genogroup [6]. This region is ideal for designing broadly reactive primers and probes for real-time RT-PCR (RT-qPCR) assays for high throughput screening in clinical diagnostic laboratories and for the detection of Norovirus RNA in
environmental samples (e.g. food and water).

The quality of the real time RT-PCR results is dependent on the quality of template RNA-extraction from clinical and environmental samples. The implementation of extraction controls in commercial RT-PCR duplex assays (e.g. Control-RNA in MutaREX Norovirus Kit, Immundiagnostik AG, Bensheim, Germany) minimizes the risk of false negative results due to inhibition or partial inhibition of the reverse transcription step and/or the PCR and due to processing errors during the extraction of RNA. Control RNA is added to a sample before RNA extraction with a commercial kit (e.g. High pure viral RNA Kit, Roche Diagnostics GmbH, Mannheim, Germany;  or intron viral gene spin, gerbion, Kornwestheim, Germany) and its recovery is measured subsequently in the duplex real time RT-PCR. The latest generation of commercially available Norovirus real time RT-PCR Kits is extremely sensitive and specific [18]. Therefore such tests have become the gold standard for Norovirus laboratory diagnosis in the past few years.

Enzyme immunoassays
For detection of Norovirus antigen in clinical samples, rapid assays (e.g. EIA) offer an alternative to real time RT-PCR assays. However, the development of a broadly reactive EIA for Noroviruses has been challenging because of the number of antigenically distinct Norovirus strains and the high viral load required for a positive signal in these assays. Commercial kits include pools of cross-reactive monoclonal and polyclonal antibodies. In evaluation studies, the sensitivity of these kits ranged from 36% to 80%, and specificity has ranged from 47% to 100% compared with real time RT-PCR [1, 2, 3, 9, 11, 14, 19].

Summary
Norovirus real time RT-PCR Kits offer a sensitive, specific, fast and cost effective diagnosis. Results can be generated within one hour. But clearly only real time RT-PCR Kits containing control RNA used as extraction control for process monitoring produce feasible and reliable results. RNA extraction from clinical specimens and the reverse transcription of RNA to cDNA are the most crucial steps in Norovirus RT-PCR procedures. Errors in sample preparation and/or RT-reaction can lead to false negative results in conventional RT-PCRs as well as real time RT-PCRs when internal controls (RNA or DNA) are already added to the PCR master-mix. Laboratories performing in-house RT-PCR for Noroviruses should critically evaluate their tests with regard to these high quality standards. Because of the modest performance of Norovirus Enzyme Immunoassays, particularly their poor sensitivity, they are not recommended for clinical diagnosis of Norovirus infection in sporadic cases of gastroenteritis. Negative samples will have to be confirmed by real time RT-PCR in outbreaks as well as in sporadic cases.

References
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4. Großer Gastroenteritis-Ausbruch durch eine Charge mit Noroviren kontaminierter Tiefkühlerdbeeren in Betreuungseinrichtungen und Schulen in Ostdeutschland, 09-10/2012. Epidemiologisches Bulletin Nr. 41/12: 414-417, Oct 15th, 2012
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The author
Christoph Metzger-Boddien, PhD
gerbion GmbH & Co. KG
Remsstr. 1, D-70806 Kornwestheim, Germany

FilmArray is a highly automated small instrument capable of detecting infectious agents using PCR technology. Due to its simplicity the tests could be performed in a rapid-response core laboratory by general medical technologists. This operational model has demonstrated achievements in reducing turn-around-time and thus improved patient care.

by Dr M. Xu, Dr X. Qin, Dr M. L. Astion and Dr J. C. Rutledge

Acute respiratory infection and the importance of early diagnosis
Acute respiratory infection is one of the major causes of outpatient visits and hospitalization in young children and older patients with chronic respiratory diseases. Most acute respiratory infections are caused by viral agents, whereas bacterial infections occur much less frequently. Occasionally, patients with viral infection, but without a definitive diagnosis, are given antibiotics unnecessarily. Viral respiratory infection in immunocompromised patients has significant morbidity and mortality implications, and early initiation of appropriate antiviral therapy can be life-saving. In addition, isolation of patients with viral respiratory infection plays a critical role in infection prevention. Therefore, laboratory tests providing accurate and timely determination of the infectious agents associated with respiratory diseases are crucial in clinical practice.

Methods of diagnosis of acute respiratory infection
Many diagnostic tests for respiratory viral infection are available. Point-of-care tests for detecting viral antigens have the shortest turn-around-time, usually just a few minutes. These rapid antigen tests are available for only a limited number of viruses such as influenza A (Flu A), influenza B (Flu B) and respiratory syncytial virus (RSV), though the sensitivity of rapid antigen tests is low ranging from 20–80%, with a generally acceptable specificity if the tests are used during the respiratory virus season [1]. Direct fluorescence assay (DFA) has higher sensitivity (~80%) than rapid antigen tests and reasonable turn-around-time (TAT) of a few hours [1]. However, these are  complex assays requiring specialized and experienced technologists.  Viral culture has long been considered as the gold standard for detection of respiratory viral infection with the shortcoming of requiring days for the definitive identification of viral etiology.

In the past few years, several molecular tests have been developed to detect viral RNA or DNA using the polymerase chain reaction (PCR) method. One study compared the rapid antigen test, DFA, and viral culture with RT-PCR in the detection of influenza A H1N1 2009, and found sensitivities of only 18%, 39% and 46% respectively [2]. The specificity of all methods is not significantly lower than that of realtime PCR, which is over 90%. The authors recommended that all DFA negative results should be tested with realtime PCR. Although most molecular tests using PCR technology show high sensitivity and specificity, they are technically complex, time consuming, and require specialized medical technologists to perform the tests. This type of molecular assays is usually only available in large reference laboratories or medical centres with specialized microbiology, virology, or molecular laboratories.  These specialized laboratories usually do not operate during evening and night shifts and perform these tests in batches, and, therefore, the TAT for most molecular testing is relatively long, ranging from 6 to 24 hours.

Emerging new technology
FilmArray (BioFire, previously named Idaho Technologies; Salt Lake City, UT) is a newly developed small desk-top single-specimen-flow instrument with fully automated process for detection of respiratory infectious agents by real time PCR technology [3].  The respiratory panel performed on FilmArray is able to detect 17 viral agents including adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, human metapneumovirus, rhinovirus/enterovirus, Flu A, Flu A H1, Flu A H1 2009, Flu A H3, Flu B, parainfluenza 1, 2, 3, 4, and RSV, plus Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae from respiratory specimens. The test requires only 5 minutes hands-on time of a technologist and 65 minutes total of analyser time. The testing pouch contains all the reagents for nucleic acid extraction, reverse transcription, and two steps of PCR amplification. The built-in software automatically analyses the specific melting curves of the PCR products and reports the results as positive or negative for specific infectious agents. General medical technologists with proper training are able to perform the test without any difficulties. Several comparative studies between FilmArray and other molecular tests for respiratory viral agents have shown comparable results for the detection of respiratory infectious agents [4–6].

Impact on TAT and patient care
Our rapid response core laboratory (Core Lab) is staffed by approximately 35 full-time employees (FTEs). It provides tests of general chemistry, hematology, coagulation, urinalysis, blood gas, limited therapeutic drug monitoring, and a few rapid manual tests such as monospot, pregnancy test, and sickle screen. Our Core Lab also went through a major process improvement using the Toyota production system to streamline the testing workflow [7], and  testing was designed based on a lean, single-piece flow principle without batching [7]. Using these principles we eliminated STAT testing. All the tests performed in core lab are standardized to meet a TAT of 1 hour, where TAT is defined as the time from sample receipt in the laboratory to the time the result is verified in laboratory information system. To provide 24-hour per day, 7-day per week (24/7) service to our emergency department (ED) and urgent care centre, we implemented the FilmArray respiratory panel in the Core Lab [8]. Prior to implementing the FilmArray testing, we sent our respiratory samples to a regional reference laboratory performing viral testing using the DFA method. The regional reference laboratory had an on-site facility for performing DFA testing. During the first 4 months of testing using FilmArray, we tested twice as many samples as the same time period the previous year. The average TAT was reduced from 7 hours the previous year using DFA, to 1.6 hours using FilmArray. With FilmArray, 82% of the tests were completed within 2 hours, and 95% were completed within 3 hours. Previously, with DFA, none of the tests were completed within 2 hours and only 2% of time the tests were completed within 3 hours. In addition, FilmArray detected 17 viral agents, whereas DFA detected only 8. The additional viral agents detected by FilmArray include 4 types of corona virus, 3 additional types of Flu A, parainfluenza 4, and rhinovirus/enterovirus. Although no specific treatments exist for some of the above viral agents, such as corona viruses, parainfluenza virus and rhinovirus, detection of them allowed physicians to make a specific diagnosis, which gave patients reassurance and prevented further costly diagnostic work-up and unnecessary use of antibiotics.

After implementing the FilmArray respiratory panel, we also looked at the effect of shortened TAT on patients admitted to the ED. The current guidelines for treating patients of positive Flu A and Flu B with oseltamivir recommend administering the medication within 48 hours of onset of symptoms. We found that due to the fast TAT of respiratory viral testing, more than 80% of patients admitted to the ED were given the medication or prescription in the ED or within 3 hours of discharge from the ED. This practice would have been impossible previously with DFA testing at the reference lab, which had an average of 7 hours of test TAT.

Finally, the additional clinical benefit of early detection of the infectious agents is the ability to cohort the patients effectively for appropriate isolation. As part of our hospital infection prevention policy, admission of patients with respiratory symptoms is subject to FilmArray respiratory viral screening at no charge. Clearly, the early and appropriate isolation of patients with respiratory symptoms has potential positive impact on infection prevention and overall cost savings for both patients and hospitals. One such example concerns two patients with respiratory symptoms who were scheduled for surgery. The respiratory viral testing results were negative for influenza virus for both patients, and this eliminated the need for the strict isolation procedures, such as wearing masks for staff and using negative pressure for the operating room, that would have had to have been used in the absence of test results.

Financial consideration
Although the price of FilmArray respiratory viral panel is slightly higher than that of other conventional PCR methods, the labour saving due to its simplicity is substantial and offsets the supply costs. In addition, the sample requirement for FilmArray test is a nasal swab rather than a nasal wash, which was the sample of choice for the DFA respiratory viral assay. It is much easier for nursing staff to collect a nasal swab than a nasal wash. In addition, the nasal wash creates an aerosol that mandates room cleaning and 30-minute room closure before the next use. The cost saving for a busy ED room time is difficult to calculate but is significant. One report examined the financial consequence of reducing ED boarding (the length of time a patient stays in the ED) and found that a 1-hour reduction in ED boarding time would have resulted in $9693 (~£6058) to $13,298 (~£8311) of additional daily revenue [9].

Future trends
The simplicity of the FilmArray assay gives it the potential to expand in small general laboratories. Currently, BioFire Diagnostics Inc. is developing gastrointestinal, blood culture ID, and sepsis panels using FilmArray technology. The current major drawback of FilmArray is its restriction to single-sample throughput. The further improvement to provide higher throughput will expand its utility in high-volume clinical laboratories.

In summary, due to its simplicity and clinical utility, the FilmArray is the first multiplex molecular test that has entered the general clinical laboratory, rather than a specialized laboratory. This marks a new era in laboratory medicine. FilmArray significantly improves the diagnosis and care of patients with respiratory infections. Overall, new and emerging technologies like FilmArray will allow more infectious agents to be detected earlier and more accurately by instruments situated in general core laboratories rather than in specialized laboratories, thereby speeding results from a 7/24 operations.

References
1. Takahashi H, Otsuka Y, Patterson BK. Diagnostic tests for influenza and other respiratory viruses: determining performance specifications based on clinical setting. J Infect Chemother 2010; 16: 155–61.
2. Ganzenmueller T, Kluba J, Hilfrich B et al. Comparison of the performance of direct fluorescent antibody staining, a point-of-care rapid antigen test and virus isolation with that of RT-PCR for the detection of novel 2009 influenza A (H1N1) virus in respiratory specimens. J Med Microbiol 2010; 59: 713–7.
3. Poritz MA, Blaschke AJ, Byington CL et al. FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection. PLoS One 2011; 6: e26047
4. Loeffelholz MJ, Pong DL, Pyles RB et al. Comparison of the FilmArray Respiratory Panel and Prodesse real-time PCR assays for detection of respiratory pathogens. J Clin Microbiol 2011; 49: 4083–8.
5. Rand KH, Rampersaud H, Houck HJ. Comparison of two multiplex methods for detection of respiratory viruses: FilmArray RP and xTAG RVP. J Clin Microbiol 2011; 49: 2449–53.
6. Pierce VM, Elkan M, Leet M et al. Comparison of the Idaho Technology FilmArray system to real-time PCR for detection of respiratory pathogens in children. J Clin Microbiol 2012; 50: 364–71.
7. Rutledge J, Xu M, Simpson J. Application of the Toyota Production System improves core laboratory operations. Am J Clin Pathol 2010; 133: 24–31.
8. Xu M, Qin X, Astion ML et al. Implementation of FilmArray respiratory viral panel in a core laboratory improves testing turn-around-time and patient care. Am J Clin Pathol Jan. 2013, In press.
9. Pines JM, Batt RJ, Hilton JA, et al. The financial consequences of lost demand and reducing boarding in hospital emergency departments. Ann Em Med 2011; 58: 331–40.

The authors
Min Xu, MD, PhD
Xuan Qin, PhD
Michael L. Astion, MD, PhD
Joe C. Rutledge, MD
Department of Laboratories, Seattle Children’s Hospital,
4800 Sand Point Way NE, A6901
Seattle, WA 98105, USA
E-mail: min.xu@seattlechildrens.org