Blood doping benefits endurance athletes (notoriously, but not only, cyclists) by raising the red blood cell (rbc) count or haematocrit, and so increasing the oxygen supply to the muscles. It is one of the most difficult types of drug abuse to detect. Awareness of blood doping was raised in the popular press recently when comments were made about the impressive nature of China’s Ye Shiwen’s Olympic gold medal wins and with Lance Armstrong’s (cycling’s famous winner of seven Tours de France after surviving advanced testicular cancer) sudden decision to drop his fight against the US Anti-Doping Agency’s drug charges. Hematocrit levels can be raised by a variety of methods ranging from legal altitude training, to the banned use of autologous blood transfusions and erythropoietin (EPO) injections.
Detection of these banned methods is extremely difficult and the fight against them is being waged in a number of ways. The UCI’s (cycling’s governing body) lines of defence include simply demonstrating possession of banned substances and monitoring hematocrit levels, with a limit set at 50% (normal being 41–50% for men).
Some early success was had with testing urine to distinguish pharmaceutical EPO from the nearly identical natural hormone by isolectric focusing, though its accuracy has been questioned with claims that it is not possible to distinguish pharmaceutical EPO from other unrelated proteins that are present in urine after strenuous exercise or as the result of sample degradation and bacterial contamination.
At present, tests that provide indirect evidence of autologous blood transfusion (where the athlete withdraws and then re-injects his own blood) are under development and involve looking at the ratio of immature to mature red blood cells and might also include the measurement of 2,3-bisphophoglycerate (2,3-BPG). As 2,3-BPG degrades over time, stored blood used for autologous transfusions would have less than fresh blood and so levels of 2,3-BPG lower than normal may then indicate blood doping by this method. The presence of plasticizers in the blood (from the IV bags in which blood is stored) has also been used as evidence of blood doping.
While these advances in the detection of blood doping are being made it is tempting to think that we have got there, that the cheats will be caught. However, in the high-stakes world of elite athletes this would be a naive hope: the possibility of athletes subjecting themselves to EPO gene therapy – so called gene doping – has been suggested and methods for the detection of transgenic DNA following in vivo gene transfer are already being developed.

Recent advances in the understanding of the compositions and structures of allergens now make it possible to use allergenic components instead of allergenic extracts in allergy testing.
In this interview, Jean-Charles Clouet, Director of Assay Business Development & Scientific Marketing at Siemens Healthcare Diagnostics, discusses the role of these molecular allergens in allergy diagnosis.

Q. What is the prevalence of allergies, and are there any geographical disparities?
Allergic diseases and asthma represent a growing and major healthcare challenge worldwide, as reported by the recent World Allergy Organization (WAO) White Book. [1] The authors confirm the steady increase of allergic diseases during the last decades that now affect approximately 30–40% of the industrialized world’s population, with an especially high percentage among the youngest subjects (40–50% of school-aged children are sensitized to one or more common allergens).

For example, allergic rhinitis is one of the most common allergic conditions, impacting roughly 500 million people of all social classes and ages globally. [2] In Europe, a study showed allergic rhinitis prevalence at approximately 25%. [3,4] It is important to note that, although direct costs induced by allergic rhinitis are limited, the condition affects subjects’ quality of life and has significant impact on performance at work or school. Therefore, its overall economic impact is probably underestimated. [2]

Another recent study, this one surveying more than 38,000 children (up to 18 years old) in the United States, reported that 8% had food allergy, including a rate of about 6% for those aged 0-2 years, and more than 8.5% for those aged 14-18 years. [5] Worldwide, it is estimated that 220–520 million people may suffer from food allergy. [1]

These statistics illustrate the high prevalence of allergy worldwide and why the World Health Organization (WHO) ranks allergy as the fourth most common global chronic disease. [6]

Q. What are the most common clinical manifestations of allergy that demand further testing?
Subjects suffering from allergic diseases tend to develop IgE-mediated immune reactions to normally harmless substances called allergens. These can include tree pollens, grasses and weeds; foods; mites; animal danders; molds; insects; and drugs. Associated clinical manifestations range from mild to severe and affect the upper and lower airways, gastrointestinal tract and skin. The consequent allergic diseases may include rhinitis, asthma, allergic conjunctivitis, atopic eczema, food allergy, insect allergy, drug allergy and anaphylaxis. Some can even be fatal, in the cases of allergic reactions to certain foods, insect venoms or drugs.

The model for the “Allergy March” published in the late 1990s emphasized that the most common forms of allergic diseases in early infancy are gastrointestinal symptoms and skin conditions (e.g., atopic dermatitis) caused by food proteins, such as hen’s egg and cow’s milk. [7] Additionally, IgE reactivity to food allergens in early infancy is a strong predictor for reactivity to respiratory allergens later in childhood. Other forms, such as allergic rhinitis and reactions to aeroallergens, happen later in life (1–10 years).

In 2003, the European Academy Of Allergy and Clinical Immunology (EAACI) published a position paper on allergy in children recommending testing for all subjects with severe, persistent or recurrent “allergic symptoms” (irrespective of age), along with those requiring a prophylactic treatment,. Proposals to select relevant allergens based on the subject’s age were provided. [8] Additional position papers are available for other forms of allergic reactions, such as drug allergy (causing 20% of deaths due to anaphylaxis) or insect allergy (fatal reactions in up to 50% of individuals with no documented history of reaction). [3]

Q. What are the current testing methods?
The objectives of allergy diagnosis are to identify both the symptoms’ origin (i.e., is the reaction IgE-mediated?) and the offending allergen(s). Allergy diagnosis is multi-factorial and includes a detailed case history and in vivo (i.e., skin tests) and/or in vitro (i.e., allergen-specific IgE measurements) testing. For some allergens (e.g., foods), oral challenges may also be performed to support diagnosis of food allergy.

Skin tests and blood tests, performed by allergists and laboratories respectively, present their own advantages and limitations. Skin tests are highly sensitive, with results immediately available for the patient. However, patients must discontinue medications (e.g., antihistamine) prior to testing, and interpretations of skin-test results are highly subjective and depend largely on operator skills. In vitro tests have the advantage of providing precise, quantitative results for each allergen, validated through extensive internal and external quality-control procedures and programs.

It is important to note that “allergens” used for in vivo and in vitro testing procedures are still primarily allergenic extracts. Obtained by extraction of proteins from crude allergenic sources, these extracts consist of a mixture of known and unknown proteins. Due to molecular-biology techniques and research begun in the 1980s, it is now possible to better understand compositions and structures of allergens, to classify them into families of proteins and to obtain for a significant number of them more qualified and standardized materials called “molecular allergens” or “allergenic components.” Whether highly purified in native form from the allergenic source or produced via recombinant protein expression techniques, molecular allergens have ushered in a new era in allergy diagnosis.

Q. What is the role of molecular allergens in allergy testing?
Allergenic extracts allow the detection of specific IgE directed against an allergenic source. In contrast, molecular allergens permit detection of precisely specific IgE directed against the disease-eliciting component(s) of the allergenic source. Therefore, measurements of specific IgE against molecular allergens yield additional key information that cannot be obtained by testing allergenic extracts. In particular, use of molecular allergens can help allergists define a more personalized and relevant sensitization profile for each patient.

For example, testing with molecular allergens makes it possible to determine if a patient’s sensitization is genuine (i.e., specific to one allergenic source) or comes from a cross-reactivity to proteins that have similar structures and are present in different sources. This is an important consideration when assessing a patient’s risk of reaction to some allergic sources and recommending appropriate avoidance measures. Allergenic molecules can also help clinicians assess the severity of a patient’s allergic reaction and, in the case of food allergy, decide whether or not to perform an oral food challenge. Finally, allergenic molecules can help clinicians identify patients who will benefit from immunotherapy treatment and decide which allergens should be used for treatment.

Q. Is this new technology likely to shift the burden of allergy testing towards the lab and away from in vivo (i.e., skin-based) testing?
In vitro testing is an accurate complement or alternative to skin testing for most allergens. However, despite their growing number, molecular allergens are not yet available for all types of allergens. Using allergenic extracts in conjunction with in vitro and/or skin testing is still the only option for a large number of
allergenic sources.

Q. In your view, what is the ideal sequence of tests to optimize the early diagnosis and treatment of allergy?
First of all, it’s important to raise awareness among the public and in the physician community of the importance of early diagnosis to better prevent and treat allergic diseases. Equally important is educating both clinicians and the public on the availability of new diagnostic tools, such as molecular allergens, along with the best way to leverage them to improve patient care.

An “ideal sequence” should begin with a careful and detailed case history taken by an allergist. This is critical in deciding whether further testing is necessary. Also, the results of specific IgE measurements should always be analysed in conjunction with the patient’s clinical history, since an allergen sensitization does not necessarily imply a clinical responsiveness.

If the clinical history suggests an allergic reaction, the clinician generally will request detection of specific IgE against the suspected allergens. Testing will then be performed using allergenic extracts in most cases. The reason is that allergen extracts are complex heterogeneous mixtures made of major and minor allergenic determinants. In some instances, a few molecular allergens will be enough to replace the corresponding allergenic extract. Unfortunately, this is not the case for sources with more complex compositions and in situations in which as-yet unidentified determinants (even minor ones) could be of clinical importance for particular patients. Also, as already mentioned, allergenic components have not yet been developed for a large number of allergenic extracts.

Therefore, in the majority of cases, testing allergenic extracts as a first step is the best or perhaps the only option to detect all sensitized patients. If specific IgE against the allergenic extract are found, relevant molecular allergens should then be tested to provide more information, as explained above.

References
1. World Health Organization. White Book on Allergy 2011-2012 Executive Summary. By Prof. Ruby Pawankar, MD, PhD, Prof. Giorgio Walkter Canonica, MD, Prof. Stephen T. Holgate, BSc, MD, DSc, FMed Sci, and Prof. Richard F. Lockey, MD.
2. Bousquet J, et al. Allergic rhinitis and its impact on asthma (ARIA) 2008 update (in collaboration with the World Health Organization, GA(2)LEN and AllerGen). Allergy. 2008;63 Suppl 86:8-160.
3. Bauchau V, Durham SR. Prevalence and rate of diagnosis of allergic rhinitis in Europe. Eur Respir J. 2004;24:758–764.
4. Bauchau V, Durham SR. Epidemiological characterization of the intermittent and persistent types of allergic rhinitis. Allergy. 2005;60:350–353.
5. Gupta, R, et al. The prevalence, severity and distribution of childhood food allergy in the United States. Pediatrics. 2011;10.1542/ped.2011-0204.
6.I nternational classification of diseases (ICD). http://www.who.int/classifications/icd/en/.
7. Kulig M, Bergmann R, Klettke U, Wahn V, Tacke U, Wahn U. Natural course of sensitization to food and inhalant allergens during the first 6 years of life. J Allergy Clin Immunol. 1999;103:1173–1179.
8. Host, et al. Allergy testing in children: why, who, when and how? Allergy. 2003:58:1-11.

Malaria is an acute and life threatening infection in individuls with no previous immunity. Symptoms are nonspecific and cannot be distinguished from those of influenza or severe bacterial infections. All febrile patients should thus be asked if they have been travelling over the past six months and if so whether the journey was to a malaria endemic area.
Microscopic examination of Giemsa stained thick and thin blood films remains the gold standard, but rapid tests using antigen-capture assays are increasingly used where access to expert microscopy is not available. The appropriate use of rapid tests and their limits are discussed.

by Dr Eskild Petersen

Malaria is caused by a protozoan parasite and five species can infect humans: Plasmodium falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. Humans are infected by bites of Anopheles mosquitoes and humans are the reservoir hosts except in the case of P. knowlesi, which is transmitted from monkeys and is only seen in South East Asia. Infection with P. falciparum shows the highest mortality and drug resistance is much more common in P. falciparum compared to P. vivax, and not a problem in the other malaria species. P. ovale and P. vivax have persistent liver forms, hypnozoites, which may reactivate, usually within six months after infection, and give rise to a malaria attack.

Malaria in Europe
Malaria was endemic in Europe up to the middle of the 20th century [Table 1]. Presently malaria is almost exclusively imported, although a number of Plasmodium vivax cases were seen in Greece in 2011, probably introduced with migrant workers from endemic areas [1]. It is estimated that between 10,000 and 15,000 cases of malaria are imported into Europe every year, which makes the recognition of symptoms and knowledge of appropriate diagnosis important.

A special risk group is immigrants resident in Europe who visit their home countries where malaria is found. The proportion of imported malaria cases in immigrants in Europe has increased from a reported 14% more than 10 years ago to 86% in more recent studies [2]. More than five million African immigrants could be living in Europe, one third of whom are from Sub-Saharan Africa [3], and children of immigrants are particularly at risk [4].

Mortality of imported malaria
The mortality from imported Plasmodium falciparum malaria cases varies from 0.4% in a large cohort from France [5], up to 5% in a recent cluster of cases imported from The Gambia [6]. Malaria infection in non-immunes is an emergency which requires prompt diagnosis and treatment while asymptomatic malaria in immigrants raises other public health issues.

Clinical symptoms
Individuals without immunity, i.e. persons who have not lived in malaria endemic countries for a long time, normally have a febrile illness with an acute onset. The symptoms include fever, malaise, muscle and joint pains, headache and rarely respiratory distress and diarrhoea. Malaria infection can be complicated by bacterial septicaemia. As the infection progresses there can be drowsiness, coma, kidney failure, disseminated intravascular coagulation and low blood pressure, and in the non-immune the mortality of untreated P. falciparum malaria is probably more than 50%.

P. falciparum in non-immunes does not usually follow a regular cyclic pattern and the fact that fever is not cyclic with a 48 or 72 hour cycle cannot be used to exclude malaria. Malaria in non-immunes is a medical emergency and diagnosis should be performed without delay.

In semi-immunes the clinical symptoms may be much more discrete and the development more subtle. Immunity to malaria is not a sterile immunity and a low level parasitaemia is seen in semi-immune individuals, ie. individuals from malaria endemic areas [7]. A special risk group is pregnant women from malaria endemic areas who are at greater risk of clinical malaria during pregnancy [8].

Malaria parasites may persist in asymptomatic immigrants long after their arrival in the host country, and malaria can be transmitted, for instance by blood transfusion or organ transplantation.

Who should be tested for malaria?
Diagnostic tests for malaria infection should be performed in any febrile patients who have a history of exposure, which includes patients with a history of travel in malaria endemic areas, as defined by the WHO.

However, rare modes of transmission mean that patients with fever but without a travel history to endemic areas should be tested. This includes so called ‘airport malaria’ where Anopheles mosquitoes carrying malaria parasites are transported in an airplane, leave the destination and take a blood meal from someone living close to the airport [9,10]. Malaria parasites can be transmitted in blood when sharing instruments used for intravenous drug abuse [11]. Transmission of malaria by blood transfusions from asymptomatic carriers is a huge problem in tropical Africa [12] and febrile patients with a history of receiving blood transfusion from a donor in a malaria endemic area should be suspected of having malaria until it is proven otherwise.

Diagnostic procedures for detecting malaria parasites
Traditionally malaria diagnosis rests on the microscopic examination of thick and thin blood films, but over the past decades, rapid tests based on antigen capture are increasingly used. However, rapid test have pitfalls and parasite density must be measured and followed to monitor the response to treatment. Thus microscopy is still a mandatory skill in institutions taking care of malaria patients.

Microscopic examination of Giemsa stained thick blood films remains the gold standard because it is rapid, easy to perform and sensitive [13] with a sensitivity down to five parasites per microlitre of blood [14]. Microscopy and counting of malaria parasites in patients are mandatory to assess the response to treatment and must be available at centres managing patients with malaria.

Rapid test are available which show a 100% sensitivity down to a parasite density level of 200 parasites per microlitre, equivalent to a parasitaemia of approximately 0.004% [15]. Molecular diagnosis by polymerase chain reaction (PCR) can detect parasites down to a density of 0.01 parasites per microlitre after a lysis procedure, and 1 parasite per microlitre without lysis [16]. However, PCR analysis is not instantly available around the clock so in practice diagnosis relies on rapid diagnostic tests and microscopy of Giemsa stained thick blood films.

Rapid tests are increasingly used in medical centres with limited access to experienced microscopists. However, a rapid test cannot determine the parasite density and rapid tests have limitations. False negative results in patients with very high parasite densities have been described, probably due to the so called ‘pro-zone’ phenomena known from other diagnostic tests [17, 18]. The problem seems to be limited to tests based on detection of the Histidine Rich protein 2, HRP2, and not tests based of detection of Plasmodia LDH, Lactate Dehydrogenase [15, 17]. Mutations in the HRP2 gene may also result is false negative results [19, 20]. All species ie. P. falciparum, vivax, ovale and malariae and P. knowlesi, will be found with tests based on the detection of pan-malarial aldolase antigen aldolase and LDH antigens [21]. P. ovale can be divided in variant and classic P. ovale [22], and variant P. ovale is not picked up in HRP2 based rapid diagnostic tests [23].

Thus clinicians using rapid tests should be instructed that no test so far is 100% reliable. In order to reduce the risk of false negative results, testing should be performed at least twice with 24 hours in between and preferable three times within a 24 hours interval. Variant P. ovale and P. knowlesi infections will be detected by rapid tests, which include those incorporating the pan plasmodia antigens Aldolase or Lactate Dehydrogenase antigens [15, 24]. The latest results of the WHO multicentre evaluation of different rapid diagnostic tests showed that the best performance was found in tests based on a combination of the HRP2 and PLDH proteins [15].

References
1. Danis K et al. Euro Surveill 2011;16:19993.
2. Jelinek T et al. Clin Infect Dis 2002; 34:572-576.
3. Eurostat. European Commission. Katya Vasileva. Population and social conditions. 34/2011. Available at: http://epp.eurostat.ec.europa.eu/cache/ITY_OFFPUB/KS-SF-11-034/EN/KS-SF-11-034-EN.PDF
4. Stäger K et al. Emerg Infect Dis 2009; 15:185–91.
5. Bruneel F et al. PLoS One 2010; 5(10):e13236.
6. Jelinek T et al. Euro Surveill.- 2008;13:19077.
7. Wertheimer ER et al. Emerg Infect Dis 2011;17:1701-3.
8. D’Ortenzio E et al. Emerg Infect Dis 2008;14:323-6.
9. Thang HD et al. Neth J Med 2002;60:441-3.
10. Tatem AJ et al. Malar J 2006;5:57.
11. Chau TT et al. Clin Infect Dis 2002;34:1317-22.
12. Noubouossie D et al. Transfus Med 2012;22:63-7
13. Bowers KM et al. Malar J 2009;8:267.
14. Petersen E et al. Am J Trop Med Hyg 1996; 55:485-489.
15. WHO. Rapid Diagnostic Tests. Results of round 3.http://www.who.int/tdr/publications/tdr-research-publications/rdt_round3/en/index.html Geneva 2011 (Accessed 17th March 2012).
16. Mahajan B et al. Transfusion 2012 Feb 10. doi: 10.1111/j.1537-2995.2011.03541.x. [Epub ahead of print].
17. Luchavez J et al. Malar J 2011;10:286.
18. Gillet P et al. Malar J 2011;10:166.
19. Koita OA et al. Am J Trop Med Hyg 2012;86:194-8.
20. Baker J et al. PLoS One 2011;6:e22593.
21. Chiodini PL et al. Trans R Soc Trop Med Hyg 2007; 101:331-337.
22. Sutherland CJ et al. J Infect Dis 2010;201:1544-1550.
23. Tordrup D et al. Malar J 2011;10:15.
24. Hellemond JJ van et al. Emerg Infect Dis 2009;15:1478–1480.
25. Bruce-Chwatt LJ, Zulueta J de. The rise and fall of malaria in Europe. Oxford University Press. 1980.

The author
Dr Eskild Petersen
Department of Infectious Diseases
Aarhus University Hospital
Skejby
Aarhus
Denmark
Tel +45 7845 2817
e-mail: joepeter@rm.dk