Nasal Provocation Test in The Diagnosis of Allergic Rhinitis

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Nasal Provocation Test in the Diagnosis of Allergic Rhinitis
Chapter · March 2012

DOI: 10.5772/39069 · Source: InTech
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10
Nasal Provocation Test in

the Diagnosis of Allergic Rhinitis
Graça Loureiro, Beatriz Tavares, Daniel Machado and Celso Pereira
Immunoallergy Department, Coimbra University Hospital
Portugal
1. Introduction
The specific provocation tests, since its introduction by Blackley in 1853, have been widely
used in the investigation of pathophysiological mechanisms, immunological and
therapeutic aspects of allergic disease, as they mimic the response to allergen exposure,
under controlled conditions. However, it has not been broadly used in the diagnosis of
allergic disease in clinical practice, because of the lack of standardization of the
methodology and the need of other complementary diagnostic tests for monitoring
specific provocation tests. Nevertheless, the importance of such test is enormous in many
circumstances, since it is the only method that can establish the exact etiology of allergic
disease. Although the usefulness of these tests has not been questioned, the need to
standardize the methodology for monitoring the response has been stressed. In this
review, these aspects will be discussed.
2. Allergic rhinitis
Rhinitis is generally subdivided into two groups: allergic and non-allergic. It has been
estimated that allergic rhinitis has a high prevalence in the general population (5 to 20%),
and non-allergic rhinitis alone is thought to affect more than 200 million people worldwide.
So, this is a very common but under diagnosed disease. The correct diagnosis has an
enormous impact in public health, since it would involve several health and economic
benefits (Bousquet & ARIA Workshop Group, 2001).
Allergic rhinitis is an IgE mediated inflammatory chronic disease affecting nasal mucosa,
characterized by the presence of itching, rhinorrea, sneezing and congestion (Bousquet &
ARIA Workshop Group, 2001). The diagnosis of allergic rhinitis is based mostly in clinical
evidence. In fact, a positive correlation between the clinical history and the allergen
sensitization is usually enough to support the diagnosis of allergic rhinitis and its aetiology.
However, in some circumstances (table 1), additional approaches are required to reach a
correct diagnosis in allergic rhinitis patients, namely nasal provocation test (NPT). Indeed, the
specific NPT is the method of choice for the reproducibility of the allergic reaction, and it is
indicated when discrepancies arise in the assessment of a patient’s medical history and the
results of skin and/or serological tests, as reviewed by several authors (Litvyakova LI &
Baraniuk JN. 2001; Loureiro, 2001; Dordal et al, 2011; Mellilo, 1997; Naclerio & Norman, 1998).
154 Allergic Rhinitis
 Multissensitized patients
 Local allergic rhinitis
Clinical practice
 Occupational allergic rhinitis
 Correlation between allergy and other morbidities
 Mechanisms of allergic reaction
Investigational research  Mechanisms of immunotherapy
 Efficacy of new treatments
Table 1. Indications for NPT: clarifying the pathogenesis and diagnostic evidence, in
particular situations of allergic rhinitis
2.1 Multissensitized patients

Atopic patients are frequently sensitized to multiple allergens. In some circumstances,
clinical history is not clearly related to allergen specific IgE. A NPT could be performed to
differentiate the relevant allergenic aetiology in multissensitized patients, since these
patients need specific therapeutic approaches.
2.2 Local allergic rhinitis

Patients with allergic rhinitis have allergen-specific IgE demonstrable both systemically as
well as local IgE produced in the nasal mucosa. On the other hand, the concept of non
allergic rhinitis is supported by negative skin tests. However, in a subset of patients who
have positive NPT to allergens despite having a negative skin prick test, it has been
hypothesized that these patients have localized allergic rhinitis. Huggins made the first
description of local allergic rhinitis (Huggins & Brostoff J, 1975). Recently, several studies
have strengthened the existence of this allergic disorder and the immunological mechanisms
involved in the immediate and late responses to NPT have been described (Kim & Jang,
2010; López S et al, 2010; Rondón et al, 2007, 2009, 2010a, 2010b). A type 2 helper T cell
inflammatory pattern in nasal secretions in response to allergen exposure was
demonstrated. Accordingly, local production of IgE and mast cell / eosinophil activation
with its inflammatory mediators was also founded in these patients. These findings support
the hypothesis of a localized inflammatory response and the concept of local allergic rhinitis.
As discussed, local allergic rhinitis involves nasal production of specific IgE in the absence
of atopy. Evidence of this entity is supported by suggestive clinical symptoms and a positive
NPT. So it is a useful tool for detecting patients with local allergic rhinitis in previously
diagnosed idiopathic / non-allergic rhinitis patients, as defended by several authors and
evidenced by our group (Loureiro et al, 2011). In our experience, the specific NPT
reproduced the clinical manifestations in some patients, supporting the concept of local
allergic rhinitis in a subset of patients with perennial rhinitis. We studied 15 patients with an
average age of 22.2±14.8 years (77.7% were female) with typical clinical symptoms of
perennial rhinitis, negative skin prick test to common aeroallergens and negative specific
IgE. The period of symptoms evolution was 5.37±3.9 years. A Dermatophagoides specific NPT
(BialAristegui, Bilbao, Spain) was performed with clinical monitoring. Total nasal symptom
scores were assessed using a validated questionnaire and a positive NPT was considered if a
score of 5 or greater was recorded (Linder, 1988). The NPT was considered positive in 8
patients. Several studies proved that house dust mites could have a pro-inflammatory
activity independent of IgE (Fujisawa et al, 2008; Gregory et al, 2009; Hammad et al, 2009;
Wong et al, 2006). This fact could explain the positive result in NPT, in our study however,
Nasal Provocation Test in the Diagnosis of Allergic Rhinitis 155
all patients were negative to a non-specific NPT. Despite the few number of patients
included, our data highlight the need for the most complete diagnostic approach. The
correct differential diagnosis with non-allergic rhinitis is crucial for therapeutic purposes,
since some of these misdiagnosed patients may benefit of specific immunotherapy. Indeed,
in our findings, all the patients with the diagnosis of local allergic rhinitis were submitted to
specific immunotherapy, with clinical improvement (data not published).
Because the concept of local allergic rhinitis is based in positive NPT, some authors
emphasize the need to standardize this procedure to better understand its usefulness in the
diagnostic approach of this new entity. It has still controversial aspects to be defined, as
discussed by some authors (Alvares & Khan, 2011; Khan 2009). In a review of the studies
that evaluated patients with negative skin tests using NPT, these authors argued that several
aspects could explain the different data in the literature. For instance, the prevalence ranges
from 0% to 100% of skin test negative individuals. This wide range in prevalence could be
explained by the differences in methodology (allergen manufacturers, concentrations, and
numbers of allergens tested) and, perhaps most importantly, criteria for a positive nasal
challenge. In another review of the literature, the concept of entopy was also considered
controversial (Forester & Calabria, 2010). In spite of this, they recognize that there are a large
number of non-allergic rhinitis patients for whom current treatment regimens are
suboptimal, considering the need to better understand the subjacent immunological
mechanisms to achieve an optimal diagnosis and treatment in this subset of patients.
2.3 Occupational allergic rhinitis

The occupational exposure to immunogenic substances, such as chemicals and biologic
products is enormous in the workplace, since it is the place were people spend more time.
Despite an increasing estimated prevalence of 5 to 15%, occupational allergic diseases,
namely occupational rhinitis it is still underestimated. Several factors are pointed, including
the worker reluctance to complain and the failure to diagnose. More than 400 substances
have been implicated as cause of occupational respiratory allergy. It is recognized that
exposure to these substances can result in increased nasal hyperreactivity and can
predispose to occupational rhinitis. It presents a major impact in quality of life, as well in
professional performance. Further the legal impact, a correct etiologic identification in
occupational allergic rhinitis as an enormous impact in the natural history of this disease.
Indeed it is assumed that occupational rhinitis coexists and it may precede occupational
asthma. Despite this, occupational asthma has been better evaluated than occupational
rhinitis, both in epidemiological and physiopathological approaches.
The real incidence and prevalence of occupational disease is not known. Occupational
disease has been recognized by physicians and epidemiologists. However, there are a few
publications about occupational rhinitis. NPT is an fundamental diagnostic approach of
occupational Allergic Rhinitis (Loureiro, 2008).
New allergens (high molecular weight as well as low molecular weight agents) are
continuously being described in occupational asthma and/or rhinitis. Standardized extracts
for skin testing are not available. A complementary diagnostic approach in occupational
rhinitis, to better recognise and early diagnose this disease, includes specific NPT with
clinical and functional monitoring. In fact, NPT is the ideal methodology to confirm or
refute the diagnosis of occupational allergic rhinitis because it confirms the clinical
symptoms and its causality. For instance, using NPT our group could reached the correct
etiologic diagnosis of the first case of fungal lipase allergy in a patient not sensitized to
amylase working in the pharmaceutical industry (Loureiro, 2009). It has well known that
occupational allergy to lipase has been reported in the detergent industry (Brant et al, 2004,
2006; Lindstedt et al, 2005; van Kampen et al, 2000). While the main allergenic enzyme in the
pharmaceutical industry is amylase, there have been reports of lipase sensitization, albeit
without clinical relevance (Park et al, 2002; Zentner et al, 1997). The NPT was the supporting
approach methodology to obtain this diagnosis confirmation, in our patient. Several cases of
occupational allergic rhinitis are described in the literature, based directly on positive NPT,
both in confirming the diagnosis and the etiological identification. The NPT reproduces the
nasal symptoms and can be performed on the workplace, or under controlled conditions in
hospital environment (Gosepath et al, 2005; Hytonen & Sala 1996; Hytonen et al, 1997;
Litvyakova & Baraniuk, 2001; Loureiro, 2008). In a relevant study, 507 NPT were performed
in 165 patients and the authors concluded that NPT is an essential, easy and safe tool in the
diagnosis of allergic occupational rhinitis (Airaksinen et al, 2007). Recently, there has been a
growing scientific interest in work-related rhinitis, because the relationship to asthma has
been evaluated (Vandenplas, 2010). Considerations of the epidemiology of work-related
rhinitis (both occupational rhinitis and work-exacerbated rhinitis) and its medico-legal
implications have stressed the need to better identify this entity. Recent consensus have
been presented to better define, classify and diagnosis occupational rhinitis, emphasizing
the importance of NPT (EAACI Task Force on Occupational Rhinitis, 2008; Moscato et al,
2011; Dordal et al, 2011).
2.4 Investigational research

The applicability of NPT on investigational research is widely described in the literature,
namely in the study of several aspects of allergic disease, namely the mechanisms of allergic
reaction, the mechanisms of immunotherapy, the efficacy of new treatments and also in the
study of the link between allergy and other morbidities, namely ENT diseases.
In a prospective controlled study, the possible role of nasal allergy in chronic disease of the
maxillary sinuses was evaluated using NPT combined with radiography and ultrasonography
(Pelikan, 2009). It was concluded that nasal allergy might be involved in some patients with
chronic sinusitis. In these patients the NPT was a useful diagnostic tool and allowed to achieve
a better diagnostic of co-morbidity and, consequently, therapeutic measures.
Otitis media with effusion (OME) is a very prevalent disease, particularly in children. The
OME pathogenesis is considered multifactorial, and it has been related to viral upper
respiratory tract infection and eustachian tube disfunction. Allergy has been implicated in
OME pathogenesis by several authors, but it is a matter still controversial. It has been
assumed that there is insufficient evidence of therapeutic efficacy or a causal relationship
between allergy and OME. For instance, 123 children with OME (and 141 controls) were
submitted to NPT. The prevalence of the allergic rhinitis in children with OME did not differ
significantly when compared to control subjects, and the abnormalities in Eustachian tube
function were the same in both groups (Yeo et al, 2007). A recent review of literature
pointed to a strong possibility of allergy as a risk factor for OME. Thus patients with allergic
rhinitis should be evaluated for OME and patients with OME should be considered for an
allergy evaluation. Allergy should be treated aggressively in these patients, because OME
has important and severe sequelae (Lack et al, 2011; Skoner et al, 2009). Our group studied
34 children with diagnosis of adenoids hypertrophy with or without OME, with 7.601.76
years. They were submitted to skin prick test with common aeroallergens battery. 24 were
sensitized to Dermatophagoides pteronyssinus. The link between allergy and OME was
evaluated in each patient with Dermatophagoides pteronyssinus specific NPT (BialAristegui,
Bilbao, Spain). The NPT was monitored using symptom scores and it was considered
positive if a total score ≥ 5. The NPT was positive in 20.8% of the sensitized children. The
therapeutic management of these patients included immunotherapy with clinical
improvement, supporting the link between allergy and OME in a subset of patients.
Concerning investigational research in the yield of the allergic disease, NPT has been widely
used to better understand the underlying mechanisms. Pereira C, 2009 showed that the cell
response starts at an early stage, in parallel with the immediate allergic response. The IgE
mediated response induces immunolymphatic involvement of the adjacent structures. This
amplifies the allergic response to locoregional lymphoid organs, while leukocyte
recirculation involves the primary lymphoid organs (thymus and bone marrow). These
central organs are responsible for the systemic immune response induced by a focused
allergen challenge, in this case, a nasal challenge.
3. Nasal provocation test

The NPT is an “in vivo” diagnostic method that mimics the allergen natural exposure. The
allergic reaction is triggered by allergenic exposure, and symptoms are recorded. Although
not standardized, it is an extremely helpful method as it has several important indications in
the diagnosis of allergic rhinitis (table 1), as described above. Indeed, specific allergen
challenge tests are still the gold standard for allergic diseases diagnosis, being an important
tool to assess the treatment outcomes. Moreover, they have been essential in research,
namely in the progressive understanding of the pathophysiology, immunology and
pharmacotherapy of allergic diseases.
The first allergen challenge was performed in 1873, by Blackley, who elicited a nasal
response after an application of fresh pollen to the membrane of his own nostrils (Blackley,
1873). After this first NPT, several consensus and guidelines have been published trying to
achieve a better diagnostic approach of allergic disease and its knowledge (Dordal et al,
2011; Druce & Schumacher, 1990; Gosepath et al, 2005; Litvyakova & Baraniuk, 2001, 2002;
Lund et al, 1994; Malm et al, 2000; Mellilo et al, 1997; Schumacher, 1992).
3.1 Methodology
The anterior section of the inferior turbinate allows direct and visible application of the
allergen extract, with consequent allergic reaction development (Dordal et al, 2011;
Litvyakova & Baraniuk, 2001; Melilo et al, 1997; Naclerio & Norman, 1998). Despite the
availability of the published international consensus guidelines, several difficulties are
described in the assessment of the technique standardization, namely the type of allergen
extracts to be used (lyophilized, aqueous or paper disc), the dose of allergen (which defaults
to increase the doses) and the technique of administration of allergen (drops, micropipette to
extract volumes, paper disks impregnated with solutions, nebulized extracts). The NPT
should only be performed after a pharmacological washout period, namely H1-
antihistamines, benzodiazepines, corticosteroids and mastocyte stabilizers. It should be
performed at least 4 weeks after an undercurrent infectious disease and avoidance of
exercise. Room conditions of temperature and humidity must be fulfilled. Aqueous solution
and lyophilized powder are the most common commercial allergen extract presentations. In
most studies it is administered unilaterally by various methods: spray (without propellant
gas), instillation (pipette, dropper, syringe) or application of small pieces of cotton or paper
discs with impregnated allergen. The use of different concentrations is recommended,
therefore the dose-response could be evaluated and hence the real sensitivity to that allergen
can be assessed. The starting dose for the NPT must be calculated from the minimum
concentration used for skin prick tests that induces a wheal diameter of 3mm. Alternatively
the initial concentration recommended could be 1 / 100 of the concentration that induced a
positive skin prick test. After establishing the initial concentration, it should be scheduled a
progressive increment of doses. All NPT should be initiated with the previous
administration of saline, to evaluate a possible irritant effect. The interval between
administrations of the allergen at different concentrations should be performed in 15 to 60
minutes. The terminus of the procedure occurs when there are symptoms of rhinitis or signs
of mucosal inflammation. The application of the allergen must occur at the level of the
previous section of the inferior turbinate, which is easily accessible, while the patient is
asked a nasal expiration. The duration of expiration is not established, but the objective is to
minimize bronchial inhalation. Several reviews in the literature analyse a variety of
techniques and approaches, dosing and concentration of allergen extracts, delivery systems,
and also the outcome-evaluation method (Dordal et al, 2011; Litvyakova & Baraniuk,
2001,2002; Tantilipikorn et al, 2010). In our experience we used commercial extracts
prepared in an aqueous solution administered as a spray, directly to the anterior section of
the inferior turbinate. The starting dose for the NPT was the concentration that induced a
wheal diameter of 3mm in each patient.
3.2 Monitoring
The response to NPT is clinical and laboratory assessable. Several parameters could be used to
evaluate the immediate and late allergic response, namely the symptoms score, the evaluation
of nasal congestion (nasal Peak Inspiratory Flow Rate (nPIFR), rhinomanometry, acoustic
rhinometry) and inflammatory cells / mediators analysis in nasal secretions, as reviewed in
the published consensus. None of the methods that evaluate the response to NPT is
standardized. In many publications the assessment of nasal response is based exclusively on
symptom scores. Some authors suggested objective measurements together with symptom
scoring. Thus, the response to NPT should be determined by the combination of symptom
scores and / or rhinomanometry (Dordal et al, 2011; Litvyakova & Baraniuk, 2001).
3.2.1 Clinical symptom scores

Despite symptom scores is a qualitative and subjective method, it is the most used to
evaluate the response to NPT, both in clinical practice and investigational research, since it
mimics a spontaneous allergic response. To assess the nasal response to NPT, it could be
used a score based on a visual analog scale (Bachert, 1997) or scales of semi-quantitative and
subjective clinical assessments (Lebel et al, 1988; Linder, 1988). Usually our group uses the
symptom scoring scaling according to Litvyakova & Baraniuk, 2001. Simple rating scales
from 0 to 3 are used, for each nasal or non-nasal symptom, with defined criteria such as 0 =
no symptoms, 1 = mild symptoms (symptoms that are present but not particularly
bothersome), 2 = moderate symptoms (symptoms that are bothersome but do not interfere
with daily activities), and 3 = severe symptoms (symptoms that are bothersome and
interfere with daily activities or disturb sleep). Even though the known individual
variability and the variability between patients, several authors have been tried to
standardized the symptom score. In all reports, symptom scores are compared with
objective parameters, supporting the relevance of the use of the symptom score in the
monitoring of NPT. For instance, 155 patients with allergic rhinitis to Dermatophagoides were
submitted to NPT to evaluate the optimal cut-off values of symptom changes after NPT for
predicting perennial allergic rhinitis, as well as the nPIFR evaluation (Chusakul et al, 2010).
In another study, the symptom score change and acoustic rhinometry values were
combined, before and after NPT in 208 patients with allergic rhinitis and in 222 controls
(Kim & Jang, 2011).
3.2.2 Methods to evaluate nasal congestion: Nasal Peak Inspiratory flow rate,
rhinomanometry and acoustic rhinometry
The assessment of nasal congestion could be evaluated by subjective parameters (symptom
score) and by an objective and quantitative method. Several techniques are available for
assessing changes in nasal airflow resistance, patency, and nasal cavity geometry. Such
techniques provide objective measurement of nasal congestion, namely nPIFR,
rhinomanometry and acoustic rhinometry. These methods provide an objective and
quantitative measurement whose value is based on the comparison of results over procedures
(diagnostic or therapeutic) in each individual. In spite of this, standardized methodologies
assessing functional abnormalities are not sufficiently developed (Nathan et al, 2005).
Comparison of results between different patients is not yet standardized. Recently, several
studies have been tried to standardize these methods as they can be useful in clinical practice
and applied as a diagnostic tool in allergic rhinitis (Chusakul et al, 2010; Kim & Jang, 2011).
Nasal Peak Inspiratory Flow Rate. This technique assesses the nasal airflow. It is easy to
perform and inexpensive, but it is difficult to reproduce because is partially dependent on
lung capacity (Wihl & Malm, 1988). Some studies demonstrated that nPIFR values correlate
with airway resistance, but this is impracticable in case of intense rhinorrea (Holmstrom et
al, 1990; Jones et al, 1991).
Rhinomanometry. This standardized technique measures the resistance to the airflow in nasal
cavities (Schumacher, 1989). Increases in resistance in one or both nasal cavities have been
considered as an objective parameter in positive responses to NPT (Clement, 1984; Kirerleri
et al, 2006). The technique depends on patient cooperation and it cannot be used in cases of
septum perforation, intense rhinorrhea or nasal obstruction (Nathan et al, 2005).
Acoustic rhinometry. This is a sound-based technique used to evaluate the nasal geometry,
which measures nasal cavity area and volume. It has been validated by comparison to
measurements with computed tomography and magnetic resonance imaging. It is used to
diagnose and evaluate therapeutic responses in conditions such as rhinitis and to measure
nasal dimensions during NPT (Keck et al, 2006; Kim et al, 2008; Uzzaman et al, 2006).
Acoustic rhinometry is easy to perform and reproducible, but there are no reference values
(Corey et al, 1998). It requires little cooperation from the patient, so it could be a useful
method for children, and it is not affected by the presence of rhinorrhea or intense nasal
obstruction. However, it cannot be applied in cases of septal perforation. Some
interpretation caution should be made, when assessing changes in NPT. The nasal cavity
volume between 2 cm and 6 cm is the most important parameter, because it corresponds to
the head of the turbinate, while the nasal cavity volume between 6 cm and 10 cm provides
information about the sinuses and ostia. The intrinsic bias of the nasal cycle should not be
overlooked, consequently, the cross-sectional areas and volumes of both nasal cavities
should be measured after NPT (Gotlib et al, 2005).
When comparing both techniques, acoustic rhinometry does not seem to be a better
diagnostic method than active rhinomanometry in the monitoring of NPT (Keck et al, 2006).
3.2.3 Laboratorial measurements: Inflammatory allergic mediators and cells

The analysis of nasal cytology is essential to distinguish non-inflammatory from
inflammatory nasal diseases. Additionally, the pattern of inflammatory mediators reflects
the underlying immunological response. So the analysis of these inflammatory allergic
mediators and cells are useful in the assessment of the response to NPT, namely in the
diagnosis of allergic disease and in the evaluation of the treatment efficacy. Indeed, the NPT
has been used to characterize and to clarify the immunological mechanisms involved in
allergic reaction, and reciprocally, known inflammatory allergic mediators and cells have
also been used to diagnose allergic rhinitis (for example local allergic rhinitis, as mentioned
above) and to monitor the response to NPT. Allergic rhinitis is an allergen-induced IgE-
mediated inflammatory disease of the nasal mucosa. Several inflammatory mediators
(histamine, tryptase, ECP, leucotrienes, cytokines and chemokines) are involved and the
cellular infiltrate is characterized of mast cells, basophils, eosinophils and T cells. The
usefulness of nasal cytology depends on several factors, namely obtaining adequate
specimens, appropriate samples staining, and the materials interpretation.
3.2.3.1 Methods to collect nasal samples
Various techniques have been used for obtaining, processing, evaluating, and interpreting
nasal specimens. The different methods for collecting samples are nasal lavage, nasal swab,
nasal brushing, nasal curettage, nasal biopsy and collection of nasal secretions, allowing the
assay of cells and inflammatory mediators. Several comparative studies show the usefulness of
these non-standardized different methods. Each technique has advantages and disadvantages,
so the selection of each method must be carefully decided. Description of the different
techniques was reviewed elsewhere, in detail. (Dordal et al, 2011; Howarth et al, 2005).
Nasal lavage. Naclerio first described this technique (Naclerio et al; 1983). This is a frequently
used method to collect and to identify cells and inflammatory mediators. It has been used in
research studies. In addition to nasal lavage, the collection of nasal secretions could be
analyzed to look for both cellular and inflammatory mediators.
Nasal brushing and Nasal biopsy should be performed on the inferior turbinate, to obtain
cellular samples. Nasal brushing is usually performed at the middle third of the inferior
turbinate, with easy sampling of the superficial of nasal mucosa. Nasal biopsy is usually
performed on the lower part of the inferior turbinate, requires anaesthesia, and reaches
deeper tissues. However it cannot be systematically repeated because it is traumatic.
3.2.3.2 Difficulties in assessment of inflammatory response
These techniques helped to attain the actual knowledge about the characteristics of allergic
disease. However, its usefulness in the evaluation of the response to NPT is restricted to
research trials, in order to better understand immunological allergic mechanisms and effects
of new therapies. In clinical practice, the assessment of these inflammatory parameters is not
enough to evaluate the response to NPT.
In our Immunoallergy Department, we performed a study to evaluate the concentrations of

the chemokines, eotaxin and RANTES, in nasal lavage and analyze the applicability of the
determination of chemokines in nasal secretions as a parameter of immune response to
specific nasal provocation test (Loureiro et al, 2003). We included 17 patients with allergic
rhinitis to Parietaria judaica (64% male; 36.311.2 years old). All the patients were submitted
to NPT with Parietaria judaica commercial extract (Leti, Madrid, Spain) outside the pollen
season. Nasal lavages were performed, before, 30 minutes and 6 hours after NPT, for
quantification of inflammatory mediators. NPT response was monitored through symptom
score. The NPT was positive in all patients, reproducing the clinical reactivity to the
allergen, with a peak in the symptom scores at the first minute with subsequent decreasing
till the sixth hour. Eotaxin was not measurable in any of the nasal lavage specimens
collected. The chemokine RANTES levels were 4.2±2.1pg/ ml before NPT and 3.96±0.98
pg/ml and 3.90±0.99 pg/ml in the specimens collected at 30 minutes and 6 hours after NPT
respectively. These results did not correlate with symptoms progression during NPT. This
could be interpreted as a discrepancy between the time of sampling and the dynamics of
inflammatory mediators in response to NPT.
In the same group of patients, during the same procedure, we also analysed the tryptase and
ECP levels, in nasal lavage, as immunological markers of immediate and late response in
allergic reaction, respectively (Loureiro et al, 2004). Tryptase was detected in only three
patients. Nasal brushings were also performed to harvest cells. Cellular phenotyping (CD3,
CD4, CD8 and CD 125) was assayed by flow citometry, before and 6 hours after NPT, to
recognize the cellular dynamics during NPT. Our findings showed an increase in CD3 and
CD8 cells in all patients. In a subset of patients submitted to immunotherapy we observed a
CD4 cells increase and a CD125 cells decrease, after NPT, while the other patients not
submitted to immunotherapy does not showed any dynamic alterations in these cells (figure
1). The differences observed in each group could be explained by different therapeutic
approaches in each group. However the dynamic cellular changes after NPT were not as
expected. These findings could be explained by premature sampling before cellular
trafficking occurred. Another possible explanation is the insufficiency of these sampling
methods to harvest the sufficient cellular infiltrate.
80 70
60
60
50
40 40
20 30
20
0 10
before NPT 6 hours after 0
NPT before NPT 6 hours after NPT
(a) (b)
Fig. 1. Nasal cell typing before and after nasal provocation test (NPT) (% of cells): A - in a
group of patients submitted to immunotherapy for one year; B - in a group of patients not
submitted to immunotherapy (Legend:  - CD3; - CD4; -CD8; - CD125)
In another study conducted by our group, 21 allergic patients were submitted to

Dermatophagoides pteronyssinus specific NPT (BialAristegui, Bilbao, Spain). Secretions,
namely tears and nasal secretions, were collected after NPT and inflammatory mediators,
such as interleukins and chemokines were measured (data not published). These
inflammatory mediators were measurable only in 21% of the tear samples and in 71.5% of
the nasal secretion samples. According to these findings, nasal secretions recovery could be
acceptable to be considered as an objective tool in the evaluation of inflammatory mediators.
However we could not find out a pattern of mediator release since the inflammatory
mediators were inconsistently detected in the different samples.
Although the analysis of immunological parameters has been described as an objective
approach to monitor the response to NPT, in our experience, these laboratorial
measurements are difficult to perform because of the scheduling of sampling. Additionally,
the cost-effectiveness of these procedures does not allow its implementation in the clinical
practice. It should be reserved to investigational research.
3.2.4 Assessment on nasal Nitric Oxide

Determination of nasal Nitric Oxide (nNO) provides an indirect measure of the
inflammation of the nasal mucosa. A decrease in nNO levels with NPT coincided with
maximal symptom intensity, in five patients with pollen-induced allergic rhinitis
(Kharitonov et al, 1997). Although nNO promises as a diagnostic non-invasive management
tool, its value in nasal pathology is still not clear, mainly due to the lack of standardization
of the test. Different methods of measurement have been used in published studies and the
results reported are not comparable (Dordal et al, 2011).
3.3 Criteria for positive NPT

Besides diverse combined criteria have been discussed in the literature none of them are
standardized criteria to define NPT positivity. This is summarized in the Table 2.
4. NPT as a diagnostic approach in respiratory allergy

The first allergen provocation test was performed in 1873, by Blackley, who elicited a nasal
response after an application of fresh pollen to the membrane of his own nostrils (Blackley,
1873). Currently, the indications of NPT are widely known. In this manuscript, the
applicability of NPT as a diagnostic tool of allergic rhinitis was discussed. But the usefulness of
NPT is not restricted to the diagnostic approach of allergic rhinitis. Supported by the concept
of “one airway, one disease”, several studies have pointed out that the NPT is a good alternative
to Bronchial Provocation Test (BPT), even in the absence of nasal symptoms. In spite of BPT
being a standardized diagnostic tool, it is not frequently used in clinical practice because of its
technically and methodologically requirement. Indeed, NPT is safer and better tolerated
Assessment of nasal
Reference Description of positivity criteria
response
Δ ≥ 4, considering Δ = (obstruction score
Hytonen et al, 1997 Symptom score + rhinorrhea score) after NPT -
(obstruction score + rhinorrhea score)
Lebel Symptom Score Scale: Positive if ≥
Lebel B et al, 1988 Symptom score
5 (maximum possible score 11 points)
Assessment of nasal
Reference Description of positivity criteria
response
Linder Symptom Score Scale: Positive if
Linder A, 1988 Symptom score
≥ 5 (maximum possible score 13 points)
Terrien et al, 1999 nPIFR assessment Fall in nPIFR ≥ 40%
Cimarra & Robledo, 2001 Rhinomanometry Airflow resistance increases by 100%
MCA and nasal cavity volume vary by
Valero & Picado, 2000 Acoustic rhinometry
25-30%
At least two of the following criteria:
Combined symptom five sneezing, runny nose, nasal
Álvarez Eire et al, 2006
score and nPIFR congestion documented by a decrease ≥
20% of nPIFR
A 40% reduction in airflow at 150 Pa in
active anterior rhinomanometry,
Combined symptom
regardless of the symptom score,
Gosepath et al, 2005 score and
or
rhinomanometry
a 20% reduction of in airflow at 150 Pa
with a symptom score of more than 2
a 30% increase in the symptom score
Combined symptom
using a visual analog scale and a 30%
Rondón C et al, 2007 score and acoustic
reduction in nasal cavity volume by
rhinometry
acoustic rhinometry
1) symptom score change: more than 2
points in the case of nasal obstruction
Combined symptom and more than 1 point for the case of
Kim & Jang, 2011 score and acoustic rhinorrea or itching; 2) more than 24.5%
rhinometry change of total nasal volume and 3)
more than 20% change of the minimal
cross-sectional area.
Combined nasal 0.5 mL (0.5 g) of nasal secretion with 5
Wihl, 1986 secretions amount and or more sneezes and a >20% reduction
nPIFR in nPIFR
30 minutes after NPT:
100 mg of nasal secretion with a 15%
Combined nasal decrease in MCA and 50% increase in
secretions amount, nasal airflow resistance;
Pirila & Nuutinen, 1998
rhinomanometry and 60 minutes after NPT:
acoustic rhinometry 210 mg of nasal secretion with a 30%
decrease in MCA and 100% increase in
nasal airflow resistance
Combined acoustic 29% decrease in MCA and 26% decrease
Ganslmayer et al, 1999
rhinometry and nPIFR in nPIFR
Table 2. Some criteria do define NPT positivity, adapted from Dordal et al, 2011.
method in asthmatic patients than BPT (Hervás et al, 2011; Marcucci et al, 2007; Oddera et al,
1998). So, NPT has been used to the diagnosis of asthma, as reviewed by Olive Pérez, 1997.
Thus, based on the united airways disease concept, the NPT could be considered as a model of
specific provocation test that is easy and quick to perform, in the demonstration of the
immediate and late phase response of type I hypersensitivity reaction. It is well known that the
nose is an integral part of the upper airway, and anatomically related to several airway
structures, such as ears and paranasal sinuses, and as well the eyes. There is an
epidemiological relationship between rhinitis and asthma. Rhinitis and asthma are often
associated, rhinitis typically precedes the development of asthma and can contribute to
insufficient asthma control (Compalati et al, 2010). On the other hand, in cross-sectional and
longitudinal studies, the vast majority of patients with asthma have rhinitis, and rhinitis is a
major independent risk factor for asthma (Togias, 2003). Treating allergic rhinitis would
probably ameliorate other associated upper airway diseases such as acute rhinosinusitis, nasal
polyposis, adenoidal hypertrophy, and OME (Marple, 2010). In addition to improve allergic
rhinitis outcome, the treatment of subjacent inflammatory disorder reduces asthma-associated
health care consuming. A close interaction between the nose and contiguous or distant organs
was described and it has been progressively clarified, supporting this epidemiological and
clinical relation (Baroody, 2011). The upper and lower airways are not anatomically and
functionally distinct areas (Slavin, 2008). It is currently established that the impaired function
of the upper airways causing nasal obstruction, retention of secretions, and disturbed
conditioning of the inspired air plays an important role in the development of lower airway
symptoms (Virchow, 2005). There are important relationships between both the nose and the
paranasal sinuses and asthma. Apart from the intrinsic physiological interaction, extensive
evidence exists to sustain the concept that the respiratory system functions as an integrated
unit (Krouse, 2008), where rhinitis and asthma are manifestations of one syndrome, the chronic
allergic respiratory syndrome, in both parts of the respiratory tract (Togias, 2003). It has been
described that parallel immunopathological processes involve the upper airway generally
occur in conjunction with lower airway diseases, and diffuse inflammation often affects
mucosal surfaces of the middle ear, nose, sinuses, and tracheobronchial tree simultaneously
(Krouse, 2008). Recent studies show that the deposition of allergen into the lower respiratory
tract leads to increased inflammation of the upper respiratory tract, even if the patients are
only suffering from allergic rhinitis (Virchow, 2005). Additionally, studies indicate that
treatment of the upper respiratory tract inflammation reduces the manifestation of allergen-
associated symptoms in the lower respiratory tract, and also have preventive effects if started
early on the disease evolution (Bousquet & ARIA Workshop Group, 2001). Both asthma and
allergic rhinitis have now been recognized as inflammatory diseases with similar
manifestations in the mucous membranes of the upper (nose and paranasal sinuses) and lower
respiratory tract (Virchow, 2005). There is increasing evidence that even in patients with
rhinitis who do not have asthma, sub-clinical changes in the lower airways and inflammatory
mediators can be detected (Compalati et al, 2010). These and other findings support that
allergic diseases have a systemic component (Virchow, 2005). The interactive mechanisms of
allergic rhinitis and associated conditions highlights the relevance of a bidirectional "unified
airway" respiratory inflammation model. Currently, it is accepted that IgE mediated allergic
reactions are not confined to the area where the trigger occurred, inducing a secondary
systemic immune response (Braunstahl, 2005, 2006; Togias, 2004). The systemic inflammation
is produced after local allergic reactions (Togias, 2003). The link between local exposure to
allergen and distant response has been clarified. Although some authors defend that this
systemic response could result from allergen entering in the systemic circulation from the local
of exposure (Hens et al, 2007) this could activate circulating basophils, inducing an
anaphylactic reaction, which is a rare condition (Togias, 2004). Both systemic cell circulation
and the nervous system activation are two major ways through which local allergic reactions
propagate. Mast cell mediators locally released, increase the expression of adhesion molecules
on postcapillary venules. This can lead to homing of circulating leukocytes, which may
infiltrate distant tissues. This cell recirculation and focalization makes the IgE mediated
allergic disease a dynamic and systemic process. Pereira C showed that this cell response starts
at an early stage, in parallel with the immediate allergic response (Pereira, 2009). The IgE
mediated response induces immunolymphatic involvement of the adjacent structures. This
amplifies the allergic response to loco-regional lymphoid organs, while circulating leukocytes
recirculation compromises the primary lymphoid organs (thymus and bone marrow). These
central organs are responsible for the systemic immune response induced by a localized
allergen challenge, in this case, a nasal challenge (Pereira, 2009). The nervous system activation
could be involved by, any or both pathways, namely neurogenic inflammation and neuronal
reflexes. Neurogenic inflammation is characterized by specific neuromediators closely related
to neuro-immune-endocrine system, and it is both a stimulus to and a consequence of allergic
inflammation. The naso-nasal and the naso-ocular reflexes are some examples of the role of the
nervous system in the propagation of the allergic disease. They seem to be predominantly
mediated by parasympathetic and cholinergic pathways, respectively (Baroody et al, 1994,
2008). Histamine release during the acute response to allergen and substance P seem to have
an important role in these neural mechanisms (Baroody et al, 1994, 2008; Fujishima et al 1997;
Micera et al, 2008; O’Meara et al, 2005; Sheahan et al, 2005). Multiple evidences support a close
interaction and influence of the nose on contiguous and distant organs via neural reflex and
systemic inflammatory processes (Baroody, 2011). In summary, a local triggered allergenic
inflammation is systematically extended, with the early connection of the immune central
organs. Independently of the involved pathway, immediate symptoms are clinically
manifested.
Besides the limitations of NPT, this is a feasible and easily method to be performed, since
the nasal cavities provide easy access to specific provocation. The concept of "One airway, one
disease" allows assuming the similarity of response to the provocation of both the upper and
lower airways, so the nasal allergic reaction could be accepted as predictor of bronchial
response. Supported by the concept of the bidirectional "unified airway" respiratory
inflammation, a local provocation test is useful in the diagnosis of allergic respiratory
disease. Concerning these aspects, the NPT is the method of choice for the reproducibility of
the allergic reaction (Litvyakova & Baraniuk, 2001; Loureiro, 2001; Mellilo, 1997; Naclerio &
Norman, 1998). Thus the NPT may be considered a model of respiratory provocation test,
easy to perform, in the demonstration of the immediate and late phase of type I
hypersensitivity reaction.
5. Characterization of NPT score symptom response

According to all the mentioned above, the clinical symptom score is widely used in clinical
practice, alone or associated to objective measurements of nasal obstruction, namely nPIFR,
rhinomanometry and acoustic rhinometry. The other methods, such as immunological

measurements, should be reserved to research procedures related to the investigation of
inflammatory network. However, due to the lack of standardization of parameters in the
monitoring of NPT response, its reproducibility remains to be defined. The main problem
includes the great variability of the responses in each patient and between patients.
Although this is an important limitation, concerning NPT response interpretation, the
symptom score has been used in the description of positive criteria to NPT response.
As pointed out above, many authors use the symptom score as a method of monitoring and
criteria for positivity in response to the NPT. According to the great variability in each
patient and between patients, it has been assumed the absence of pattern of response to the
NPT. The attempt to standardize this methodology was characterized by the symptom score
quantification, through the use of symptoms scaling. One of the most important limitations
of this symptoms scaling, is the overemphasis on nasal obstruction, since firstly not all
patients value the perception of this symptom, and secondly, when it is present, it can result
from concomitant obstructive and inflammatory causes. Besides there is no clinical pattern
of response to NPT, our data showed a response profile, which can not be accepted as
standard, but it can be useful in monitoring the NPT, namely in the evaluation of the
dynamics of the response to NPT, as described bellow.
5.1 Clinical symptom score pattern

In our experience, the symptom score has supported the positivity of NPT. We analysed that
the most frequent and intense symptoms occurred within the first 30 minutes after NPT,
agreeing to immediate phase of allergic reaction. From all the studies conducted by our
group, we did not observe a clinical score symptom pattern. However, we describe a clinical
symptom score profile, which was frequent and was characterized by the presence of nasal
and extra-nasal symptoms within the first 30 minutes, with a peak at 5 minutes.
10
8
6
4
2
0
before 5 minutes 30 minutes 6 hours
NPT
Fig. 2. Score symptoms after Parietaria judaica specific nasal provocation test (NPT) (legend:
 total score;  nasal score;  non-nasal score).
Indeed, in a group of patients allergic to Parietaria judaica, as described above, specific NPT
was performed and a symptom score was recorded. The figure 2 presents the total, nasal and
non-nasal symptom scores. The higher total score of symptoms was recorded at the fifth
minute with progressively decreasing symptoms till 30 minutes and then till 6 hours. Each
nasal symptom followed this pattern. The non-nasal symptoms showed a different pattern,
having a lower score, with similar values at both the fifth and the 30th minutes, followed by a
decline till 6 hours. Looking at the score of each nasal symptom (Figure 3), except for nasal
obstruction, all of them followed the response pattern of total symptoms score, with a peak of
symptoms at the fifth minute. Sneezing was the predominant symptom at the fifth minute,
while nasal obstruction was the predominant symptom at the 30th minute and the sixth hour.
2.5
1.5
0.5
0
before 5 minutes 30 6 hours
NPT minutes
Fig. 3. Nasal symptom score after specific Parietaria judaica nasal provocation test (NPT):
evolution of each nasal symptom (Legend:  nasal congestion;  pruritus;  sneezing; 
rhinorrea).
In another study mentioned above, the Dermatophagoides pteronyssinus specific NPT were
performed in 34 children with OME. Those who had positive NPT, showed a response
dynamics characterized by a rapid increase of symptoms score till a peak at the 5th minute
(monitored till 1 hour), as shown in figure 4. Looking at the score of each nasal symptom
(Figure 5), except for nasal pruritus, all followed the response pattern of total symptoms
score, with the peak of symptoms at the fifth minute.
12
10
8
6
4
2
0
before 1st 5th 30th 1 hour
NPT minute minute minute
Fig. 4. Score symptoms after Dermatophagoides pteronyssinus specific nasal provocation test
(NPT); (Legend:  total score;  nasal score;  non-nasal score).
3
2.5
2
1.5
1
0.5
0
before 1st 5th 30th 1 hour
NPT minute minute minute
Fig. 5. Nasal symptom score after specific Dermatophagoides pteronyssinus nasal provocation
test (NPT): evolution of each nasal symptom (Legend:  nasal congestion;  pruritus; 
sneezing;  rhinorrea).
Beyond the description of symptoms score obtained during NPT, it is also important to
compare them with the usual symptoms described by the patient. This looks particularly
relevant in the diagnosis of local allergic rhinitis.
In our study related to local allergic rhinitis diagnosis, we included 15 patients with typical
clinical symptoms of perennial rhinitis, negative skin prick test to common aeroallergens
and negative specific IgE, as mentioned above (Loureiro G et al, 2011). The patients had an
average age of 22.214.8 years, 77.7% were female. A Dermatophagoides pteronyssinus specific
NPT was performed with clinical monitoring. Total nasal symptom scores were assessed
using a validated questionnaire and a positive challenge was considered if a score of five or
greater was recorded. NPT supported the diagnosis of local allergic rhinitis in a group of
patients previously diagnosed with “non-allergic rhinitis”. They presented a period of
symptoms evolution of 5.373.9 years. The symptom scores reported during natural
exposure and after NPT are shown in figure 6. During natural exposure, the nasal total score
was 6.22.05. Nasal congestion was always reported and it had the highest recorded value
(2.80.35). The highest nasal recorded value during NPT was 6.42.19. Nasal congestion and
pruritus were always reported and this second symptom had the higher recorded value
(2.40.5). None of the 15 patients had conjunctivitis or asthma. Furthermore, in the 8 patients
that had positive NPT, extra-nasal symptoms were recorded, namely conjunctival
symptoms, oropharyngeal pruritus, cough and dyspnea, although with lower values.
Concerning the occurrence of non-nasal symptoms, the major non-nasal symptoms
observed were those localized in the conjunctiva, followed by oropharyngeal pruritus.
Dyspnea and cough were recognized rarely. Non-nasal symptoms were documented in 20
up to 100% of the positive NPT performed, considering the different studies conducted in
our Immunoallergy Department.
Natural NPT
exposure
During natural exposure, The highest nasal

the nasal total score was recorded value during
6.2±2.05 NPT was 6.4±2.19
Nasal congestion was Nasal congestion and

always reported and it pruritus were always
had the highest recorded reported and the latest
value (2.8±0.35) had the higher recorded
value (2.4±0.5)
Fig. 6. Symptom scores reported during natural exposure and after nasal provocation test
(NPT).
5.2 Comparison of Dermatophagoides pteronyssinus nasal provocation test versus

conjunctiva provocation test
Our group and others authors have been using clinical scores to evaluate NPT response.
According to our findings described previously, in respect to the symptoms scores pattern in
response to NPT, we conducted a study to characterize the clinical response to NPT comparing
to conjunctiva provocation test (CPT). As CPT is easy to perform and systemic reactions are
uncommon, some authors have studied the concordance between NPT and CPT in the
diagnosis of allergic rhinitis (Andersen et al, 1996; Leonardi et al, 1993; Malmberg et al, 1978;
Petersson et al, 1986; Riechelmann et al, 2003) and asthma (Mosbech et al, 1987) using clinical
score symptoms and/or objective methods. However, we are not aware of any publication
describing the clinical pattern of NPT and CPT responses, neither about its comparison.
Our aim was to compare the dynamics of clinical responses induced by NPT and CPT, using
a clinical score system.
5.2.1 Material and methods

5.2.1.1 Subjects
We studied two groups of voluntary adult patients, referred to our outpatient Immunoallergy
Department, with Dermatophagoides pteronyssinus (Dp) allergic rhinitis/rhinoconjunctivitis
with or without associated bronchial asthma, according to ARIA (Bousquet & ARIA
Workshop Group, 2001) and GINA guidelines, respectively. All patients were clinically
stable at the time of the study. Patients with past or ongoing immunotherapy for
Dermatophagoides, an exacerbation of allergic disease or a respiratory tract infection in the
last month, a nasal surgery in the last 3 months or nasal pathology such as polyps or a
deviated nasal septum, were excluded. H1-antihistamines and costicosteroids, either nasal
or oral, were withheld for 2 weeks and 4 weeks prior to the challenge test, respectively. All
patients underwent the challenge between January and February of 2009, a period of low
natural exposure to mites in Portugal. A Dp NPT was performed in 21 patients and the
conjunctival provocation test (CPT) was performed in the other 21 patients. The local ethics
committee approved the study and all the participants gave written informed consent before
entry. A respiratory function test (pletismography using Master screen Body Jaeger®) was
performed by all the participants, before specific provocation tests, with all presenting a
baseline FEV1  80% and FEV1/FVC  80. After provocation, all patients were asked for the
presence of dyspnoea, thoracic oppression, wheezing or cough.
5.2.1.2 Specific nasal and conjuctival provocation tests
A skin prick test aqueous extract of Dp with a 5 mg/ml concentration (23 g/ml of Der p
1, BialAristegui, Bilbao, Spain), with 1/1, 1/10, 1/100 and 1/1000 dilutions were
performed; negative and positive controls were performed in all patients, according to
standardized procedures (Dreborg & Frew A, 1993). The concentration used to specific
provocation was the minimum that induced a prick test wheal at least equal to that
induced by histamine, which curiously was the 1/10 dilution in all patients. Specific NPT
with Dp extract were performed in the morning and after an adaptation to room
temperature for 30 minutes, in both groups. NPT was performed with unilateral nasal
application of 2 consecutives puffs (total volume of 160 l) of the Dp extract to the inferior
nasal turbinate of the less congested nostril, using a nasal applicator spraying and patients
were asked to perform apnoea during the allergen spraying. CPT consisted in unilateral
ocular application of 1 drop (50 l) of the Dp extract in the inferior and external quadrant
of the bulbar conjunctiva. Nasal and eye symptoms were recorded at the 1st and 5th
minutes after specific provocation tests, using a clinical score system to assess the
response (Linder A, 1988).
5.2.1.3 Clinical score scaling
Clinical responses were evaluated using a nasal clinical score (NCS) and an ocular clinical
score (OCS), at the 1st and the 5th minutes. An adaptation of the previously used NCS
(Linder A, 1988) and OCS (Mortemousque, 2007) were applied. A total clinical score (TCS),
representing the sum of NCS (range: 0-15) and OCS (range: 0-13) was also used, ranging
from 0 to 28 points. Rhinorrhea, sneezing, itchy nose, itchy ear/throat, nasal obstruction,
watery eyes, redness of eyes and burning of eyes were rated on a scale from 0 to 3 points (0,
none; 1, mild; 2, moderate; 3, severe). Itchy eyes were scored from 0 to 4 points (0, none; 1,
mild; 2, moderate; 3, severe; 4, very severe). A positive response to NPT was considered
when NCS 3 (Linder A, 1988) and to CPT when OCS 5 (Mortemousque, 2007). Clinical
evaluation was interrupted after the 5th minute to collect humours for further investigation
to determine inflammatory markers within a research investigation of immunologic
mechanisms in allergic disease (Pereira, 2011, in press).
5.2.1.4 Statistical analysis
Statistical analysis were performed using SPSS® Statistics 17.0 software. Comparisons
between NPT and CPT were studied using Chi-Square test. Intra-groups differences
between the 1st and the 5th minutes after provocation were analyzed using a Mann-Whitney
U-test. A statistical significant difference was assumed with p < 0.05.
5.2.2 Results
Demographical and clinical data are presented in Table 3. Table 4 shows the number of
patients that presented nasal and ocular responses at the 5th minute, induced by NPT and
CPT, as well as the number of positive challenges at the 1st and the 5th minutes. A
progressive increase in clinical score was observed in both provocations. The NPT
progressive response was linear while for the CPT it was exponential, as shown in figures 7.
CPT response was stronger than NPT at the 5th minute, achieving borderline significance
(p=0.05). Clinical score results for NPT and CPT are shown in Table 5.
14 14
12
9 10
8
4 6
4
2
-1
before 1st m inute 5th m inute 0
NPT before CPT 1st minute 5th minute
(a) (b)
Fig. 7. Dynamics of symptoms score in response to: A – NPT (Linear progression); B – CPT
(Exponential progression) Legend:  - Total Symptom score;  - Nasal symptom score;  -
Non-nasal symptom score)
The most frequent symptoms were nasal obstruction, itchy ear/throat and itchy nose, for
NPT, and ocular hyperaemia and burning eyes, for CPT in all patients. In NPT, nasal
obstruction was observed in 100% of the group. CPT induced ocular hyperaemia and
burning eyes in all patients. There were neither bronchial symptoms nor systemic reactions
in any of the provocation tests.
The highest scores were reached by nasal obstruction and rhinorrhea in NPT and by ocular
hyperaemia in CPT. The average intensity of each sign/symptom at the 5th minute is shown
in figure 8.
NPT CPT
n 21 21
Average age (years) 28.0  9.0 28.1  5.7
Gender ♀ (%) 57.1 66.7
Rhinitis (n) 20 16
Rhinoconjunctivitis (n) 1 5
Associated asthma (%) 42.8 90.5
Cutaneous reactivity to Dp (mm) 6.5  2.1 8.6  3.6
Specific IgE to Dp (KU/L) 29  24.9 36.3  37.2
Disease evolution (years) 13  10 12.3  8.5
Table 3. Demographical and clinical data of patients submitted to NPT and CPT (Legend:
NPT – nasal provocation test; CPT – conjunctiva provocation test)
NPT CPT p
Nasal response at 5th min 21 (100%) 20 (95.2%) ns
Ocular response at 5th min 10 (47.6%) 21 (100%) 0.0001
Number of positive challenges:
1st min 15 6
0.005
5th min 21 21
Table 4. Frequency of nasal and ocular symptoms at the 5th minute and NPT and CPT
outcomes at the 1st and the 5th minutes (Legend: NPT – nasal provocation test; CPT –
conjunctiva provocation test; ns - not significant).
Comparing NPT and CPT, in the first one the response was faster at the 1st minute (p=0.005)
while for CPT it was stronger at the 5th minute (p=0.05).
Although the inoculation of allergen was unilateral, NPT induced bilateral nasal symptoms
in 100% and bilateral ocular symptoms in 47.6%. On the other hand, CPT induced unilateral
ocular symptoms in 100% and bilateral nasal symptoms in 95.2%. There were neither
bronchial symptoms nor systemic reactions.
rhinorrea
nasal obstruction
nasal itching
sneeze
palate itching
ocular erythema
chemosis
lacrimation
ocular itching
0 1 2 3
Fig. 8. The average intensity of each sign/symptom at the 5th minute; (Legend:  - Nasal
provocation test;  - Conjunctiva provocation test)
5.2.3 Discussion
Although the importance of the objective monitoring of specific provocation tests is
unquestionable, its applicability in clinical practice is not always possible. Usually it is limited
to the evaluation of only one symptom, such as nasal patency by nasal peak flow, acoustic
rhinometry and/or rhinomanometry (Nathan et al, 2005); however it is not always the most
perceived symptom by patients. Clinical scoring systems, even though more subjective, reflect
NPT CPT p
Total
1st min 5.2  3.8 4.7  3.6 ns
5th min 9.9  4.4 12.7  4.4 ns (0.05)
5th - 1st 4.6  4.57 8.0  3.87 0.011
Nasal (NCS)
1st min 4.28  2.6 1.24  2.1 <0.0001
5th min 8.29  2.9 4.95  2.8 0.001
Ocular (OCS)
1st min 0.95  1.8 3.4  3 <0.0001
5th min 1.57  2.3 7.7  3 <0.0001
Table 5. Clinical score results for NPT and CPT (Legend: NPT – nasal provocation test; CPT
– conjunctiva provocation test; ns – not significant).
all symptoms, are easy and costless to apply in clinical practice. The validity and
reproducibility of CPT based on clinical score systems were demonstrated in several studies
(Abelson et al, 1990; Moller et al, 1984; Mortemousque, 2007; Rimas et al, 1992).
According to our findings, we can describe a dynamic response profile to specific
provocation. In our study, NPT response at the 1st minute was faster than CPT (p=0.005),
with 15/21 patients presenting a positive NPT versus 6/21 patients with positive CPT. We
speculate that this can eventually be explained by the existence of particular characteristics
in nasal and ocular mucosa, resulting in differences related to the contact with the allergen
and/or the time response of type I hypersensitivity. The NPT progressive response was
linear whereas CPT one was exponential, till the 5th minute of response.
On the other hand, CPT response was stronger at the 5th minute when comparing to NPT,
achieving borderline significance (p=0.05). This corroborates other results related to the
evaluation of patient discomfort of NPT versus CPT using a visual-analogue scale, with a
higher discomfort being appointed to CPT (Riechelmann et al, 2003). Apparently, these
results are different from the study of Malmberg et al, 1978, in which the conjunctiva of 55%
of the patients that underwent both NPT and CPT, using sequentially diluted allergen
solutions, was less sensitive to allergen challenge than nasal mucosa. However, the intensity
of the positive CPT response was not described in this study. Our patients submitted to CPT
had higher specific IgE values, but it is unlikely that this could explain the higher intensity
symptoms score. The absence of a direct correlation between the degree of allergen
sensitization and the severity of clinical symptoms is well known.
As expected by direct allergen exposure, the higher intensity of nasal response was induced
by NPT, while CPT was responsible for the higher intensity of ocular response.
At the 5th minute, procedures to collect secretions were performed, and consequently the
clinical evaluation of the response to specific provocation tests was disrupted. However,
patients were clinically monitored till 4th hour. Interestingly, after the 5th minute, the
intensity of the conjunctival response rapidly decreased while a similar intensity of nasal
response persisted for a longer period. This data is not shown because the procedures for
collection of secretions could alter the dynamic of response.
Even though the allergen was unilaterally inoculated, NPT induced bilateral nasal
symptoms in 100% and bilateral ocular symptoms in 47.6%. On the other hand, CPT
induced unilateral ocular symptoms in 100% and bilateral nasal symptoms in 95.2%. This is
in accordance with previous studies and can be explained by different mechanisms
mentioned above (Section 4. NPT as a diagnostic approach in respiratory allergy). An
additional explanation for the higher number of patients with nasal symptoms induced by
CPT, when comparing with the number of patients in whom NPT induced ocular
symptoms, is the direct contact of the inoculated allergen with the nasal mucosa, through its
passage via naso-lacrimal duct.
This study describes, for the first time to our knowledge, the clinical patterns of NPT and
CPT responses, using a clinical score system. NPT is faster than CPT and has a linear
progression, while CPT has an exponential progression and has a stronger response. The
induction of both nasal and ocular responses by NPT or CPT, corroborates the systemically
response triggered by local allergen application. Although both methodologies can elicit
extra-local symptoms, these are safe procedures. Finally, these data support the applicability
of CPT in the diagnosis of allergic rhinitis, even in the absence of ocular signs/symptoms,
surpassing some NPT limitations (such as nasal polyps or deviated nasal septum) and
decreasing specific challenge risk.
6. Conclusion
The specific provocation tests have been widely used in the investigation of
pathophysiological mechanisms, immunological and therapeutic aspects of allergic disease,
since they mimic the response to allergen exposure, under controlled conditions. It is well
known that NPT has limitations, but it has been helpful to a better clarification of the
underlying mechanisms of allergic reaction, and also to recognize the systemic framework
of allergic disease. The usefulness of NPT is focused in the diagnosis of allergic rhinitis itself,
but it has also a relevant role in the diagnosis of allergic respiratory disease. The upper and
lower airways do not exist as anatomically and functionally distinct areas. There are
important relationships between both the nose and the paranasal sinuses, and asthma. These
epidemiological, clinical and immunopathologic concordance between allergic rhinitis and
asthma supports the concept of bidirectional "unified airway" respiratory inflammation
model. Multiple evidence supports a close interaction and influence of the nose on
contiguous and distant organs via neural reflex and systemic inflammatory processes.
In clinical practice, NPT plays a central role in the diagnosis of allergic rhinitis in some
circumstances, as described. This is the only method that could establish the correct
aetiology of the allergic disease, namely local allergic rhinitis and occupational rhinitis. The
specific therapeutic implications emphasize the attempt to reach the most complete
diagnostic approach.
The monitoring of the response to NPT is not standardized, but several parameters have
been used, for example symptom scores. Our data suggest that the clinical symptom pattern
to NPT develops has a dynamic response which is characterized by a linear progression of
symptoms intensity till a 5th minute peak. The prevalence of non-nasal symptoms had a
great variability in the studies performed by our group. Those symptoms had a lower score
comparing to nasal symptoms. In our opinion, the symptom score is a valuable method to
monitor the NPT response.
7. Acknowledgements
We would like to acknowledge Dr Borja Bartolomé, Bial Aristegui, I&D Department, Bilbao,
Spain; Dr António Martinho & Dr Artur Paiva, PhD, Histocompatibility Center, Coimbra,
Portugal.
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Nasal Provocation Test in the Diagnosis of Allergic Rhinitis

Chapter · March 2012

Daniel Machado Oliveira Celso Pereira

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Nasal Provocation Test in

2.1 Multissensitized patients

2.2 Local allergic rhinitis

2.3 Occupational allergic rhinitis

2.4 Investigational research

3. Nasal provocation test

3.2.1 Clinical symptom scores

3.2.3 Laboratorial measurements: Inflammatory allergic mediators and cells

In our Immunoallergy Department, we performed a study to evaluate the concentrations of

In another study conducted by our group, 21 allergic patients were submitted to

3.2.4 Assessment on nasal Nitric Oxide

3.3 Criteria for positive NPT

4. NPT as a diagnostic approach in respiratory allergy

5. Characterization of NPT score symptom response

rhinomanometry and acoustic rhinometry. The other methods, such as immunological

5.1 Clinical symptom score pattern

During natural exposure, The highest nasal

Nasal congestion was Nasal congestion and

5.2 Comparison of Dermatophagoides pteronyssinus nasal provocation test versus

5.2.1 Material and methods

Braunstahl, GJ. (2005). The unified immune system: Respiratory tract–nasobronchial

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