Tailoring the Ruggiero-Klinghardt Protocol to Immunotherapy of Autism

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© 2018 Nicola Antonucc i, Dietrich Kli nghardt, Stefan ia Pacini and Marco Ruggiero. This open access article is distributed
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American Journal of Immunology
Original Research Paper
Tailoring the Ruggiero-Klinghardt Protocol to
Immunotherapy of Autism
1Nicola Antonucci, 2Dietrich Klinghardt, 3Stefania Pacini and 3Marco Ruggiero
1Biomedical Centre for Autism Research and Treatment, Bari, Italy
2Sophia Health Institute and Klinghardt Academy, Woodinville, WA USA
3Silver Spring Sagl, Arzo-Mendrisio, Switzerland
Article history
Received: 02-03-2019
Revised: 17-04-2019
Accepted: 24-04-2019
Corresponding Author:
Marco Ruggiero
Silver Spring Sagl, Arzo-
Mendrisio, Switzerland
Email: info@bravo-europe.com
Abstract: Here, we describe the adaptation to the field of autism of an
original procedure denominated the "Ruggiero-Klinghardt Protocol" (RK
Protocol), a procedure that represents a paradigm change with significant
implications for chronic conditions where immunotherapy may prove
effective; that is from silent infections to neurodegenerative diseases,
autism and cancer. In the context of autism, the modified RK Protocol that
we propose here serves the purpose of discovering hidden infections that
may be associated with autism and contribute to its symptoms. This notion
is consistent with the observation that immune modulating molecules are
effective in autism treatment. In the RK Protocol modified for autism, we
introduce the Autism Treatment Evaluation Checklist (ATEC), a more
objective and sophisticated method of evaluation compared with the
Clinical Global Impression of Improvement scale that was previously used,
independently of the RK Protocol, to evaluate the effectiveness of
immunotherapy of autism. The modifications that we present in this study
take advantage of the experience that has accumulated in the two years after
the publication of the original RK Protocol. Similarly to the original RK
Protocol, this new version offers the advantage of being safe and relatively
inexpensive since it does not require sophisticated instruments; because of
this, it can be implemented in different parts of the world. We envisage that
implementation of the RK Protocol modified for autism may contribute to
decrease the burden of the disease as it enables prevention, early diagnosis
and treatment on a large scale.
Keywords: Ultrasound, Autonomic Response Testing, Immune System,
Imaging, Neuroinflammation, Autism
Introduction
The Ruggiero-Klinghardt Protocol (RK Protocol)
described in 2017 in the American Journal of
Immunology (Ruggiero and Klinghard 2017), was
developed with the goal of improving the sensitivity of
diagnosis and efficacy of therapy in chronic conditions.
Although this protocol was originally developed for
persistent Lyme disease, we realized that the full
protocol, or parts of it, may be useful in other chronic
conditions, including autism, with particular reference to
optimization of immunotherapeutic approaches. We
recently described clinical cases of autism successfully
treated with an immunotherapeutic approach that yielded
significant results leading to complete normalization of
some hallmark symptoms of autism (Antonucci et al.,
2019a). Here we propose an adaptation of the RK
Protocol to immunotherapy for autism. The protocol that
will be illustrated below in detail is composed of a
sequence of diagnostic and therapeutic procedures that
aim at increasing sensitivity and specificity of diagnosis
and at assessing and optimizing the efficacy of
treatments. The diagnostic arm of the protocol described
here is based on Autonomic Response Testing (ART)
and diagnostic ultrasonography. ART is a manual
biofeedback technique developed by Dr. Klinghardt that
has the objective of evaluating the presence and/or
persistence of spirochete and other infectious and non-
infectious noxae that may be found in conjunction with
and possibly responsible for, persistent Lyme disease.

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ART may be considered an evolution and an
improvement of the approach that was proposed by
Omura in 1985 and validated in several trials that
include two randomized-order blinded registered clinical
trials. ART developed by Dr. Klinghardt is currently
utilized by scores of independent researchers for
diagnosis and treatment of a variety of diseases ranging
from Lyme to cancer (for details and references
pertaining to ART, please see Ruggiero and Klinghardt,
2017). As far as the use of ultrasonography in the present
protocol is concerned, this technique can be utilized to
refine the diagnostic hypotheses suggested by ART or it
can be performed independently of ART. In the latter
case, here we describe the areas of the body that we
recommend to study because of their potential
involvement in the pathogenesis of autism. Among the
organs and systems to be evaluated by ultrasonography,
the brain and the meninges, examined by transcranial
ultrasonography as originally reported in Bradstreet et al.
(2014), are of utmost importance as evidence
accumulates linking autism with impaired circulation of
meningeal lymph and consequent accumulation of extra-
axial fluid inside the cranial cavity (Bradstreet et al.,
2014; 2015; Shen et al., 2018). It is worth noticing that
transcranial ultrasonography has a number of advantages
over Magnetic Resonance Imaging (MRI) that is the
technique recently utilized by Shen et al. (2018) to study
extra-axial fluid accumulation in children at risk for
autism. At variance with MRI, transcranial
ultrasonography does not require sophisticated
instruments or dedicated structures, is relatively
inexpensive, is simpler and faster and can be performed
even on hyperactive children without the need of
sedation. In addition, it provides results in real time, can
be easily repeated to evaluate progression of the disease
or results of treatments and, thanks to the portability of
modern ultrasound systems, it can be performed in
places where MRI machines are not available. In short,
the transcranial ultrasonography that we first described
in Ruggiero et al. (2013) and was then applied to the
field of autism (Bradstreet et al., 2014), is a much more
versatile technique that can be performed with ease on a
high number of subjects in any part of the world.
Protocol
Human Subjects
Please notice: All methods described here, are
routinely implemented as common medical procedures.
No experimental procedure, either diagnostic or
therapeutic is described in this protocol. The originality
consists in the logic and in the sequence of the
application of procedures. All procedures described in
the protocol are to be performed by certified Therapists
according to laws and rules regulating medical
treatments in each Country. The procedures here
proposed can be performed with common, commercially
available, medical devices that are approved for medical
use in an authorized medical structure.
Structure of the Protocol
The protocol is here described as a sequence of
diagnostic and therapeutic steps. Each step includes
several sub-steps. The order of the sequence of some
sub-steps may be varied according to individual needs.
For example, in step 3 "Diagnostic Ultrasonography",
organs and regions of the body can be studied in a
sequence different from the one here proposed. It is
important, however, to study all the organs and the
regions of the body listed in the step.
1. General and Autism-Specific Evaluation and
Assessment
1.1. Perform traditional medical examination involving
the collection of anamnesis, the study of previous
laboratory and imaging results and objective
examination
1.2. Perform a specific evaluation for autism. For
example, the diagnostic criteria for autism adopted
at the Biomedical Centre for Autism Research and
Treatment are:
1.2.1. Marked impairment in the use of multiple
nonverbal behaviors such as eye-to-eye gaze,
facial expression, body postures and gestures to
regulate social interaction
1.2.2. Failure to develop peer relationships
appropriate to developmental level
1.2.3. Lack of spontaneous seeking to share
enjoyment, interests or achievements with other
people (e.g., by a lack of showing, bringing or
pointing out objects of interest)
1.2.4. Lack of social or emotional reciprocity
1.2.5. Delay in, or total lack of, the development of
spoken language
1.2.6. In individuals with adequate speech, marked
impairment in the ability to initiate or sustain a
conversation with others
1.2.7. Stereotyped and repetitive use of language or
idiosyncratic language
1.2.8. Lack of varied, spontaneous, make-believe play
or social imitative play appropriate to
developmental level
1.2.9. Encompassing preoccupation with one or more
stereotyped and restricted patterns of interest
that is abnormal either in intensity or focus.
1.2.10. Apparently inflexible adherence to specific
nonfunctional routines or rituals
1.2.11. Stereotyped and repetitive motor mannerisms
(e.g., hand or finger flapping or twisting, or
complex whole-body movements)

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1.2.12. Persistent preoccupation with parts of objects
As a practical example, the patients described in
Antonucci et al. (2019a) met six or more criteria from
this list and had been diagnosed by either a child
neurologist or developmental psychologist, in addition to
receiving the evaluation of the clinician at the
Biomedical Centre for Autism Research and Treatment:
1.3. Autism Treatment Evaluation Checklist (ATEC).
Review the ATEC that has to be compiled by
parents before any type of intervention. The
ATEC is a 77-item diagnostic assessment tool
where a questionnaire, available in several
different languages, is compiled by a parent. It has
been demonstrated effective in evaluating
interventional effects in autism as well as in
following behavioral development over periods of
time. It evaluates specific areas that are pertinent
to the disorder such as speech/language and
communication (section 1); sociability (section 2);
sensory and cognitive awareness (section 3);
physical/health behavior (section 4)
1.4. Collect biological samples for laboratory tests
before performing ultrasonography, either
diagnostic or therapeutic
1.4.1. Follow the instructions of the specialized
laboratory as far as the modalities for collection
of biological samples (urine, stools, blood,
serum, breath) are concerned
2. Autonomic Response Testing (ART)
2.1. Perform ART to examine all the aspects pertaining
to the autonomous response and not only muscle
strength or resistance (for details see Ruggiero and
Klinghardt, 2017)
2.2. Narrow the spectrum of diagnostic hypotheses and
identify organs or region of the body that will be
studied by diagnostic ultrasonography in step 3
2.3. Record the findings of ART that will be used for
comparison in step 6
2.4. Repeat steps 2.1 - 2.3 with a different couple of
Therapist/Assistant; evaluate and record
consistency and reproducibility
3. Diagnostic Ultrasonography
3.1. Use an ultrasound system with echo-color-Doppler
application and with a linear and a convex
transducer
3.2. Study the organs or the regions of the body
indicated by ART
3.2.1. If ART is not implemented, study the following
organs and record all images and
measurements. Record as many images as
possible at different levels of magnification
3.2.1.1. Temporal lobes of the brain through the
temporal squama using a linear probe as
described in Ruggiero et al. (2013)
3.2.1.1.1. Study the meninges, cortex, gray matter
and extra-axial fluid looking for cortical
dysplasia, extra-axial fluid accumulation
and other brain anomalies as described in
Bradstreet et al. (2014) and confirmed by
Shen et al. (2018)
3.2.1.1.2. Figure 1 as an example of transcranial
ultrasonography with the identification of
cortical dysplasia
3.2.1.2. Thyroid
3.2.1.2.1. Look for homogeneity of echostructure,
nodules, cysts and pattern of
vascularization
3.2.1.2.2. Figure 2 as an example of a significant
increase in blood flow that suggests chronic
viral infections or autoimmune processes
3.2.1.3. Salivary glands and deep cervical nodes as
described in Ruggiero and Klinghardt (2017).
and in Antonucci et al. (2019b)
3.2.1.3.1. Look for homogeneity of echostructure and
vascularization of the salivary glands and
for enlarged nodes. Measure the nodes and
document their vascularization and
echostructure
3.2.1.4. Carotid artery, jugular vein and vagus nerve,
bilaterally
3.2.1.4.1. Study arterial and venous blood flow and
vagus nerve echostructure as described in
Ruggiero and Klinghardt (2017)
3.2.1.4.2. Figure 3 as an example of peculiar blood
flow in the carotid artery
3.2.1.5. Spleen and abdominal organs as described in
Ruggiero and Klinghardt (2017)
3.2.1.5.1. Study dimensions, morphology and
echostructure of the spleen as well as blood
flow in the hilum and periphery
3.2.1.5.2. Figure 4 as an example of normal
morphology and echostructure of the
spleen showing normal blood flow
3.2.1.6. Look for abnormalities in morphology,
dimensions, echostructure, vascularization and
blood-flow of abdominal organs
4. Application of Therapeutic Ultrasound
4.1. Select the appropriate pulsed sequence, frequency
and duration of treatment
4.1.1. For the spleen select pulsed sequence indicated
as 50%, frequency of 1 MHz, for 3 min

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4.1.2. For the deep cervical nodes and the vagus nerve,
pulsed sequence indicated as 20%, frequency of
3.3 MHz, for 90 seconds on each side of the neck
4.1.3. For other nodes identified by ART and/or
ultrasonography, pulsed sequence indicated as
20%, frequency of 3.3 MHz, for 90 seconds in
correspondence of each node.
4.1.4. For the brain, pulsed sequence indicated as
10%, frequency of 3.3 MHz, for 90 seconds on
each side of the head using the temporal
acoustic window as described in Ruggiero et al.
(2013) and Bradstreet et al. (2014)
4.2. Apply therapeutic ultrasound with slow circular
movements in order to direct the ultrasound waves
to the targeted organ or structure. Use abundant gel
4.3. Invite the patient to exercise or to breathe slowly
and deeply for about 5 minutes after the last
application of therapeutic ultrasound.
5. Manual Lymphatic Drainage.
5.1. Perform manual lymphatic drainage as described
in Antonucci et al. (2019b).
5.2. Instruct parents or tutors to perform manual
lymphatic drainage every night before sleep.
Fig. 1: Transcranial ultrasonography. Squama temporalis, meninges, subarachnoid space with extra-axial fluid and cortex are visible.
On the right, a putative lesion compatible with cortical dysplasia as described in Bradstreet et al. (2014) can be appreciated.
This figure refers to step 3.2.1.1.2. of the protocol. This image is from the personal archive of one of the Authors and is
presented as an example of transcranial ultrasonography; it does not refer to any of the clinical cases mentioned in this study
Fig. 2: Ultrasonography of the thyroid. In the left panel, the longitudinal projection of the thyroid lobe in B-mode shows an
inhomogenous, rather hypoechoic appearance. In the right panel, echo-color-Doppler shows abnormal increase in blood flow.
This finding is compatible with inflammatory processes that, in the case of the thyroid, are often due to viral or autoimmune
noxae. This figure refers to step 3.2.1.2.2. of the protocol. This image is from the personal archive of one of the Authors and is
presented as an example of ultrasonography of the thyroid; it does not refer to the any of the clinical cases mentioned in this study

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Fig. 3: Ultrasonography of the neck: carotid artery, jugular vein, vagus nerve. Left panel: Right side of the neck, B-mode. The carotid
artery and the jugular vein are clearly visible. The vagus nerve appears as a small triangular structure located posteriorly
inside the carotid sheath between the common carotid artery and the internal jugular vein. Right panel: Left side of the neck,
echo-color-Doppler. Blood flow in the carotid artery and jugular vein is clearly visible. Arterial blood flow appears red
whereas venous blood flow appears blue according to the rule of thumb designated Blue Away Red Towards (BART). The
blue area inside the carotid artery may indicate a condition known as pulsus bisferiens. This figure refers to step 3.2.1.4.2. of
the protocol. This image is from the personal archive of one of the Authors and is presented as an example of ultrasonography
of the neck with study of blood flow; it does not refer to the any of the clinical cases mentioned in this study
Fig. 4: Ultrasonography of the spleen. In this example, dimensions, morphology, echostructure and pattern of vascularization of the
spleen appear normal. This figure refers to step 3.2.1.5.2. of the protocol. This image is from the personal archive of one of
the Authors and is presented as an example of ultrasonography of the spleen with study of blood flow; it does not refer to the
any of the clinical cases mentioned in this study
6. Second ART.
6.1. Perform ART; compare the results with those
obtained in step 2 and record the results and the
comparisons.
6.2. Evaluate and record consistency and
reproducibility with a different couple of
Therapist/Assistant and compare the results with
those obtained in step 2.4
7. Biological Sample Collection.
7.1. Six hours after step 4, collect biological samples
as in step 1.4
8. Individualized Treatment
8.1. Treat the patient according to the standard of care
indicated for each specific condition identified
with the previous steps

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8.2. Perform ART to fine-tune the choice of specific
drugs, supplements or procedures
8.2.1. Confirm the potential efficacy of the treatment in
the preceding step, using diagnostic
ultrasonography as described in the following step
8.2.1.1. Find below examples of effects to be expected
using an immunotherapeutic approach (imuno)
as described in Antonucci et al. (2019a)
8.2.1.1.1. Increase in splenic blood flow due to
activation of macrophages with the release
of nitric oxide as described in Ruggiero et al.
(2014).
8.2.1.1.2. Reduction of signs of neuroinflammation
as observed by transcranial
ultrasonography (step 3.2.1.1.1.).
8.3. Apply targeted therapeutic ultrasound as described
in step 4 with the goal of exploiting the known
therapeutic effects of pulsed ultrasound that
comprise anti-inflammatory effects, enhanced
lymphatic drainage and optimization of drug
uptake and utilization.
9. Evaluation of Efficacy and Assessment of End-point
9.1. Repeat steps 1 to 8 to evaluate the efficacy of the
treatment and its end-point
9.1.1. Second ATEC. Review ATEC compiled after
implementation of this protocol or other
therapeutic intervention. In the following sub-
steps, examples of results evaluated by ATEC
using an immunotherapeutic approach
(imuno) as described in Antonucci et al.
(2019a) are reported. In this example, the
second ATEC was performed after 8 weeks of
treatment with imuno
9.1.1.1. "… the behavioral symptoms described by the
queries "bed-wetting", "wets pants/diapers",
"soils pants/diapers", "diarrhea", "constipation",
"eats too much/too little", "not sensitive to pain"
improved from "moderate" to "not a problem",
thus indicating complete normalization of these
very significant symptoms of autism."
(Antonucci et al., 2019a).
9.1.1.2. "… the behavioral symptoms described by the
queries "shows no affection", "fails to greet
parents", "avoids contact with others", "dislikes
being held/cuddled" improved from "very
descriptive" to "not descriptive", thus indicating
complete normalization of these significant
symptoms of autism." (Antonucci et al., 2019a).
9.1.1.3. "… In section 2, that is the subscale for sociability,
improvements were observed for the symptoms
described by the queries "does not imitate",
"disagreeable/not compliant", "indifferent to
being liked" where the answers showed
improvement from "somewhat descriptive" to "not
descriptive", thus indicating normalization of
behaviors." (Antonucci et al., 2019a)
9.1.2. Adapt and/or modify the therapeutic approach
based on review of ATEC and other results
Discussion
Three of the critical steps in the original RK Protocol
as described in Ruggiero and Klinghardt (2017) were
represented by ART, diagnostic and therapeutic
ultrasound. ART was used to achieve different purposes.
Thus, the initial ART had the goal to identify the organs
or the regions of the body that required further
investigation; to narrow the diagnostic hypotheses and to
provide information on the underlying pathology i.e., the
presence of pathogens, neoplastic cells, abnormal cells or
toxicants. ART was then repeated; this second ART had
the scope of evaluating whether therapeutic ultrasound
had been successful in mobilizing pathogenic noxae
from sanctuaries or reservoirs making them "visible" to
the Therapist performing ART. Thirdly, ART for
specific patient treatment served the purpose of fine-
tuning the therapy. Another critical step was represented
by the use of therapeutic ultrasound that, in the context
of the original RK Protocol, had the role to "squeeze" at
the cellular and molecular level the organs or the
tissues that may have offered a place to hide to
pathogens or other noxae. The original RK Protocol was
designed as a recursion of diagnostic procedures that
served to confirm with each other and the goal of
achieving accurate and early diagnosis in elusive
conditions and provide for individualized treatment.
The original RK Protocol borrowed the "shock and
kill" approach that is used to eliminate the reservoirs of
HIV that are responsible for the latency and persistence
of the virus. The "shock and kill" strategy pursues the
goal of stimulating HIV replication in a latent viral
reservoir; at first sight, such a strategy may appear
counterintuitive as the objective of pharmacological
antiretroviral therapies is to block, not to stimulate, HIV
replication. However, the rationale behind this approach,
as in the RK Protocol, is to render the hidden virus
"visible" to the immune system and to the chemical
drugs (Melkova et al., 2017). Thus, the scope of the
original RK Protocol was to render pathogens, toxicants,
neoplastic cells or cells infected by viruses that would
otherwise be inaccessible to diagnostic and therapeutic
tools, "visible" so that they can be identified and fought
by the Therapist and by the body's immune system.
When the original RK Protocol was developed, it was
envisaged that future applications would have been in the
field of neurodegenerative and neurodevelopmental
conditions with particular reference to autism. In the
context of autism, the modified RK Protocol that we

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propose here might serve the purpose of discovering
hidden infections that may be associated with autism and
contribute to its symptoms. This notion, in conjunction
with the observation that immune modulating molecules
were effective in autism, can be found in the seminal
paper by Bradstreet et al. (2012). In 2019, we were able to
confirm those results with a more potent compound,
(imuno) that yielded impressive clinical results. At
variance with the paper by Bradstreet et al. (2012) our
results in 2019 were confirmed by the ATEC, a more
objective and sophisticated method of evaluation
compared with the Clinical Global Impression of
Improvement scale that was used in 2012. It was based on
this observation that we decided to include the ATEC in
the modified RK Protocol adapted for autism that is here
described. In addition, it is worth noticing that the effects
of immune modulating strategies that work on the
immune-neuro-endocrine axis, such as imuno, may be
slow and progressive; since these approaches function by
rebalancing physiological mechanisms, the effects may go
unnoticed unless specifically addressed. The ATEC has
been introduced in the modified RK Protocol precisely to
address these aspects since it provides a useful tool to
objectively assess the efficacy of the treatment.
The original RK Protocol published in 2017 represented
a novelty in the field of diagnostics and therapeutics
because it aimed at achieving a higher degree of precision
by combining in an integrated and logically sequential
manner, techniques and procedures that have been used for
decades. The modifications that we present in this study are
meant to tailor the protocol to the specific field of autism
and take advantage of the experience that has accumulated
in the two years after the publication of the original RK
Protocol. Similarly to the original RK Protocol, this new
version offers the advantage of being safe and relatively
inexpensive since it does not require sophisticated
instruments or dedicated structures; because of this, it
can be easily implemented in any part of the world.
The latter consideration bears relevance in the
context of prevention, early diagnosis and treatment of
autism. As demonstrated by Shen et al. (2018),
accumulation of cerebrospinal extra-axial fluid is a
reliable brain anomaly that can be observed relatively
early, that is before the onset of clinical symptoms. Study
of extra-axial fluid accumulation may serve the purpose of
identifying children at risk for developing autism, thus
enabling implementation of early interventions aimed at
preventing the development of the disease. Among
suitable interventions, the immunotherapeutic approaches
described by Antonucci et al. (2019a; 2019b) appear most
promising as they address the major pathogenetic factors
responsible for the symptoms of autism. However, the
majority of clinical settings and therapists have no easy
access to MRI for studying extra-axial fluid
accumulation, thus limiting applicability of this
technique to large-scale prevention, early diagnosis and
treatment of autism. The RK Protocol modified for
autism, on the contrary, can be implemented in every
medical office of the world as it does not require
expensive or sophisticated instruments but a common
ultrasound system that has the additional advantage of
being portable. We envisage that implementation of the
RK Protocol modified for autism may contribute to
decrease the burden of the disease as it enables prevention,
early diagnosis and treatment on a large scale.
Acknowledgement
The Authors wish to thank the parents of the autistic
children whose cases are reported here for their priceless
collaboration. The Authors wish to thank Ms. Daniela
Deiosso for inspiring discussion and relentless support
and the Therapists at the Sophia Health Institute for their
precious collaboration. The Authors wish to express their
gratitude for the human and scientific legacy of Dr.
Bradstreet whose insight inspires their work.
Conflict of Interest
Nicola Antonucci is the founder of the Biomedical
Centre for Autism Research and Treatment, a private
clinic. Dietrich Klinghardt is the inventor of ART and
the founder of the Klinghardt Institute, the Klinghardt
Academy, the Institut fuer Neurobiologie and the Sophia
Health Institute, a private clinic. Dr. Klinghardt consults
for several companies producing supplements and other
remedies. Marco Ruggiero is the inventor of a number of
supplements and, together with Stefania Pacini, developed
the supplement mentioned in this study (imuno). Neither
he, nor Dr. Pacini, had any prior knowledge of the
therapies being used, nor of the details of any patient
whose clinical outcomes are summarized in this study.
Marco Ruggiero is member of the Editorial Board of The
American Journal of Immunology and is waived from the
Article Processing fee for this contribution; he receives no
remuneration for his editorial work.
Authors' Contribution
Nicola Antonucci: Performed all the diagnostic and
therapeutic procedures whose results are described in
this study.
Dietrich Klinghardt: Is the inventor and developer
of ART.
Marco Ruggiero and Stefania Pacini: Wrote the
first draft of this paper, provided critical input and
assisted in revising and improving the paper.

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Advisory
No information in this paper is presented by the
Authors as direct medical advice to any individual.
Caregivers, researchers and interested parties should
research all information given. Beginning any significant
biomedical or other interventions that may impact
physiology or making changes to an established regimen
should be discussed with the patient’s physician in
advance. Standard of care for each pathology must be
followed as well as rules and regulations established by
Health Authorities of each Country.
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