Tailoring the Ruggiero-Klinghardt Protocol to Immunotherapy of Autism

Corresponding Author: Marco Ruggiero Silver Spring Sagl, ArzoMendrisio, 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.


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 noninfectious noxae that may be found in conjunction with and possibly responsible for, persistent Lyme disease.
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 extraaxial 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.

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.     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 substeps, 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 finetuning 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 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.