Estrogens as a Possible Therapeutic Strategy for the Management of Neuroinflammation and Neuroprotection in COVID-19.

The Coronavirus disease 2019 (COVID-19) affects several tissues, including the central and peripheral nervous system. It has also been related to signs and symptoms that suggest neuroinflammation with possible effects in the short, medium, and long term. Estrogens could have a positive impact on the management of the disease, not only due to its already known immunomodulator effect, but also activating other pathways that may be important in the pathophysiology of COVID-19, such as the regulation of the virus receptor and its metabolites. In addition, they can have a positive effect on neuroinflammation secondary to pathologies other than COVID-19. The aim of this study is to analyze the molecular mechanisms that link estrogens with their possible therapeutic effect for neuroinflammation related to COVID-19. Advanced searches were performed in scientific databases as Pub- Med, ProQuest, EBSCO, the Science Citation index, and clinical trials. Estrogens have been shown to participate in the immune modulation of the response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to this mechanism, we propose that estrogens can regulate the expression and activity of the Angiotensin-converting enzyme 2 (ACE2), reestablishing its cytoprotective function, which may be limited by its interaction with SARS-CoV-2. In this proposal, estrogens and estrogenic compounds could increase the synthesis of Angiotensin-(1-7) (Ang-(1-7)) that acts through the Mas receptor (MasR) in cells that are being attacked by the virus. Estrogens can be a promising, accessible, and low-cost treatment for neuroprotection and neuroinflammation in patients with COVID-19, due to its direct immunomodulatory capacity in decreasing cytokine storm and increasing cytoprotective capacity of the axis ACE2/Ang (1-7)/MasR.

Role of the MicroRNAs in the Pathogenic Mechanism of Painful Symptoms in Long COVID: Systematic Review.

The ongoing pandemic of COVID-19 has caused more than 6.7 million tragic deaths, plus, a large percentage of people who survived it present a myriad of chronic symptoms that last for at least 6 months; this has been named as long COVID. Some of the most prevalent are painful symptoms like headache, joint pain, migraine, neuropathic-like pain, fatigue and myalgia. MicroRNAs are small non-coding RNAs that regulate genes, and their involvement in several pathologies has been extensively shown. A deregulation of miRNAs has been observed in patients with COVID-19. The objective of the present systematic review was to show the prevalence of chronic pain-like symptoms of patients with long COVID and based on the expression of miRNAs in patients with COVID-19, and to present a proposal on how they may be involved in the pathogenic mechanisms of chronic pain-like symptoms. A systematic review was carried out in online databases for original articles published between March 2020 to April 2022; the systematic review followed the PRISMA guidelines, and it was registered in PROSPERO with registration number CRD42022318992. A total of 22 articles were included for the evaluation of miRNAs and 20 regarding long COVID; the overall prevalence of pain-like symptoms was around 10 to 87%, plus, the miRNAs that were commonly up and downregulated were miR-21-5p, miR-29a,b,c-3p miR-92a,b-3p, miR-92b-5p, miR-126-3p, miR-150-5p, miR-155-5p, miR-200a, c-3p, miR-320a,b,c,d,e-3p, and miR-451a. The molecular pathways that we hypothesized to be modulated by these miRNAs are the IL-6/STAT3 proinflammatory axis and the compromise of the blood-nerve barrier; these two mechanisms could be associated with the prevalence of fatigue and chronic pain in the long COVID population, plus they could be novel pharmacological targets in order to reduce and prevent these symptoms.

Neuropathic Pain in Parkinson's Disease.

Pain is a well-recognized and important non-motor manifestation in Parkinson disease (PD). Painful or unpleasant sensations in PD can be classified as musculoskeletal, dystonia, akathisia, radicular, and central or primary pain; the last two are associated with neuropathic pain. Particularly, neuropathic pain in PD has not been fully clarified; therefore, it goes somewhat unnoticed, and the affected patients do not receive adequate pain treatment. The main purpose of this literature review was to identify the incidence of neuropathic pain in PD and the involvement of dopamine of this type of pain by the integration of different lines of investigation. In this review, a search was conducted using PubMed, ProQuest, EBSCO, Medline, EMBASE, and the Science Citation index for studies evaluating pain in patients with PD. The inclusion criteria were as follows: original articles that evaluated incidence and possible mechanism of neuropathic, central, and radicular pain in PD. Nine studies related to the incidence of neuropathic pain in PD suggest the activation of cerebral areas, such as the cortex, striatum, amygdala, thalamus, raphe nuclei, and locus coeruleus. Neuropathic pain is related to altered levels of dopamine, serotonin, and norepinephrine; these neurotransmitters are related to the sensitive and emotional dimensions of pain. Dopamine could cause hypersensitivity to pain, either indirectly through modulatory effects on affective pain processing and/or directly by affecting the neural activity in key areas of the brain that modulate pain. A considerable proportion of patients with PD suffer neuropathic pain; however, it has been disregarded, this has led to an inability to achieve an adequate treatment and a decrease in pain to improve the quality of life of these patients. We consider that neuropathic pain in PD is possibly induced by neurophysiological changes due to the degradation of dopaminergic neurons.

Trends in Gliosis in Obesity, and the Role of Antioxidants as a Therapeutic Alternative.

Obesity remains a global health problem. Chronic low-grade inflammation in this pathology has been related to comorbidities such as cognitive alterations that, in the long term, can lead to neurodegenerative diseases. Neuroinflammation or gliosis in patients with obesity and type 2 diabetes mellitus has been related to the effect of adipokines, high lipid levels and glucose, which increase the production of free radicals. Cerebral gliosis can be a risk factor for developing neurodegenerative diseases, and antioxidants could be an alternative for the prevention and treatment of neural comorbidities in obese patients. AIM: Identify the immunological and oxidative stress mechanisms that produce gliosis in patients with obesity and propose antioxidants as an alternative to reducing neuroinflammation. METHOD: Advanced searches were performed in scientific databases: PubMed, ProQuest, EBSCO, and the Science Citation index for research on the physiopathology of gliosis in obese patients and for the possible role of antioxidants in its management. CONCLUSION: Patients with obesity can develop neuroinflammation, conditioned by various adipokines, excess lipids and glucose, which results in an increase in free radicals that must be neutralized with antioxidants to reduce gliosis and the risk of long-term neurodegeneration.

Correction: Long, S. SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives. Viruses 2020, 13, 1923.

The author is no longer affiliated with the original institution "AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA", so the author wishes to change the affiliation to "Independent Researcher, Clarksburg, MD 20871, USA" [...].

The emerging role of ultrasound in detecting interstitial lung disease in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the potential role of US in the detection of ILD in a cohort of patients with RA. METHODS: Patients with diagnosis of RA were consecutively enrolled. All patients underwent pulmonary examination, laboratory data, DLCO measure, chest HRCT and radiographs, and US examination. A healthy group was included as control group. US was performed according the 14-intercostal space scanning protocol using the following semiquantitative scale [0=normal (≤5 B-lines); 1=slight (≥6 and ≤15 B-lines); 2=moderate, (≤16 and ≥30 B-lines); 3=severe (≥30 B-lines)]. RESULTS: A total of 74 RA patients and 74 healthy controls were included. Thirty of 74 patients (40.5%) showed US signs of ILD with respect to the healthy controls (3 subjects, 4.1%) (P<0.001); whereas HRCT showed ILD in 27 (36.4%) of 74 patients. Among the 30 patients that showed US findings of ILD, 17 (56.6%) were asymptomatic from respiratory view-point. The sensitivity and specificity of US were 92% and 89% respectively. A positive correlation between US and HRCT findings were found (P<0.001) whereas no correlation was found with chest radiographs and DLCO findings. Positive association between US findings and DAS28-ESR, anti-CCP and RF (P<0.01 for each respectively) was found. Feasibility, represented by the mean time spent to perform the pulmonary US assessment was 7.8minutes (±SD 1.2, range 6 to 10minutes). CONCLUSIONS: Our results support the potential of US in detect accurately ILD in patients with RA and provide a rationale to consider it as a friendly screening tool to be implemented in early phases of the disease.

Potential Neuroprotective Effect of Cannabinoids in COVID-19 Patients.

The global pandemic caused by the SARS-CoV-2 virus began in early 2020 and is still present. The respiratory symptoms caused by COVID-19 are well established. However, neurological manifestations that may result from direct or indirect neurological damage after SARS-CoV-2 infection have been reported frequently. The main proposed pathophysiological processes leading to neurological damage in COVID-19 are cerebrovascular disease and indirect inflammatory/ autoimmune origin mechanisms. A growing number of studies confirm that neuroprotective measures should be maintained in COVID-19 patients. On the other hand, cannabinoids have been the subject of various studies that propose them as potentially promising drugs in chronic neurodegenerative diseases due to their powerful neuroprotective potential. In this review, we addresses the possible mechanism of action of cannabinoids as a neuroprotective treatment in patients infected by SARS-CoV-2. The endocannabinoid system is found in multiple systems within the body, including the immune system. Its activation can lead to beneficial results, such as a decrease in viral entry, a reduction of viral replication, and a reduction of pro-inflammatory cytokines such as IL-2, IL-4, IL-6, IL-12, TNF-α, or IFN-c through CB2R expression induced during inflammation by SARS-CoV-2 infection in the central nervous system.

Ultrasensitive detection and quantification of viral nucleic acids with Raindance droplet digital PCR (ddPCR).

Sensitive detection of viral nucleic acids is critically important for diagnosis and monitoring of the progression of infectious diseases such as those caused by SARS-CoV2, HIV-1, and other viruses. In HIV-1 infection cases, assessing the efficacy of treatment interventions that are superimposed on combination antiretroviral therapy (cART) has benefited tremendously from the development of sensitive HIV-1 DNA and RNA quantitation assays. Simian immunodeficiency virus (SIV) infection of Rhesus macaques is similar in many key aspects to human HIV-1 infection and consequently this non-human primate (NHP) model has and continues to prove instrumental in evaluating HIV prevention, treatment and eradication approaches. Cell and tissue associated HIV-1 viral nucleic acids have been found to serve as useful predictors of disease outcome and indicators of treatment efficacy, highlighting the value of and the need for sensitive detection of viruses in cells/tissues from infected individuals or animal models. However, viral nucleic acid detection and quantitation in such sample sources can often be complicated by high nucleic acid input (that is required to detect ultralow level viruses in, for example, cure research) or inhibitors, leading to reduced detection sensitivity and under-quantification, and confounded result interpretation. Here, we present a step-by-step procedure to quantitatively recover cell/tissue associated viral DNA and RNA, using SIV-infected Rhesus macaque cells and tissues as model systems, and subsequently quantify the viral DNA and RNA with an ultrasensitive SIV droplet digital PCR (ddPCR) assay and reverse transcription ddPCR (RT-ddPCR) assay, respectively, on the Raindance ddPCR platform. The procedure can be readily adapted for a broad range of applications where highly sensitive nucleic acid detection and quantitation are required.

In pursuit of sensitivity: Lessons learned from viral nucleic acid detection and quantification on the Raindance ddPCR platform.

Sensitive PCR detection of viral nucleic acids plays a critical role in infectious disease research, diagnosis and monitoring. In the context of SARS-CoV-2 detection, recent reports indicate that digital PCR-based tests are significantly more sensitive than traditional qPCR tests. Numerous factors can influence digital PCR reaction sensitivity. In this review, using a model for human HIV infection and the Raindance ddPCR platform as an example, we describe technical aspects that contribute to sensitive viral signal detection in DNA and RNA from tissue samples, which often harbor viral reservoirs and serve as better predictors of disease outcome and indicators of treatment efficacy.

SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives.

SARS-CoV-2, the etiologic agent at the root of the ongoing COVID-19 pandemic, harbors a large RNA genome from which a tiered ensemble of subgenomic RNAs (sgRNAs) is generated. Comprehensive definition and investigation of these RNA products are important for understanding SARS-CoV-2 pathogenesis. This review summarizes the recent progress on SARS-CoV-2 sgRNA identification, characterization, and application as a viral replication marker. The significance of these findings and potential future research areas of interest are discussed.

Putative mechanism of neurological damage in COVID-19 infection.

The recent pandemic of the coronavirus infectious disease 2019 (COVID-19) has affected around 192 countries, and projections have shown that around 40% to 70% of world population could be infected in the next months. COVID-19 is caused by the virus SARS- CoV-2, it enters the cells through the ACE2 receptor (angiotensin converting enzyme 2). It is well known that SARS-CoV-2 could develop mild, moderate, and severe respiratory symptoms that could lead to death. The virus receptor is expressed in different organs such as the lungs, kidney, intestine, and brain, among others. In the lung could cause pneumonia and severe acute respiratory syndrome (SARS). The brain can be directly affected by cellular damage due to viral invasion, which can lead to an inflammatory response, by the decrease in the enzymatic activity of ACE2 that regulates neuroprotective, neuro-immunomodulatory and neutralizing functions of oxidative stress. Another severe damage is hypoxemia in patients that do not receive adequate respiratory support. The neurological symptoms that the patient presents, will depend on factors that condition the expression of ACE2 in the brain such as age and sex, as well as the mechanism of neuronal invasion, the immune response and the general state of the patient. Clinical and histopathological studies have described neurological alterations in human patients with COVID-19. These conditions could have a possible contribution to the morbidity and mortality caused by this disease and may even represent the onset of neurodegenerative activity in recovered patients.The recent pandemic of the coronavirus infectious disease 2019 (COVID-19) has affected around 192 countries, and projections have shown that around 40% to 70% of world population could be infected in the next months. COVID-19 is caused by the virus SARS- CoV-2, it enters the cells through the ACE2 receptor (angiotensin converting enzyme 2). It is well known that SARS-CoV-2 could develop mild, moderate, and severe respiratory symptoms that could lead to death. The virus receptor is expressed in different organs such as the lungs, kidney, intestine, and brain, among others. In the lung could cause pneumonia and severe acute respiratory syndrome (SARS). The brain can be directly affected by cellular damage due to viral invasion, which can lead to an inflammatory response, by the decrease in the enzymatic activity of ACE2 that regulates neuroprotective, neuro-immunomodulatory and neutralizing functions of oxidative stress. Another severe damage is hypoxemia in patients that do not receive adequate respiratory support. The neurological symptoms that the patient presents, will depend on factors that condition the expression of ACE2 in the brain such as age and sex, as well as the mechanism of neuronal invasion, the immune response and the general state of the patient. Clinical and histopathological studies have described neurological alterations in human patients with COVID-19. These conditions could have a possible contribution to the morbidity and mortality caused by this disease and may even represent the onset of neurodegenerative activity in recovered patients.

Comparing intraoperative parathyroid identification based on surgeon experience versus near infrared autofluorescence detection - A surgeon-blinded multi-centric study.

BACKGROUND: Near infrared autofluorescence (NIRAF) detection has previously demonstrated significant potential for real-time parathyroid gland identification. However, the performance of a NIRAF detection device - PTeye® - remains to be evaluated relative to a surgeon's own ability to identify parathyroid glands. METHODS: Patients eligible for thyroidectomy and/or parathyroidectomy were enrolled under 6 endocrine surgeons at 3 high-volume institutions. Participating surgeons were categorized based on years of experience. All surgeons were blinded to output of PTeye® when identifying tissues. The surgeon's performance for parathyroid discrimination was then compared with PTeye®. Histology served as gold standard for excised specimens, while expert surgeon's opinion was used to validate in-situ tissues. RESULTS: PTeye® achieved 92.7% accuracy across 167 patients recruited. Junior surgeons (<5 years of experience) were found to have lower confidence in parathyroid identification and higher tissue misclassification rate per specimen when compared to PTeye® and senior surgeons (>10 years of experience). CONCLUSIONS: NIRAF detection with PTeye® can be a valuable intraoperative adjunct technology to aid in parathyroid identification for surgeons.

Intersticiopatía pulmonar subclínica en pacientes con esclerosis sistémica. Estudio piloto sobre el papel del ultrasonido / Subclinical Interstitial Lung Disease in Patients with Systemic Sclerosis. A Pilot Study on the Role of Ultrasound

Introducción: La enfermedad pulmonar intersticial (EPI) es una complicación común de la esclerosis sistémica (ES). El empleo de la tomografía computarizada de alta resolución (TACAR) se ve muy limitado, y el ultrasonido pulmonar (USP) puede ser un instrumento alternativo para la evaluación de la EPI. Objetivo: Determinar la validez del USP en la detección temprana de la EPI en pacientes con ES. Métodos: Se incluyeron 68 pacientes con ES≥18 años sin síntomas respiratorios. Un reumatólogo valoró el estado respiratorio subclínico, otro reumatólogo, cegado a la evaluación clínica realizó el USP. Para determinar la validez concurrente se realizó una TACAR. Resultados: Un 41,2% de pacientes mostró EPI por USP, a diferencia de los controles sanos (4,8%) (p=0,0001). Las variables asociadas con los hallazgos de EPI al USP fueron anticuerpos anti-centrómero (p=0,005) y la puntuación de piel RSS (p=0,004). Se encontró una correlación positiva entre los hallazgos de EPI por USP y TACAR (p=0,001). La sensibilidad fue del 91,2% y la especificidad de 88,6%. Una buena confiabilidad entre observadores de los hallazgos por USP fue observada (k=0,72). Conclusiones: Al ser una herramienta alternativa válida, confiable y factible, consideramos que el USP puede ser implementado para la detección temprana de EPI en ES.(AU)

Introduction: Interstitial lung disease (ILD) is a common comorbidity present in patients with systemic sclerosis (SSc). Employment of high-resolution computed tomography (HRCT) is very limited and lung ultrasound (LUS) can be an alternative tool for the early evaluation of ILD. Objective: To determine the validity of LUS in the early detection of ILD in patients with SSc.Methods: Sixty-eight patients with SSc ≥18 years without respiratory symptoms were included. A rheumatologist rated the subclinical respiratory condition, another rheumatologist blinded to the clinical assessment performed the LUS. To determine validity HRCT was performed as well. Results: Prevalence of ILD in SSc patients was 41.2% in contrast to the 4.8% healthy controls (P=.0001). Variables associated with LUS and HRCT findings were anti-centromere antibodies (P=.005) and the Rodnan skin score (P=.004). A positive correlation was present between the findings of HRCT and LUS (P=.001). Sensitivity and specificity were 91.2% and 88.6% respectively. Good reliability in the LUS findings was found between observers (k=.72). Conclusions: By proving to be a valid, trustworthy and feasible alternative tool, we consider that LUS can be implemented for the early detection of ILD in SSc.(AU)

Development of a reverse transcription droplet digital PCR (RT-ddPCR) assay for sensitive detection of simian immunodeficiency virus (SIV).

BACKGROUND: Simian immunodeficiency virus (SIV)-infected rhesus macaques constitute an excellent model of human HIV infection. Sensitive detection of SIV RNA in cell and tissue samples from infected animals subjected to treatment regimens becomes especially critical in determining which therapeutic attempts are successful, and consequently, which interventions should be prioritized in HIV cure research. RESULTS: In this report, we describe the design and testing of a Raindance ddPCR platform-based, sensitive SIV reverse transcription droplet digital PCR (RT-ddPCR) assay by exploring the combinations of various priming conditions and reverse transcriptases, and testing one-step vs. two-step procedures, to eliminate background signal(s) and enable detection and quantification of low level target signals. CONCLUSIONS: Similar reaction conditions and assay validation procedures can be explored for potential development of additional assays for other applications that require sensitive detection of low-level targets in RNA samples.

Subclinical Interstitial Lung Disease in Patients with Systemic Sclerosis. A Pilot Study on the Role of Ultrasound. / Intersticiopatía pulmonar subclínica en pacientes con esclerosis sistémica. Estudio piloto sobre el papel del ultrasonido.

INTRODUCTION: Interstitial lung disease (ILD) is a common comorbidity present in patients with systemic sclerosis (SSc). Employment of high-resolution computed tomography (HRCT) is very limited and lung ultrasound (LUS) can be an alternative tool for the early evaluation of ILD. OBJECTIVE: To determine the validity of LUS in the early detection of ILD in patients with SSc. METHODS: Sixty-eight patients with SSc ≥18 years without respiratory symptoms were included. A rheumatologist rated the subclinical respiratory condition, another rheumatologist blinded to the clinical assessment performed the LUS. To determine validity HRCT was performed as well. RESULTS: Prevalence of ILD in SSc patients was 41.2% in contrast to the 4.8% healthy controls (P=.0001). Variables associated with LUS and HRCT findings were anti-centromere antibodies (P=.005) and the Rodnan skin score (P=.004). A positive correlation was present between the findings of HRCT and LUS (P=.001). Sensitivity and specificity were 91.2% and 88.6% respectively. Good reliability in the LUS findings was found between observers (k=.72). CONCLUSIONS: By proving to be a valid, trustworthy and feasible alternative tool, we consider that LUS can be implemented for the early detection of ILD in SSc.

Neurophysiological Mechanisms Related to Pain Management in Bone Tumors.

BACKGROUND: Primary and metastatic bone tumor incidence has increased in the previous years. Pain is a common symptom and is one of the most important related factors to the decrease of quality of life in patients with bone tumor. Different pain management strategies are not completely effective and many patients afflicted by cancer pain cannot be controlled properly. In this sense, we need to elucidate the neurophysiology of cancer-induced pain, contemplating other components such as inflammation, neuropathies and cognitive components regarding bone tumors, and thus pave the way for novel therapeutic approaches in this field. AIM: This study aims to identify the neurophysiology of the mechanisms related to pain management in bone tumors. METHODS: Advanced searches were performed in scientific databases: PubMed, ProQuest, EBSCO, and the Science Citation index to get information about the neurophysiology mechanisms related to pain management in bone tumors. RESULTS: The central and peripheral mechanisms that promote bone cancer pain are poorly understood. Studies have shown that bone cancer could be related to neurochemicals produced by tumor and inflammatory cells, coupled with peripheral sensitization due to nerve compression and injury caused by tumor growth. The activity of mesolimbic dopaminergic neurons, substance P, cysteine/ glutamate antiporter, and other neurochemical dynamics brings us putative strategies to suggest better and efficient treatments against pain in cancer patients. CONCLUSION: Cancer-induced bone pain could include neuropathic and inflammatory pain, but with different modifications to the periphery tissue, nerves and neurochemical changes in different neurological levels. In this sense, we explore opportunity areas in pharmacological and nonpharmacological pain management, according to pain-involved mechanisms in this study.

The Mechanisms of Action of Botulinum Toxin Type A in Nociceptive and Neuropathic Pathways in Cancer Pain.

BACKGROUND: Botulinum toxin type A (BoNT-A) is widely employed for cosmetic purposes and in the treatment of certain diseases such as strabismus, hemifacial spasm and focal dystonia among others. BoNT-A effect mainly acts at the muscular level by inhibiting the release of acetylcholine at presynaptic levels consequently blocking the action potential in the neuromuscular junction. Despite the great progress in approval and pharmaceutical usage, improvement in displacing BoNT-A to other pathologies has remained very limited. Patients under diagnosis of several types of cancer experience pain in a myriad of ways; it can be experienced as hyperalgesia or allodynia, and the severity of the pain depends, to some degree, on the place where the tumor is located. Pain relief in patients diagnosed with cancer is not always optimal, and as the disease progresses, transition to more aggressive drugs, like opioids is sometimes unavoidable. In recent years BoNT-A employment in cancer has been explored, as well as an antinociceptive drug; experiments in neuropathic, inflammatory and acute pain have been carried out in animal models and humans. Although its mechanism has not been fully known, evidence has shown that BoNT-A inhibits the secretion of pain mediators (substance P, Glutamate, and calcitonin gene related protein) from the nerve endings and dorsal root ganglion, impacting directly on the nociceptive transmission through the anterolateral and trigeminothalamic systems. AIM: The study aimed to collect available literature regarding molecular, physiological and neurobiological evidence of BoNT-A in cancer patients suffering from acute, neuropathic and inflammatory pain in order to identify possible mechanisms of action in which the BoNT-A could impact positively in pain treatment. CONCLUSION: BoNT-A could be an important neo-adjuvant and coadjuvant in the treatment of several types of cancer, to diminish pro-tumor activity and secondary pain.

Development and optimization of a simian immunodeficiency virus (SIV) droplet digital PCR (ddPCR) assay.

Accurate and sensitive quantification of rebound competent HIV that persists despite combination antiretroviral treatment (cART), including in latently infected cells (i.e., viral reservoir), is critical for evaluating cure strategies for decreasing or eliminating this reservoir. Simian immunodeficiency virus (SIV)-infected Rhesus macaques are an important non-human primate (NHP) system for studying potential cure strategies as they model many key aspects of human HIV-infection including the persistence of a latent viral reservoir in resting memory CD4+ T cells in animals receiving prolonged cART. In this report, we describe the design and testing of a sensitive SIV droplet digital PCR (ddPCR) assay through exploring the combination and optimization of different probe systems (including single, double quencher probes and minor groove binder (MGB) probes) and reaction conditions to eliminate background signal(s), ensure distinct target signal cluster separation from non-target signals, and enable detection and quantification of low level authentic target signals. Similar reaction conditions and assay validation procedures can be explored for potential development of additional assays for other applications that require sensitive detection of low-level targets in a large background of nucleic acid input derived from cell or tissue sources.