Rodent models for oral microbiome research: considerations and challenges- a mini review.

Rodent models have been commonly employed in oral microbiota research to investigate the relationship between bacteria and oral disease. Nevertheless, to apply the knowledge acquired from studies conducted on rodents to a human context, it is crucial to consider the significant spatial and temporal parallels and differences between the oral microbiota of mice and humans. Initially, we outline the comparative physiology and microbiology of the oral cavity of rodents and humans. Additionally, we highlight the strong correlation between the oral microbiome of rodents and genetic makeup, which is influenced by factors including vendor, husbandry practices, and environmental conditions. All of these factors potentially impact the replicability of studies on rodent microbiota and the resulting conclusions. Next, we direct our attention toward the diversity in the microbiome within mice models of disease and highlight the diversity that may potentially affect the characteristics of diseases and, in turn, alter the ability to replicate research findings and apply them to real-world situations. Furthermore, we explore the practicality of oral microbial models for complex oral microbial diseases in future investigations by examining the concept of gnotobiotic and germ-free mouse models. Finally, we stress the importance of investigating suitable techniques for characterizing and managing genetically modified organisms. Future research should consider these aspects to improve oral microbiome research's translational potential.

Differential Expression of Immunohistochemical Markers in Ameloblastoma & Ameloblastic Carcinoma: A Systematic Review and Meta-analysis of observational studies.

Background: Differentiating between ameloblastoma (AB) and ameloblastic carcinoma (AC) is difficult, especially when AB has atypical cytological characteristics or an uncommon clinical history. This systematic review and meta-analysis aimed to elucidate the differential expression of immunohistochemical markers between AB and AC. Methods: We conducted a thorough search of PUBMED and SCOPUS according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to identify cross-sectional studies that compared the expression of immunohistochemical markers in AB and AC. We used a random-effects model to analyze the risk ratios and their corresponding 95% confidence intervals (CIs). The quality of the included studies was assessed using the Newcastle-Ottawa scale. The Egger's test was used to assess publication bias. Results: In total, 301 articles were identified. After excluding irrelevant titles and abstracts, 86 articles were selected for full-text review. We categorized the 41 markers into proliferative and non-proliferative markers. Among non-proliferative markers, nuclear markers were differentially expressed in AB and AC. SOX2 was the only marker that significantly differentiated AB and AC, with an RR of -0.19 (CI 0.10-0.36, I2=0). Conclusion: The current evidence suggests the significance of SOX2 in differentiating between AB and AC, warranting prospective confirmation in well-defined extensive studies. We highlight the paucity of high-quality replicated studies of other markers in this field. Collaborative efforts with standardized techniques are necessary to generate clinically useful immunohistochemical markers.

Assessment of reproducibility and influence of different dental specialties on functional implant prosthodontic score: A prospective clinical study analyzing single-unit implant crowns.

AIM: Implant therapy in partially edentulous patients has become the most adapted and predictable treatment modality. The success rate of implants has been measured in terms of esthetic, biological, and technical factors such as radiographic bone loss, prosthetic complications, and stability. Despite the existence of several indices for the esthetic assessment of implant crowns, a need for functional evaluation of the implant crown with an objective and reproducible score has arisen. The study aims to validate the reproducibility of the functional implant prosthodontic score (FIPS) and the influence exerted by different dental specialties while evaluating posterior single-unit implant crowns. STUDY SETTING AND DESIGN: This was a prospective clinical study. MATERIALS AND METHODS: Fifteen patients with cement-retained single-implant crowns in the posterior region of the jaws were included. Eight examiners, two prosthodontists, two periodontists, two oral surgeons, and two orthodontists evaluated 15 photographs of single-unit implant crowns during the 1-year follow-up examination. The examiners assessed the photographs for FIPS, which includes five parameters for objectively evaluating the single-unit implant crowns. Assessments were performed twice at a gap of 4 weeks. STATISTICAL ANALYSIS USED: Pearson's correlation with a 95% confidence interval was calculated for the intra-examiner and the Kruskal-Wallis test for inter-examiner reproducibility. RESULTS: The mean total FIPS scores for all included examiners were 7.133 for time T1 and 7.074 for time T2, showing a strong Pearson correlation coefficient for intra-examiner reproducibility. No significant difference was analyzed among different specialties with statistically significant values of the Kruskal-Wallis test. CONCLUSION: Intra- and inter-examiner analysis showed very consistent results during the reproducibility assessment of FIPS. The results validated the use of FIPS as a long-term predictive functional evaluation tool for the single-implant crowns in posterior sites irrespective of the effect of different dental specialties. It could be used for risk estimation and prognosis for long-term survival and performance of implant crowns.

Primary Tuberculosis of Buccal and Labial Mucosa: Literature Review and a Rare Case Report of a Public Health Menace.

Tuberculosis (TB) is a chronic granulomatous infectious disorder, caused by Mycobacterium tuberculosis. Despite the recent advancements in antitubercular therapy (ATT), it remains a global public health concern. TB is a leading infectious cause of global mortality, second only to coronavirus disease 2019 (COVID-19). TB of the oral cavity is an uncommon occurrence and may be classified as a primary and secondary form. The primary tubercular lesions are extremely rare, as the intact oral squamous epithelium resists the entry of tubercle bacilli. The commonest oral TB lesion is solitary ulceration with undermined edges, usually on the tongue, that does not exhibit healing with conservative therapies. Owing to the atypical presentation, the oral TB lesions often go unnoticed during clinical examination; hence, an oral physician should be familiar with the various oral manifestations of TB. A timely diagnosis coupled with interdisciplinary treatment is the key to combat disease dissemination. This manuscript aims to report a rare case of primary tuberculosis of the buccal and labial mucosa in a 43-year-old immunocompetent male patient. Buccal and labial mucosa are the infrequently affected sites for primary oral TB lesions. A detailed literature search carried out on the Google Scholar and PubMed search engines revealed only fifteen case reports and two case series of primary tuberculosis of the buccal mucosa and labial mucosa.

Estimation of Cancellous Changes Using Fractal Analysis in Patients with Periodontitis.

Periodontal disease is a broad term given when the periodontium is affected by inflammation. There are several methods to diagnose periodontitis, but no method to quantify the cancellous bone is presently used. For this purpose, a non-invasive tool that is efficient in analyzing bone quality called fractal analysis can be employed. The objective of the study was to utilize fractal dimension (FD) to evaluate cancellous patterns of interproximal alveolar bone using a digital intraoral periapical radiograph (IOPAR) in different clinical presentations of periodontitis classified according to the latest classification by the World Workshop for Periodontal and Peri-Implant Diseases and Conditions, 2017 (WWDC, 2017). The study aimed to numerically calculate the periodontitis changes in the cancellous bone around the affected tooth on an IOPAR using fractal analysis and to provide an additional criterion for the staging of periodontitis. In this cross-sectional observational study, 75 patients were selected and equally allotted to five groups based on the staging system proposed by the WWDC, 2017. The region of interest was selected on the IOPA radiograph of the tooth with the site having the most significant clinical attachment loss, and FD was calculated using Image J analysis. The association of gender and age with stages of periodontitis was studied using the chi-square test of independence. A comparison of % bone loss and fractal dimension among stages was studied by using the non-parametric Kruskal Wallis test. The relationship between % bone loss and fractal dimension within stages and gender was studied by using the Karl Pearson correlation. A receiver operating characteristic (ROC) curve analysis was performed for FD value as a marker of periodontitis patients. We demonstrated that the FD values decreased significantly with the increase in stages (p < 0.0001). The overall relationship between % bone loss and FD showed a significantly strong negative correlation of -0.739 (p < 0.0001), except for stages III and IV. FD can be utilized as a quantitative method for detecting cancellous bone changes in different stages of periodontitis, aiding in diagnosing periodontitis.

Comparison of Immunohistochemical Markers in Oral Submucous Fibrosis and Oral Submucous Fibrosis Transformed to Oral Squamous Cell Carcinoma-A Systematic Review and Meta-Analysis.

The objective of the study was to compare the expression of immunohistochemical (IHC) markers of oral submucous fibrosis (OSMF) (non-transformed group) to those of oral squamous cell carcinoma (OSCC) transformed from OSMF (transformed group). The search for comparative cross-sectional studies was carried out in PubMed and Scopus abiding to the PICO criteria, where expression of IHC markers in OSMF were compared with that of OSCC transformed from OSMF. The cellular distribution, number of positive cases, staining intensity, and mean immunoreactive score (IRS) of each IHC marker were evaluated in both groups. A total of 14 studies were included in the systematic review, in which immunoexpression of 15 epithelial and 4 connective tissue biomarkers were evaluated. Expression of ß1-integrin, OCT-3, CD1a, CD207, survivin, Dickkopf-1, COX-2, hTERT, CTGF, MDM2, Ki-67, and α-SMA were increased during transformation of OSMF to OSCC. Conversely, expression of PTEN and lysyl oxidase decreased during transformation of OSMF to OSCC. Expression of a group of epithelial markers, such as COX2, hTERT, CTGF, survivin, MDM2, and p53, was 38 times lower in the non-transformed group cases compared to transformed group cases (95% CI: 58% to 10%; p = 0.01; and I2 = 90%). Meta-analysis of all markers involved in cell metabolism/apoptosis, which included ß1-integrin along with the above markers also suggested 42 times lower expression in the non-transformed group as compared to the transformed group (95% CI: 58% to 10%; p = 0.01; and I2 = 90%). Sub-group analyses on cytoplasmic and nuclear epithelial markers were inconclusive. Meta-analysis of connective tissue markers was also inconclusive. No publication bias was found. Instead of delving into numerous markers without a strong basis for their use, it is advisable to further study the markers identified in this study to explore their clinical utility.

Can Immunoexpression of Cancer Stem Cell Markers Prognosticate Tongue Squamous Cell Carcinoma? A Systematic Review and Meta-Analysis.

The objective was to evaluate the association of the immunoexpression of cancer stem cell (CSC) markers with clinicopathological and survival outcomes in tongue squamous cell carcinoma (TSCC) patients. This systematic review and meta-analysis [PROSPERO (CRD42021226791)] included observational studies that compared the association of clinicopathological and survival outcomes with CSC immunoexpression in TSCC patients. Pooled odds ratios (ORs) and hazard ratios (HRs) with 95% confidence intervals (CI) were used as outcome measures. Six studies identified the association with three surface markers (c-MET, STAT3, CD44) and four transcription markers (NANOG, OCT4, BMI, SOX2). The odds of early-stage presentation were 41% (OR = 0.59, 95% CI 0.42-0.83) and 75% (OR = 0.25; 95% CI 0.14-0.45) lower in CSC and SOX2 immuno-positive cases than immuno-negative cases, respectively. The odds of well-differentiated tumors in transcription marker immuno-positive cases were 45% lower compared to immuno-negative cases (OR = 0.55, 95% CI 0.32-0.96). The odds of positive lymph nodes were 2.01 times higher in CSC immuno-positive cases compared to immuno-negative cases (OR = 2.01, 95% CI 1.11-3.65). Mortality in immuno-positive cases was 121% higher than that in immuno-negative cases (HR = 2.21; 95% CI 1.16-4.21). Advanced tumor staging and grading, lymph node metastasis, and mortality were significantly associated with positive immunoexpression of CSC markers.

A Comprehensive Overview of Epidemiology, Pathogenesis and the Management of Herpes Labialis.

Herpes labialis remains exceedingly prevalent and is one of the most common human viral infections throughout the world. Recurrent herpes labialis evolves from the initial viral infection by herpes simplex virus type 1 (HSV-1) which subsequently presents with or without symptoms. Reactivation of this virus is triggered by psychosocial factors such as stress, febrile environment, ultraviolet light susceptibility, or specific dietary inadequacy. This virus infection is also characterized by uninterrupted transitions between chronic-latent and acute-recurrent phases, allowing the virus to opportunistically avoid immunity and warrant the transmission to other vulnerable hosts simultaneously. This review comprehensively evaluates the current evidence on epidemiology, pathogenesis, transmission modes, clinical manifestations, and current management options of herpes labialis infections.

Spindle Cell Hemangioma of Lower Lip: The Fourth Case Report with Review of Literature.

Background: Spindle cell hemangioma previously known as spindle cell hemangioendothelioma is a benign vascular tumour with rare presentation in head and neck. Presentation in lip is even rarer with three cases reported previously. Method: This report describes a case of spindle cell hemangioma presented as an asymptomatic growth on lower lip of a 32-year-old male. Clinicopathological characterization of this case along with previously reported 15 cases of spindle cell hemangioma of head and neck were conducted. Result: Microscopic evaluation shows a well-circumscribed vascular neoplasm of spindled and epithelioid endothelial cells. Large ectatic thin-walled vascular spaces were seen engorged with RBCs. The neoplasm was CD31 positive. Slight predilection for female gender and young age were observed. Minimal possibility of recurrence was also observed. Conclusion: Spindle cell hemangioma needs to be considered in the differential diagnosis of vascular tumours of head and neck to avoid misdiagnosis of aggressive vascular neoplasms.

Lymphangioma of the Lower Lip-A Diagnostic Dilemma: Report of a Rare Case with a Brief Literature Review.

Hamartomas are tumor-like deformities typified by cellular propagation indigenous to the original site, although they display growth arrest without the possibility for further growth. Various hamartomatous oral lesions include hemangiomas, lymphangiomas, nevi, odontomas, Cherubism, etc. Lymphangiomas are benign, developmental hamartomatous entities typified by abnormal proliferation of lymphatic vessels. They are usually congenital, and more than 90% of cases occur by 2 years of age, with a rare occurrence in adults. They have a site affinity for the head and neck, and oral lesions are relatively uncommon. The dorsum of the tongue is the commonest oral site of predilection; however, the lip is a rare site of involvement. Hereby, we present an uncommon case of lymphangioma of the lower lip in a 45-year-old male patient, who reported to our hospital with an asymptomatic lower lip submucosal mass present for the last 3 years. Our case is unique as it occurred on the lower lip of a 45-year-old male. A detailed history and clinical evaluation, ultrasonography, and histopathology confirmed the diagnosis of lymphangioma.

Perspective of nuclear fractal dimension in diagnosis and prognosis of oral squamous cell carcinoma.

Background: Owing to the restricted predictive value of conventional prognostic factors and the inconsistent treatment strategies, several oral squamous cell carcinoma (OSCC) patients are still over-treated or under-treated. In recent years, computer-assisted nuclear fractal dimension (nFD) has emerged as an objective approach to predict the outcome of OSCC. Objective: This study is an attempt to find out the differences in nFD values of epithelial cells of normal tissue, fibroepithelial hyperplasia, verrucous carcinoma, and OSCC. Further effort to evaluate the predictive potential of nFD of tumor cells for cervical lymph node metastasis (cLNM) was also assessed. Methodology: Formalin-fixed paraffin-embedded blocks of OSCC tissues of patients treated with neck dissection were collected. Photomicrographs of H-&E-stained sections were subjected to the image analysis by ImageJ and Python programming to calculate nFD. The association of categorical variables with nFD was studied using cross-tabulation procedure and the Fisher exact test. Receiver operating curve analysis was performed to find out cutoff value of nFD. A logistic regression model was developed to test the individual and combined predictive potential of grading and nFD for cLNM. Results: A significant difference between the mean nFD of healthy cells and malignant epithelial cells was observed (P = 0.01). nFD was not found to be an independent predictor of cLNM, although nFD and grading together demonstrated significant predictive potential (P = 0.004). Conclusion: nFD combined with grading can predict lymph node metastasis in OSCC. To the best of our knowledge, this is the first study of its kind.

Are Survival Outcomes Different for Young and Old Patients with Oral and Oropharyngeal Squamous Cell Carcinoma? A Systematic Review and Meta-Analysis.

This systematic review and meta-analysis aims to address whether age can be a determinant of overall survival (OS), disease-free survival (DFS), recurrence, distant metastasis (DM) and second primary (SP) in surgically treated oral and oropharyngeal squamous cell carcinoma (OOPSCC). A total of 4981 cases and 44254 controls from 25 comparative observational studies were included in the analysis. A significantly better OS (matched subgroup analysis: OR 1.64; 95% CI 1.31-2.04, overall analysis: OR 1.48; 95% CI 1.09-2.01) was observed in young patients compared to older adults, with heterogeneity ranging from moderate to severe. Worse DFS (unmatched subgroup analysis OR 0.43; 95% CI 0.27-0.68) was observed in young patients compared to older adults with minimal to moderate heterogeneity. The frequency of recurrence (OR 1.49; 95% CI 1.10-2.02) and DM (OR 1.83; 95% CI 1.10-3.03) was significantly higher in the young patients, as found in unmatched and matched subgroup analysis, with the least heterogeneities. Young age can be considered as an independent prognostic factor for recurrence and distant metastases in OOP-SCC. Larger and methodologically robust observational studies with longer follow-up are needed to establish the definitive role of age as an independent prognostic factor on OS and DFS in OOPSCC.

Amelanotic melanoma of the palate: report of a case.

Primary amelanotic melanoma is an infrequent occurrence in the oral cavity. Owing to the high rate of local invasion and distant metastasis, oral amelanotic melanoma (OAM) carries a very poor prognosis. The absence of pathognomonic clinical and routine histological features in OAM is the reason for diagnosticdelay, which further worsens the prognosis. This case report discusses the masquerading nature of OAM that was clinically and histologically mimicking several malignant neoplasms. This case also demonstrates the poor prognosis of OAM. The objective of presenting this case is that the diagnostic delay of OAM can be avoided through enhanced clinical awareness and subsequent appropriate immunohistochemical investigations, in addition to the routine H&E-stained histopathological evaluation.

Innocuous presentation of ameloblastic carcinoma.

Ameloblastic carcinoma is a rare malignant odontogenic neoplasm that exhibits diverse clinical and radiological presentations. In fact there are several differential diagnoses during histopathological evaluation too. Lack of adequate reports could not establish the predominant demographic, clinical and radiological presentations. For the same reasons, the role of adjuvant radiotherapy and chemotherapy is also unsubstantiated yet. This case discusses the innocuous clinical and radiological presentation of ameloblastic carcinoma in a 55-year-old man where the diagnostic confirmation was achieved through histopathological evaluation. The differential diagnoses, treatment and follow-up details of this case are discussed in light of the previous published case reports and systematic reviews of case reports in an attempt to increase the sensitisation among dentists towards ameloblastic carcinoma.

A Plausible Proposition of CCL20-Related Mechanism in Fusobacterium nucleatum-Associated Oral Carcinogenesis.

OBJECTIVE: The objective of this prospective observational case-control study is to evaluate the prevalence of Fusobacterium nucleatum in the tissues of oral squamous cell carcinoma (OSCC). Reconnoitering the CCL20-related mechanism of carcinogenesis in Fusobacterium nucleatum-positive OSCC is another objective. METHODOLOGY: Tissues from 50 OSCC patients and 30 healthy oral tissues were collected. The prevalence of Fusobacterium nucleatum was evaluated in both tumour and healthy tissue by polymerase chain reaction. The immunohistochemistry of OSCC tissues was conducted to evaluate the difference in the expression of CCL20 between Fusobacterium nucleatum-positive and -negative OSCC tissues. RESULTS: Fusobacterium nucleatum was significantly (p < 0.001) prevalent in OSCC tissues (74%), compared to healthy tissues (26%). No association of Fusobacterium nucleatum or CCL20 immuno-expression with any clinical or histopathological features of OSCC was observed. While the intensity of CCL20 immuno-expression did not differ (p = 0.053), the CCL20-positive cell population was significantly different (p = 0.034) between Fusobacterium nucleatum-positive and -negative OSCC. CONCLUSION: Fusobacterium nucleatum is possibly prevalent in oral cancer tissues in the Indian population. By using immunohistochemistry, this is the first study to propose that the carcinogenesis in Fusobacterium nucleatum-positive OSCC may be CCL20-related. The findings enrich the knowledge of mechanisms involved in Fusobacterium nucleatum-mediated oral carcinogenesis.

Endoplasmic reticulum & mitochondrial calcium homeostasis: The interplay with viruses.

Calcium ions (Ca2+) act as secondary messengers in a plethora of cellular processes and play crucial role in cellular organelle function and homeostasis. The average resting concentration of Ca2+ is nearly 100 nM and in certain cells it can reach up to 1 µM. The high range of Ca2+ concentration across the plasma membrane and intracellular Ca2+ stores demands a well-coordinated maintenance of free Ca2+ via influx, efflux, buffering and storage. Endoplasmic Reticulum (ER) and Mitochondria depend on Ca2+ for their function and also serve as major players in intracellular Ca2+ homeostasis. The ER-mitochondria interplay helps in orchestrating cellular calcium homeostasis to avoid any detrimental effect resulting from Ca2+ overload or depletion. Since Ca2+ plays a central role in many biological processes it is an essential component of the virus-host interactions. The large gradient across membranes enable the viruses to easily modulate this buffered environment to meet their needs. Viruses exploit Ca2+ signaling to establish productive infection and evade the host immune defense. In this review we will detail the interplay between the viruses and cellular & ER-mitochondrial calcium signaling and the significance of these events on viral life cycle and disease pathogenesis.

The Potential of Light Microscopic Features of the Oral Mucosa in Predicting Post-mortem Interval.

OBJECTIVES: The post-mortem interval (PMI) refers to the amount of time elapsed between death and discovery of the body. This study aimed to evaluate light microscopic cellular changes in the oral mucosa and identify the potential of this method for predicting PMI. METHODS: This prospective study was conducted between July 2016 and January 2018 at the Institute of Dental Sciences, Siksha 'O' Anusandhan University, Bhubaneswar, India. A total of 150 post-mortem (including 75 gingival and 75 buccal mucosa samples) and 40 ante-mortem (including 20 gingival and 20 buccal mucosa samples) tissue samples were compared using haematoxylin and eosin, periodic acid-Schiff (PAS) and van Gieson stains. Microscopic changes in the epithelium and connective tissue were categorised according to PMI stage as early (<12.5 hours since death), intermediate (12.5-20.5 hours since death) or late (>20.5 hours since death). RESULTS: Most epithelial cellular changes occurred early, except for arc-shaped nuclei and epithelial shredding which were intermediate and late changes, respectively. However, microscopic changes in the connective tissue were only observable at ≥12.5 hours. There was a progressive decrease in intensity in van Gieson stains and an increase in intensity in PAS stains as PMI increased. Several microscopic features were found to be significant predictors of PMI including epithelial homogenisation, cytoplasmic vacuolation, nuclear degeneration, arc-shaped nuclei, chromatin clumping, red blood cell clumping and lysis, melanin incontinency, myofibril degeneration, salivary gland acini degeneration and epithelial connective tissue separation (P <0.050 each). CONCLUSION: These findings indicate that microscopic evaluation of the oral mucosa may be helpful for PMI prediction.

Differences in the bacteriome of swab, saliva, and tissue biopsies in oral cancer.

Microbial dysbiosis has been implicated in the pathogenesis of oral cancer. We analyzed the compositional and metabolic profile of the bacteriome in three specific niches in oral cancer patients along with controls using 16SrRNA sequencing (Illumina Miseq) and DADA2 software. We found major differences between patients and control subjects. Bacterial communities associated with the tumor surface and deep paired tumor tissue differed significantly. Tumor surfaces carried elevated abundances of taxa belonging to genera Porphyromonas, Enterobacteriae, Neisseria, Streptococcus and Fusobacteria, whereas Prevotella, Treponema, Sphingomonas, Meiothermus and Mycoplasma genera were significantly more abundant in deep tissue. The most abundant microbial metabolic pathways were those related to fatty-acid biosynthesis, carbon metabolism and amino-acid metabolism on the tumor surface: carbohydrate metabolism and organic polymer degradation were elevated in tumor tissues. The bacteriome of saliva from patients with oral cancer differed significantly from paired tumor tissue in terms of community structure, however remained similar at taxonomic and metabolic levels except for elevated abundances of Streptococcus, Lactobacillus and Bacteroides, and acetoin-biosynthesis, respectively. These shifts to a pro-inflammatory profile are consistent with other studies suggesting oncogenic properties. Importantly, selection of the principal source of microbial DNA is key to ensure reliable, reproducible and comparable results in microbiome studies.

Two novel genomic DNA sequences as common diagnostic targets to detect Cryptosporidium hominis and Cryptosporidium parvum: Development of quantitative polymerase chain reaction assays, and clinical evaluation.

Introduction: Cryptosporidium is an intestinal parasite responsible for gastroenteritis. Conventional diagnosis of Cryptosporidium is made by microscopy. The most frequent molecular detection method for this parasite is polymerase chain reaction (PCR). The objective of the present study was to identify the novel DNA targets and development of PCR-based assays for the specific detection of two major human infecting species Cryptosporidium parvum and Cryptosporidium hominis. Methodology: Sensitive and specific SYBR green quantitative PCR (qPCR) and TaqMan qPCR assays were developed and validated at both diagnostic and analytical level using the new identified targets TU502HP-1 and TU502HP-2. Results: Assay validation results showed that the newly developed real-time PCR assays are 100% specific with a reliable limit of detection. Overall repeatability and reproducibility of these assays showed good quality results over intra- and inter-laboratory analysis. Conclusion: Novel target-based qPCR assays can be rapid an efficient tool for simultaneous detection of a C. parvum and C. hominis. These genes could also be utilized for the development of innovative DNA-based Point-of-Care test development.