Advances in Point of Care Diagnostics for Rapid Pathogen Detection.pptx

This presentation explores the latest advancements in Point-of-Care (PoC) diagnostics for rapid pathogen detection, highlighting innovative technologies, emerging trends, and real-world applications. It covers biosensors, microfluidics, CRISPR-based detection, and AI-driven diagnostics, emphasizing their role in early disease detection, outbreak control, and personalized medicine. The slides provide insights into the speed, accuracy, and accessibility of modern PoC diagnostic tools, transforming healthcare by enabling on-site testing and real-time results.

1. Advances in Point of Care Diagnostics for Rapid Pathogen Detection Presentor - Dr Punith Kumar ESIC MC & PGIMSR, Bengaluru Moderator- Dr Praveen Kumar

2. LAYOUT Introduction Need for Rapid Diagnostic Tests Need for POCT Past & Present Methods Requirements & Procedure Lateral Flow Immunoassay Spectrum of POCT Technical Considerations Bacterial/Viral/Fungal/Parasitic POCT Biomarkers Advantages & Disadvantages Advances Microfluidics Plasmonic Technologies Biosensors & Nanotechnology Nucleic Acid Based Detection Molecular POCT Challenges Conclusion

3. REFERENCES Gavina K, Franco LC, Khan H, Lavik J-P, Relich RF. Molecular point-of-care devices for the diagnosis of infectious diseases in resource-limited settings - A review of the current landscape, technical challenges, and clinical impact. J Clin Virol [Internet]. 2023;169(105613):105613. Available from: http://dx.doi.org/10.1016/j.jcv.2023.105613 Kozel TR, Burnham- Marusich AR. Point-of-care testing for infectious diseases: Past, present, and future. J Clin Microbiol [Internet]. 2017;55(8):2313–20. Available from: http://dx.doi.org/10.1128/JCM.00476-17

4. Hansen GT. Point-of-care testing in microbiology: A mechanism for improving patient outcomes. Clin Chem [Internet]. 2020;66(1):124–37. Available from: http://dx.doi.org/10.1373/clinchem.2019.304782 Zumla A, Al-Tawfiq JA, Enne VI, Kidd M, Drosten C, Breuer J, et al. Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections--needs, advances, and future prospects. Lancet Infect Dis [Internet]. 2014;14(11):1123–35. Available from: http://dx.doi.org/10.1016/S1473-3099(14)70827-8

5. Advice on the use of point-of-care immunodiagnostic tests for COVID-19 [Internet]. Who.int. [cited 2025 Feb 5]. Available from: https://www.who.int/news-room/commentaries/detail/advice-on-the-use-of-point-of-care-immunodiagnostic-tests-for-covid-19 Aborode AT, Adesola RO, Scott GY, Arthur-Hayford E, Otorkpa OJ, Kwaku SD, et al. Bringing lab to the field: Exploring innovations in point-of-care diagnostics for the rapid detection and management of tropical diseases in resource-limited settings. Adv Biomark Sci Technol [Internet]. 2025;7:28–43. Available from: http://dx.doi.org/10.1016/j.abst.2025.01.001

6. Shang M, Guo J, Guo J. Point-of-care testing of infectious diseases: recent advances. Sens Diagn [Internet]. 2023;2(5):1123–44. Available from: http://dx.doi.org/10.1039/d3sd00092c ChatGPT [Internet]. Chatgpt.com. [cited 2025 Feb 5]. Available from: https://chatgpt.com/ Gemini Advanced - Gemini 2.0 [Internet]. Gemini. [cited 2025 Feb 5]. Available from: https://gemini.google/advanced Deepseek.com. [cited 2025 Feb 5]. Available from: https://www.deepseek.com/

7. The growing burden of infectious diseases worldwide necessitates rapid detection for effective disease control and management. Point-of-care (POC) diagnostics enable early detection at or near the site of patient care, reducing reliance on centralized laboratory testing. Recent technological advancements have improved accuracy, portability, and affordability of these diagnostics, making them indispensable in modern healthcare.

8. What is Point-of-Care Diagnostics? POC diagnostics refer to medical diagnostic tests performed at or near the site of patient care with immediate results. Characteristics: Rapid turnaround time, user-friendly operation, minimal sample processing, and high specificity/sensitivity. Advantages: Decentralized testing, improved patient management, reduced burden on healthcare infrastructure. Common applications: Detection of infectious diseases (HIV, TB, COVID-19), chronic disease monitoring (diabetes, cardiac biomarkers), and use in emergency and disaster settings.

9. As coined by the WHO, the fundamental criterions of POC diagnostics are denoted to be “ASSURED” A ffordable S ensitive S pecific U ser-friendly R apid and robust E quipment-free D eliverable to end user

10. What are the Challenges in Traditional Pathogen Detection? Time-Consuming Processes: Culture-based methods can take 24-48 hours or longer . Complexity: Requires specialized laboratories, trained personnel, and expensive equipment. Delayed Treatment: Slows clinical decision-making, leading to poorer patient outcomes. .

11. Why Rapid Pathogen Detection? Early detection reduces morbidity, mortality, and transmission of infectious diseases. Essential for antimicrobial stewardship and preventing overuse of antibiotics Infectious diseases remain a leading cause of morbidity and mortality, particularly in resource-limited settings. Conventional diagnostic methods, including culture-based techniques, ELISA, and PCR, are time-consuming and require specialized infrastructure. Rapid POC testing enhances timely decision-making, reduces the risk of disease transmission, and supports timely initiation of appropriate therapies.

12. PAST F irst large-scale use of the immunoassay for the diagnosis of infectious disease was in a report in 1917 by Dochez and Avery that pneumococcal polysaccharide can be detected by immunoassay of serum and urine from patients with lobar pneumonia . In a prescient comment, the authors suggested that antigen detection could enable a rapid diagnosis of infection

13. One of the first tests to be introduced was the detection of Streptococcus pyogenes by throat swab, leading to the prescription of antibiotic treatment when the test was positive and no treatment when the test was negative. Furthermore, for a long time, gynecologists performed tests to detect bacterial vaginosis (Nugent test) and used direct microscopic observation to detect the presence of Trichomonas vaginalis, Gram negative bacteria, or gonococci

14. The promise of immunoassays such as ELISA and RIA for the diagnosis of disease prompted numerous individuals and biotechnology companies to find the means to perform rapid tests at the POC. Early steps included the use of capillary migration in cellulose acetate sheets as the structural foundation for an immunoassay and the ability to couple antibodies to colloidal gold or latex particles .

15. Several companies then developed technologies that led to the present lateral flow immunoassay (LFIA) platform . The home pregnancy test that uses the lateral flow format provided clear evidence of the value of the format for at-home use of antigen testing. In turn, the rapid test for the diagnosis of streptococcal pharyngitis popularized the LFIA technology for the diagnosis of infectious diseases.

16. PRESENT Most POC rapid diagnostics use the LFIA platform. The LFIA platform is extremely versatile. The detection of high-molecular-weight antigens requires an antibody pair where an antibody to one analyte epitope is labeled with a reporter, such as colloidal gold, and a capture antibody to a second epitope on the same analyte is immobilized on the lateral flow strip .

17. In an antigen-capture sandwich format , the intensity of the signal at the test line is proportional to the concentration of the analyte. Sandwich immunoassays are the foundation for POC tests for infectious diseases that detect microbial products in clinical samples, e.g., the group A streptococcal cell wall carbohydrate.

18. The detection of low-molecular-weight analytes with a single antigenic determinant requires a competitive format . In these assays, the intensity of the test line is inversely proportional to the analyte concentration. Examples of assays using competitive formats include many immunoassays for the detection of drugs of abuse.

19. Finally, the LFIA format can be used for the detection of patient antibodies to target antigens. In this instance, the target antigen is immobilized on the strip, and the binding of patient antibody is detected by the use of a labeled reporter, such as a second antibody. Examples of serological assays in the LFIA format include tests for HIV-1/2 or hepatitis C virus

20. Specimen Requirements and Procedure There are 3 primary stages in the POCT process: Pre-Analytical Analytical Post-Analytical.

21. The pre-analytical phase occurs before running the POCT on a sample and involves collection, transport, preparation, and loading. The analytical phase is the stage in which the actual testing sequence of a POCT is conducted. The post-analytical phase begins when testing is complete, and an obtained result is available.

22. Lateral Flow Immunoassay

23. Lateral Flow Immunoassay

24. Lateral flow tests or strip tests rely on the binding of a microbial antigen present in the clinical sample to a primary antibody conjugated to gold or a fluorescent marker. The antibody antigen complex then migrates either under the effect of a lysis buffer or by capillarity in a solid substrate. The antibody-antigen complex is then captured by a secondary antibody, leading to the appearance of an initial color band, while the excess primary antibody, conjugated to gold beads, continues migrating to a second point of capture with tertiary antibodies, leading to the appearance of a second color band.

25. The test reading is taken within 15 min, and interpretation is based on visualization of the first band (present equals positive, and absent equals negative) and visualization of the second band (present equals an interpretable test result, and absent equals an uninterpretable test result).

26. Technica l : T raining Education and visual acuity of staff Adequate storage Kits: temperature, humidity Samples: lysis can occur during mixing or storage Sample requirements Volume required for testing Whole blood/serum/plasma

27. Proper instructions for using the kits Preparation of dilutions of sample/buffer can affect uniformity and flow due to cell lysis and aggregation of parasitized cells Misinterpretation of test lines may lead to false positives or false negatives: visual acuity, experience, quality of kit

28. Technica l : Performance Depends On The Design Formats Radioimmunoassay Molecular assays Lateral flow immunoassay: most popular – dipstick, strips, cards, pad, wells, cassettes Ease of use and training requirements for the health workers For blood safety, kits that include lancets and alcohol swabs are preferred Cassettes and cards are easier to use than dipsticks in field conditions; dipsticks may be suitable in laboratory settings Internal controls Proper instructions for preparation of samples, diluents, and interpretation of results Poor visibility of test bands can lead to erroneous results

29. BACTERIAL POCT Organism Method Used Group A Streptococcus (GAS) LFIA GAS antigen Treponema pallidum (syphilis) LFIA Antibodies to T. pallidum Borrelia burgdorferi (Lyme disease) LFIA IgG and IgM antibodies to B. burgdorferi Helicobacter pylori LFIA IgG antibodies to H. pylori   Biochemical Urease enzyme activity   LFIA H. pylori antigen  Salmonella sps  LFIA  OMP antigen Treponema pallidum LFIA uses preserved avian erythrocytes coated with extracted antigens of T. pallidum (Nichols strain)

30. VIRAL POCT Organism Method Used Infectious mononucleosis LFIA Heterophile antibodies Influenza types A and B LFIA Influenza type A and B antigens   Biochemical Neuraminidase enzyme activity Respiratory syncytial virus LFIA Respiratory syncytial virus antigen HIV-1 and HIV-2 LFIA Antibodies to HIV-1/2   LFIA HIV-1 antigen, antibodies to HIV-1/2 HIV-1 LFIA Antibodies to HIV-1 Influenza type A LFIA Influenza type A antigen Influenza type B LFIA Influenza type B antigen Adenovirus LFIA Adenoviral antigen Hepatitis C virus LFIA Antibodies to hepatitis C virus Covid LFIA SARS COV-2 antigen

31. POCT IN Parasitology

32. Organism Method Used Gardnerella vaginalis, Bacteroides spp., Prevotella spp., and Mobiluncus spp Biochemical Sialidase enzyme activity Trichomonas vaginalis  LFIA T. vaginalis antigen Plasmodium falciparum LFIA  An infection with Plasmodium falciparum (falciparum malaria) can be detected with rapid tests to two specific antigens—histidine-rich protein 2 (HRP2) and parasite-specific lactate dehydrogenase ( pLDH ).

33. Organism Method Used Entamoeba histolytica lateral flow immunoassays or ELISA tests Entamoeba histolytica antigen or specific antibodies Giardia duodenalis LFIA lateral flow immunoassay, fluorescent immunoassay, or immunochromatographic assay for the detection of antigens or antibodies. Toxoplasma gondii LFIA   Leishmaniasis LFIA   Trichinella ELISA-based excretory-secretory (E/S) antigen that has been purified from the larvae of infected pigs. Filariasis Antigen antibody   Echinococcosis Taeniasis and cysticercosis, Schistosomiasis IgG- ELISA   Paragonimiasis LFIA P. heterotremus  ES antigen

34. POCT In Mycology POCT Target IFI Specimen Aspergillus IgG LFA Antibody (IgG, IgM, IgE & IgA) CPA Serum Coccidioides antibody Antibody (IgG and IgM) Coccidioidomycosis Serum        

35. POCT Target IFI Principle Specimen CrAg LFA Antigen Cryptococcal meningitis, cryptococcosis Uses the anti-GXM(detection of cryptococcal antigen ( CrAg ), a component of the cryptococcal polysaccharide capsule glucoronoxymannan (GXM)) monoclonal antibodies—the test uses two monoclonal antibodies and can recognize all four GXM serotypes (A–D) Whole blood, serum, plasma, CSF, urine Histoplasma antigen LFA Antigen Progressive disseminated histoplasmosis H & M antigens Serum, urine Aspergillus GM LFA Antigen IPA Galactomannan (GM) is a polysaccharide antigen that exists primarily in the cell walls of Aspergillus species. GM may be released into the blood and other body fluids even in the early stages of Aspergillus invasion, and the presence of this antigen can be sustained for 1 to 8 weeks BAL fluid, serum Aspergillus specific LFD Antigen IPA An immunochromatographic assay using a mouse monoclonal antibody, JF5, which binds to an extracellular glycoprotein antigen from Aspergillus spp., only secreted during active growth. Bronchoalveolar lavage fluid, serum

36. Potential molecular POCTs, such as the loop-mediated isothermal amplification (LAMP) assay for Histoplasma capsulatum (H. capsulatum DNA)and proximal ligation assay for Aspergillus- (detects Aspergillus mannoprotein). have been developed and evaluated but are not yet commercially available

37. Host biomarkers/ Non-Pathogen-Specific Diagnosis One approach for differentiating between viral and bacterial infections is to examine host biomarkers that respond differently to both types of pathogens CRP is the most well-studied inflammation biomarker for predicting bacterial infections with POC testing. Plasma levels of CRP rise after 4 hours to 6 hours of bacterial infection and peak at 36 hours. Procalcitonin levels rise 3 hours after infection and peak within 24 hours.

38. Cytokines, such as interleukin-6 and interleukin-8, have also been investigated as potential informative markers. However, cytokines have short half-lives, so levels rise and fall back to baseline relatively quick (within 6 hours), and can have variable changes in response to different types of bacteria Other biomarkers are known to increase in response to viral infections. These include tumour necrosis factor–related apoptosis-inducing ligand interferon (IFN) gamma-induced protein 10 (IP-10) myxovirus resistance protein A ( MxA ).

39. Biomarkers for fungal pathogens: Galactomannan Detection- (aspergillus ) Mannan- (candida) β-(1,3)- D-Glucan- (pan fungal ) Pathogen Nucleic Acids : A drawback using pathogen nucleic acids as biomarkers is the inability to differentiate between infection and colonization. Antibodies : The presence of anti-pathogen antibodies can serve as biomarkers to evaluate the infectious state. Pathogen Proteins : All pathogens causing infectious diseases carry proteins, such as capsid and envelope proteins

40. Circulating microRNAs : MicroRNAs (miRNAs) are non-coding RNA molecules with small sizes (~20 nt ), and function to post-transcriptionally regulate gene expression extracellular miRNAs are extremely stable in body fluids including plasma, serum, urine, saliva, and semen, protected by RNA-binding proteins, high density lipoprotein particles and lipid vesicles It has also been reported that two pairs of plasma miRNAs (miR-495-3p in combination with let-7b-5p, miR-151a-5p, or miR-744-5p; and miR-376a-3p in combination with miR-16-5p) can be potentially used as biomarkers for HIV-associated neurological disorders (HAND)

41. Advantages And Disadvantages Of Rapid Microbiological Tests

42. Advantages Immediate initiation of specific antibiotic therapy is possible. Reduction in unnecessary antibiotic consumption Reduction in selection pressure Immediate recognition of infection chains Reduction in pre-analytical interference Extension of diagnostic instrumentarium ; independent of culture Better compliance with patients who are difficult to reach

43. Disadvantages Older POCT systems perform more poorly (before the introduction of immunochromatographic techniques) Lack of data on pathogen sensitivity Increased risk of operator becoming infected Operator's qualifications may be inadequate. Double or multiple infections are more likely to be overlooked than in culture. Necessity of performing measures for quality control

44. Advances

45. Microfluidics Microfluidics is a technology used to manipulate very small volume of fluids, offering precise, programable, spatial and temporal control of the fluids . Through microfluidics technology, samples and reagents can be transported, mixed, and reacted in specific micro chambers in a precisely controlled manner . It is naturally an ideal platform for POC test development with many desired features such as automation, integration, and miniaturization

46. Microfluidic devices can provide a fully integrated POC device for sample processing, fluid handling, and signal generation Microfluidics-based devices use channels to transport small amounts of fluid by actuation forces. On-chip immunoassays have many similarities to the standard LFIA, ELISA, or molecular diagnostics platforms; however, the use of microfluidic technologies reduces assay complexity and enables multiplex analysis.

47. Plasmonic Technologies Plasmonics studies the interaction between light and the conductive electrons of metallic nanomaterials . Common plasmonic metals include gold, silver and aluminum. Various plasmonic nanomaterials have been designed and fabricated to target POC applications owing to their label-free nature, facile optical tunability and high sensitivity to surrounding medium

48. Based on the sensitivity of the Surface Plasmon Resonance (SPR) to the changes in the dielectric properties of the surrounding medium, and the enhancement of the electromagnetic (EM) field in proximity to noble metal nanostructures, two important classes of plasmonic sensors have evolved: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS) sensors.

49. Key Technologies in POC Diagnostics Microfluidics and Lab-on-a-Chip: Miniaturized systems capable of handling small volumes of fluids for high-throughput analysis. Lateral Flow Assays (LFAs): Paper-based diagnostics commonly used in home tests like pregnancy and COVID-19 tests. Nucleic Acid-Based Detection (PCR, LAMP): Advanced molecular detection techniques enabling high-sensitivity pathogen identification. Biosensors and Nanotechnology: Utilization of nanomaterials and bioengineered sensors for enhanced detection sensitivity. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) -based diagnostics: Exploiting gene-editing technology for rapid, specific pathogen identification.

50. Microfluidics and Lab-on-a-Chip Working Principle: Utilizes microscale fluid manipulation for biochemical reactions on a single chip, enabling automation and high-speed analysis. Advantages: Reduced reagent consumption, faster reaction times, potential integration with digital health systems. Real-world Applications: Portable devices for detecting sepsis biomarkers, viral RNA detection, and antibiotic susceptibility testing.

51. Lateral Flow Assays (LFAs) Mechanism: Antibody-antigen interactions drive visual signal development, commonly using gold nanoparticles. Advantages: Cost-effective, easy-to-use, no laboratory infrastructure required. Innovations: Multiplexing capabilities for detecting multiple pathogens simultaneously, smartphone-based result interpretation, and integration with cloud-based tracking systems.

52. Nucleic Acid-Based Detection (PCR, LAMP) PCR (Polymerase Chain Reaction): Gold standard for nucleic acid amplification, requiring thermocyclers. LAMP (Loop-mediated Isothermal Amplification): A field-deployable alternative to PCR that provides rapid amplification under isothermal conditions, enhancing portability. Examples: Rapid COVID-19 testing kits using LAMP, handheld PCR devices for field diagnostics.

53.  Biosensors and Nanotechnology Biosensors: Devices combining a biological recognition element (e.g., enzymes, antibodies) with a transducer to convert biological signals into readable data. Nanotechnology Applications: Nanoparticle-based signal amplification, quantum dots for fluorescence detection, and graphene-based electrochemical sensors for real-time monitoring. Examples: Wearable biosensors for continuous pathogen monitoring, highly sensitive tuberculosis detection kits.

54. CRISPR-Based Diagnostics Principle: CRISPR-Cas enzymes specifically recognize and cleave target DNA/RNA sequences, enabling highly specific pathogen detection. Advantages: High specificity, rapid turnaround, potential for integration with portable devices. Notable Platforms: SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing ), DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) for viral diagnostics, including SARS-CoV-2 and Zika virus.

55.  Challenges in POC Diagnostics Analytical Sensitivity & Specificity: Ensuring reliable detection of low pathogen loads without cross-reactivity. Cost & Accessibility: Balancing affordability with advanced technology integration for widespread deployment. Regulatory Approvals: Variability in global regulatory frameworks (FDA, CE, WHO prequalification) impacting market entry. Data Integration: Need for electronic health record (EHR) compatibility and telemedicine integration.

56. Future Directions and Innovations AI & Machine Learning: Enhancing diagnostic accuracy through automated interpretation of results. Smartphone-based Diagnostics: Leveraging mobile devices for real-time analysis, cloud storage, and remote consultation. Wearable Biosensors: Development of non-invasive, continuous monitoring systems for real-time pathogen detection. Advances in Sample Collection: Development of painless micro-needles, breath-based diagnostic tools, and saliva-based POC assays.

57. CONCLUSION The major strength of LFIAs and POC molecular assays is their ability to provide diagnostic information during the initial patient visit. These tests are most valuable in cases where the choice of treatment for a patient is time sensitive and where particular treatments or actions would be triggered by the test results (e.g., fluconazole treatment for a CrAg -positive AIDS patient or hospital admittance for very young infants with RSV).

58. The major weaknesses of current POC tests are sometimes low clinical sensitivity in the case of the LFIA and both cost and infrastructure requirements in the case of POC molecular assays. Accessible and rapid tests that can provide an initial diagnosis at the POC are a powerful tool for effective patient care, antibiotic stewardship, and outbreak containment.

59. Case Studies and Real-World Applications COVID-19 POC Diagnostics: Lessons learned from global deployment of rapid antigen and molecular tests. Malaria & Tuberculosis Detection: Impact of rapid POC tests in reducing mortality and improving treatment outcomes in resource-limited settings. Veterinary & Environmental Applications: Role of POC diagnostics in detecting zoonotic diseases and monitoring waterborne pathogens.

60. Key Takeaways: POC diagnostics are revolutionizing pathogen detection by providing rapid, accessible, and accurate testing solutions. Global Health Impact: Potential for mitigating disease outbreaks, improving patient management, and reducing healthcare costs. Call to Action: Need for continued research, interdisciplinary collaboration, and investment in scalable, cost-effective technologies.

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