[01.14c] Didactics 1_ Innovations in Microbiology and Parasitology Diagnostics V2

detection, containment, and control of disease

What three aspects of infectious diseases are challenged by the dynamic nature of microbiology, clinicians, laboratory personnel, and public health officers?

a rapid and accurate diagnostic paradigm

What is crucial for the detection, containment, and control of infectious diseases, as highlighted by past experiences like the Covid-19 pandemic?

detect and understand the microbial world

What has increasingly improved due to innovations in infectious disease testing?

Non-prescription over-the-counter point of care infectious disease tests, Mass Spectrometry-based detection of host response, and implementation of artificial intelligence and machine learning

What three new innovations were introduced during the Covid-19 pandemic to identify infected individuals?

limited local distribution, in research and developmental stages locally, and limitations in geographic scope and capability

What are the limitations of the innovations discussed in the presentation within the Philippine setting?

Direct detection of microbial particles or indirect host reaction

What does laboratory diagnosis require a demonstration of?

viral, bacterial, fungal, or parasitic agents

What types of agents are demonstrated in tissues, fluids, or excreta of the host for laboratory diagnosis?

detection, identification, and determination of antimicrobial susceptibility

What are clinical microbiology laboratories responsible for regarding pathogens?

Microscopic visualization of pathogens in clinical material or growth of microorganisms in the laboratory

On what two traditional methods has the detection of pathogenic agents largely relied?

reliable but time-consuming

What is a characteristic of direct phenotypic methods for pathogen identification?

fermentation profiles for bacteria, cytopathic effects in tissue culture for viral agents, and microscopic morphology for fungi and parasites

What are examples of phenotypic characteristics used for identification in traditional methods?

Protein Analysis and Genotypic Techniques

What two examples of new methods are increasingly becoming standard for microorganism detection, quantitation, or identification, replacing traditional methods?

improve the effectiveness of treatments and avoid long-term complications

What can early and accurate diagnosis of an infectious disease achieve for the infected patient?

precise laboratory confirmation

What allows for a definitive diagnosis, leading to improved clinical outcomes, reduced treatment costs, shorter hospital stays, and strengthened antimicrobial stewardship?

prevent or stop an outbreak

What can early diagnosis help to do, preventing undiagnosed patients from unknowingly transmitting disease?

antibiotic resistance

To what do widespread overuse and misuse of antibiotics contribute?

when antibiotics are an appropriate treatment and when they are not

What can diagnostic tests determine regarding antibiotic use?

expedient de-escalation from broad-spectrum agents to targeted antimicrobial therapy

What does rapid identification of microorganisms in clinical samples enable?

disruption of normal flora, toxic side effects of drugs, and selective pressure

What three risks of antibiotics are minimized by switching to tailored therapy?

epidemic potential or insufficient/no counter-measures

What two criteria does WHO use to advise research and development initiatives for prioritizing diseases?

Ebola Virus Disease, Lassa Fever, Crimean-Congo Haemorrhagic Fever, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Nipah Virus Infection, Rift Valley Fever, Zika Virus Disease, Coronavirus Disease (COVID-19), and Disease X

What are the current priority diseases for R&D initiatives according to WHO?

HIV/AIDS, Global Health Security (SARS-CoV-2), Tuberculosis, Influenza, and Vaccine-Preventable Diseases

What are the health priorities of the CDC in the Philippines?

first light microscope (1716), Koch's postulates (1890), and Polymerase Chain Reaction (PCR) (1983)

What three historical innovations are mentioned as benefiting the field of infectious diseases testing?

speed, efficiency, and portability

What three aspects of pathogen detection technologies have later discoveries aimed at improving?

simple laboratory-based microscopy, microbiological culture, and PCR

Beyond what three traditional methods has infectious disease testing evolved today?

Serology, Mass spectrometry, and a broad range of molecular techniques like sequencing and PCR

What are examples of direct pathogenic detection methods under rapid immunoassays that have become commonplace?

limited to some of the newest trends in diagnostic microbiology and parasitology that would have the most practical use in clinical management and public health response

What is the scope limitation of the discussion in the presentation regarding diagnostic microbiology and parasitology?

Medical testing at or near the site of patient care

What is the definition of Point-of-Care Tests (POCT)?

handheld, portable, transportable, and benchtop devices

What were the traditional formats for POCTs?

development of bedside rapid molecular tests, proliferation of wearable health monitoring devices, and growth of direct-to-consumer and over-the-counter testing

What three dramatic changes have been observed in POCT related to infectious disease testing over the last 10 years?

rapid screening or definitive pathogen identification

For what purposes were cellphone-based technologies developed for use in the field?

Legionnaires disease

For what disease were cellphone-based technologies successfully used in Europe?

HIV and bacterial pathogens

For what infections were cellphone-based technologies used in some middle resource ASEAN member states?

Holomic Rapid Diagnostic Reader (for lateral flow assay) and Fluorescent Microscopy Platform (detects single nanoparticles and viruses)

What are two examples of cellphone-based technologies mentioned?

bright field fluorescence, dark field transmission, and polarized microscopy modes

Into what can a cellphone dongle POCT be converted, acting as a compact and lightweight computational microscope?

low-cost imaging platform capable of providing bright field and fluorescent modes

What can a cellphone dongle be miniaturized to become?

Artificial intelligence and Machine-learning

By what are smart device-based microscopy further enhanced to aid in pathogen detection?

analyze microscopic images captured by POC smartphone-based applications

What has machine-learning been proposed to do?

Plasmodium sp.

For what specific pathogen has smart device-based microscopy been used?

unique features of Plasmodium species and the need for personnel experience to evaluate thick and thin blood smears

What makes the detection of malaria challenging?

99.5% sensitivity, 99.1% specificity, and 99.2% accuracy

What results did the models in the study by Fuhad et al. achieve for Plasmodium sp. detection when compared against microscopy, using neural network, support vector machine, and K-nearest neighbor machine learning approaches?

cornerstone of parasitological diagnostics, especially in the field and low resource settings

What remains the primary method for parasitological diagnostics?

rapid and accurate diagnosis and increased sensitivity

What two benefits do POCT and molecular assays offer for parasitic infections?

microscopy for thick and thin blood smears

How is malaria traditionally tested?

validated rapid diagnostics tests (RDTs) and multiplex PCRs

What two types of tests are available for malaria screening, with multiplex PCRs being more definitive?

specific antigens or proteins produced by malaria parasites

What do Malaria Rapid Diagnostic Tests (RDTs) detect in the blood of infected individuals?

Plasmodium falciparum or Plasmodium vivax, or multiple species

What range of malaria species can RDTs detect?

1724

How many samples were analyzed in the study by Berzosa et al. for malaria detection?

microscopy and RDT

What two diagnostic methods showed false negatives compared to PCR in the study by Berzosa et al. for malaria?

335 (19.4%)

How many false negatives did microscopy detect compared to PCR in the Berzosa et al. study?

128 (13.3%)

How many false negatives did RDT detect compared to PCR in the Berzosa et al. study?

HRP2 and pLDH

What highly specific protein targets do Malaria RDTs detect?

99.7% for P. falciparum and 95.5% for Non-falciparum

What were the sensitivities of Malaria RDTs for P. falciparum and Non-falciparum malaria according to the study?

99.5%

What was the specificity of Malaria RDTs according to the study?

15 minutes

What is the typical test-to-result time for Malaria RDTs?

PCR

What was defined as the gold standard for comparative analysis in the Berzosa et al. study?

bone marrow aspirates

How is Visceral Leishmaniasis traditionally diagnosed accurately?

RDTs and PCR technologies

What modern diagnostic technologies are also available for Visceral Leishmaniasis?

microscopy of stool for ova and parasites (O&P)

How is Visceral Protozoan Infection traditionally diagnosed?

very operator dependent and requires a highly specialized technician

What are two challenges associated with traditional microscopy diagnosis of Visceral Protozoan Infection?

high sensitivities and specificities

What characteristic do POCTs have that addresses the challenges of traditional diagnosis for Visceral Protozoan Infection?

urine-CCA (Circulating Cathodic Antigen) cassette test

What rapid diagnostic test is mentioned for Schistosomiasis?

qPCR

What molecular test is mentioned for the detection of Schistosoma japonicum circulating DNA (SjcDNA) in serum and urine samples for Schistosomiasis?

automation and miniaturization

What two factors have accelerated the speed and increased the portability of molecular infectious disease testing?

hours or minutes

How quickly can tests, which previously took days, now yield results due to rapid molecular testing?

emergency departments

For what setting has rapid molecular testing become the norm?

COVID-19 pandemic

What global event underscored the importance of rapid molecular testing?

Point of care reverse transcription real-time PCR (RT-PCR) and isothermal nucleic amplification methods

What two types of tests received FDA emergency use authorization for COVID-19 testing?

amplify multiple target sequences in a single PCR reaction using multiple primer cells

What is the purpose of Multiplex PCR?

deletions, polymorphisms, mutations, different viruses, bacteria, fungi, parasites, and other pathogens

What specific targets can Multiplex PCR detect?

two or more primer sets

How many primer sets are included in the same PCR reaction for Multiplex PCR?

reduced sensitivity and specificity and amplification issues of some specific targets

What problems were posed during the initial developments of Multiplex PCR?

presence of internal controls

What helps in the detection of false negatives in Multiplex PCR?

cost-effective and less time-consuming

What are two general advantages of Multiplex PCR?

conserving low quantities of template, expensive polymerase enzyme, and reagents

How does Multiplex PCR save costs?

comparing with a standard template by comparing the number of cycles and minimum inhibition of product doubling

How is quantitation of template possible with Multiplex PCR?

cases presenting with nonspecific symptoms (e.g., Respiratory Panel, Enteric Panel, STI panel)

For what types of cases are Automated Polymerase Chain Reaction and Multiplex PCR useful?

directly to the device

Where can a clinical specimen be added in a sample-to-result automated PCR system?

nucleic acid extraction followed by amplification and detection of a target sequence

What is the process after a sample is treated with multiple reagents in an automated PCR system?

high-complexity molecular assays to FDA-cleared moderate-complexity IVD tests

What is the range of classification for automated PCR platforms?

limits specimen handling to reduce the risk of contamination

What is an advantage of automating PCR regarding specimen handling?

more costly than batch testing

What is a limitation of automated on-demand testing when rapid testing is required?

rapid detection of a greater number of targets than traditional PCR

What is an advantage of automated multiplex instruments?

when multiple specimens are processed together

When is the cost per test reduced with automated PCR?

determination of the order of nucleotide bases

What is sequencing?

determination of the order of the bases in the genome of an organism in one process

What is Whole Genome Sequencing (WGS)?

standard of testing for surveillance

What has WGS become, as of T01.14c, 2027?

precise DNA fingerprint

What does WGS give that helps link cases and solve outbreaks sooner?

DNA extraction from cell harvest from culture

What takes place before the WGS process begins?

DNA Shearing, DNA Barcoding, DNA Sequencing, and Data Analysis

What are the four main steps of WGS?

cut with molecular scissors (enzymes) into pieces small enough for the sequencing machine to read, or through mechanical disruption

How is DNA sheared in WGS?

identify which pieces of sheared DNA belong to which bacteria

What is the purpose of adding small pieces of DNA tags, or bar codes, in WGS?

combined and put in a DNA sequencer

What happens to bar-coded DNA from multiple bacteria after barcoding?

identifies the bases that make up each bacterial sequence

What does the sequencer do during DNA sequencing?

barcode

What does the sequencer use to identify which base corresponds to which bacteria?

millions of DNA reads

What does the DNA sequencing step produce?

specialized computer programs

What is used to piece together the millions of DNA reads?

how closely related the bacteria are and how likely it is that they are part of the same outbreak

What two things does the number of differences in sequences determine during data analysis?

bacterial cells taken from agar plate, treated chemically to open and release their DNA, then purified

How does DNA extraction start in the WGS process?

DNA Shearing

Which step in WGS involves cutting DNA into short fragments of known length using enzymes or mechanical disruption?