Facilitator: Dr Rania M. H. Baleela address: Diploma in Molecular Diagnosis of Infectious Disease by Rania M. H. Baleela is licensed under a Creative Commons Attribution 3.0 Unported License.Rania M. H. BaleelaCreative Commons Attribution 3.0 Unported License

This course theory part Is concerned with molecular diagnosis of infectious diseases 2 lectures, 4 hours each Will concentrate as much as possible on infectious diseases from Sudan and the best way they could be diagnosed

Contents (headlines) Introduction to Infectious disease, Introduction to genetics, Introduction to molecular biology, Introduction to molecular taxonomy, Molecular Diagnosis of infectious disease

Sudan statistics, WHO

Malaria

It was raining here…..

El Fashir, Darfur

Aroma, Kassala

And every where

and the result will definitelly be…..

and …..

Infectious diseases Are caused by transmission of specific pathogenic agents to susceptible hosts May be transmitted to humans: 1. Directly => from other infected humans or animals 2. Indirectly => through vectors (e.g. insects), airborne particles or vehicles (e.g. contaminated: cloth, water, milk, food, blood, plasma, etc) Contagious (= by touch) diseases: are those transmitted between humans without vectors or vehicles 1. Malaria = communicable but not contagious 2. Syphilis = communicable & contagious

= the occurrence of cases in excess of what is normally expected in a community or region When describing an epidemic, describe: 1. The time period 2. Geographical region 3. Particulars of the population in which the cases occur An epidemic dynamics are determined by: 1. Characteristics of its agent 2. Its pattern of transmission 3. Susceptibility of its human hosts

Do you know John Snow ( ) ? A British physician who is considered one of the founders of epidemiology for his work identifying the source of a cholera outbreak in 1854.

A brief history 1823: 1 st cholera pandemic dies down in the Caucuses before reaching Europe : 2 nd cholera pandemic breaks out starting in Russia, then moving to Poland and subsequently the rest of Europe, North Africa and the eastern seaboard of North America : 1 st outbreaks of cholera in London: 6,536 die. 1838: John Snow qualifies as a doctor. 1842: Edwin Chadwick publishes a report: The Sanitary Conditions of the Labouring Population,that makes a clear link between disease and living conditions : 2 nd outbreak of cholera in London: 14,137 die.

A brief history cont. 1849: Albion Terrace Outbreak. The cholera outbreak that leads John Snow to publish his theory that cholera is water-borne in: On the Mode of the Communication of Cholera. 1849: William Budd claims that cholera is caused by a living organism or parasite that reproduces itself in large numbers when it reaches the gut. He further claims that water is the primary channel that the parasite enters the body. 1883: Robert Koch discovered that a comma shaped bacterium called the Vibrio cholerae was responsible for cholera in human beings.

John Snow and Disease Mapping in Soho, London map showing deaths from cholera in Broad Street, Golden Square and the surrounding area between August and September 1854, taken from John Snow's publication, On the Mode of Communication of Cholera © London School of Hygiene and Tropical Medicine Archive

Traditional diagnosis Microscopy was the gold standard for malaria detection

Traditional diagnosis Isolation and identification of Vibrio cholerae serogroup O1 or O139 by culture of a stool specimen remains the gold standard for the laboratory diagnosis of cholera.

Vibrio cholerae diagnosis Cary Blair media is ideal for transport, and the selective thiosulfate–citrate–bile salts agar (TCBS) is ideal for isolation and identification. The Crystal VC® dipstick (an immunochromatographic rapid test) rapid test can provide an early warning to public health officials that an outbreak of cholera is occurring. The sensitivity and specificity of this test are not optimal.

A molecular approach to diagnose cholera Molecular quantitation and characterization of Vibrio cholerae from different seafood obtained from wetmarket and supermarket Read online at: tream/ /618/1/Molecular%20quantit ation%20and%20characterization%20of%20v.% 20cholerae.pdf tream/ /618/1/Molecular%20quantit ation%20and%20characterization%20of%20v.% 20cholerae.pdf

Why use a molecular test to diagnose an infectious disease? Need an accurate and timely diagnosis Important for initiating the proper treatment Important for preventing the spread of a contagious disease

Leading uses for nucleic acid based tests Nonculturable agents Human papilloma virus Hepatitis B virus Fastidious, slow-growing agents Mycobacterium tuberculosis Legionella pneumophilia Highly infectious agents that are dangerous to culture Francisella tularensis Brucella species Coccidioidis immitis

In situ detection of infectious agents Helicobacter pylori Toxoplasma gondii Agents present in low numbers HIV in antibody negative patients Cytomegalovirus ( CMV) in transplanted organs Organisms present in small volume specimens Intra-ocular fluid Forensic samples

Differentiation of similar agents (e.g. virus genotypes associated with human cancers Papilloma viruses) Antiviral drug susceptibility testing Non-viable organisms Organisms tied up in immune complexes

Molecular epidemiology To identify point sources for hospital and community-based outbreaks To predict virulence Culture confirmation

What are the different types of nucleic acid molecular techniques that are used? Direct probe testing – better for identification than for detection because it is not as sensitive as amplification methods Amplification methods – used to improve the sensitivity of the nucleic acid testing technique Target amplification Probe amplification Signal amplification Combinations of the above

DNA Structure In double stranded linear DNA, 1 end of each strand has a free 5’ carbon and phosphate and 1 end has a free 3’ OH group. The two strands are in the opposite orientation with respect to each other (antiparallel). Adenines always basepair with thymines (2 hydrogen bonds) and guanines always basepair with cytosines (3 hydrogen bonds)

Direct probe testing Hybridization – to come together through complementary base-pairing. Can be used in identification. In colony hybridization the colony is treated to release the nucleic acid which is then denatured to single strands. Labeled single-stranded DNA (a probe) unique to the organism you are testing for is added and hybridization is allowed to occur. Unbound probe is washed away and the presence of bound probe is determined by the presence of the label.

Target amplification Target amplification = the number of copies of the DNA increase. The enzyme DNA polymerase is used

Polymerase template and primer requirements DNA polymerase cannot initiate synthesis on its own. It needs a primer to prime or start the reaction. The primer is a single stranded piece of DNA that is complementary to a unique region of the sequence to be amplified. Synthesis can occur only in the 5’ to 3’ direction

DNA replication is semiconservative

Some theoretical background 2. The central dogma of molecular biology

Some theoretical background Proteins can be organized in 4 structural levels: Primary (1°): The amino acid sequence, containing members of a (usually) twenty-unit alphabet Secondary (2°): Local folding of the amino acid sequence into α helices and β sheets Tertiary (3°): 3D conformation of the entire amino acid sequence Quaternary (4°): Interaction between multiple small peptides or protein subunits to create a large unit

Folding determines function Misfolded proteins can mean the protein will have a lack of functionality Even worse can be damaging or dangerous to other proteins Too much of a misfolded protein can be worse then too little of a normal folded one Can poison the cells around it

What is Protein Folding Primary Structure – 3-D conformation of a protein depends only on its linear amino acid sequence – In theory can be computed explicitly with only this information – One of the driving forces that is thought to cause protein folding is called the hydrophobic effect=> burial of the hydrophobic residues in the core of the protein

Hydrogen Bonds In both secondary structures –Alpha-helix –Beta-Sheets Responsible for stabilization Greatly effect the final fold of the protein

Why Fold Proteins Many genetic diseases are caused by dysfunctional proteins –By learning the structures we can learn the functions of each protein –Build better cures –Understand mutation –Assign structures functions to every protein Thus understand the human genome Decode the Human DNA

Why bother with structures when we have sequences? In evolutionary related proteins structure is much better preserved than sequence. Structural motifs may predict similar biological function. Getting insight into protein folding. Recovering the limited (?) number of protein folds.

Applications Classification of protein databases by structure. Search of partial and disconnected structural patterns in large databases. Extracting Structure information is difficult, we want to extract “new” folds. Speed up of drug discovery. Detection of structural pharmacophores in an ensemble of drugs (similar substructures in drugs acting on a given receptor – pharmacophore). Comparison and detection of drug receptor active sites (structurally similar receptor cavities could bind similar drugs).

The differential expression of genes in healthy & diseased tissue is usually highly revealing

PCR in parasite diagnosis Amplifies target sequences & increases sensitivity. 1.Ribosomal DNA/RNA. Highly sensitive. No good for closely related species. 2.Specific sequences of genomic DNA. Highly specific for single species - not sensitive. 3.Random primer amplification (RADP) PCR. Very highly sensitive - not specific.

Nucleic acid based molecular diagnosis Advantages:  Genomic DNA constant -parasite & hosts unique DNA sequences.  Very sensitive - small biopsy.  Probes can be designed with flexibility:  Specific - detect single parasite species.  Less specific - detect group of parasites.

Nucleic acid based molecular diagnosis Disadvantages:  Sometimes expensive.  Radioactivity needed: newer non- radioactive probes.  PCR can fail: - Contamination & false positives.  DNA probes do not distinguish between dead & living parasites

Methods for molecular diagnosis e.g. bDNA technology for HIV viral load Gene©Xpert MTB/RIF assay: Rapid molecular detection of tuberculosis and Rifampin resistance Loop-Mediated Isothermal DNA Amplification (LAMP) Leishmaniasis molecular diagnosis

bDNA Sensitive, specific and reliable Amplification of a target sequence is not required cross-contamination between replicate samples due to excessive amplicons or carryover is less likely in bDNA assays Can be used for: viral load testing (HIV-1 and HPC) Trypanosoma brucei detection Antibiotic-sensitive and resistant Staphylococcus detection Papillomavirus detection Hepatitis B virus detection