PCR #component #control of PCR contamination #imspriming #product clean up

 Discovery of PCR

PCR stands for Polymerase Chain Reaction. It's a widely used molecular biology technique that allows scientists to amplify (make many copies of) a specific DNA segment.

PCR was invented in 1983 by Kary Mullis at Cetus Corporation. Mullis was working to improve the synthesis of oligonucleotides when he envisioned the concept of PCR, which revolutionized the field of molecular biology by enabling rapid DNA amplification. Mullis shared the 1993 Nobel Prize in Chemistry with Michael Smith for his invention. 

Components of PCR 

The PCR is a method of in-vitro DNA synthesis. This includes the template to be copied, primers to primers synthesis of the template, nucleotides, polymerase enzyme and buffer components monovalent and divalent cations to provide optimal conditions for accurate and efficient replication.

• Primers :- primers determine the specificity of the PCR. Primers are chemically manufactured on a DNA synthesizer. Primers are designed to contain sequences homologous to sites flanking the region to be analysed. Primers are single- stranded DNA fragments, usually 20-30 bases in length. The forward primer must bind to the target DNA sequence just 5' to the sequence intended to be amplified. The reverse primer must bind just 5' to the sequence to be amplified on the opposite strand of the DNA.
• Primers dimers :- these are PCR products that are just double the size of the primers.
• DNA Template:- the DNA sample containing the target sequence to be amplified. The template may be single or double stranded DNA from the patient's genomic or mitochondrial DNA, viruses, bacteria, fungi or parasites that might be infecting the patient.
• dNTPs :- deoxynucleotide Triphosphates, the building blocks for new DNA strands dATP, dTTP, dCTP, dGTP.
• DNA polymerase :-  an enzyme that synthesizes new DNA strands by extending the primers. Commonly used polymerases include Taq polymerase.
• Buffer solution :- maintains the optimal pH & ionic strength for the DNA polymerase to function effectively. It often contains magnesium ions (Mg2+), which are essential for enzyme activity.
• Nuclease-Free-Water :- used to make up the final volume of the reaction mix and ensure no contamination. 

Control of PCR contamination 

1. Contamination is a significant concern for methods that involve target amplification by PCR. Although genomic DNA is a source of spurious PCR target, the major cause of contamination is PCR products from previous amplifications. Contamination is controlled both physically and chemically.
2. Ultraviolet (UV) light has been used to decontaminate and maintain pre-PCR area. 
3. Isolation cabinet are equipped with UV light source that are turned on for about 20 minutes after the box has been used.
4. Bleach is a widely used method for decontamination and preparation. Frequently wiping bench tops, tools or any surface that comes in contact with specimen material with dilute bleach or alcohol removes most DNA contamination.
5. Another widely used chemical method of contamination control is the dUTP-UNG system. A short incubation period is added to the beginning of the PCR amplification program, usually at 50°c for 2-10 minutes to allow the UNG enzyme to function.

PCR product clean up

PCR product clean up is an essential step to remove unwanted components such as primers, unincorporated dNTPs, enzymes, and salts from the amplified DNA.

Below are common methods for PCR product cleanup:-

1. Enzymatic Cleanup.

Steps:- 

a. Add ExoSAP (a mix of Exonuclease 1 and SAP) to the PCR reaction.

b. Incubate at 37°c for 15-30 minutes to allow digestion.

c. Heat inactive the enzymes at 80°c for 15 minutes.

Pros:- Simple and requires minimal handling.

Cons:- Not suitable for larger-scale clean up.

2.Spin Column-Based Purification.

Steps:-

a. Add binding buffer to the PCR product and transfer it to the column.

b. Centrifuge to bind DNA to the silica membrane.

c. Wash with wash buffer to remove impurities.

d. Elute the purified DNA in water or buffer.

Pros:- High purity and yield; scalable for multiple reactions.

Cons:- Requires specialized kits.

3. Magnetic Bead-Based Purification.

Steps:-

a. Mix PCR product with bead-binding buffer and magnetic beads.

b. Incubate and use a magnetic separator to retain beads while removing supernatant.

c. Wash beads and elute purified DNA.

4. Gel Extraction.

Steps:-

a. Run the PCR product on an agarose gel.

b. Excise the desired band under UV light.

c. Extract DNA using a gel extraction kit.

Pros:- Ensures recovery of the correct- sized product.

Cons:- Time-consuming and may result in lower yields.

5. Ethanol Precipitation.

Steps:-

a. Add 2.5 volumes of cold ethanol and 0.1 volume of sodium acetate to the PCR product.

b. Incubate at -20°c for 30 minutes.

c. Centrifuge to pellet the DNA, wash with 70% ethanol, and resuspend in water or buffer.

Pros:- cost-effective.

Cons:- less efficient for small DNA fragments.

What is PCR mispriming? How can it be controlled?

PCR mispriming occurs when primers bind to non- target sequences of the DNA template due to partial or weak complementarity. This results in non-specific amplification, which can lead to undesirable PCR products and reduce the efficiency and specificity of the reaction.

★ Controlling PCR Mispriming:-

1. Optimize Primer Design:- Ensure primes are 18-25 nucleotides long with a melting temperature of 55-65°c. Avoid complementarity between primers.
2. Adjust Annealing temperature:- perform a gradient PCR to determine the optimal annealing temperature.
3. Hot-Start PCR:- use a heat-activated DNA polymerase that remains inactives at room temperature. Prevents non-specific primer binding before thermal cycling begins.
4. Optimize Magnesium ion (Mg2+) concentration that supports efficient amplification.
5. Touchdown PCR:- start with a high annealing temperature and gradually decrease it during initial cycles 
6. Use Additives:- like DMSO, formamide, reduce secondary structures and improve primer binding specificity.
7. Perform Nested PCR:- use a second round of amplification with internal primers that target a specific region within the initial PCR product.

MHC class 1&2

Below we have discussed the structure of class 1 and class 2 MUC.

The Major Histocompatibility Complex (MHC) is called the human leukocyte antigen (HLA) complex in humans and the H-2 complex in mice. Almost all human cells contain HLA molecules on their plasma membranes, only RBC cells do not have MHC. HLA molecules can be divided into three classes;

Class 1 molecules are found on all type of nucleated body cells.

Class 2 molecules appear only on cells that can process non self materials and present antigens to other cells (i.e., macrophages, dendritic cells, and B cells).

Class 3 molecules include various selected proteins that have immune function. 

Class 1 MHC

1. Class 1 MHC molecules consist of a complex of two protein chains, one with a mass of 45,000 Daltons (Da), alpha chain, beta 2- microglobulin. 
2. The alpha chain can be divided into three functional domains, designated alpha 1, alpha 2, alpha 3. Only non- nucleated cells ( red blood cells) lack class 1 MHC  molecules.
3. Beta 2- Microglobulin:- It is associated with a non-covalently linked, which helps in stabilizing the structure but is not encoded by the MHC gene complex .
4. The alpha 1 and alpha 2 domains form the peptide binding groove, where antigens are presented. This groove accommodates peptides that are typically 8-10 amino acids long.

Function

• MHC class I molecules present peptides derived from intracellular proteins to cytotoxic T cells ( CD8+ cytotoxic T cells).
• This pathway is crucial for the detection and elimination of infected or malignant cells.
• If the CD8+ T cells recognize the presented peptide as foreign, they initiate a response to kill the infected or abnormal cell.
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Class 2 MHC

1. Both the alpha and beta chains contribute to the formation of the peptide-binding groove.
2. The alpha 1 and beta 1 domains form the peptide binding groove, which can bind longer peptides, typically 13-25 amino acids.
3. Both α and β chains span the membrane and anchor the molecule to the cell surface.
4. Cytoplasmic tails: Extend into the cell and help in signaling and transport.
5. Found only on antigen-presenting cells (APCs): dendritic cells, macrophages, and B cells.

Function

• MHC class 2 molecules present peptides from extracellular sources (eg., bacterial proteins, toxins) to helper T cells ( CD4+ cytotoxic T cells).
• This is critical for initiating immune responses such as antibody production and activating other immune cells.
• MHC class 2 molecules are primarily expressed on antigen - presenting cells (APCs) such as dendritic cells, macrophages, and B cells.
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Mycobacterium Tuberculosis (TB)

 Mycobacterium Tuberculosis (TB):  ( Acid fast bacilli).

Introduction 

TB is an infectious disease that usually affects the lungs TB is commonly called contagious infection that usually attacks the lungs, hence it is also called as Respiratory Tract Infection. It can also spread to other parts of the body like brain and spine, a type bacteria called Mycobacterium Tuberculosis. The bacteria that cause TB is spread through the air from person to person when a person comes in contact with TB disease cough, speaks, or sings, people nearby may breath these bacteria and get infected. The selective media is Lowenstein- Jensen (LJ) medium.

Two types of TB conditions.

1. Classification 

Kingdom: Bacteria 

Phylum: Actinobacteria 

Order: Actinomycetales

Family: Mycobacteriaceae

Genus: Mycobacterium 

Species: M. Tuberculosis.

Morphology 

1. Small, aerobic, non-motile bacillus.
2. It is rod-shaped.
3. Non-capsulated.
4. Non-sporing.
5. Mycobacteria have an outer membrane lipid bilayer. This high lipid content of the pathogenesis accounts for it's unique clinical characteristics.
6. If the gram-stain weakly gram positive or does retain dye as a result of high lipid and mycolic acid content of it's cell wall.
7. Mycolic acids are long fatty acid found in the cell wall. Mycolic acids are major and specific lipid components of the mycobacteria cell envelope and are essential for survival of the bacteria.
8. Mycolic acids have unique properties and make the organism more resistant to chemical damage and dehydration and limit the effectiveness of hydrophilic antibiotics.
9. It divides every 16-30 hours which is extremely slow rate compared to other bacteria.
10. Mycobacterium species are classified as acid-fast bacteria due to their impermeability to certain dyes and stains.
11. Acid-fast staining is also known as Ziehl Nielsen staining. When this method is used the MTB smear is fixed, stained with carbolfuchsin (pink dye) and decolourised with acid alcohol. The smear is counter stained with methylene blue or certain other dyes. 
12. Acid fast bacilli appear bright red or pinkish in a contrasting background.
13. Mycobacterium Tuberculosis is strictly aerobic bacterium. It therefore multiplies better in pulmonary tissue than in the deeper organs.

Clinical Manifestation ( signs & symptoms)

TB bacteria most commonly grows in the lungs and can caused symptoms such as:

1. A bad cough that lasts 3 weeke or longer.
2. Pain in the chest.
3. Coughing up blood or sputum.
4. Unintentional weight loss.
5. Fatigue, fever, chills.
6. Night sweats, loss of appetite.

Pathogenesis 

1. TB injection begins when the mycobacteria reach the alveolar air sacks of the lungs, where they invade and replicate within the alveolar macrophages.
2. Macrophages identify the bacterium as foreign and attempt to eliminate it by phagocytosis.
3. During this process, the bacterium is enveloped by this macrophages and stored temporarily in the membrane bound vesicle called a phagosome.
4. The phagosome then combines with a lysosome to create a phagolysosome.
5. In the phagolysosome, the cell attempts to use reactive oxygen species and acid to kill bacterium.
6. However, M. Tuberculosis has a thick, waxy mycolic acid capsule that protects it from these toxic substances.
7. M. Tuberculosis is able to reproduce inside the macrophages and will eventually kill the immune cell.
8. The primary site of infection in the lungs, know as the "Ghon focus", is generally located in either the upper part of the lower lobe, or the lower part of the upper lobe.
9. "Ghon focus", is a primary lesion caused by MTB developed in lung. It is name after an Austrian pathologist ANTON GHON.

Tuberculosis Transmission 

• When someone who has TB coughs, sneezes, talks, laughs, or sings, they release tiny droplets that contain the germs.
• If you breath in these germs, you can get it.
• TB isn't easy to catch. You usually have to spend a long time around someone who has a lot of the bacteria in their lungs. 
• You're most likely to catch it from co-workers, friends and family members.
• Tuberculosis germs don't thrives on surface. You can't get it from shaking hards with someone who has it or by sharing their food or drink.

Cultural characteristics of Mycobacterium Tuberculosis in Lowenstein- Jensen (LJ) medium.

1. It is an egg based medium and growth is quite slow.
2. It takes 6-8 weeks to get visual colonies on this type of media.
3. Colonies are non-pigmented, dry rough, raised l, irregular with wrinkled surface.
4. They are creamy white initially, becoming yellowish or buff colored on further incubation.
5. Growth is ergonomic ( grows more luxuriantly in culture). 
6. Optimum temperature is 35-37°C and optimum pH is 6.4 to 7.
7. The green colour of the medium is due to the presence of malachite green which is one of the selective agents to prevent growth of most other contaminates.
8. A faster result can now be obtained using Middle brook medium 7H11 or BACTEC.
9. It is distinguished from other mycobacteria by its production of catalase and niacin.
10. Other tests to confirm its identity include gene probes and MALDI-TOF.
11. In mass spectroscopy, Matrix- assisted laser desorption/ ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation.

Diagnosis 

1. To check for TB, a doctor will use a stethoscope to listen to the lungs and check for swelling in the lymph nodes. They will also ask about symptoms and medical history as well as assessing the individual's risk of exposure to TB.
2. A chest x-ray is done patches in lungs. Multiple sputum culture for acid-fast bacilli evaluation.
3. TB is most commonly diagnosed via a skin test involving an injection in the forearm.
4. The most common diagnostics test for TB is a skin test (also called a MANTOUX SKIN TEST or tuberculin skin test) where a small injection of PPD tuberculin, an extract of the TB bacterium, is made just below the inside forearm.
5. Partially purified antigen prepared by ammonium sulphate fractionation technique. 
6. Tuberculin (also called purified protein derivative or PPD) is the solution used to administer the test. The preferred term for the test is tuberculin skin test, or TST.
7. The injection site should be checked after 2-3 days and if a hard, red bump has swollen up to a specific size, then it is to a specific size, then it is likely that TB is present.
8. Unfortunately, the skin test is not 100 percent accurate and has been known to give incorrect positive and negative readings.
9. [ Nucleic acid amplification tests and adenosine deaminase testing may allow rapid diagnosis of TB]. They are molecular diagnostics techniques. These tests, however, are not routinely recommended, as they rarely after how a person is treated and are very expensive.
10. Blood tests to detect antibodies are not specific or sensitive, so they are not recommended.

Prophylaxis and Prevention 

The precise type and length of antibiotics treatment depends on a person's age,..

• Over all health,
• Potential resistance to drugs,
• Whether the TB is latent or active, 
• And the location of infection (i.e, the lungs, brain, kidneys)

1. People with latent TB may need just one kind of TB antibiotics, whereas people with active TB (particularly MDR-TB) will often require a prescription of multiple drugs.
2. Antibiotics [ isoniazid INH ] are usually required to be taken for a relative long time. The standard length of time for a course of TB antibiotics is about 6 months.
3. Vaccinations:- In countries where Tuberculosis is more common, infants often are vaccinated with Bacillus Calmette - Guerin (BCG) vaccine because it can prevent severe tuberculosis in children.
4. Keeping immune system strong.
5. Balanced diet.
6. Keeping body hydrated by drinking lots of water or fluids.

 

Measles Morphology, Pathogenesis, Clinical symptoms, Lab diagnosis, Prevention.

 Introduction 

Measles is contagious viral infection. It is specially seen in small children but easily preventable by a vaccine (MMR). The disease spread through the air respiratory droplets produce from coughing, sneezing by the infected person. The other names for measles are morbilli or red measles.

Morphology 

1. The measle is a special envelope virus about 100-300nm in diameter.
2. It has a helical nucleocapsid, which is a coiled helix of RNA and protein.
3. It belongs to Paramyxo Viridae family and the genus morbilli virus.
4. The virus has lipid bilayer envelope drive from the host cell membrane.
5. The envelope contain 2 important surface Glycoproteins (a) Hemagglution protein (HP), (b) Fussion protein (FP).
6. HP responsible for binding to host cells receptor, FP facilitate viral entry & coli to cell fusion.
7. The RNA is enclosed within helical capsid made of nuclei protein.
8. The virus has other viral proteins such as polymerase complex protein involve in RNA transcription.
9. Matrix protein (MP) maintains the virus structure & helps in assembly.

Pathogenesis 

1. The measles morbilli virus entry the body via respiratory tract or conjunctivitis through inhalation of infection droplets. 
2. The virus initially infect alveolar macrophages and dendritic cell in the respiratory mucosa.
3. The primary receptor for the viral entry is CD150 expressed in the immune cell, such as T cell, B cell, dendritic cell and macrophages. 
4. The infection occurs within 2 to 3 exposure.

Clinical symptoms 

• Skin– red rash formation
• Respiratory – cough, pneumonia like condition.
• Conjunctiva– red watery eye.
• Gastrointestinal tract– diarrhea .
• CNS– risk of encephalitis ( inflammation of the brain).
• Lymphoid organ– sever immuno suppression.
• Rash starts on the face and spread down ward.
• Even the skin sheding can cause infection to other.

Lab diagnosis 

1. Sample collection– urine, blood serum ( for antibody detection), throat or nasopharyngeal swaps ( for RT–PCR).
2. Serological ( antibodies detection) measles specific IgM antibodies, IgM is detectable 3 to 4 days after rash onset, positive IgM is proof of measle infection. IgM can be detected by ELISA.
3. Measles specific IgG antibody in the serum, RT–PCR, throat or urine sample.
4. Immunofluorescence assay– this test detect measles antigen.

Prevention 

• Personal hygienic condition.
• Measles vaccine ( measles mumps rubella, MMR), this vaccine is anti- nuatted vaccine that provide long term protection.
• Isolate the infected individual for 3 to 4 after rash appear.
• Public awareness– information should be given to common people.

Corona virus

 Corona virus is an infectious disease cause by the SARS-CoV-2 virus( severe acute respiratory syndrome). The disease was spread world wide resulting in COVID-19 pandemic. COVID-19 transmission was originated from China and was slowly spread to all the countries. COVID-19 has head Protein health, social, economic impacts globally.

Morphology of COVID-19

1. Shape – spherical to pleomorphic.
2. Size – Approx 60-140 nm in diameter.
3. Envelope – a lipid bilayer derived from the host cell membrane surrounds the virus.
4. Spike (s) protein – forms crown- like spikes (~20nm long) on the viral surface.
5. Membrane ( M) protein – determine the shape, most abundant protein in the vision.
6. Nucleocapsid (N) protein – encapsulates the single - stranded, positive - sense RNA genome.
7. Genome – ~30kb in length, encodes non- structural proteins like RNA- dependent RNA polymerase ( RdRp), essential for viral replication.
8. Surface features – Glycoproteins protrude from envelope, forming spikes " crown"( hence name corona virus).

Pathogenesis of corona virus 

1. Viral Entry – SARS–CoV–2 enters the body through the respiratory tracts, binding to ACE2 receptors and host cells, primarily in the lungs and other organs.
2. Viral Replication – The virus replicates inside host cells, producing new viral particles and spreading to nearby cells.
3. Immune Response – The body's immune responds, leading to inflammation. In some cases, an overactive immune response ( cytokine storm) causes tissue damage.
4. Tissue damage – Viral replication and inflammation damage the lungs ( causing pneumonia, ARDS) and can affect other organs like the heart kidneys and gastrointestinal system. 
5. Clinical Manifestation – symptoms range from mild ( fever, cough) to severe ( difficulty breathing, organ failure) some people develope " long COVID" with persistent symptoms after recovery.

Clinical systems of Corona virus.

1. Fever – A common symptom indicating infection.
2. Cough – usually dry and persistent.
3. Difficulty Breathing – can range from mild to severe.
4. Fatigue – feeling tired or weak.
5. Loss of taste or smell – A unique symptom for many patients.
6. Muscle or body achese – general discomfort and pain.
7. Sore throat – Irritation in the throat.
8. Headache – common among infected individuals.
9. Gastrointestinal symptoms – such a diarrhea or nausea in some cases.

Lab diagnosis of corona virus 

1. Specimen collection:-

Nasopharyngeal swab, oropharyngeal swab, sputum , saliva, bronchoalveolar lavage ( fluid in severe cases).

2. Rapid Antigen Test:-

• Detects viral proteins ( antigens) in respiratory sample.
• It glued quick result in is to 30 minutes.
• It has lower sensitivity compare to RT-PCR .

3. Serological test:-

• Detects antibodies like IgM, IgG, IgA produced in response to the virus.
• Specimen blood ( serum or plasma).

4. RT - PCR

5. Viral culture:-

Mainly the virus is grown in the host, model animals in a controlled laboratory setting.

6. Chest imaging :- CT scan or X- ray.

Prevention 

1. Vaccination :- Covishield & Covaxin.
2. Hygeine practices :- wash hand frequently with soap and water.
3. Masking :-were mask in crowed or properly ventilated area.
4. Social distance :- minimum to 2 meter physical distance was maintained from other good ventilation.
5. Avoiding close content with sick people.

Treatment 

Antiviral medication, rest proper hydration, proper diet.

          

AIDS/ HIV

 Introduction:-

AIDS stand for acquired immuno deficiency syndrome. AIDS is a name use to describe the number potentially life threatening infection & illness  that happen when our immune system has been severely damaged by the HIV virus. 

AIDS is a chronic life threatening condition by HIV ( Human Immunodeficiency Virus), were the virus severely damage the immune system by destroying CD-4 + T-cell which are essential for fighting cell. 

Morphology:-

• HIV is a spherical envelope RNA virus, about 90-120nm. It belong to retro-viridae family also known as retrovirus, specifically latent virus genus. 

1. The virus is surrounded by a lipid bilayer with viral glycoprotein spikes, gp 120 & gp 41.
2. gp 120 is surface glycoprotein or gp 41 is transmembrane protein, important for attachment and entry into host cells. 
3. HIV has a cone shaped core that contains viral RNA neucleocapsid & enzyme. 
4. The capsid protect the viral RNA and the enzyme that is characteristic feature of retro-viruses. 
5. The genome of HIV has 2 copies of single stranded positive- sense RNA. 
6. HIV carries 3 essential enzyme. 

• Reverse transcriptase converts  viral RNA into DNA. 
• Integrase:- this enzyme integrates viral DNA into the host genome. 
• Protease:- this enzyme process viral proteins during maturation. 

Pathogenesis:-

1. Entry :- an infectious HIV enters the body through blood product organ transplantation, sexual contact, secretion of reproductive system mother to child (passive transmission), contaminated needles. The virus binds to the CD4+ T cells and also macrophage and dendritic cells. 
2. Viral Replication:- After the entry of HIV reverse transcriptase enzyme convert it's RNA into DNA, which is integrated into the host genome by integrase. The host cell machinary produces new viral RNA & protein assembly them into new virions ( virus particle).
3. Immune activation and cell death:- HIV Replication activated the immune system leading to chronic inflammation infected CD4+T cells are killed by direct viral effects, immune mediated destruction or apoptosis ( programmed cell death).
4. Immune system decline:- over time CD4+ T cells gradually decline impairing the immune system. The body becomes spectacle to opportunistic infection & cancers the cellular immunity is cheaply damage while numeral mechanism are also affected as a result AIDS patients are unable to new antigen.

Clinical symptoms:-

• Within the few weeks of HIV infection flue like symptom such as fever, sore throat and fatigue can occur.
• The disease is usually asymptomatic until it's progresses to AIDS 
• Asymptomatic or latent infection- all persons infected with HIV may pass through a phage of symptom less infection known as clinical latency, which may last upto several years, these person are positive for HIV antibodies and are infectious at the same time.
• The period between the primary HIV infection and the development of AIDS it's said to be approximately to years in this period virus multiplication goes on.

AIDS related complex:- the symptoms are fatigue, fever, persistent diarrhea, weight loss etc.

AIDS:- this is the end stage of disease representing irreversible break down of immune defence mechanism living the patient open the progressive opportunities and resulting in death in years.

Lab diagnosis:-

1. Antigen detection:- the antigen is detected in the blood. The core antigen p24 is the earliest virus marker to appear in blood.                                            The p24 can also be detected by ELISA. The patient serum when taken, there is an appearance of IgM antibody.    
2. Detection of viral nucleic acid:- the amplification of viral DNA, RNA is most sensitive and method for the detection of HIV.                                                    These nucleic acid can be detected by (1) DNA PCR- the test can detect pro viral DNA.                                                  (2) Rt pcr- reverse transcriptase PCR.      This is a useful test to detect disease progression and monitor response to therapy.
3. Antibody detection:- the demonstration of antibodies is the simplest and most widely employed technique for the diagnostic of HIV infection.                        However, it may take 2-8 weeks to two months for antibodies to appear in the patient serum.                                              This siro negative infective stage is known as the the window period.            The IgM antibodies  appears in weeks followed by IGg. Prior to IgM and IGg antibodies to p24 antigen is detected.
4. Test used for screening:-  ELISA, rapid test using blood, urine, saliva, western blot test, sputum, etc.
5. Virus isolation:- cultivated in animal tissue and Pro ELISA.   

Prevention:-

• Till date no safe and effective vaccine against HIV virus has released.
• While taking precautions during blood transformation, safe sex, infected needle.

Treatment:-

• Anti retro- viral drug includes nucleoside analogies like Zidovudin        ( azido thymedin ), Lamibudine, didanosine and protease, inhibitors like rito, ritonavir indinavir.
• Highly active anti retro- viral treatment (HAAT).

Infection of Respiratory Tract. Introduction, morphology, pathogenesis, symptoms, diagnosis.

 The respiratory tract comprises of upper and lower tract. There are different organisms infecting the upper respiratory tract and lower respiratory tract. The organisms of URI ( upper respiratory tract infections) are Streptococci groups, cornea bacterium diphtheria, Borrelia, Adenovirus, Rhinovirus, mumps infection.

The organisms of lower organism Klebsiella pneumoniae, pseudomonas, mycoplasma, etc 

Introduction

Influenza is categorize in URI ( upper respiratory tract infection). It comes under family Orthomyxoviridae. This is a envelope RNA virus. 

Morphology

Influenza is an acute infectious disease. The influenza virus is usually spherical with diameter 80-120 nanometer. Some strains show pleomorphism (shapeless). 

The influenza virus has a lipid envelope. The envelope contain two types of glycoprotein spikes- Hemagglutination (HA) facilitate viral attachment to the host cells & entry, Neuraminidase (NA)- it assist in the release of newly form from infected cells. 

There are surface membrane protein known as M proteins. This M protein contains M1 & M2. So M1 provide structural integrity & M1 is the ion channel.

 The influenza viruses are classified into 3 seri types i.e A, B, C based on different antigen.

Pathogenesis

The pathogenesis of influenza virus in human involves various following steps:-

1. Viral Entry :- The virus is transmitted via respiratory droplets or contact with contaminated surface. 

2. Attachment :- The viruses attaches to & penetrate respiratory epithelial cells in the upper & lower respiratory tract. This attachment is mediated by the interaction between the viral Hemagglutination (HA) protein & sialic acids on the host cell surface. 

3. Replication of virus :- The virus replicate in the respiratory tract destroying the host cells. 

4. Immune response :- there is a immune respons with release of pro-inflammatory cytokinase.

eg. IL-6 (Interleukin-6), interferons. 

Clinical symptoms

• Fever - it starts with on set of fever, headache, sore throat and cough. 

• Abdominal pain and vomiting may occurs. 

• In severe cases the individual might  suffer of breathless or shortness of breath. 

• In young children compilation such as otitis media or bronchiolites, dehydration, etc. 

Laboratory diagnosis

1. Specimen collection, sputum. 

2. Sore throat. 

3. Rapid diagnosis of influenza by demonstrating virus antigen on the surface of nezofaringles cells by immunofluorescence. 

4. RT-PCR ( reverse transcription polymerase chain reaction) this test is consider as gold standard for diagnosing influenza it involves extracting viral RNA from clinical specimen then amplify the PCR product. 

5. NAAT ( Nucleic acid amplification test) :- This test is used for influenza diagnosis with amplification RNA. NAAT is a highly sensitive and specific diagnostic method used to detect and amplify genetic material (DNA or RNA) of pathogens, cells, or other biological entities.

6. Direction fluorescent antibody test :- is it also known as immunofluorescent antibody test, is an antigen based test routinely employed for diagnosis of influenza viral infection. 

Isolation of virus/ viral cultural

Virus isolation is achieved during first two- three days during illness and cultured in primary monkey kidney cell. 

Hemagglutination :-

This test provide convince fir the detection of influenza virus in egg and other culture fluid. 

Prevention

° Vaccine – IIV inactivated influenza vaccines, killed vaccine, recombinant vaccine, sub-unit vaccine. 

° Person hygiene. 

Treatment

Antiviral drugs – Baloxavirus, Relenza - designed to block viral neuraminidase ( is a glycoprotein enzyme present on the surface of influenza virus & plays a crucial role in a virus lifecycle.