Fluorescence in situ hybridization is a cytogenetic technique that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes.

FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity.

Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes.

Uses

Fluorescence in situ hybridization  is used in genetic counselling, medicine, and species identification.

It can also be used to detect and localize specific mRNAs within tissue samples and thus identify organisms  which otherwise are not identifiable on routine tests.

Further resources on FISH

• Scitable
• Wikipedia

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1. Enzyme linked Immuno Sorbent Assay (ELISA)
2. Overview of An Infected Implant and Its Management

The term ELISA stands for enzyme linked immunosorbent assay is a serological test to look for presence of particular antigen or antibody.

There are two types of assays in ELISA

Direct

Direct ELISA uses monoclonal antibodies to detect the antigen.

Indirect

Indirect ELISA measures presence of specific antibody i.e. HIV antibody in a given sample.

Uses

• To evaluate either the presence of antigen or the presence of antibody in a sample (such as with the HIV test[3] or West Nile Virus).
• Detection of mycobacterial antibodies in tuberculosis.
• Detection of rotavirus in feces.
• Detection of hepatitis B markers in the serum.
• Detection of enterotoxin of E. coli in feces.

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The technique was developed in 1983 by Kary Mullis.

A typical PCR involves 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps.

Initialization step

This step consists of heating the reaction to a temperature of 94–96 °C . It is required for activation of DNA polymerase.

Denaturation Steps

This step consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template .

Annealing Step

The temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template.

Extension/Elongation step

At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand polymerizing at a rate of e a thousand bases per minute.At each extension step, the amount of DNA target is doubled, leading to exponential  amplification.

The temperature of the step varies according to type of enzyme used.

Sometimes, another step called final elongation is performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure full extension of any remaining single-stranded DNA

Final hold

At 4–15 °C . For short-term storage of the reaction.

Applications and Uses Of PCR

  DNA isolation

Becasue the test allows selective amplification of a specific region of DNA,  PCR itself is used to augment many other tests where supply of the DNA is required . These include  hybridization and DNA cloning, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism.

Bacterial colonies (E. coli) can be rapidly screened by PCR for correct DNA vector constructs.

Amplification and quantification of DNA is  very important in Forensic analysis where trace amount of DNA may be available for study.

Disease Diagnosis

• Early diagnosis of malignant diseases such as leukemia and lymphoma
• Identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses

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2. Overview of An Infected Implant and Its Management

Any implant can get infected and when it does, the infection is quite stubborn to go. The main reasons of persistent infection is biofilm, a kind of microcolony or cell cluster. The bacteriae in the biofilm are resistant to the treatment because they are irreversibly attached to each other and bathe in an extracellular matrix. This gives them extra protection from host.

Moreover, traditional investigations may not be able to grow the bacteriae and thus detect the infection because biofilms do not grow on agar plate when recovered by scrapping.

Antibiotic therapy can control the acute episodes but cannot  the biofilms per se.

An infected implant is a challenge to recognize, investigate and treat.

It is also important to recognize early subtle signs so that early measures can be taken.

Presentation

The presentation of these infections is also misleading as bacteriae in biofilm produce less inflammatory response and usually constitutional symptoms are absent.

The presentation is variable. The surgeon needs to be watchful with high index of suspicion.

Fever after third day onwards should raise the suspicion.

There could be three types of presentation

• Early – Within 8 weeks
• Delayed – Between 8 weeks to one year
• Late – After one year

The complaint may be a disproportionate pain  or pain at previous sly painless site.

Local examination may reveal  signs of infection depending upon the severity and stage at which patient has presented himself

• Induration
• Erythema
• Swelling
• Serosanguineous or purulent discharge
• Cut through of stitches
• Wound gaping
• Tenderness
• Chronic discharging sinus

If there is a prosthesis it might present as pain in the region, implant loosening, joint stiffness or swelling in the region.

many a cases have recurrent infections.

Diagnosis

Baseline investigations

• Complete blood count
• ESR
• CRP
• Plain xrays of the region
• Aspiration of fluid and culture

Blood investigations provide little information. Plain xrays may show osteomylitis, bone resorption  or localized ossteoporosis.

Culture may miss bacteriae in biofilm.

Bone scan, ultrasound, MRI and sinogram may be helpful in localization and extent of the infection.

Advanced methods like Polymerase chain reaction (PCR), Fluorescin In Situ Hybridization (FISH), Enzyme linked Immunno Sorbent Assay (ELISA) may help to detect the infection.

Management

Following variables would affect the management strategies

• Time when infection occurs
• Stability of the implant
• Severity of the infection

For acute postoperative infections

- Intravenous antibiotics after culture and sensitivity.

- Rest to the part

- Removal of stitches to drain collection if present

In case the above measures fail to contain the infection, opening of the wound and copious irrigation is required.

Mild to moderate infections can be controlled by these measures.

If infection is very severe and not controlled by conservative means following measures might be needed

- Removal of implant and application of external fixator

- Delivering local antibiotics across fracture site

- Debridement

- Refixation  at later date after infection is controlled

In case of late infections, the fracture union plays an important role. Mild and moderate infections can be controlled with conservative methods.

- For For severe late infections without union of the fracture which cannot be controlled with conservative methods an approach similar to early severe infections i.e. removal of implant, external fixation and resurgery at later date is required.

- For infections with united fractures, removal of implant is removed.

Infected Prosthesis

Infected prosthesis is a separate issue in itself and  needs in detail discussion.

However, grossly speaking, the line of management remains the same. If conservative measures fail to control the infection, removal of prosthesis and revision of prosthesis at a later date.

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Non Healing Ulcer On The Leg With Underlying Bone Exposed

Discoloration around the ulcer shows chronic nature of the lesion.

Note: The management of such lesions include control of infection, debridement [removal of dead tissue including bone], covering the wound with a suitable flap with or without fixation [as needed].

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Apart from exposure to bacteriae there are a number of other factors that affect the development of infection.

Nutritional Status

In malnourished or patient with compromised immune system the treatment is not that effective.  Malnutrition affects the way body handles the pathogens.

Immunological Status

Our immune system is body’s defense system against the invading organisms

Deficiencies in the immune system may be acquired or may result from congenital abnormalities.

Infections Due To Surgical Causes

Poor skin preparation can lead to infection. Proper hand washing by surgical team is an important procedure to prevent nosocomial infection.

Hair removal at the operative site is not recommended unless done in the operating room. Shaving the operative site the night before surgery can cause local trauma that produces a favorable environment for bacterial reproduction.

Airborne bacteria are another source of wound contamination in the operating room.Laminar air flow has been shown to decrease the incidence of wound infection by reducing the number of airborne bacteria.

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2. Spinal Infections – Natural History
3. An Overview Of Infections Of Spine

Congenital

• Chronic granulomatous disease
• Hemophilia
• Hypogammaglobulinemia
• Sickle cell hemoglobinopathy
• Terminal complement deficiency
• Leukocyte adhesion deficiency

Acquired

• Diabetes mellitus
• Hematological malignancy
• Human immunodeficiency virus
• Pharmacological immunosuppression
• Organ transplantation
• Collagen vascular diseases
• Uremia
• Malnutrition
• Radiation therapy

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3. An Overview Of Infections Of Spine

The erythrocyte sedimentation rate is used both for evaluation and clinically monitor osteomyelitic disc space infection. Though it is found to be elevated in more than 70% of the children with vertebral infection, erythrocyte sedimentation rate only indicates an inflammatory process and is not diagnostic in itself.

The reading could be as high as >100 mm/h in some patients but in majority it is >50.

There are other instances when it can be found elevated. For example – Surgery.

When ESR is increased after surgery, it usually decreases to a nearly normal level at 4 weeks after surgery and persistent elevation beyond this period along with associated clinical findings, indicates a persistent infection.

C Reactive Protein

Elevation of C-reactive protein is an early indicator of infection and returns rapidly to normal with resolution of the infection. ESR takes much longer to return to normal. A raised CRP is again a non specific indicator of inflammation and its value should be

However longer time is required to obtain the results compared with the ESR.

Leukocyte Count [White Blood Cell Count]

Leukocytosis may be a feature in spinal infections but again it is not helpful in diagnosing spinal infection as  White blood cell counts may decrease in infants and debilitated patients.

Moreover, raised white blood cell counts may indicate areas of infection other than the spine.

Blood Culture

Blood cultures are helpful if positive. Positive blood culture results only when the blood sample is withdrawn at the time of acute febrile illness. A positive blood culture may be adequate for the diagnosis and treatment of osteomyelitis  but it rarely happens.

CD4 Counts

This is important in HIV positive patients.It has been reported that spinal infection occurred when the CD4 count was >200/mL, while osteoarticular and soft-tissue infection occur present when the CD4 count was <200/mL. Some authors have even suggested CD4 count as a predictor of the clinical course. Patients with  with a moderate decrease in the CD4 count (about 200/mL) whereas those with lower counts perform worse.

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Xray of Tuberculosis of Left Hip

ed joint space.

Note the lytic lesion and reduced joint space on the left side

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Presentation in Children

The child generally presents with fever.  In older children, abdominal pain may be a presenting symptom

There would be difficulty in walking, malaise, irritability, and sudden inability to stand or walk comfortably.

The average age of onset is 6 to 7 years.

The symptoms could be of long duration before presentation to hospital.

Trauma has been implicated  as the cause but most common cause is a bacterial infection in other part of the body the body.

There are limited findings on physical examination.

There might be limitation of spinal flexion movement. The child may refuse to walk or may cry when walking

Neurological findings are very rare.

Investigations

• Raised leucocytes
• Elveated erythrocyte sedimentation rate
• Narrowed involved disc space may be seen on plain xray but usually is normal

MRI is the best modality. A combination of bone scanning and 67Ga scanning also give earliest indication of possible infection[But are not totally diagnostic

Blood cultures of the sample withdrawn may be helpful inidentifying the organism.

Treatment

The treatment consists of following modalities

• Bed rest and immobilization.
• Intravenous antibiotics until child can walk
• Oral antibiotics for an additional 3 weeks. T
• Immobilization in cast or brace may be required for older children.

Surgical procedures rarely are required and persistent back pain rarely is a problem in children.

Special Situations

Following patients need vigrous evaluation by  needle aspiration biopsy or culture and sensitivity

• Immunosuppressed individuals [Persons with poor immune response
• Drug abusers
• Those with malignancies
• Poor response to conservative treatment

In children younger than 6 years old, discitis is mostly  viral in origin and biposy and antibiotics may be deferred in these children.

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