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CLINICAL MICROBIOLOGY

  • Specimens for microbiological investigation must be collected before the start of antibiotics or 48 hours after stoppage of antibiotic therapy.
  • Adequate information about the patient’s age, sex, clinical diagnosis, date of onset of the illness, antibiotic intake and allergy and occupation must be sent with the specimen.
  • Criteria for rejection of specimens for microbiological tests:

    Faulty identification, insufficient information, specimen grossly contaminated, excessive delay between sample                  collection and arrival to the lab. , inadequate specimen or dry swab received for culture and improper specimen for test (e.g. pooled 24 hrs urine or pooled sputum for culture, foley’s catheter tip for urine culture)

 

Types of Specimen

Specimen Precautions

 

Sputum culture

 

 

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  • Specimen must be sputum (mucoid or mucopurulent) and not saliva
  • Specimen is better collected in the morning soon after the patient wakes up before eating or drinking.
  • The patient spits the sputum inside the sterile cup and the container is closed immediately and outer surface is to be disinfected within 70% alcohol. 
  • Immediate delivery of the specimen to the laboratory and never refrigerate specimen.

Sputum for ZN stain

  • 3 successive morning sputum samples.

Throat and Mouth swabs

  • Under a good source of light, using a tongue depressor swab the tonsillar surface, soft palate and posterior pharyngeal wall using a sterile cotton swab, do not contaminate the swab with saliva.
  • For 8 hours before swabbing the patient must not be treated with mouth antiseptics or gurgles.

Endotracheal tube

  • After removal of the tube, the tip is cut by a sterile scissor and put inside a sterile container and sent to the lab within 30 minutes.

Nasopharyngeal aspirate

  • Gently pass a sterile catheter through one nostril as far as the nasopharynx.
  •  Aspirate the mucopus with sterile syringe and send it immediately to the microbiology laboratory.

Ear discharge

  • Whenever possible aspirate a small amount of the discharge in a sterile container or collect a specimen on a sterile dry cotton wool swab.
  • Send the swab immediately to the microbiology laboratory.

Eye specimens

  • Are cultured as soon as possible (bed side inoculation of the culture plates are preferred).The discharge is collected from the lower conjunctival fornix using a dry sterile cotton wool swab. 

Pus from wound, abscesses, burns and sinueses

  • is collected at the time the abscess is incised and drained, or after ruptured naturally, avoid contamination with normal skin flora.
  • Specimen is collected before an antiseptic dressing is applied.
  • Using a sterile technique, aspirate or collect from a drainage tube up to 5 ml of pus or use a sterile cotton wool swab to collect the sample from infected site.
  • Pus for anaerobes: aspirate in a syringe and ask lab. for a transport media

Urogenital Specimens

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Types of Specimen

Specimen Precautions

Uretheral specimen

  • Cleanse around the uretheral opening using a swab moistened with sterile physiological saline.
  • Gently massage the urethera from above downwards and collect the sample of pus on a sterile cotton wool swab.
  • The patient should not have passed urine preferably for 2 hours before sample collection.
  •  Collect pus also on a slide and send it with the swab for Gram stain.

Prostatic discharge

  • Urine sample is collected before and after prostatic massage for analysis.
  • Cleanse around the uretheral opening using a sterile cotton wool swab moistened with sterile physiological saline.
  • After prostatic massage the discharge is collected in a sterile container.

Semen

  • A period of abstinence 3-5 days should be followed.
  • The whole ejaculate is placed in a sterile container, unmixed with urine or water and sent within 30 min to the lab.If the sample is to be collected in a condom, the condom should be washed prior to ejaculation with sterile saline.

Cervical specimens

  • Avoid sexual activity 2 days before sampling.
  • Moisten a vaginal speculum with sterile warm water and insert into the vagina
  • Cleanse the cervix using a swab moistened with sterile physiological saline
  • Pass a sterile cotton wool swab into the endocervical canal and gently rotate the swab to obtain the specimen
  •  Send immediately to the laboratory.
  • Better to send two swabs one for culture and the other for microscopic examination
  • For Chlamydia Ag and mycoplasma detection, use special swabs supplied by lab.

Vaginal discharge

  • Vaginal douches are stopped on the day of sampling
  • Collect a sample of the discharge on a sterile cotton wool swab.
  • The specimen must be collected from the posterior vaginal pouch under speculum.
  • Alternatively, aspirate the discharge from the posterior vaginal fornix by a syringe or sterile Pasteur pipette.

Urine samples

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Midstream urine (MSU)

  • Give the patient a sterile, wide-necked, leak-proof container.
  • Female patient should be instructed to cleanse the area around the uretheral opening with clean water, dry the area, pass the urine while the labia are held apart and collect the midstream urine.
  • About 20ml urine should be collected.
  • Send the mid stream urine sample immediately, if not possible refrigerate at 4ºC. it delay more than 2 hrs, boric acid is added to urine with final concentration 1.8%.

Urine culture for TB

  •  First early morning urine specimen is collected over 3 successive days.

Urine for ZN stain

  • 3 successive morning samples

Urethritis

  • Collect the first voided urine.

Infants

  • Tap above the symphysis pubis (1 hr after feeding); 1 tap /sec for 1 min and then urine is collected using adhesive bags and clean-catch specimen.
  • Supra-pubic aspiration

Catheter urine

  • If the patient is catheterized, urine is collected from the catheter tube proximally after clamping, before it enters the catheter bag using a sterile syringe after disinfecting the site of entry of the syringe with 70 % alcohol and left to dry.

Fecal samples

  • Feces for microbiological examination should be collected during the acute stage of diarrhea.
  • Give the patient a clean, dry, disinfectant-free bedpan or suitable wide-necked container in which to pass the specimen.
  • Ask the patient to avoid contaminating the specimen with urine.
  • 2-3 samples on subsequent days are preferred.
  • Transfer a portion of the specimen (about a spoonful), especially that contains mucus, pus, or blood into a clean-dry, leak-proof container.
  • If the specimen contains worms or worm segments, transfer to a separate container.

Rectal swab

  • If there is difficulty in obtaining a stool sample, but the swab should contain a fecal material.

Cerebrospinal fluid

 

 

 

 

 

 

 

  • Disinfect the skin with Betadine (povidone-iodine) and collect the sample in 3 sterile screw capped tubes.
  • Send immediately to the lab., specify if anaerobes are suspected and never refrigerate the sample.

Blood

  • The phlebotomist should wear gloves.
  • Patient preparation: the vein from which the blood is to be drawn is chosen before the skin is disinfected. Blood is not to be drawn from catheters (central or peripheral). In case if catheter related blood stream infection is suspected: a sample is drawn from the catheter and another sample from a peripheral vein.  70% isopropyl alcohol then povidone iodine is applied to the skin over the suspected vein by means of cotton tipped application or cotton ball from inwards outwards and left to dry.
  • Blood is not allowed to clot. Single needle technique is recommended. The rubber stopper of the culture bottles should be also disinfected.
  • Sample volume: 5-10 ml of blood per 50-100 ml blood culture bottle.
  • Number of cultures: two or three blood cultures are sufficient to achieve maximum sensitivity.
  • Timing: optimal time 1-2 hrs before onset of chills, before antibiotic intake, if antibiotherapy is to be started, a larger volume of blood (from different venepunture sites using two different syringes is inoculated in two different bottles, if the patient is already on antibiotics, blood is drawn just before intake of the next dose.

Effusions

  • Collect 2-3 ml in a sterile tube or syringe and send immediately to the lab.
  • Can be collected on citrate (if sample clotting is a limitation)
  • Specify if anaerobes are suspected and never refrigerate samples.

 

 

 

Tissue biopsy

  • The sample should be placed in a sterile, leak proof container without formalin and sent to the lab within 30 min.

 

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FUNGAL SPECIMENS

  • Samples are collected in a clean, dry and sterile container.
  • Hair, nails and skin scales can be collected in a sterile petridish.
  • Samples are sent to the lab immediately, if delay is suspected, samples are not to be refrigerated. 

Suppurative lesions

  • The skin is disinfected by 70% alcohol.
  • The sample is collected within a sterile syringe better than swabbing.

Urine and sputum

  • First morning samples on 3 successive days.

Skin, nail and hair

  • Skin: The skin is disinfected by 70% alcohol and the inflammatory red margin is scrapped by a sterile scalpel or edge of a microscopic glass slide.
  • Nails: scrapping under the nails to obtain softened material from the nail bed.
  • Hair: areas with alopecia , scales or fluorescence under wood’s light  are sampled (hair is cut neat its follicle by a scissor)

 

SEROLOGY

SEROLOGY

Test Name

  Sample

Test Interference

Remarks

  • Anti-streptolysin O titer (ASO)

serum

Only 25% of strept. Pyoderma are positive.TB and chronic liver diseases give false positive results as well as lipemic or contaminated samples.

Rise1 week after infection

Return to normal in 6 months-1year

  • Brucella standard agglutination test

serum

 

 

  • CRP (qualitative)

Serum

Increases with estrogen, oral contraceptives.

Decreases with corticosteroids and other anti-inflammatory drugs

Increases in inflammatory conditions (infections and collagen diseases), tissue damage and malignancy.

  • CRP(semi-quantitative)

Serum

Increases with estrogen, oral contraceptives.

Decreases with corticosteroids and other anti-inflammatory drugs

 

 

 

  • CMV-IgM

Serum

 

 

  • CMV-IgG

serum

 

 

  • Epstein-Barr virus (VCA IgM)

Serum

 

IgM and IgG  are present during the acute phase, decline by 1-2 months.

  • Epstein-Barr virus (VCA IgG)

serum

 

IgM and IgG  are present during the acute phase, decline by 1-2 mo. IgG titers  persist at lower levels for few years

  • Herpes simplex virus II (IgM-IgG)

Serum

 

Acute infection Cross-reactivity betweenHSV1 and HSV2 and varicella zoster antigen

should be considered. IgM antibody may persist for 8 wks.

Antibodies may be present in the CSF of patients with CNS infection

  • Measles antibody (IgM)

Serum

 

 

  • Measles antibody (IgG)

serum

 

Immunity

  • H pylori antigen

stool

 

 

 

  • Adenosine deaminase

 

Serum, fluid and CSF

 

 

  • Rubella Ab (IgM-IgG)

Serum

False positive IgM antibodies (Parvovirus, CMV infections and Rheumatoid factor positive). A positive Ig M result is confirmed with a rising IgG titre after 2 weeks.

In naturally acquired infection IgM& IgG are present. In congenital Rubella infant's antibody at delivery: fetal IgM , IgG&IgA and maternal IgG.

  • Toxoplasma Ab (IgM-IgG)

Serum

 

 

 

  • VDRL

Serum

False positive results in collagen diseases, infectious mononucleosis, leprosy, malaria, pregnancy and addiction.

Positive within 1-3 months after chancre appears, becomes negative after effective treatment

  • Treponema pallidum haemagglutination test

serum

False positive in non venereal treponemas.

Remains positive after effective treatment. May be negative very early in the disease or in the late latent stage.

  • Widal test

Serum

 

Early intake of antibiotic may cause lower titre of “O”antibody. Salmonella food poisoning, chronic liver disease and autoimmune disorders may give false positive results.

 

Test should be repeated for rising titre. Rise in “O” antibodies indicate a recent infection. Rise in “H” and negative “O”: past infection or antibiotic intake. 

 

 

 

 

MOLECULAR TESTS

Test Name

Sample

Test Interference

Remarks

  • CMV PCR

EDTA blood

 

Plasma is stored at -70◦C

  • TB PCR

EDTA blood, body fluids, sputum, pus

 

Samples are stored at -70◦C