Examination of sputum

Sputum  Sputum is a thick fluid produced in the lungs and in the adjacent airways A sputum culture is a test to detect and identify bacteria or fungi that infect the lungs or breathing passages. 

Possible pathogens BACTERIA Gram positive Streptococcus pneumoniae Staphylococcus aureus Streptococcus pyogenes Also Mycobacterium tuberculosis Gram negative Haemophilus influenzae Klebsiella pneumoniae Pseudomonas aeruginosa Proteus species Yersina pestis Moraxella catarrhalis Also Mycoplasma pneumoniae, and Legionella pneumophila.

Mycobacterium tuberculosis Infection of the lungs with M. tuberculosis causes pulmonary tuberculosis. Tuberculosis is the commonest HIV-related, causing about 30% of deaths in AIDS patients. The development of tuberculosis in HIV co-infected persons accelerates progression to full-blown AIDS. Tuberculosis is difficult to diagnose by sputum examination in those co-infected with HIV when there is marked immunosuppression resulting in diffuse infiltration without cavitation. When immune responses are less suppressed, typical pulmonary caseating granulomas and cavities form, and AFB can usually be detected in sputum.

Bacteria that cause respiratory tract infection S. pneumoniae and H. influenzae are the commonest causes of acute respiratory tract infections in tropical countries. S. pneumoniae causes lobar pneumonia and bronchopneumonia in young children (especially when malnourished), in those co-infected with HIV (major HIV-related pathogen), the elderly, the bed-ridden and other debilitated persons.

Bacteria that cause respiratory tract infection S. aureus, S. pyogenes, and H. influenzae are often secondary invaders in patients with influenza virus pneumonia. H. influenzae is associated with acute and chronic bronchitis and chest infections in post-surgical patients and the elderly. S. aureus can produce a severe pneumonia (with a tendency to form abscesses), especially in children and following influenza.

Bacteria that cause respiratory tract infection P. aeruginosa is more commonly found in patients with chronic lung cavities or as a complication of treatment with immunosuppressive drugs. ● K. pneumoniae may be found with E. coli and yeasts as a complication of antibiotic therapy. ● Moraxella catarrhalis can cause upper and lower respiratory tract infections, mostly in adults with pre-existing respiratory disease and those with immunodeficiency.

Bacteria that cause respiratory tract infection Y. pestis (highly infectious) can be found in the sputum of patients with pneumonic plague. The specimen may contain blood. ● Mycoplasma pneumoniae  ( M. pneumoniae) causes primary atypical pneumonia. ● Legionella pneumophila (L. pneumophila) causes Legionnaire’s disease, a severe and often fatal form of pneumonia.

Bacteria that cause respiratory tract infection Eosinophils can be found in the sputum of patients with allergic respiratory conditions such as asthma. Commensals Sputum as it is being collected passes through the pharynx and the mouth. It therefore becomes contaminated with small numbers of commensal organisms from the upper respiratory tract and mouth.

COLLECTION AND TRANSPORT OF SPUTUM Sputum for microbiological investigation is collected and transported as follows: In a hospital with a microbiology laboratory 1 Give the patient a clean (need not be sterile), dry, wide-necked, leak-proof container, and request him or her to cough deeply to produce a sputum specimen.

COLLECTION AND TRANSPORT OF SPUTUM Caution: When a sputum specimen is being collected, adequate safety precautions must be taken to prevent the spread of infectious organisms and to avoid contaminating the outside of the container. Use a phenol-containing disinfectant to wipe the outside of the container after collecting the specimen. Important: The specimen must be sputum, not saliva. Sputum is best collected in the morning soon after the patient wakes and before any mouth-wash is used. When pulmonary tuberculosis is suspected, up to three specimens may need to be examined to detect AFB. Mucopus aspirated from the nasopharynx When it is not possible to get sputum from children with suspected pneumonia or bronchopneumonia, pathogens can often be isolated from mucopus aspirated from the nasopharynx.

COLLECTION AND TRANSPORT OF SPUTUM 2-Label the container, and complete a request 3- When pneumonia or bronchopneumonia is suspected, deliver the sputum to the laboratory with as little delay as possible because organisms such as S. pneumoniae and H. influenzae require culturing as soon as possible. Note: Specimens for the isolation of S pneumoniae and H. influenzae must never be refrigerated.

When pneumonic plague is suspected: Deliver the sputum to the laboratory as soon as possible. Make sure the specimen is marked HIGH RISK.

In a health centre for dispatch to a microbiology laboratory 1 Collect the sputum in a container supplied by the microbiology laboratory .Follow the technique and observe the precautions mentioned under the hospital collection of sputum. 2 To ensure the survival of pathogens such as S. pneumoniae and H. influenzae, transfer a purulent part of the sputum to a cotton-wool swab, and insert it in a container of Amies transport medium. Label the container using a lead pencil. Amies medium will help the pathogens to survive and avoid the overgrowth of fast-multiplying commensals. 3 Send the sputum specimen and swab with a request form to reach the microbiology laboratory within 6 hours.

LABORATORY EXAMINATION OF SPUTUM sputum specimens should be examined in a biological safety cabinet 1- Describe the appearance of the specimen Describe whether the sputum is: Purulent: Green-looking, mostly pus Mucopurulent: Green-looking with pus and mucus Mucoid: Mostly mucus Mucosalivary: Mucus with a small amount of saliva When the sputum contains blood, this must also be reported.

LABORATORY EXAMINATION OF SPUTUM When the sputum is mostly saliva, report the specimen as ‘Unsuitable for microbiological investigation’ and request another specimen. Note: Before culturing sputum, many laboratories examine a wet preparation or Field’s stained smear microscopically for cells. When large numbers of squamous epithelial cells (often covered with bacteria) are present and only a few or no pus or macrophage cells, this indicates that the specimen is unsuitable for culturing.

2- Examine the specimen microscopically Gram smear Using a piece of stick, transfer a purulent part of the sputum to a glass slide, and make a thin smear. Allow the smear to air-dry in a safe place. Fix Examine the smear for pus cells and predominant bacteria. Look especially among the pus cells for: ● Gram positive diplococci (capsulated) that could be S. pneumoniae . The

● Gram positive cocci in groups that could be S ● Gram positive cocci in groups that could be S. aureus , but not often seen. ● Gram negative rods and cocco-bacilli that could be H. influenza, particularly when these are the predominant organisms. ● Gram negative capsulated rods that could be K. pneumoniae, but not often seen. ● Gram negative diplococci in and between pus cells that could be M. catarrhalis

Gram stained smears of sputum must be reported with caution Gram stained smears of sputum must be reported with caution. Cocci, diplococci, streptococci, and rods may be seen in normal sputum because these organisms form part of the normal microbial flora of the upper respiratory tract. Note: When pus cells are present but no bacteria are seen in a Gram stained smear, this may indicate the presence of microorganisms such as M. tuberculosis, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophilia or viruses.

Ziehl-Neelsen smear to detect AFB the NaOC1 concentration technique is also safer for laboratory staff. Because NaOC1 kills M. tuberculosis,

Eosin preparation when an allergic condition requires investigation Transfer a small amount of sputum to a slide. Add a small drop of alkaline eosin solution, mix, and cover with a cover glass. Using the 10 and 40 objectives with the condenser iris closed sufficiently to give good contrast, examine the preparation for eosinophils*. *Eosinophils can be easily differentiated from pus cells because they contain bright red-staining granules and a bilobed nucleus. Free eosinophilic granules may be seen in the preparation and occasionally elongated refractile Charcot Leyden crystals (formed from the material of dead eosinophils). Large numbers of eosinophils in sputum can also be found with paragonimiasis.

Giemsa or Wayson stained preparation when pneumonic plague is suspected Fix the sputum smear with methanol for 5 minutes. Stain using Giemsa technique or rapid Wayson’s technique Bipolar Caution: Y. perstis is highly infectious (Hazard Group 3 pathogen), therefore handle specimens with great care. Laboratory-acquired infections can occur following accidental inoculation or inhalation of the organisms. Minimize procedures that create aerosols and whenever possible carry out procedures in a safety cabinet.

Culture the specimen To obtain as pure a culture as possible of a respiratory pathogen it is necessary to reduce the number of commensals inoculated. Ways of reducing commensal numbers include washing the sputum free from saliva or liquefying and diluting it. The technique using saline-washed sputum is described. The dilution technique requires a liquefying agent such as dithiothreitol (Sputolysin, Sputasol ) which is expensive and unstable.

The procedure for the routine liquefaction of sputum is as follows: The sputum is expectorated into a sterile Universal container or other wide mouthed screw-capped bottle Add approximately 5 times the volume of 0.85% saline and agitate to free the sputum from adherent saliva. Remove the saline with a sterile Pasteur pipette. To the washed sputum, add an equal volume of Sputasol solution Shake the mixture well, place in a 37°C water bath and incubate, with periodic shaking, until liquifaction is complete Inoculate on to a suitable culture medium. For the total cell count, place a drop of the liquefied sputum in a haemocytometer for enumeration. For a differential cell count, fix a dried smear in methyl alcohol and stain with haematoxylin and eosin or with Lieshmann stain. The working solution of Sputasol will remain stable for at least 48 hours stored at 2-8°C. An investigation into the survival of respiratory pathogens in specimens that had been stored for 48 hours at 4°C following homogenisation using Sputasol, showed that the organisms remained viable and, when necessary, treated specimens could be successfully re-cultured8.

Blood agar and chocolate agar – Wash a purulent part of the sputum in about 5 ml of sterile physiological saline. – Inoculate the washed sputum on plates of: ● Blood agar, see No. 16 ● Chocolate (heated blood) agar, see No. 16 Inoculation technique to reduce commensal numbers: Using the technique described in subunit 7.4 (to inoculate a whole plate of agar), flame the loop in between each spread. This will help to obtain a pure growth of the pathogen in the areas of the 3rd and 4th spread. – Add an optochin disc to the blood agar plate within the area of 2nd spread. This will help to identify S. pneumoniae (see subunit 7.18.4).

Availability: Optochin discs can be purchased economically from Mast Diagnostics (see Appendix 11) in vials of 100 discs, code D-42. They have a shelf-life of about 2 years. – Incubate the blood agar plate aerobically and the chocolate agar plate in a carbon dioxide enriched atmosphere (see subunit 7.4). ADDITIONAL Culture of sputum for M. tuberculosis Culturing and susceptibility testing of M. tuberculosis are usually undertaken in a Tuberculosis Reference Laboratory, mainly for surveillance purposes, to determine levels of drug resistance, and to manage treatment failures and relapses (see also subunit 7.18.28). Culture of sputum when pneumonic plague is suspected Isolation of Y. pestis is described in subunit 7.18.22.