Examination of Sputum

A sputum test, also known as sputum examination or sputum analysis, is a laboratory procedure used to evaluate the material expelled from the lungs, bronchi, trachea, and larynx through coughing. Sputum primarily consists of mucus along with various cellular and non-cellular components derived from the host. During expectoration, the sputum sample can become contaminated with normal bacterial flora and cells from the pharynx and mouth. This test is crucial for diagnosing respiratory infections and chronic conditions affecting the airways and lungs.

Examination of sputum is mainly carried out for:

1. Identification of causative agent or organism associated with a particular suspected infection of the lower respiratory tract, e.g.
   • Suspected tuberculosis
   • Pneumonia especially if severe or in an immunocompromised host
   • Pneumocystic carinii pneumonia in HIV-positive patients
   • Suspected fungal infection
   • Infective exacerbation of a chronic disease like bronchiectasis
2. Cytological examination for the investigation of viral infections (viral inclusions in cytomegalovirus and herpes simplex infections), fungal infection, asbestosis and malignant cells.

How to Collect a Sputum Sample

1. Ideally, a sputum sample should be collected in the morning, as secretions tend to accumulate overnight. The sample should be obtained soon after the patient awakens and before any mouthwash or food intake.
2. The sputum sample should be collected in a sterile, clean, dry, and wide-mouthed plastic container equipped with a securely fitting screw cap. The container must be made of unbreakable or break-resistant plastic and be leak-proof to prevent desiccation and aerosol formation. It should have a capacity of approximately 30 ml.
3. The patient should be instructed to take a deep breath 2-3 times, filling their lungs, then cough deeply and expel the sputum into the plastic container. A volume of approximately 2-5 ml of sputum should be collected. Samples consisting solely of saliva, which appear watery, clear, and foamy, are not acceptable for laboratory investigations; in such cases, a new sample must be collected. The container, containing the sputum sample, should be securely capped and properly labeled.

Induction of Sputum

If the patient is unable to expectorate sputum spontaneously, sputum induction can be achieved by inhalation of an aerosol containing 15% sodium chloride (NaCl) and 20% propylene glycol (C₃H₈O₂) for 20 minutes. Sputum can also be induced by inhaling distilled water combined with chest physiotherapy or by inhaling nebulized hypertonic saline.

For microbiological examination, the sputum sample should be sent to the laboratory immediately. Allowing the sputum to stand can lead to the rapid proliferation of contaminating bacterial flora from the throat and oral cavity, resulting in inaccurate results. Additionally, pathogenic organisms, particularly Haemophilus influenzae, do not survive for long periods in collected samples. Therefore, sputum samples for bacterial culture should not be refrigerated.

If the sample is to be transported to a remote laboratory for mycobacterial culture, sputum should be collected in 25 ml of the following solution:

• N-acetylpyridinium chloride 5 gm
• Sodium chloride 10 gm
• Distilled water 1000 ml

Appearance of Sputum

The physical appearance of sputum is often indicative and symptomatic of underlying pathological processes, as outlined below:

1. Bloody: Indicates hemoptysis, which can be associated with conditions such as pulmonary tuberculosis, bronchogenic carcinoma, bronchiectasis, lung abscess, pulmonary infarction, and mitral stenosis.
2. Bloody and gelatinous (red currant jelly): Suggestive of Klebsiella pneumonia.
3. Rusty: Characteristic of pneumococcal lobar pneumonia.
4. Purulent and separating into three layers upon standing: Indicative of lung abscess or bronchiectasis.
5. Copious amounts of purulent sputum: Associated with bronchopleural fistula, lung abscess, or bronchiectasis.
6. Green: Typically signifies a Pseudomonas infection.
7. Pink, frothy (with air bubbles): Indicative of pulmonary edema.

Microbiological Examination of Sputum

Microbiological examination of sputum are designed with the primary objective of identifying the causative agents responsible for lower respiratory tract infections and various other respiratory disease. Additionally, these tests serve as invaluable tools in monitoring the efficacy of clinical interventions. Foremost among these diagnostic modalities is the sputum culture, which assumes paramount importance, particularly in cases where pneumonia is suspected. This test is instrumental in elucidating the microbial etiology, pinpointing the specific bacteria or fungi responsible for airway or pulmonary infections. Complementing this diagnostic approach is sputum smear microscopy, serving as the foundational step in the comprehensive analysis of sputum specimens within the laboratory setting.

The sputum sample typically contains adulterations and contaminants from the normal flora inhabiting the pharynx and oral cavity. Below is a list of the normal flora commonly found in the pharynx and oral cavity.

• Gram-positive microorganisms: Diptheroids, streptococci (S. pneumoniae, S. viridans), staphylococci (S. epidermidis, S. aureus), lactobacilli, enterococci, Yeasts (Candida spp.), micrococci.
• Gram-negative microorganisms: Coliforms, Haemophilus spp; Neisseria spp; Moraxella catarrhalis, fusobacteria.

Gram staining

Pathogenic organisms found in sputum include—

• Gram-positive: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae.
• Gram-negative: Klebsiella pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Yersinia pestis, Pseudomonas aeruginosa.

For the bacteriological examination of sputum, the sample should be processed in the laboratory within an hour of collection. A small portion of the sputum is transferred to a sterile Petri dish, and its physical characteristics are meticulously observed. From the purulent section of the sputum, a thin smear is prepared on a grease-free, sterile glass slide using a clean stick. The slide is then air-dried, fixed, and stained with Gram's stain. Samples that are entirely watery, mucoid, white, or frothy often reveal squamous epithelial cells covered with clusters of bacteria, indicating that the specimen primarily contains secretions from the oral cavity and throat. Such samples are deemed unsuitable for bacteriological examination (see Figure 1). Additionally, culture is not conducted if there are fewer than 10 polymorphonuclear neutrophils per epithelial cell.

Because of the presence of various contaminating Gram-positive and Gram-negative microorganism deriving from throat and mouth (normal bacterial flora), Gram-stained smear of sputum should be elucidated carefully.

Morphological appearance of bacterial cells on Gram stained smear is redolent of a particular microorganism as follows:

• Gram-negative diplococci, both intra- and extracellular: Moraxella catarrhalis.
• Gram-positive yeast cells with budding and pseudohyphae: Candida.
• Gram-positive diplococci with surrounding clear space (capsule): S. pneumoniae (see Figure 2).
• Gram-negative coccobacilli: H. influenzae.
• Gram-positive cocci in grape-like clusters: S. aureus.
• Large granules with center gram-negative and periphery gram-positive: Actinomyces.

Bacteriological Culture

For the absolute identification of microorganisms, culture media are inoculated with a floccule of the purulent portion of the sputum. A sputum sample is deemed unsuitable for bacterial culture if it contains more than 25 squamous epithelial cells per low-power field. An ideal sputum sample for bacterial culture should contain bronchial epithelial cells, numerous neutrophils (more than 5 per high-power field), alveolar macrophages, and fewer than 10 squamous epithelial cells per high-power field.

To minimize contamination by the normal bacterial flora, saliva is washed away from the sputum using sterile normal saline. The washed sputum is then inoculated onto a blood agar plate and a chocolate agar plate (heated blood agar). The chocolate agar plate is incubated in an environment with elevated carbon dioxide (CO₂), while the blood agar plate is incubated aerobically. After an incubation period of 18 hours, the inoculated agar plates are examined for bacterial growth. If the growth is insufficient, an additional 24 hours of incubation is recommended. Antibiotic sensitivity testing is performed only if the bacterial growth is significant.

Examination of Sputum for Mycobacterium tuberculosis

Tuberculosis represents a significant public health challenge in both Pakistan and India. Early diagnosis of pulmonary tuberculosis is critical as it facilitates timely initiation of treatment, which not only promotes cure but also curtails the transmission of the disease to others. In recent years, the incidence of tuberculosis has surged, prompting the World Health Organization to declare it a global emergency. Additionally, there is a growing prevalence of multi-drug resistant tuberculosis, exacerbating the public health crisis.

The Mycobacterium tuberculosis complex includes M. tuberculosis, M. africanum, and M. bovis, all of which are the causative agents of tuberculosis in humans. Other mycobacterial species are classified as non-tuberculous mycobacteria.

There are two primary approaches to the identification of tuberculosis.

1. Direct test: This involves detection of M. tuberculosis or its components
2. Indirect test: This consists of detection of cellular or humoral immune response to tuberculosis infection.

Direct tests for the detection of tuberculosis on sputum sample are as follows:

1. Examination of sputum smear
   • Ziehl-Neelsen technique
   • Fluorescence microscopy
2. Molecular Method
3. Culture on standard media
4. Commercial automated culture system

Examination of Sputum Smear

To detect Mycobacterium tuberculosis, it is essential to examine a minimum of three sputum samples collected on separate occasions, with at least one early morning sample included. A thin sputum smear should be prepared on a clean, sterile, and grease-free glass slide from the yellowish, grayish, opaque, or blood-tinged portion of the sputum. In cases where children cannot expectorate sputum, a sample of fasting gastric juice may be aspirated and examined similarly to sputum.

The smear is stained using the Ziehl-Neelsen technique and examined under an oil immersion lens with a standard light microscope. If a fluorescent microscope is available, the smear can alternatively be examined after staining with a fluorochrome, such as auramine O or auramine-rhodamine.

Ziehl-Neelsen Staining of Sputum Smear

This method is simple, rapid, and cost-effective. It is primarily employed for:

1. Diagnosing pulmonary tuberculosis (as cases with positive sputum smears are significant sources of infection spread).
2. Determining the effectiveness of treatment or identifying treatment failure.
3. Evaluating the response to anti-tuberculosis therapy.

A Ziehl-Neelsen-stained sputum smear is considered positive if 5000-10000 tubercle bacilli per milliliter are present. The technique's sensitivity is reported to be 60-80%. The likelihood of detecting tubercle bacilli increases with the examination of multiple sputum samples or the use of the bleach concentration technique. In this method, a concentrated sodium hypochlorite (NaOCl) solution is added to the sputum sample, liquefying the mucus and killing the mycobacteria. The sample is then allowed to sediment overnight (or undergoes centrifugation), after which a thin smear is prepared from the sediment, stained, and examined.

With Ziehl-Neelsen staining, mycobacteria appear as bright red, straight, or slightly curved rods (0.2-0.5 μm in width and 2-4 μm in length) against a green or blue background (see Figure 3). Both acid- and alcohol-fast mycobacteria are referred to as acid-fast bacilli (AFB). At least 100 fields must be examined before reporting the smear as negative. If acid-fast bacilli are observed, their quantity should be documented.

A negative sputum smear does not exclude the diagnosis of tuberculosis, as the smear may be of poor quality, the organisms may be few in number, or the sputum sample may not have been collected properly.

Fluorescence Microscopy

A thin sputum smear is prepared and stained with a fluorochrome, such as auramine O or auramine-rhodamine. This smear is then examined under a fluorescence microscope, where mycobacteria appear as bright yellow against a dark background (see Figure 4). This technique is notably simple and rapid, as the sputum smear is examined under a low-power lens. It is particularly useful when the organisms are few in number. However, it is essential to confirm any positive sputum smear with the Ziehl-Neelsen stain, due to the high rate of false-positive results associated with the fluorescent staining technique.

Molecular Method

There are two primary methods for the molecular diagnosis of tuberculosis in sputum samples:

1. Detection of Mycobacterium tuberculosis in isolates from culture using nucleic acid probes.
2. Direct detection of Mycobacterium tuberculosis in sputum samples.

M. tuberculosis can be rapidly detected directly in sputum samples by identifying specific DNA sequences unique to it. In the M. tuberculosis complex, the targeted DNA sequence is IS 6110, as it is exclusively found within this complex. Multiple copies of this sequence are present in the genome of M. tuberculosis. This method allows for the detection of 10-1000 organisms per milliliter of sputum. Other specific DNA and RNA sequences of the M. tuberculosis complex can also be targeted.

However, laboratory cross-contamination, particularly due to aerosolized PCR products, can lead to unreliable and false-positive results. PCR amplifies DNA sequences of both dead and live bacilli, making it unsuitable for evaluating response to therapy. Additionally, this test is expensive. Therefore, PCR-based assays should be interpreted in conjunction with clinical features, Ziehl-Neelsen sputum smear findings, and the presence of tuberculosis in other family members.

Sputum Culture (Conventional)

For the definitive diagnosis of tuberculosis, a pure culture technique is employed. Mycobacterium tuberculosis is isolated from the culture of a sputum sample. Sputum culture is typically undertaken for the following purposes:

1. Identification of specific species, especially if an organism other than M. tuberculosis is suspected, which is important for understanding the incidence, distribution, and control of diseases.
2. Drug susceptibility testing.
3. Diagnosis in patients who exhibit distinctive radiological and clinical features of tuberculosis but have negative sputum smears.

Sputum culture is more sensitive than sputum smear examination, capable of detecting 10 to 100 microorganisms per milliliter of sputum. Its sensitivity for tuberculosis identification is 80-85%, and its specificity is 98%. Despite its reliability, the procedure is expensive and time-consuming, requiring approximately six weeks for results, with drug susceptibility testing taking even longer. Additionally, prior decontamination of sputum is necessary to eliminate normal bacterial flora.

Contaminating bacteria can rapidly grow and degrade the culture medium before the tubercle bacilli begin to grow. Therefore, decontamination of the sputum sample with 4% sodium hydroxide is required to inhibit these contaminants.

The standard culture media for isolating M. tuberculosis are:

1. Solid media: Agar-based (Middlebrook 7H10 or 7H11) or egg-based (Lowenstein-Jensen medium).
2. Liquid media: Middlebrook 7H9, Middlebrook 7H12.

The most commonly used solid medium is the Lowenstein-Jensen medium, which requires up to six weeks for visible mycobacterial growth. Further biochemical tests are performed to identify the species.

Commercial Automated Culture Systems

Nowadays, rapid automated culture systems are commercially available, capable of providing results within two weeks, as opposed to the six weeks required with standard media. However, these procedures are expensive. Examples of such systems include the BACTEC™ 460TB system (see Figure 5) and the BACTEC™ 9050 automatic blood culture analyzer, developed by Becton-Dickinson Diagnostic Instrument Systems, Maryland, USA. These instruments are highly sensitive and can detect Mycobacterium tuberculosis in clinical samples. In this method, a broth containing radiolabeled ¹⁴C-palmitate is utilized. Mycobacteria metabolize the ¹⁴C-palmitate, producing radiolabeled ¹⁴CO₂, which is subsequently detected by the instrument.

Examination of Sputum for Organisms Other Than Tubercle Bacilli

The following interpretations are applicable when infection by specific organisms is suspected:

• Paragonimus: Saline wet mount of sputum to identify eggs.
• Histoplasmosis: Giemsa-stained smear.
• Pneumocystis carinii: Bronchoalveolar lavage fluid stained with Giemsa and silver stains (see Figure 6).
• Yersinia pestis (pneumonic plague): Giemsa-stained smear.
• Aspergillus: Potassium hydroxide wet mount of sputum.
• Yeast-like organisms on Gram’s smear: Culture on Sabouraud dextrose agar.

Cytological Examination of Sputum

Cytological examination of sputum is typically conducted for the diagnosis of bronchogenic carcinoma. It can also be useful in identifying fungi, protozoa, asbestos bodies, and viral inclusions such as those of Cytomegalovirus and Herpes simplex virus.

For cytological examination, an early morning sputum sample is preferred. To diagnose lung cancer, it is recommended to collect a sputum sample daily for the first five consecutive days. This approach increases the likelihood of detecting malignant cells (see Figure 7).

A sputum sample can be either spontaneously produced or artificially induced. If the patient is unable to expectorate sputum spontaneously, inhalation of an aerosol containing 15% sodium chloride (NaCl) and 20% propylene glycol (C₃H₈O₂) for 20 minutes can effectively induce expectoration, typically resulting in an adequate sputum sample.

The sputum sample should be sent to the laboratory immediately after collection without any fixative. If the sample needs to be transported to a remote laboratory, prefixation with Saccomanno’s fixative is recommended. This involves collecting the sputum in a mixture of 2% carbowax and 50% ethyl alcohol.

In the laboratory, a thin sputum smear is prepared on a clean, sterile, grease-free glass slide from the yellowish, grayish, opaque, or blood-tinged portion, or from tissue fragments in the sputum, and then stained using the Papanicolaou technique. The sputum sample is considered adequate for cytological examination if bronchial epithelial cells or alveolar macrophages are present in the smear.

The average sensitivity of sputum examination for detecting malignant cells is approximately 65%. Sensitivity increases under the following conditions:

• The tumor is large.
• The lesion is centrally located rather than at the lung periphery.
• The carcinoma is of the squamous type rather than adenocarcinoma or small cell carcinoma.
• An increased number of sputum samples are examined.