Preparations for Diagnosing Infection

Specimen Collection

Laboratory diagnosis of diseases begins with the collection of a clinical specimen for examination or processing in the laboratory.

Learning Objectives

Describe how laboratory diagnosis of disease begins with the collection of a clinical specimen for examination and processing

Key Takeaways

Key Points

  • Specimen collection requires withdrawing blood, cerebrospinal fluid, collecting urine, or swabs from mucosal surfaces.
  • Specimen collection is performed using aseptic techniques to ensure sterility of the sample and avoid contamination from bacteria or other bodily fluids.
  • The types of biological samples accepted in most clinical laboratories are: serum samples, virology swab samples, biopsy and necropsy tissue, cerebrospinal fluid, whole blood for PCR, and urine samples. These are collected in specific containers for successful processing in the laboratory.

Key Terms

  • PCR: polymerase chain reaction
  • necropsy: The pathological dissection of a corpse; particularly to determine cause of death. Applicable to the examination of any life form.
  • biopsy: The removal and examination of a sample of tissue from a living body for diagnostic purposes.

Laboratory diagnosis of an infectious disease begins with the collection of a clinical specimen for examination or processing in the laboratory.

The laboratory, with the help of well-chosen techniques and methods for rapid isolation and identification, confirms the diagnosis.

It has been observed that the most important and frequent factor affecting laboratory analysis, even in a well-functioning laboratory, is not the laboratory investigation itself but specimen preparation and errors in identification or labeling. Proper collection of an appropriate clinical specimen is, hence, the first step in obtaining an accurate laboratory diagnosis of an infectious disease.

Applying one’s knowledge of microbiology and immunology for the collection, transportation and storage of specimens is as important as it is in the laboratory. For starters, the interpretation of the observation may be misleading if the specimen is inadequate.

There are several types of specimens recommended for diagnosis of immunological diseases including: serum samples, virology swab samples, biopsy and necropsy tissue, cerebrospinal fluid, whole blood for PCR, and urine samples.

Serum is the preferred specimen source for serologic testing. Blood specimens are obtained aseptically using approved venipuncture techniques by qualified personnel. Specimens are allowed to clot at room temperature and then are centrifuged. Serum is transferred to tightly-closing plastic tubes and stored at 2 – 8°C before shipment–which should always be prompt. Acute serum should be collected at the onset of symptoms. Convalescent specimens should follow two to four weeks later. Paired sera are tested together.

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Venipuncture: Performed to draw blood sample using a vacutainer

Plasma is also collected for a very limited number of tests. Lipemic, hemolyzed, or contaminated sera may cause erroneous results and should be avoided as should repeated freeze-thaw cycles.

Another type of specimen used for disease diagnosis is cerebrospinal fluid (CSF). This should be transported in tightly-closing plastic tubes. Refrigerated CSF is acceptable for a limited number of serologic tests; however, if PCR is to be performed for the viral panels, the specimen must be frozen and shipped on dry ice. CSF specimens should be clear of any visible contamination or blood. A lumbar puncture (or LP, and colloquially known as a spinal tap) is performed to collecte CSF. This consists of the insertion of a hollow needle beneath the arachnoid membrane of the spinal cord in the lumbar region to withdraw cerebrospinal fluid for diagnostic purposes or to administer medication.

Immediate Direct Examination of Specimen

Following collection in appropriate containers, clinical specimens undergo a rapid examination in the laboratory.

Learning Objectives

Describe how immediate direct examination of a specimen is useful to determine microscopic and macroscopic morphology

Key Takeaways

Key Points

  • Immediate direct examination methods depend on the nature of the specimen.
  • Diagnostic laboratory techniques include direct testing using a microscope, and immunological or genetic methods that provide immediate clues as to the identity of the microbe or microbes in the sample.
  • Phenotypic methods include the examination of microscopic, macroscopic, and biochemical characteristics of a pathogen.

Key Terms

  • biopsied: to remove and examine a sample of tissue from a living body for diagnostic purposes.

Immediate Direct Examination of Specimen

For specimen collection at sites that normally contain resident microflora, care should be taken to sample only the infected site and not the surrounding areas. For example, throat and nasopharyngeal swabs should not touch the tongue, cheek, or saliva. Saliva is an especially undesirable contaminant because it contains millions of bacteria, of which are normal flora. Sputum, the mucous secretion that coats the lower respiratory surfaces, especially the lungs, is discharged by coughing or taken by a catheterization to avoid contamination with saliva. Also the mucous lining of the vagina, cervix, or urethra can be sampled with a swabbed or applicator stick.

Additional sources of specimens are the vagina, eye, ear canal, nasal cavity (all by swab), and diseased tissue that has been surgically removed (biopsied). Urine is taken aseptically from the bladder with a catheter. Another method called the “clean catch” is taken by washing the external urethra and collecting the urine in midstream. Some diagnostic techniques require first-voided “dirty catch” urine. Sterile materials such as blood, cerebrospinal fluid, and tissue fluid must be taken by sterile needle aspiration.

Diagnostic laboratory techniques include direct testing using a microscope, immunological, or genetic methods that provide immediate clues as to the identity of the microbe or microbes in the sample, and cultivation, isolation, and identification of pathogens using a wide variety of general and specific tests (such as blood or other fluids).

Most tests fall into two categories: presumptive data, which place the isolated microbe in a preliminary category such as genus, and more specific, confirmatory data, which provide more definitive evidence of a species. Some diseases are diagnosed without the need to identify microbes from specimens. Serological tests on a patient’s serum can detect signs of an antibody response. One method that clarifies whether a positive test indicates current or prior infection is to take two samples several days apart and see if the antibody titer is raising. Skin testing can pinpoint a delayed allergic reaction to a microorganism. These tests are important in screening the general population for exposure to an infectious agent such as rubella or tuberculosis.

The main phenotypic methods include the direct examination of specimens, observing the growth of specimen cultures on special media, and biochemical testing of specimen cultures.

MICROSCOPIC MORPHOLOGY

Traits that can be valuable aids to identification of cell shape and size, Gram-stain reaction, acid-fast reaction and special structures, including endospores, granules, and capsules. Electron microscopes can pinpoint additional features such as cell wall flagella, pili, and fimbriae.

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Microscopic examination of E. coli: Gram negative E. coli examined under the microscope from a patient with urinary tract infection.

MACROSCOPIC MORPHOLOGY

Traits that can be assessed with the naked eye are also useful in diagnosis. These include the appearance of colonies, including texture, shape, size, pigment, speed of growth, and patterns of growth in broth and gelatin.

PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERISTICS

Enzymes and other biochemical properties of bacteria are reliable and stable expressions of the chemical identity of each species. Diagnostic tests exist for determining the presence of specific enzymes and assessing nutritional and metabolic activities.

Test examples include: fermentation of sugars, capacity to digest or metabolize complex polymers such as proteins and ploysaccharides; production of gas; presence of enzymes such as catalase, oxidase, and decarboxylase; and sensitivity to antimicrobial drugs.

CHEMICAL ANALYSIS

This involves analyzing the types of specific structural substances that the microorganism contains, such as the chemical composition of peptides in the cell wall and lipids in the membrane.

Cultivation of Specimen

Following direct examination, clinical specimens are cultivated to generate more confirmatory data.

Learning Objectives

Describe how direct microscope observation of a fresh or stained specimen is one of the most rapid methods of determining its characteristics

Key Takeaways

Key Points

  • The success of pathogen identification and treatment depends on how the specimen is collected, handled, and stored.
  • Diagnostic laboratory techniques include direct testing using a microscope, and immunological or genetic methods that provide immediate clues as to the identity of the microbe or microbes in the sample.
  • Following direct testing, cultivation, isolation, and identification of pathogens using a wide variety of general and specific tests is required.

Key Terms

  • mannitol: A polyhydroxy alcohol, an isomer of sorbitol, used as an artificial sweetener.
  • MacConkey agar: A culture medium designed to grow Gram-negative bacteria and differentiate them from lactose fermentation.

Cultivation of Specimen

The success of pathogen identification and treatment depends on how the specimen is collected, handled, and stored. It is also critical that the pathogen is isolated in a pure culture first. Direct microscope observation of a fresh or stained specimen is one of the most rapid methods of determining characteristics. Stains most often employed for bacteria are the gram stain, though they do not work on some organisms.

The direct florescence antibody (DFA) test can highlight the presence of the microbe in patient specimens by means of labeled antibodies. This test is useful for bacteria such as syphilis spirochete, which are not readily cultivated in a laboratory, or if a rapid diagnosis is essential for the survival of a patient.

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Blood culture: This blood is cultured in a bottle to detect bloodstream infections.

In most cases, specimens are also inoculated into differential media that define such characteristics as fermentation patters (mannitol salt and MacConkey agar) and as reactions in blood (blood agar). A patient’s blood is usually cultured in a special bottle of broth that can be periodically sampled for growth. Work must be done from isolated colonies or pure cultures, as working with mixed or contaminated cultures gives misleading and inaccurate results. From such isolates, clinical microbiologists obtain information about a pathogen’s microscopic morphology and staining reactions, culture appearance, motility, oxygen requirements, and biochemical characteristics.

Serological testing uses in-vitro diagnostic testing of serum, has a high degree of specificity and sensitivity, and is based on the specificity an antibody has for its antigen. These techniques do not necessitate a cultivation step. Serum can be directly used in agglutination, precipitation, complement fixation, fluorescent microscopy, and enzyme-linked assays. Results of specimen analysis are entered in the patient’s summary chart.

DNA Analysis Using Genetic Probes and PCR

Genotyping of pathogenic isolates provides valuable support during investigations of suspected outbreaks and when tracing infectious diseases.

Learning Objectives

Describe how genetic probes can be used to detect unique nucleotide sequences within the DNA or RNA or a microorganism

Key Takeaways

Key Points

  • Hybridization can identify a bacterial species by analyzing segments of its DNA.
  • Genetic probes are small fragments of DNA or RNA that are complementary to the specific sequences of DNA from a particular microbe.
  • This approach is most useful in the detection of infections due to microorganisms that are difficult to culture.

Key Terms

  • polymerase chain reaction: A technique in molecular biology for creating multiple copies of DNA from a sample; used in genetic fingerprinting etc.

Genetic probes are based on the detection of unique nucleotide sequences with the DNA or RNA of a microorganism. Once such a unique nucleotide sequence, which may represent a portion of a virulence gene or of chromosomal DNA, is found, it is isolated and inserted into a cloning vector ( plasmid ), which is then transformed into Escherichia coli to produce multiple copies of the probe. The sequence is then reisolated from plasmids and labeled with an isotope or substrate for diagnostic use. Hybridization of the sequence with a complementary sequence of DNA or RNA, follows cleavage of the double-stranded DNA of the microorganism in the specimen. The use of molecular technology in the diagnoses of infectious diseases has been further enhanced by the introduction of gene amplication techniques, such as the polymerase chain reaction (PCR) in which DNA polymerase is able to copy a strand of DNA by elongating complementary strands of DNA that have been initiated from a pair of closely spaced oligonucleotide primers. This approach has had major applications in the detection of infections due to microorganisms that are difficult to culture (e.g., the human immunodeficiency virus), or that have not as yet been successfully cultured (e.g., the Whipple’s disease bacillus).

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Mycobacterium Tuberculosis: a Mycobacterium tuberculosis culture revealing the organism’s colonial morphology.

It is well established that genotyping of pathogenic isolates provides valuable support for the investigation of suspected outbreaks, the detection of unsuspected transmission, the tracing of infectious agents within a community, and the identification of possible sources of infection for newly diagnosed cases. At the national or international level, fingerprinting allows strains from different geographic areas to be compared, and the movement of individual strains to be tracked. Fingerprinting technique requires high-quality genomic DNA, which is not only difficult to prepare but also requires culturing of the organism, resulting in a long turnaround time. In addition, fingerprint interpretation and matching can be complicated and require sophisticated computer software for large-scale analysis.

In contrast, nucleic acid amplification-based assays do not require culturing of the organisms, allowing the analysis of samples in real time. In many PCR-based typing assays, the target DNA of interest is amplified and labeled by PCR, and the labeled products are hybridized to an array of immobilized diagnostic probes. This method has been successfully used for the detection of mutations in drug resistance genes of Mycobacterium tuberculosis, and for Mycobacterium species identification. Spoligotyping, a reverse dot blot assay that detects the presence of a series of unique spacers in the direct repeat (DR) locus, meets the need for a simple and rapid method by which to distinguish M. tuberculosis complex strains. However, spoligotyping has significantly less discriminatory power than fingerprinting.

Nucleic Acid Sequencing and rRNA Analysis

Nucleic acid sequencing and rRNA analysis consist of comparing nitrogen bases in rRNA.

Learning Objectives

Describe how the 16SrRNA gene can be used for phylogenetic studies and in medical microbiology for bacterial identification

Key Takeaways

Key Points

  • This method is effective in identifying general group differences of pathogens.
  • 16S rRNA gene sequences contain hypervariable regions that can provide species -specific signature sequences useful for bacterial identification.
  • This method can also be fine-tuned to identify pathogens at the species level.

Key Terms

  • ribosomes: Large and complex molecular machine, found within all living cells, that serves as the primary site of biological protein synthesis.

Sixteen S ribosomal RNA (or 16S rRNA) is a component of the 30S small subunit of prokaryotic ribosomes. It is approximately 1.5kb (or 1500 nucleotides) in length. The genes coding for it are referred to as 16S rDNA, and are used in reconstructing phylogenies. Multiple sequences of 16S rRNA can exist within a single bacterium.

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Ribosomal RNA: Structure and shape of the E.coli 70S ribosome. The large 50S ribosomal subunit (red) and small 30S ribosomal subunit (blue) are shown with a 200 Ångstrom (20 nm) scale bar. For the 50S subunit, the 23S (dark red) and 5S (orange red) rRNAs and the ribosomal proteins (pink) are shown. For the 30S subunit, the 16S rRNA (dark blue) and the ribosomal proteins (light blue) are shown.

The 16SrRNA gene is used for phylogenetic studies, as it is highly conserved between different species of bacteria and archaea. Carl Woese pioneered this use of 16S rRNA. In addition, mitochondrial and chloroplastic rRNA are also amplified. Unfortunately, while primers can be defined to amplify this gene from single genomes, this method is not accurate enough to estimate the diversity of microbial communities from their environments. Principal limits are the lack of real universal primers; DNA amplification biases and reference database selection impact the annotation of reads.

Paradoxically, methodological denial is now a rule in published articles that use 16S rRNA gene amplicon surveys to study unknown microbial communities. In these articles, one pair of primers (although many of them are designed, and provide different results) is used to amplify a region of the 16S rRNA gene. In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid and cheap (while inaccurate) alternative to phenotypic methods of bacterial identification. Although it was originally used to identify bacteria, 16S sequencing was subsequently found to be capable of reclassifying bacteria into completely new species, or even genera. It has also been used to describe new species that have never been successfully cultured.