Theory and Practicalities of Bacterial Diagnostic Methods - Report Example

Theory and Practicalities of Bacterial Diagnostic Methods: A Critique Bacteria are among the most important human pathogens. They are the etiologic agents for tuberculosis, malaria, diarrhoea, most sexually transmitted diseases, and typhoid fever, to name a few (Kayser, Bienz, Eckert, & Zinkernagel, 2005). These diseases can reach epidemic proportions and cause millions of death yearly, especially in the less developed countries. Since there are numerous bacterial species and strains, effective treatment of a bacterial disease starts with the identification of the etiologic agent. Initially, a symptomatic patient is observed for characteristic signs and symptoms from which a presumptive clinical diagnosis is made. However, a laboratory analysis is necessary to validate the clinical diagnosis. Laboratory analysis starts with specimen collection, mostly body fluids (sputum, blood, urine, faeces, and tissue). The specimens are tested for microorganisms that may cause the illness, and to provide information about the effectiveness of antibiotics used against the microorganism after it has been identified. Methods used in bacterial diagnosis fall under four broad categories (Washington, 2008): 1. Microscopy 2. Culture in selective media, followed by biochemical tests 3. Serologic tests (e.g. agglutination, complement fixation, ELISA) 4. Genetic methods (DNA, RNA hybridization, markers, PCR) These methods may be used independently of each other for the rapid identification of bacteria. However, two or more techniques may be deemed necessary in special cases and to verify the original diagnosis. 1. Microscopy Microscopy is the direct viewing of the microorganism after it has been stained with special dyes or after it has conjugated with specific fluorochromes (Kayser, Bienz, Eckert, & Zinkernagel, 2005). Most common dyes are the Gram stain, which differentiates Gram-positive from Gram-negative bacteria, and acid-fast stain. Microscopic examination will also reveal the shape of the bacterial cells and the associations with neighbouring cells. Microscopy offers a rapid technique, with minimal requirement of a compound binocular microscope equipped with low-power (1OX), high-power (40X), and oil immersion (1OOX) objectives, 10X wide-field oculars, a mechanical stage, and a good light source (Washington, 2008). For fluorescent microscopy, an exciter barrier filter, darkfield condenser, and ultraviolet light source are necessary. The sensitivity of the microscopic method is usually dependent on the number of organisms that are obtained from the specimen and the morphological uniqueness of the microorganism as it appears under the microscope. As an example, the detection of Mycobacterium tuberculosis in sputum smear microscopy is insensitive, laborious, and time-consuming (Buijtels, et al., 2008). An acid-fast smear of concentrated sputum must have at least 104 colony forming units (CFU) of tubercle bacilli per millilitre of sputum for successful detection (Washington, 2008). Likewise, there is only 75% detection of the Gram-negative coccobacilli in cerebrospinal fluid from children with Haemophilus influenzae meningitis, because in some patients the CFU per millilitre of cerebrospinal fluid is less than 104. The type of specimen and sampling method could also affect the sensitivity of microscopic diagnosis. The fluorescent antibody stain for Chlamydia trachomatis is more sensitive when endocervical cells are obtained using a cytobrush rather than a swab. Furthermore, the stage of the disease when the specimen is collected also affects sensitivity (Washington, 2008). However, sensitivity and specificity can both be improved with the collection of better specimens. 2. Culture in selective media followed by biochemical tests The culture of etiologic microorganisms is another method for confirming the cause of an infection (Washington, 2008), and the only method which can measure reaction to antibiotics (Solomon, Peeling, Foster, & Mabey, 2004). Bacteria are cultured in either liquid or solid media. If the colonizing bacteria are made up of one species, liquid media has greater sensitivity; however, mixed cultures have to be subcultured to solid media for separate processing. These steps plus succeeding biochemical assays take time and make the culture method quite slow. Clinical specimens from bacterial infections are often a mixture of aerobic, facultative anaerobic, and anaerobic bacteria, thus, these specimens are usually inoculated into several general-purpose, differential, and selective media, which are then incubated under aerobic and anaerobic conditions, at 35 - 37°C. Differential media incorporate one or more carbohydrates, with a suitable pH indicator. Selectivity of culture media is possible by the addition of antimicrobial agents that allow only the growth of the bacteria resistant to the inhibitors. Microbial growth in cultures is demonstrated by the appearance of turbidity, gas formation, and discrete colonies in broth and on agar. These form the bases for the identification of the bacteria. Some bacteria are easy to characterize, while others (most Gram-negative bacilli) require twenty tests for the biochemical characterizations (Washington, 2008). For some bacteria like Mycobacterium tuberculosis, culture is complicated and slow with a sensitivity of only 80 to 85% (Buijtels, et al., 2008). This is a disadvantage for microorganisms that have the potential to cause disease outbreaks, because culturing will not meet the demand for fast identification. The general view that culture is slow can change with the introduction of chromogenic agars, which can produce results within 24 hours, without the added expense that is associated with molecular tests (Malhotra-Kumar, et al., 2008). With chromogenic agar, detection depends on the incorporated colourless chromogenic substrate that is cleaved by the targeted bacterial enzyme. Once cleaved, an insoluble colour substance is formed, which allows identification of the bacteria possessing the specific enzyme. Many chromogenic substrates are already commercially available for the detection of methicillin resistant Staphylococcus aureus (MRSA). 3. Serologic and immunologic tests If a suspected microbial agent is difficult to isolate in culture, serologic and immunological techniques can be used for identification. These approaches make use of the specific antibody response to the infecting microorganism. Bacterial antigens can be detected using enzyme-linked immunosorbent assay (ELISA) latex particle agglutination and coagglutination (Washington, 2008). The principle of ELISA, which is the most used method to detect microbial antigens, is the use of an antibody, which is specific for a bacterial strain. This antibody is fixed to a solid phase, either latex or metal beads, or bound to the inside of the wells of an ELISA plate. The specific bacteria or antigen, if present in the specimen, will bind to the antibody. The next step is to add a second antibody, which is specific for the antigen; this second antibody is bound to an enzyme that can react with a substrate to produce a coloured product. The substrate is added and wells that have the coloured product are positive for the bacteria of interest. The advantages of immunological techniques are rapidity, fast turn-over, and results visibility. However, the main disadvantage of serology in diagnosis is the observed lag between the start of the infection and development of antibody response to the infecting microorganism. There are some cases that the high titre of IgM and IgG antibodies in the patient’s serum could be from a past infection due to another organism (Washington, 2008). Immunosuppression in some patients could also lead to the absence of antibody response; this is another limitation in serology as a diagnostic tool. Aside from ELISA, other methods in serodiagnosis are: microagglutination, flow cytometry (FC), indirect immunofluorescence assay (IIFA) and Western blotting (WB). An assessment in their efficiencies in detecting the antibodies IgG, IgA, and IgM from clinically proven infections with Francisella tularensis was performed (Porsch-Ozcurumez et al., 2004). The diagnostic sensitivity and specificity were 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. Therefore, ELISA, western blotting, and microagglutination are reliable serodiagnostic tools. Other tools that have been developed based on the antigen-antibody relationship are lateral flow tests, or rapid tests that are available as commercial kits. However, these kits give subjective results (Cho, 2007). Newer rapid immunologic tests are fluorescence polarization assay, surface Plasmon resonance imaging, and protein-chip-based assays. 4. Genetic methods The use of molecular techniques is based on the specific complementary pairing of strands of nucleic acids (DNA and RNA), and their capacity to reproduce nucleic acid strands via the polymerase chain reaction or PCR. Molecular techniques that have been employed in bacterial diagnostics are the following: ribotyping or characterization of bacteria based on rRNA sequences (Flandroisa & Mignard, 2006), restriction fragment length polymorphism, DNA hybridization, DNA sequencing, PCR and DNA microarray (Kayser, Bienz, Eckert, & Zinkernagel, 2005) (Tomioka, et al., 2005). The polymerase chain reaction (PCR) has enhanced the diagnosis of infectious diseases. This approach has led to the easy and reliable detection of infections caused by bacteria that are difficult to culture or that have not yet been successfully cultured. Broad-range bacterial PCR can detect microorganisms that are not frequently found or have unknown origins. However, PCR has not been widely used in clinical diagnosis due to risk in contamination and the amount of time needed to prepare the samples for PCR and analysis of the PCR products (Wu, et al., 2008). The development of molecular probes has led to the DNA hybridization techniques where the DNA of the bacteria hybridizes its complement from a known bacterial species or strain (Washington, 2008). Use of DNA microarrays have been developed, like the Lab-on-chip (STMicroelectronics, 2005), which has the capacity to amplify clinically relevant samples by PCR using the microreactors buried in MEMS chip. The feedback on nucleic acid amplification tests (NAATs), which are probe and PCR-based tests, have been mixed, depending on the organism. As an example, NAATS have more superior accuracy in diagnosing tuberculosis (TB) when applied to respiratory samples compared to other body fluids. Compared to commercial tests, nucleic acid amplifications tests were better at ruling out TB, but not as good at ruling it in (Dinnes, et al., 2007). Thus, for diagnosing TB, the nucleic acid amplification tests cannot be relied upon to rule out TB but can be the first-line test for ruling in TB. This has also been observed in other studies (Thwaites, et al., 2004). Ziehl-Neelsen (ZN) staining, the Gen-Probe amplified Mycobacterium tuberculosis direct test (MTD), and culture with 341 cerebrospinal fluid specimens from 152 adults were compared for sensitivity and specificity. Results showed that ZN staining of the cerebrospinal fluid for the rapid diagnosis of tuberculosis is as good as MTD, much faster, and less expensive. The nucleic acid tests were very useful in detecting and characterizing DNA from Chlamydia trachomatis, the causative agent of eye disease trachoma. Nucleic acid amplification tests were found to be superior due to their straightforward sampling requirements, high sensitivity, and high specificity (Solomon et al., 2004). However, the nucleic acid amplification tests only detect chlamydial nucleic acid and not the presence of other viable organisms during time when sample was collected. False positive results can arise from remnant of previous infections. The use of NAATs for N. Gonorrhoea diagnosis presents a big challenge and has limitations like cost, risk that there will be carryover contamination, and inhibition. False positives are a major consideration because of the frequent horizontal genetic exchange within the Neisseria genus, which lead to the acquisition of N. gonorrhoea genes in commensal Neisseria (Whiley, Tapsall, & Sloots, 2006). NAAT false-negative results happen due to sequence variation in some gonococcal populations. DNA microarrays, where many bacterial gene sequences are imprinted on a chip, are a means of identifying the presence of more than one bacterial species in one sample. This technique has been used to detect bioterrorism, where blood can be used to carry bacterial pathogens for bioterrorism acts (Tomioka, et al., 2005). The use of molecular techniques removes the reliance on culture methods; are faster, more sensitive, more discriminating and have higher specificity. The major disadvantage would be the cost since the reagents and equipment used are more expensive. There is also a need to improve methods for fastidious organisms. References Buijtels, PC, Willemse-Erix, HM, Petit, P, Endtz, H, Puppels, G, Verbrugh, HA, van Belkum, A, van Soolingen, D and Maquelin, K 2008, ‘Rapid identification of mycobacteria by Raman spectroscopy’, Journal of Clinical Microbiology, vol. 46, pp. 961-965. Cho, S 2007, ‘Current issues in molecular and immunological diagnosis of tuberculosis’, Yonsei Medical Journal, vol. 48, pp. 347-399. Dinnes, J, Deeks, J, Kunst, H, Gibson, A, Cummins, E, Waugh, N, Drobniewski, F and Lalvani, A 2007, ‘A systematic review of rapid diagnostic tests for the detection of tuberculosis infection’, Health Technology Assessment, pp. 1-196. Flandroisa, S & Mignard, J 2006, ‘16S rRNA sequencing in routine bacterial identification: a 30-month experiment’, Journal of Microbiological Methods, vol. 67, no. 3, pp. 574-581. 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Whiley, D, Tapsall, J & Sloots, T 2006, ‘Nucleic acid amplification testing for Neisseria gonorrhoeae: an ongoing challenge’, Journal of Molecular Diagnostics, vol. 8, no. 1, pp. 3-15. Wu, Y, Chen, L, Wu, C, Shang, SQ, Lou, JT, Du, LZ and Zhao, ZY 2008, ‘Gram stain-specific-probe-based real-time PCR for diagnosis and discrimination of bacterial neonatal sepsis’, Journal of Clinical Microbiology, vol. 46, no. 8, pp. 2613-2619. Read More