Molecular Methods in Diagnosis of Fastidious organisms

I- Fastidious Bacteria
Together with virology, the diagnosis of infections due to fastidious bacteria has benefited greatly from molecular detection. Many of these fastidious bacteria have public health implications such as Mycobacterium tuberculosis, Chlamydia trachomatis, Neisseria gonorrheae and Bordetella pertussis (David J Speers., 2006)
Non-culture-based molecular testing has the advantage of avoiding the delays of days to weeks for conventional culture to allow early recognition and treatment as a public health imperative. Commercial assays are available for M. tuberculosis and Mycobacterium avium complex, C. trachomatosis, and N.gonorrhoeae.
Several nucleic acid detection technologies are in use including PCR, transcription based amplification, ligase chain reaction, strand displacement amplification and the Qβ replicase system (Yang S, Rothman RE 2004)
In conclusion, molecular diagnostic techniques have a significant role to play in clinical bacteriology, although their adoption will never replace conventional methodologies, which continue to be the cornerstone of modern bacteriological methods. Indeed, such molecular diagnostic assays may only be implemented in specialized laboratories to enhance laboratory diagnostic efficiency (Millar et al., 2003) where the use of such assays will be mainly confined to diagnosis, identification and genotyping, where current conventional approaches are grossly inadequate


1-Fastidious sexually transmitted bacteria
The introduction of molecular detection for the fastidious sexually transmitted bacteria has led to a large increase in the proportion of laboratory confirmed cases due to its increased sensitivity allowing more effective contact tracing.
In the management of sexual health traditional screening methods require speculum examination in women and urethral swabs in men. These require special equipment and cause embarrassment and discomfort, thus reducing compliance.
Molecular detection is useful since noninvasive specimens unsuitable for traditional culture, such as initial stream urine and self-collected vaginal swabs can be used. These are more convenient and acceptable increasing the compliance with testing (David J Speers., 2006)
Although molecular testing for C. trachomatis and N.gonorrhoeae does not allow monitoring of antibiotic resistance or detect other sexually transmitted diseases, urine testing has shown equivalent sensitivity and specificity to invasive specimens for detection of C. trachomatis in men and women, and for detection of N.gonorrhoeae in men when compared to urethral swabs.
In women the sensitivity and specificity of the PCR assay for N. gonorrhoeae was lower for urine compared to cervical samples, however self-collected vaginal swabs may help in this regard.
The PCR assay for C. trachomatis has equal sensitivity for vaginal and cervical swabs and a transcription mediated amplification assay has been approved by the U.S. FDA for testing C. trachomatis and N. gonorrhoeae from vaginal specimens (Cook RL, Hutchison SL et al., 2005)
In remote areas, molecular methods have the advantage of being performed on dry swabs with little degradation of the DNA during transit compared to the difficulties of transporting samples in specialised transport medium to preserve viability.
In addition, molecular methods can test for multiple genital pathogens such as C. trachomatis, N. gonorrhoeae, the Donovanosis agent and the genital mycoplasmata from the same swab.
2- Mycobacteriology
Mycobacteriology has been aided by the introduction of molecular methods. However, it is important to note that molecular detection of M. tuberculosis is one of the few examples where conventional culture remains more sensitive.
This is possibly due to the difficulty in releasing the DNA from the bacterial cells during the extraction process. Despite this limitation, molecular detection of M. tuberculosis has a definite role as it allows confirmation of acid-fast bacilli seen on microscopy with up to 98% sensitivity in pulmonary tuberculosis within a day compared to two weeks or more by culture.
Specimens that are smear-negative have a much lower chance of molecular confirmation, with reported sensitivities as low as 40% (Bergmann JS, Woods GL.1996).
In addition to direct detection from clinical specimens, molecular methods can confirm a positive culture within a day compared to approximately four weeks using phenotypic methods.
This has shortened the time for laboratory confirmation of suspected tuberculosis even for smear-negative but culture-positive cases Mycobacteriology has also advanced through the use of molecular methods for the speciation of the many non tuberculous mycobacterial species (Kapur Vet al., 1995).
Detection of Mycobacterium tuberculosis nucleic acid in respiratory specimens has high specificity but relatively poor sensitivity, particularly for smear negative disease. The recent development of an integrated specimen processing and real-time PCR testing platform for M. tuberculosis and rifampicin resistance is an important advance that requires evaluation in childhood TB.( Nicol MP, Zar HJ., 2011)