Definitive diagnosis of fungal infections depends on a positive blood culture or histopathological evidence of deep-tissue invasion but the sensitivity of the blood culture, especially in invasive aspergillosis, is very low. Culture techniques are also time-consuming, taking up to several weeks - an unacceptable time for the initiation of specific treatment of fungal infection. Alternative techniques to the traditional methods, such as enzyme linked immunosorbent assay detection of the galactomannan antigen in serum and bronchoalveolar lavage (BAL) fluid, and a plasma-P-D-glucan assay, have recently been introduced. Although these techniques demonstrate good sensitivity and varying degrees of specificity in high-risk patients, they may only give positive results at advanced stages of the infection (Ascioglu et al., 2002). Real-time PCR technology, however, is a simple, quick, highly specific and sensitive method for the detection of infectious agents.
Besides fungal detection, quantification of the fungal burden is also important in monitoring the effectiveness of treatment. Several investigators recently applied and showed the effectiveness of real-time PCR technology. White et al. (2003) devised a method for rapid identification of 39 systemic infections with Candida species (within one working day) using the LightCycler® real-time PCR system. Similarly, Aspergillus fumigatus DNA in both BAL and blood samples from high-risk patients can be rapidly and specifically detected and quantified by a LightCycler® based real-time PCR assay (Spiess et al., 2003). Using an iCyler iQ® real-time PCR system, pulmonary aspergillosis has been identified in over 90% of BAL samples of high-risk patients (Sanguinetti et al., 2003). Several studies reported the application of real-time PCR in susceptibility of Aspergillus and Candida species to antifungal agents (Trama et al., 2005; Chau et al., 2004; Balashov et al., 2005).
As one of the major infectious diseases in developing world, malaria has also been targeted by rapid diagnostic testing by real-time PCR methods. Lee et al., (2002) developed a TaqMan® real-time PCR technique for malaria diagnosis that can be adapted for high-throughput rapid screening of hundreds of samples during an outbreak to prevent further transmission of malaria with a better sensitivity and specificity than those of the microscopic method. The method is also shown to be useful in monitoring the effectiveness of antimalarial therapy, especially in situations where drug-resistant strains of the parasites are prevalent (Lee et al., 2002). Recently a single reaction real-time PCR assay has been described to detect and identify all plasmodium species in 3 hours, including standard DNA sample preparation, amplification and detection, with sensitivities equivalent to microscopy (Mangold et al., 2005).
The intestinal protozoan parasite Entamoeba histolytica is endemic in a large part of the world and is considered responsible for millions of cases of dysentery and liver abscess each year. Traditionally, the laboratory detection of E.histolytica in human feces has depended on the microscopic examination of fresh stool samples. However, recently identified Entamoeba dispar is a nonpathogenic type with no requirement of treatment and cannot be differentiated morphologically from E. histolytica. In this regard, Blessmann et al. (2002) developed a sensitive, specific and quick real-time PCR method which can distinguish the two species. In another study using multiplex real-time PCR, simultaneous detection of E. histolytica, Giardia lamblia and Cryptosporidium parvum in fecal samples is successfully performed (Verweij et al., 2004).
In conclusion, real-time PCR technology offers a sensitive, specific and fast detection of fungal infections as well as parasites. Routine clinical applications of the technique in near future will result in the better control and treatment of infectious diseases.
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