Wednesday, August 17, 2011

Applications of molecular biological techniques in diagnosis of aseptic meningitis

Molecular methods for diagnosis of viral infections as etiological agents for AM are now well established in the routine virology laboratories.They replaced conventional techniques such as viral isolation or detection of a virus-specific antibody response approaches which in comparison are slow or lack sensitivity. The widespread use of PCR has improved the laboratory diagnosis and the understanding of viral etiology of different clinical syndromes. Modifications of the basic PCR technique have been used to increase the sensitivity of detection of viruses either by using nested primers or by the detection of more than one virus in an assay as by using multiplex primers (Kalvatchev et al ., 2004).

The obvious advantage of M-PCR is the ability to detect more

than one virus in a single test.M-PCR could offer the potentially cost savings especially in specimens from which several different viruses (or different genotypes of the same virus) can be recovered .Other advantages of M-PCR are its high degree of sensitivity and ability to detect non-cultivatable viruses (Kalvatchev et al ., 2004).

The wide range of viruses associated with neurological diseases include HSV, CMV, VZV, EBV, the enterovirus group (ECHO viruses, polioviruses, and coxsackieviruses) require a rapid and reliable diagnosis to provide a rational basis for chemotherapy and limit unnecessary procedures and irrelevant therapy (Schmutzhard et al ., 2004).

Multiplex PCR assay has a higher molecular sensitivity for the viruses that were amplified than the sensitivities of the duplex assays for HSV, VZV , enteroviruses and echovirus . In a comparison of the two protocols, there is approximately between 10 to 100-fold more sensitive for the detection of identical isolates of HSV-1, VZV and poliovirus by M.PCR. In addition , because the detection of these viruses is now performed with a single nucleic acid extraction and amplification screen, it is convenient and cost-effective for using a routine basis. The assay takes approximately 4 h to be completed (Steven & John , 1999).

Amplicor enterovirus PCR has been shown to be 99% reproducible and much more sensitive than culture for the diagnosis of enterovirus meningitis ( Emilia et al ., 1999).

RT-PCR differs from PCR only by the addition of a preliminary step , the initial conversion of RNA into DNA template by an RNA-dependent DNA polymerase (reverse transcriptase) ( Schutten & Niesters , 2001).

According to Uchida et al (2005) a novel qRT-PCR assay consists of two multiplex reactions. A qRT-PCR assay developed for the diagnosis of viral meningitis detects enterovirus in cerebrospinal fluid and is significantly more sensitive than viral culture.This assay is more sensitive than a conventional RT-PCR assay with the same primer sets and as sensitive as nested conventional RT-PCR (Verstrepen et al ., 2002 and Mackay et al ., 2002).

The diagnosis of a wide range of different neurological syndromes were established by a reverse transcription multiplex PCR assay. The presence of enterovirus and herpesviruses were studied in cerebrospinal fluid samples collected prospectively from 200 patients hospitalized with neurological diseases suspected of viral infection. Positive PCR results for enterovirus and neurotropic herpesvirus ( HSV and VZV) were obtained among the immunocompetent patients (35%) who presented with AM or encephalitis. Among immunocompromised patients the yield of positive PCR results was 41% and lymphotropic herpesviruses were the predominant (34%). CMV DNA was detected in patients with several clinical syndromes including encephalitis,chronic meningitis, retinitis, ventriculitis, polyradiculomyelitis, and myeloradiculitis. EBV and VZV-specific DNA sequences were
detected in patients with either encephalitis, A.M and chronic meningitis. Dual infections of CMV and HSV or CMV and EBV were established in two AIDS patients with encephalitis and polyradiculomyelitis, respectively. The applications of this RT multiplex PCR assay are extensive and may prove to be particularly valuable for the rapid and sensitive diagnosis of neurological diseases in both immunocompetent and immunocompromised patients (Casas et al ., 1999).

The sensitivities of nested PCR and real-time PCR were compared by testing serial dilutions of an HSV-2 DNA standard as well as by serial dilutions of HSV-2 isolates in CSF. Real-time PCR and nested PCR detected DNA standard and HSV-2 dilutions equally well in CSF down to 55 to 60 copies per reaction. Below this level, real-time PCR gave more positive reactions than nested PCR. When 2.5 to 12 copies/reaction were tested (44%) patients tested positive by real-time PCR compared to (8%) patients by nested PCR, combining the results of a standard and HSV-2 in CSF (Elisabeth et al ., 2007 and Schmutzhard et al ., 2004).).
O'Sullivan et al ., 2003 found that among ten samples of HSV patients were positive with real-time PCR only (three primary infection,six recurrent infection and one AM patient). Another four samples (three primary infection and one recurrent infection patients) were positive by nested PCR but negative by real-time PCR. These four samples were subsequently reanalyzed in duplicate. As a result, two samples both from primary meningitis were found to be positive by real-time PCR as inhibitory factors may influence the efficiency of PCR.
Real-time PCR makes it possible to evaluate quantitative data in relation to the clinical course and antiviral treatment. The clinical symptoms of recurrent HSM are generally milder and of shorter duration than those of primary meningitis . Accordingly, the viral loads in primary meningitis were significantly larger and the inflammatory changes in the CSF were more pronounced in primary meningitis than in recurrent episodes (Afonso et al ., 2007).

Elisabeth et al ., 2007 concluded that the sensitivity of real-time PCR for the diagnosis of HSV-2 meningitis was evaluated

and found to be high. However , it was lower in recurrent infection than primary infection. Real-time PCR for the detection of HSV-2 and VZV DNA in CSF is a quick and efficient tool for the etiological diagnosis of A.M and should be used in the first-line routine diagnosis.For more awareness of the diagnosis, a thorough history taking and the consistent use of novel diagnostic methods HSV-2 and VZV etiologies will be revealed in an even higher percentage of cases.
Although the “gold standard” for diagnosis of tuberculous meningitis (TBM) isolation of Mycobacterium tuberculosis (M. Tb), there are still several complex issues. Recently, in the diagnosis of TBM the detection of M. Tb DNA in CSF samples using PCR has been widely performed as more rapid, sensitive and specific diagnostic method. Based on Taq Man® PCR the authors developed a novel technique of internally controlled quantitative nested real-time (QNRT) PCR assay that provided a prominent improvement in detection sensitivity and quantification. The total of 43 CSF samples that were collected from 8 serial patients with suspected TBM were analyzed. The CSF samples were collected before and during standard anti-tuberculosis treatments (ATT). The QNRT-PCR assay revealed positive results in 24 out of 43 serial CSF samples (55.8%) collected during the treatment course of ATT. Moreover, the cell numbers of M. Tb analyzed by the QNRT-PCR assay decreased gradually correlating with the improvements of the patient's clinical conditions. Since the QNRT-PCR assay provides the ability to calculate a numerical value for the initial numbers of M. Tb in CSF samples, this method is an extremely useful and advanced technique for use in assessing the clinical course of TBM (Takahashi et al ., 2009).
The NASBA is a suitable alternative method to RT-PCR as it is a sensitive for the detection of viruses especially enterovirus

in CSF, respiratory and stool samples . The use of kit-based reagents will enable the laboratories to undertake molecular-based diagnostic procedures for detection of RNA viruses and they could provide results within a time frame relevant to patient management (Fox et al ., 2002).
The sensitivity of LCR in TBM was 66.7% as compared with culture.This is attributed to the varying proportion of smear-positive and in conclusion, LCR cannot replace CSF smear and culture in the diagnosis of TBM. LCR complements these 2 processes with the aim of increasing yield and obtaining early clinical diagnosis. Because of the poor negative predictive value(NPV) , a negative result does not exclude TBM. Communication with the clinician is important as a clinical diagnosis of tuberculosis may be made even if LCR is negative and M. tuberculosis cannot be isolated from the CSF. In the clinical context of subacute or chronic meningitis with CSF lymphocytic pleocytosis a positive result is strongly suggestive of TBM. Early institution of antituberculous therapy reduces the morbidity and mortality resulting from delayed treatment (Chua et al ., 2005).

PCR is a rapid sensitive and specific method for diagnosing leptospiral meningitis especially during the first few days of the disease(Chandrasekharan & Gomathi et al .,2004).

Also PCR can be used for differentiation or typing of various leptospiral isolates by the Random amplified polymorphic DNA (RAPD) technique.The size and number of amplified products depend on the particular primer of template DNA. It is used to compare inter and intraspecific differences among leptospires. This technique can be applied on purified DNA or crude extracts of cells(Chandrasekharan & Gomathi et al .,2004).

A study comparing the diagnostic yield of a real time PCR assay in CSF samples with conventional microbiological techniques for the diagnosis of neurobrucellosis in six patients with neurobrucellosis (three meningitis and three meningoencephalitis) showed that following amplification of a 223 bp sequence specific for Brucella genus, melting curve analysis was performed to verify the specificity of the PCR products. Brucella specific amplicons were easily demonstrated by their characteristic melting temperature in all the real time PCR assays. In conclusion real time PCR assay in CSF samples is more rapid and sensitive than conventional microbiological tests ( Colmenero et al ., 2005).
Recently studies based on PCR technology indicated the value of this method for early diagnosis of invasive candidiasis as real-time PCR assays are standardized, rapid, accurate and reproducible. Moreover, quantification of the fungal load is also possible (Loeffler et al ., 2000).

More-rapid approaches to detect fungal DNA have been developed.These include the PCR-enzyme-linked immunosorbent assay . However, the preparation of DNA still requires a significant amount of time and manpower. Moreover, to achieve a high sensitivity of the PCR assay, standardized DNA extraction protocols with high-quality nucleic acid purification are mandatory. The DNA from fungal cultures could be extracted by using the MagNA Pure LC technique and from blood samples in very short time. Thus, combining automated DNA extraction and real-time PCR permits results to be obtained within one working day for up to 32 samples (Juergen et al ., 2002).
PCR tests for Lyme disease have also been developed to detect the genetic material of the Lyme disease spirochete.PCR

tests are susceptible to false-positive results from poor laboratory technique. Even when properly performed PCR often shows false-negative results with blood and CSF specimens. Hence PCR is not widely performed for diagnosis of Lyme disease. However PCR may have a role in diagnosis of Lyme arthritis because it is highly sensitive in detecting ospA DNA in synovial fluid. Alternatively,serologic studies test for antibodies against Borrelia are used. High titers of either immunoglobulin G (IgG) or immunoglobulin M (IgM) antibodies to Borrelia antigens indicate disease but lower titers can be misleading. The IgM antibodies may remain after the initial infection, and IgG antibodies may remain for years (Aguero-Rosenfeld et al ., 2005).
Western blot, ELISA and PCR can be performed on blood or CSF. Though, antigen capture in the CSF is much more elusive as positive results are obtained in only 10–30% of culture positive patients. The diagnosis of neurologic infection by Borrelia should not be excluded solely on the basis of normal routine CSF or negative CSF antibody analyses(Aguero-Rosenfeld et al ., 2005).
New techniques for clinical testing of Borrelia infection have been developed such as LTT-MELISA which is capable of identifying the active form of Borrelia infection (Lyme disease). Others, such as focus floating microscopy is under investigation. Recently,chemokine CXCL13 may also be a useful marker for neuroborreliosis (Valentine-Thon et al ., 2007).
Tests based on PCR to identify treponemal DNA in clinical material are quite promising. Specificity is reported to be 95–97 % and sensitivity 91–95 %. Most tests are not validated and very expensive making routine use is impossible. In some cases

of oral or rectal chancres, when histology is non-definitive or in chancres pretreated with antibiotics, PCR may be a valuable addition (Palmer et al ., 2003).

In certain circumstances PCR may be helpful in diagnosis by demonstrating T. pallidum in tissue samples, vitreous fluid and CSF (Muller et al ., 2007).

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