CNS OIs are the most common cause of neurological diseases in AIDS patients ,–, the pathogens mainly include Cryptococcus neoformans, Mycobacterium tuberculosis, Toxoplasma gondii, CMV, JCV, EBV, VZV, HSV and various bacteria, fungi. Cryptococcus neoformans is the most common pathogen –,,. Pathogens often concurrent in AIDS patients, the symptoms and signs had no specificity, even the imaging features are also difficult to identify (Fig. 3). Thus, to comprehensively evaluate the microbiologic etiologies of CNS OIs in AIDS patients undoubtedly a great challenge in clinical work.
In terms of current routine test methods, including India ink stain, Cr Ag detection, Acid-fast stain, mycobacteria culture, immunoglobulin (Ig) G and M antibody, pathological examination of cerebral parenchymal lesions, especially the development and wide application of CSF PCR in recent years, only the identification of Cryptococcus neoformans and tuberculosis (TB) is relatively efficient –,,. Of note, the low cost of the Cr Ag lateral flow assay underscores its central role in the initial diagnosis of cryptococcal meningitis, that cannot be replaced by PCR test as the preferred assay for first episodes of cryptococcosis ,. Moreover, for TB diagnosis, the Xpert MTD/RIF assay is a fully automated nucleic acid amplification testing (NAAT) that can deliver a result in about 2 h, Which was widely thought to be efficient and cost-effective . It has been endorsed by the World Health Organization and widely deployed –.
Nevertheless, as yet, for most pathogens, especially the pathogens mainly caused cerebral parenchymal lesions, had low sensitivity and specificity means of diagnosis. In the clinical process, in most cases, the pathological examination of biopsy tissues can only exclude tumor or Mycobacterium tuberculosis, for most histopathological diagnosis only indicated inflammatory lesions, can’t ascertain the pathogen, then simply given the empiric therapy. This not only increase the risk of blind medication, but also may delay the diagnosis and treatment of AIDS related CNS diseases.
In this study, we establish a set of FFPET PCR assay system, tried to identify the microbiologic etiologies for encephalitis, in a cohort of AIDS inpatients, who are without definite diagnosis. We selected CMV, TG and JCV for the targeted pathogens, which hyperendemic and frequently caused cerebral parenchyma lesions in AIDS patients, but difficult to ascertain by routine test methods . The data suggested that the most common pathogen causing encephalitis is JCV. Our finding highlighted the utility and efficiency of the PCR assay for improving pathogen identification for CNS OIs.
JCV is the causative virus of progressive multifocal leukoencephalopathy (PML), which is a demyelinating disease of the CNS resulting from reactivation of JCV in immunocompromised patients . JCV is a ubiquitous polyomavirus that infects 50% or more of the adult population throughout the world, the virus has also been found in the brains of otherwise normal individuals .
Generally, the approach to diagnosis of PML was considered the demonstration of JCV DNA from CSF-PCR coupled with the appropriate clinical and imaging features . Nevertheless, the sensitivity of CSF-JCV-PCR dropped from 89.5% in the pre- highly active antiretroviral therapy (HAART) era to 57.5% in the HAART era, and JCV positivity tends to occur in patients with lower CD4 cell counts . Thus, the ability to detect JCV declines substantially following exposure to HAART and in the presence of higher CD4 counts, and may render laboratory-confirmed diagnosis difficult . In addition, since JC virus viremia can occur in healthy individuals, any contamination of the CSF with blood has the potential for providing a false-positive result ,. Moreover, some study showed that , despite the high sensitivity of the PCR assay, a negative PCR does not rule out PML, in some cases biopsy of the brain with PCR amplification from the brain tissue has been employed to establish the diagnosis.
The neuropathologic definitive diagnosis of PML requires demonstration of the typical histopathologic triad (demyelination, bizarre astrocytes, and enlarged oligodendroglial nuclei) coupled with the techniques to show the presence of JCV . The histopathologic triad is rather convincing evidence of the disorder as this unique cluster is not observed in other neurologic disorders. Yet, there are examples in which PML has been misdiagnosed at the time of biopsy as a glioma . Silver et al. . reported that light microscopy and immunohistochemistry techniques alone may be insufficient in establishing the etiology and PCR enhances the yield when tissues obtained by stereotactic biopsy are nondiagnostic. Hence, either the clinical, imaging features or histopathological manifestations are unconvincing or CSF PCR is negative, the brain biopsy and cerebral parenchymal lesion PCR to assist in determining diagnostic certainty in these circumstances is provided.
CMV is also a ubiquitous agent that can cause infection during the course of life at any time ,. By serology, 30–100% of the general population exhibit prior exposure to the virus . CMV remains latent in the infected host throughout life and rarely reactivates to cause clinical illness except in immunocompromised individuals –.
In AIDS patients, CMV is a major cause of morbidity and mortality . Transmission through sexual appears to be the most common route of CMV infection in adults ,. The CMV neurological disease usually occurs at CD4 cell counts < 100 cells/ul ,, which approximately coincided with our study (median value 33, range 6-189).
The routine diagnostic methods for CMV in CNS were serology, CSF-PCR and histopathology respectively. Serological test is mainly the detection of anti-CMV IgG and IgM antibody. A study reports that 93.9% of the AIDS patients were positive for anti-CMV IgG antibody, and 11.1% were anti-CMV IgM antibody positive . Nevertheless, for most of these patients, the diseases caused by CMV is so mild or asymptomatic that it is overlooked, which bring about the fact that AIDS patients are commonly positive for CMV by serology, but the CMV merely being a bystander .
Rongrong Yang et al.  detected the CMV by CSF-PCR. Among the CMV-DNA positive patients, selected 6 patients who had the brain CT or MRI examination, no patient was found with brain parenchymal lesion. That reflected this method having a high false positivity to a certain extent.
Other research reported that diagnosis of CMV infection from tissue biopsies is considered the gold standard with specificity for histopathological evaluation near 100% but with low sensitivity (23.2%) .
Toxoplasma encephalitis (TE) is a very relevant neurological disease in individuals with AIDS . This disease is caused by the intracellular protozoan parasite, TG. Immunocompetent persons with primary TG infection are usually asymptomatic, and latent infection can persist for the life of the host. In immunosuppressed patients, especially patients with AIDS, TG can reactivate and cause disease, usually when the CD4 count falls below 100 cells/ul, which also coincided with our study approximately (median value 75.5, range 12–130).
Laboratory testing is usually necessary to establish the diagnosis of TE, because the clinical manifestations of infection are so protean. Available diagnostic modalities for TG include serologic assays, molecular-based techniques (eg, PCR based assays), and histopathology.
The serologic assays mainly conclude the IgG and IgM antibody. However, false-negative serologic testing is typically seen in patients among those who are significantly immunocompromised (eg, transplant patients, especially bone marrow transplants, or those with HIV infection) ,. And a false-positive IgM test result may be related to rheumatoid factor, antinuclear antibodies, and nonspecific binding in vitro, that’s also a significant issue in serologic testing for toxoplasmosis . Likewise, although detection of IgG and IgM antibody in CSF was also widely used, the CSF antibody tests can be confounded by contamination of the CSF by serum during the lumbar puncture, or passive transfer of antibody from the blood.
The PCR assays can detect TG DNA in blood, cerebrospinal fluid, aqueous humor, and bronchoalveolar lavage fluid ,. Since there is no standardized PCR assay, the sensitivity of PCR assays varies widely (from 15 to 85 percent for blood), although specificity appears to be high (greater than 95 percent) . Depending upon another studies, the detection of TG by PCR in CSF has demonstrated high specificity (96 to 100 percent), but variable sensitivity (50 to 98 percent). Treatment also affects diagnostic sensitivity ,. Thus, a positive PCR result establishes the diagnosis of TE, but a negative one does not rule it out.
Histopathological detection of TG is in one of two forms: tachyzoites or cysts . Nevertheless, they were all rarely or difficult detected in the cerebral parenchymal lesion tissue .
The ultimate significance of PCR based detection of toxoplasmosis is that PCR can detect the DNA of parasites even when the tissues available for testing are in state of decomposition; in contrast, in histopathology it is quite difficult to identify the necrosed tachyzoites in the regions of marked generalized necrosis of the parent tissues induced by tachyzoites themselves . Moreover, the size of the sample analyzed is very important in the detection by histopathology and it is quite possible that the parasitic stage in the sample to be tested is either low or sparse and show focal distribution in the tissues or it may be all together dead. But PCR will give amplification even if the parasitic stage is dead  and/or very less in number. PCR can even detect 0.1 pg. of DNA  and even a very few tachyzoites are sufficient in accurate diagnosis no matter if they are living or dead.
Our study showed there was no patient’s WBC counts higher than 20 × 109cells/L in blood. Likewise, the WBC count in CSF did not exceed 40 cells/L in our study. This reflected from the side that blood and CSF were not so sensitive to the encephalitis, in other words, they cannot accurately reflect the severity of encephalitis.
In our study population, the total mortality rate was 42.42% (14/33), that mainly focus on the JCV and CMV patients. The patients with FFPET TG positivity were totally alive with improvement, that because all of the patients in our study were routinely received the empirical/experimental anti-toxoplasma therapy after the operation when the histopathological diagnosis only indicated inflammatory lesions. This is not only because the TE is prevalent in AIDS patients, but also the other pathogens, like CMV and JCV, have no specific, effective treatment. For this reason, the application of PCR assay for the cerebral parenchymal lesions, not only improving pathogen identification efficiency, but also lay a foundation for further research of pathological mechanism and precise treatment of CNS OIs.
In our study, the variety of specific pathogens for PCR testing was limited and there are other possible pathogens of CNS infection, but were not assessed. For instance, we excluded the Epstein-Barr virus (EBV) and herpes simplex viruses (HSV), the reasons are as follows:
EBV was not validated by additional confirmatory testing due to its ubiquitous nature and unclear significance in this immunocompromised population, as described previously ,. In line with a study by Kelly et al.  speculated that EBV might be a non-pathogenic marker of immunosuppressed population. Furthermore, another research showed that EBV was left out of an FDA-approved PCR panel, and the prevalence of EBV should be interpreted with caution, especially lacking confirmatory PCR testing . Rajasingham et al.  thought that there is little need for expansion of HSV PCR testing. Although if further patients were tested, HSV likely would have been detected, the prevalence appears quite low and testing would not be cost-effective compared with other interventions.
On account of our study is a retrospective analysis, these patients lacked the pertinent therapy at that time, so we cannot further appraise the accuracy of this method effectively. Consequently, additional studies to validate the accuracy of PCR assay in the diagnosis of cerebral parenchymal OIs are needed across the further prospective study. Our study is also limited by the relatively small sample size, so multiple pathogens infection in CNS disorder patients will be an important topic to assess with further study in larger cohorts and with subsequent validation of results.
In terms of economic effectiveness, it seems that the introduction of the PCR assay would result in more rapid and accurate diagnosis along with minimization of unnecessary testing, a shortened time to initiation of targeted therapy, and shorter hospital durations, which have a substantial cost savings in countries . Whereas, to validate the cost-effectiveness of the PCR assay method in the diagnosis and monitoring of cerebral parenchymal infections are also needed further studies.
In summary, the PCR assay offers a promising platform for the rapid and accurate diagnosis of cerebral parenchymal infections, but need further efforts to validate.