Diagnosis of central nervous system complications in HIV-infected patients

Introduction Neurological complications afflict the majority of people who are infected with HIV. These include opportunistic infections, tumours, vascular and metabolic complications, and also disorders that are unique to AIDS itself and associated with infection of the nervous system by HIV 1. Although the use of different antimicrobial treatments has led to a significant increase in the survival of AIDS patients, many of the nervous system disorders still represent a diagnostic and therapeutic challenge. The clinical diagnosis of central nervous system (CNS) diseases in patients with AIDS is based on neurological examination and neuroimaging studies. Further examinations, such as standard analysis of cerebrospinal fluid (CSF), electroencephalography and neuropsychological assessments, may also be useful. None of these investigations, however, provides specific information, and only the identification of a pathogen in the CSF or brain makes a diagnosis of aetiology possible. In some cases, conventional microbiological techniques applied to analyse CSF, such as the isolation of bacteria, mycobacteria and fungi, and the detection of Cryptococcus neoformans polysaccharide antigen, are adequate for diagnosis. CSF examination may also be helpful in demonstrating the presence of tumour cells in cases of metastatic CNS lymphoma, or in revealing Venereal Disease Research Laboratory (VDRL) test reactivity in cases of neurosyphilis, but these conditions represent only a minority of CNS complications. On the other hand, brain biopsy is indicated in limited instances, although this is often impracticable because of the critical condition of the patients. CNS diseases in AIDS patients are therefore often diagnosed on clinical grounds, by either excluding other conditions or evaluating the response to specific treatments. A definitive aetiological diagnosis can frequently only be established at post-mortem examination. Over the past decade, DNA amplification-based techniques, primarily the polymerase chain reaction (PCR), have made a massive contribution towards improving the diagnosis of many microbial diseases. The PCR identification of microbial genomes in CSF found its first successful applications in CNS infections during the early 1990s, when it was used for the diagnosis of herpes simplex encephalitis, and enteroviral and tuberculous meningitis in immunocompetent patients 2–4. Following the first investigations, CSF PCR was assessed and found to be reliable for diagnosing several infectious CNS diseases 5–7. This review will briefly describe CSF PCR principles and procedures, focus on its use in diagnosis of the various HIV-related complications whilst also considering the conventional methods for diagnosis, and discuss its use in clinical practice. PCR of cerebrospinal fluid The main property of PCR, accounting for its extraordinary sensitivity, is its ability to amplify a small quantity of target DNA molecules to produce considerably larger amounts (over 106 DNA copies), which can then be visualized by means of common laboratory procedures. The technique is based on the use of oligonucleotides (primers), which specifically recognize and anneal to the target DNA, and a thermostable DNA polymerase, which forms new DNA strains starting from single nucleotides. DNA amplification takes place during repeated cycles of heating and cooling, and these allow the denaturation of DNA, the annealing of the primer to the denatured DNA strand, and final extension of the DNA itself 8,9. In clinical microbiology, PCR is used to identify the nucleic acids of microbial pathogens that are present in samples. Several PCR protocols have been developed for each microorganism in order to obtain adequate specificity and sensitivity of detection; these may differ in terms of the primers used, choice of buffer and the heating and cooling cycling conditions. The primers are usually designed to recognize highly conserved regions of the genome in order to avoid false-negative results. They may also be complementary to sequences that are repeated many times in the microbial genome, such as insertion elements, thereby enhancing PCR sensitivity 10. The variants of classical PCR include 'multiplex' and 'nested' PCR. The former enables the identification of more than one microorganism in the same PCR reaction, using two or more primer pairs, each specific for a single agent. The latter allows the products of the first amplification to be reamplified using a second set of primers that is nested between the first set, a procedure which overcomes nonspecific amplification and increases the sensitivity of detection. Reference DNA molecules can be added to a PCR reaction in order to reveal possible inhibitors of amplification, generally substances that are already present in the samples. These reference standards are recognized by the same primers and are amplified to the same extent as the template but can be distinguished by their size or the modification of an internal sequence 9. PCR-amplified DNA can be detected by means of different procedures. The simplest technique consists of the visualization of DNA bands of the expected size after electrophoresis on agarose gel stained with ethidium bromide. Hybridization with DNA probes that are complementary to the target DNA may follow or substitute for the gel electrophoresis, after the transfer of DNA to a filter, tubes or microplates. The probes are labelled with radioisotopes or other non-radioactive molecules, which are necessary for the identification of the hybridized DNA. Hybridization not only confirms the specificity of PCR but may also enhance its sensitivity 9. Various protocols have been used for the pre-PCR preparation of CSF (Table 1). The simplest include the repeated freezing and thawing of specimens and/or heating to 95°C for 10–15 min, procedures which facilitate cell membrane disruption and release of DNA. Nucleic acids may also be extracted from CSF, to eliminate factors that potentially inhibit PCR, and/or concentrated. Its relatively simple composition, however, makes CSF one of the most easily used specimens that exist, thereby obviating in most of the cases the need for nucleic acids purification. The simplest CSF preparations are less time-consuming and reduce the risk that small amounts of nucleic acids may be lost or that the samples may be contaminated by exogenous DNA during the different stages of the nucleic acid purification process. When detecting RNA viruses or studying RNA expression, a reverse transcriptase (RT) is used to convert RNA into complementary DNA, which is in suitable form for PCR amplification 9.Table 1: Most common procedures used for cerebrospinal fluid (CSF) preparation for polymerase chain reaction (PCR).Finally, a number of quantitative and semi-quantitative methods have been established to assay the amount of DNA or RNA that is present in CSF. The latter include those based on the endpoint dilution of samples before PCR, or the direct measurement of the PCR product of amplification. The main disadvantage of these procedures is that they do not take into account the possible variations in amplification efficiency between the different samples. Quantitative assays allow a more accurate estimate of DNA and RNA levels, by means of the co-amplification of the target DNA and a reference standard. The standard represents both an internal control and a 'competitor' against which to compare the extent of the target amplification. After PCR, the amounts of both the target and the standard are calculated, using different procedures, and the ratio between them reflects the ratio existing before amplification precisely 11. Cytomegalovirus infection of the CNS Post-mortem findings of neurological disorders caused by cytomegalovirus (CMV) have been reported in up to one-third of AIDS patient autopsies 12–14. Clinico-pathological patterns include encephalitis, meningitis, myelitis, polyradiculitis and peripheral neuropathy, as well as various combinations of these forms. Micronodular encephalitis and ventriculoencephalitis account for the majority of the CMV neuropathologies 15,16. Although their clinical relevance has been underestimated in the past, it is now clear that these neurological disorders, and CMV ventriculoencephalitis in particular, represent a major cause of morbidity and mortality in AIDS patients because of their high frequency of occurrence and devastating effects on brain parenchyma 17–19. Patients with CMV encephalitis are generally in latestage of AIDS, with a common history of previous extracerebral CMV disease. Clinical presentation is of subacute encephalopathy, dominated by an impairment of alertness and cognition, and often accompanied by cranial nerve palsies, and motor and sensory deficiencies. Prognosis is poor, with death ensuing within weeks from onset, usually irrespective of antiviral treatment 17–19. Brain computed-tomography (CT) scans or magnetic resonance imaging (MRI) may show periventricular enhancement, suggestive of periventriculitis, or small focal lesions that are a possible expression of focal parenchymal necrosis 20. Standard CSF analysis may demonstrate a non-specific pattern of hypoglychorrachia, hyperproteinorrachia or mild pleocytosis 18,19. CMV can also cause severe disease of the ventral and dorsal rootlets of the cauda equina, generally associated with myelitis 15,21. Weakness, sensory loss and areflexia in the legs present subacutely, often associated with bladder or anal sphincter dysfunction, tend to evolve to an ascending paraparesis 21–23. Gadolinium-enhanced MRI may show enhancement of the lumbosacral rootlets 24. The CSF pattern is peculiar, characterized by marked polymorphonuclear cell pleocytosis and increased protein content. The prognosis is variable since this form is potentially responsive to antiviral agents 21–23. Conventional diagnostic techniques Various techniques have been used for the diagnosis of CNS complications caused by CMV, none of which have been satisfactorily sensitive. In patients with CMV encephalitis, CMV can only rarely be cultured from the CSF 15,16,25,26. Occasionally, cytological examination of the CSF may reveal typical cytomegalic cells, and the immunostaining or in situ hybridization of CSF polymorphonuclear cells may demonstrate CMV antigens or DNA 27–29. All of these techniques are more useful in patients with CMV polyradiculitis, which is usually accompanied by CSF polymorphonuclear pleocytosis 28–30. Given the lack of an adequate immune response, the demonstration of intrathecal production of CMV-specific antibody appears to be of little help in diagnosis 31. Finally, brain biopsy is of limited application in CMV encephalitis examination because there are generally no focal lesions. CSF PCR CSF PCR for the detection of CMV DNA is the most sensitive diagnostic test in CMV complications of the CNS. Its diagnostic reliability has been widely evaluated in AIDS patients 18,26,29,31–42. The majority of the investigations were case-control retrospective studies, including patients with CMV encephalitis or polyradiculomyelitis that was diagnosed at autopsy, by virus isolation from the CSF, or clinically (Table 2). In most of the studies both the sensitivity and specificity were higher than 80%, and positive and negative predictive values varied between 86–92% and 95–98%, respectively 26,42. Specificity remained high even when control patients with extracerebral CMV disease were examined 32,33. The discrepancies in sensitivity and specificity among the various studies can be ascribed to differences in methodology and the criteria for sample selection. For instance, the low specificity of 42% obtained in a study examining post-mortem CSF samples may reflect the presence of degradation products from non-CSF cells 37. When histopathology is used as the reference diagnostic standard, the time-lag between CSF sampling and death, and the modality of CNS tissue sampling and examination may also be crucial in the evaluation. Furthermore, clinical diagnoses are often based on unspecific findings, and may be uncertain as reference standard. Quantitative PCR techniques showed CSF CMV DNA levels of up to approximately 107 copies/ml. The CMV load correlated with both the extent of CMV lesions in the CNS and the pattern of infection; the highest DNA titres being found in patients with polyradiculopathy 29,38,40,43.Table 2: Polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) for cytomegalovirus (CMV) DNA in HIV-infected patients: characteristics and findings from the most relevant studies.The finding of CMV DNA in the CSF is strongly predictive of the presence of CMV lesions in the CNS and is generally associated with clinical encephalitis or polyradiculomyelitis. However, CMV DNA can be recovered from the CSF also in the presence of mild CMV lesions which are unlikely to cause relevant symptoms. Therefore, CMV DNA-positive samples could be further analysed by quantitative PCR for best interpreting CSF PCR results. Since the presence of CMV DNA in the CSF is related to virus replication in the CNS, a positive PCR result should represent an indication for an anti-CMV treatment. Given the high negative predictive-value of CSF PCR, a negative result is likely to exclude the possibility of a CMV disease of the CNS. Other CNS disorders, primarily HIV encephalopathy, should be considered in patients with subacute encephalitis and CMV DNA-negative CSF. Besides its use in pure diagnosis, the amplification of CMV DNA from the CSF has also been used to monitor the response of CMV disorders of the CNS to antiviral treatments. Preliminary studies have shown a decrease in CMV DNA titres or a clearance of CMV DNA from the CSF of patients with CMV encephalitis receiving ganciclovir 43,44. Moreover, CMV-UL97 gene mutations that confer resistance to ganciclovir have been detected by the direct sequencing of PCR products amplified from the CSF 45. Since CMV is only sporadically cultured from the CSF, the application of molecular biology techniques to examining PCR products appears to be a feasible alternative to biological assays for studies of CNS CMV strains. Herpes simplex virus infection of the CNS Herpes simplex virus (HSV) infections of the CNS are present in approximately 3% of AIDS cases that are examined at autopsy 13,46. Pathological and virological aspects differ from what is observed in immuno-competent adults, in whom herpes encephalitis classically presents with necrotizing lesions of the fronto-temporal lobes, in almost all cases caused by HSV-1. In AIDS patients neuropathological patterns include ventriculoencephalitis, focal encephalitis, and myelitis, associated with either HSV type 1 or type 2 infection 16,47,48. In the vast majority of cases, HSV is present together with CMV in the brain lesions, and the clinical and neuroradiological presen-tations are similar to those observed in patients with pure CMV encephalitis 46–48. Prognosis is poor, and HSV is often unresponsive to antiviral drugs 49,50. Conventional diagnostic techniques As HSV encephalitis is one of the most common and frightening encephalitises in immunocompetent patients, various techniques have been employed in the past in order to obtain a rapid diagnosis. CSF cultures yield positive results in fewer than 5% of immunocompetent patients with HSV encephalitis, and sporadically also in AIDS patients with neurological complications 25,51. In the immunocompetent, a humoral immune response within the CNS can be demonstrated 7–10 days after the onset of neurological symptoms, but this occurs infrequently in AIDS patients 52. Until the introduction of PCR, the identification of HSV in brain biopsies represented the only conclusive method for an in vivo diagnosis of HSV encephalitis in the immunocompetent. In AIDS patients most of the cases described have been recognized only at autopsy 46–48. CSF PCR PCR has been used for the amplification of HSV DNA in the CSF of immunocompetent patients with suspected HSV encephalitis for more than 5 years. Several retrospective and prospective studies have clearly indicated that this technique is the method of choice for an early diagnosis of HSV encephalitis 52–54. In limited instances, CSF PCR for HSV DNA has been applied to HIV-infected individuals 39,42,50,55. Its diagnostic potential has been evalu-ated in a group of 219 AIDS patients, which included six patients with HSV encephalitis. By comparing PCR data with histopathology findings at autopsy, all these six patients with HSV encephalitis were found to be PCR-positive in CSF, giving a 100% sensitivity for the assay. Specificity was 99.5%, positive and negative predictive-values were 86% and 100%, respectively. HSV-1 or HSV-2 was found in both the CSF and CNS in three cases each, and CMV was simultaneously present in five of the six patients 42. A number of HSV DNA amplification techniques are used for diagnosis of HSV encephalitis 54. In AIDS patients, in whom both HSV-1 and HSV-2 seem to contribute equally towards causing HSV encephalitis, a procedure for the identification of both viruses at the same time, i.e. multiplex PCR, can be employed 42. The identification of HSV-1 or HSV-2 DNA in the CSF of AIDS patients with encephalitis is highly suggestive of its HSV aetiological. In these cases, it is of primary importance that the possibility of dual HSV and CMV infection of the CNS is considered, in order that the correct antiviral treatment is instituted. Varicella-zoster virus infection of the CNS According to neuropathological studies, the prevalence of varicella-zoster virus (VZV) injuries of the nervous system in AIDS patients is 2–4% 13,56. The pathological patterns include multifocal or periventricular encephalitis, meningitis, myelitis, vasculopathy and combined forms 56. The clinical picture is consistent with the anatomical presentation and neuroradiology may show ischemic/haemorrhagic infarctions, and/or small oval demyelinating lesions. Typical skin eruptions often precede and sometimes also appear at the same time as the onset of the neurological disease. Treatment with aciclovir has been effective in some cases 56,57. Conventional diagnostic techniques As with HSV encephalitis, the diagnostic techniques for VZV infection of the CNS have mainly been studied in immunocompetent individuals. It is only rarely possible to isolate VZV from the CSF. The detection of intrathecally-produced VZV-specific antibodies from a patient who has also had a recent history of chickenpox or shingles has long been the most used form of diagnosis for immunocompetent patients 58,59. VZV has been cultured from the CSF in isolated patients with AIDS, but no other reliable diagnostic technique has been reported 60. CSF PCR In immunocompetent patients, the use of CSF PCR for the detection of VZV DNA has become a major tool in diagnosing neurological complications of chick-enpox and zoster, and the technique is also useful for identifying cases of VZV aseptic meningitis 61,62. In a few instances, the method has also been used in AIDS patients 39,42,57,63,64. From 500 AIDS patients with neurological complications, VZV DNA was detected in the CSF of 3% of them. However, only a minority of the patients with VZV DNA-positive CSF had encephalitis or myelomeningoencephalitis that was clearly to In the the neurological were caused by the presence of other HIV-related CNS complications, that the of VZV DNA may be associated with VZV 42. The of diagnosis of VZV CNS complications by CSF PCR for VZV DNA in patients with AIDS therefore further multifocal multifocal up to of AIDS patients 12–14. multifocal lesions, to the of the brain sometimes of the and brain of the are characteristics of is the of the infection of with the virus The clinical presentation of is it has a subacute onset, and a clinical picture that the of the lesions, often including and motor and an scans or MRI show small focal or more lesions, or enhancement Standard CSF analysis is generally effective treatment for and the prognosis is poor, with an time of survival from diagnosis of Conventional diagnostic techniques has been have antigens been in the CSF. In patients with detection of intrathecal antibody has a sensitivity and a but the of this technique in diagnosing AIDS patients has not been established Brain biopsy has been the only means of a conclusive diagnosis of for many years. However, to the of the and the prognosis of the the diagnosis of is often and only based on clinical and neuroradiological CSF PCR Over recent a of data has been an between DNA in the CSF and (Table The majority of the studies reported were comparing CSF PCR findings in patients with or the diagnoses being made either on a or on a clinical the first investigations a sensitivity for DNA PCR of studies showed a sensitivity of more than and a specificity of between and and negative predictive-values were and respectively The of the data from the different studies reflect the different criteria used for patient but may also from the use of different However, a detection of genomes for almost all the a clinical sensitivity rarely A higher was obtained using a PCR assay with a biological sensitivity of one genome, but this was associated with less specificity DNA purification procedures starting from larger CSF 500 have been used in an to the sensitivity of but these not seem to be than the simplest CSF This relatively low sensitivity may be to the that is and this could that there is a low release of in extracerebral However, the of a positive result are higher when PCR is applied to CSF specimens to death, that more virus may be in the CSF in the stages of disease Polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) for virus DNA in HIV-infected patients: characteristics and findings in the most relevant studies.The choice of the target genome appears to the of DNA detection in CSF, because of variations in The to be one of the most suitable for PCR. However, two other virus and virus can also be amplified from clinical samples by using primers in this The correct identification of therefore the hybridization of the PCR product with specific or with two of encephalitis associated with virus but not however, have been and these in a patient with AIDS and in an immunocompetent patient Given the high positive predictive of CSF PCR, the diagnosis of can the of DNA in the CSF, obviating the need for a brain The false-negative of that a negative PCR result be considered to exclude being However, it may an alternative diagnosis of HIV to be On the other hand, the clinical of a CSF examination should be repeated at a time, since the sensitivity of the technique increases with the of the disease. CSF PCR has also been applied to samples from AIDS patients with who were receiving a in a few cases, has been found to be effective in improving neurological conditions In the majority of patients there was no DNA clearance and this a lack of clinical response CNS of all AIDS patients or and CNS occurs in of these The clinical presentation and pathological patterns include meningitis, tuberculous and meningitis is characterized by the of non-specific symptoms, such as and are present in the majority of the cases, but focal neurological are less enhancement of scans or MRI is observed in approximately of the cases, and is a and tuberculous in of with CNS with variable clinical and on the and of the lesions. enhancing lesions are and may CNS In CSF analysis usually pleocytosis with and increased protein levels, as found in the CSF of patients with parenchymal lesions is more often specific the mortality of AIDS patients with CNS is the most being the clinical of HIV infection at presentation Conventional diagnostic techniques Conventional microbiological techniques that direct of on stained and the isolation of the in lack sensitivity when applied to CSF. than of CSF specimens are and cultures are positive in fewer than of the immunocompetent patients clinically to have the disease on the of other criteria Moreover, the use of cultures is time and in patients with HIV in whom in diagnosis is related to prognosis Several techniques have been applied to the CSF for the rapid diagnosis of CNS including detection of specific antibodies and antigens and demonstration of and a of by means of the specificity of these techniques is to the presence of antibody against and or application in the however, has not been evaluated CSF PCR In with conventional methods for diagnosis, nucleic acid amplification procedures have the of the sensitivity of the detection of mycobacteria in clinical as well as the time usually to identify these The clinical of these methods is of importance in infections, which are often by a low number of methods for detection of DNA in CSF, have been described in of many small of both HIV and patients The reliability of CSF PCR has been evaluated by comparing PCR findings with the results of diagnosis of CNS obtained using conventional CSF cultures in or by findings at autopsy, or clinical PCR results have been obtained in of immunocompetent patients with and in of HIV-infected patients with or tuberculous parenchymal lesions (Table Specificity of detection was was higher in HIV-infected patients than in immunocompetent patients, related to a higher of positive CSF cultures in HIV-infected patients in immunocompetent The discrepancies in the sensitivity of these studies on the of the cases but may also be because of the different techniques used, which included CSF preparation and procedures for the detection of amplified lack of two techniques have been in a a assay and a amplification assay In studies, these methods showed a sensitivity and specificity in the diagnosis of is considerably less with the use of these in the direct detection of in CSF although a