Objectification and Quantification of the Cognitive Impairment from an Existing HIV Infection or HIV Encephalopathy Using Magnetic Resonance Spectroscopy

Abstract

Some patients with HIV develop dementia. Using in vivo proton nuclear magnetic resonance (¹H-NMR) spectroscopy, it is possible to measure the metabolic changes noninvasively. In this study, it is of interest to answer the clinically relevant question of whether magnetic resonance spectroscopy is suitable for the diagnosis of HIV encephalopathy. In total, 14 HIV-positive patients were investigated by means of localized ¹H-NMR spectroscopy in the following locations: (1) the mid-parietal gray matter, (2) the parietal white matter (PWM), and (3) the frontal white matter. All patients had no other brain diseases, apart from the HIV encephalopathy. The clinical extent of HIV encephalopathy of each patient was investigated using the following tests: (1) an electroencephalogram, (2) a neurological examination and psychiatric assessment, and (3) a psychometrical test. The spectroscopic changes in the PWM were more pronounced than those in the cortex, and the myo-inositol/creatine (mI/Cr) signal showed a clear increase in the cortex. Overall, the mI/Cr ratio emerged as the most reliable and earliest parameter to indicate an HIV encephalopathy.

Introduction

The HIV infection leads to cognitive impairments of the brain. ¹ However, the pathophysiology of the emergence of neuro-AIDS is still unknown. ^2,3 For a physician, despite the known development of cognitive impairments, the early diagnosis, objectification, and quantification of the existence and extent of this impairment during infection are difficult to recognize in each individual case. Through the use of a modern medical treatment (highly active antiretroviral therapy [HAART]), the survival of HIV-positive patients has significantly increased in the past decade. Hence, the number of cognitively impaired HIV-positive patients has significantly increased. ^4,5 A combination of various evaluations, such as electroencephalogram (EEG); consulted psychometric test ⁶ ; physical, neurological, and psychiatric examinations; and history taking are necessary for the proper diagnosis of an HIV-associated disease. ^{7 –9} Nevertheless, the physician still remains unsure whether this is the correct diagnosis in many cases. A new technology, magnetic resonance spectroscopy (MRS), can help to perform the diagnosis. In this study, the value of the magnetic resonance spectroscopic investigation was compared with that of the conventional techniques. The use of MRS enables the analysis of metabolite concentrations in the brain without having physical contact with the patient. In contrast to magnetic resonance imaging (MRI), signals other than the hydrogen signal, which is obtained predominantly from water and lipids, are analyzed. Therefore, biochemical information about the function of the brain is measurable using MRS. It has previously been shown that HIV-associated dementia is associated with the changes in MRS. ^{10 –12} We aimed to determine whether this new analysis could be used clinically and whether it is applicable to patient diagnostics. To answer these important questions, in this study, conventional diagnostic procedures were examined using HIV-positive patients with different and pronounced cognitive impairments, and these impairments were compared using MRS. In addition, conventional diagnoses were made using the conventional procedure. ^{7 –9,13} Therefore, a battery of tests, which consisted of neurological and psychiatric investigations, patient history, a psychometric test (the number connection test; NCT), and the EEG were used to assess the patient. The results of this conventional procedure were summarized using a score value.

Patients and Methods

Patients

For the diagnosis of HIV encephalopathy, the Deutsche Neuro-AIDS Arbeitsgemeinschaft (DNAA, German Neuro-AIDS community) requires an investigation of the neurological status (including an EEG: slowing of the motor responses and testing of the fine motor functions) and psychological assessments (including a psychometric test or an IQ test). These elements correspond to the score values used in this study, which were established by the DNAA. ¹⁴ The inclusion criterion for the patients was HIV-positivity. The exclusion criterion was the presence of any other disease of the central nervous system (CNS).

A group of 14 patients comprised of 13 men and 1 woman, with varying ages and duration of HIV infection, was studied. All the patients were admitted to the university hospital (HIV policlinic) in Frankfurt, Germany, within a 6-month study period for the confirmation of HIV encephalopathy. Participation in this study was voluntary, and most recruits already recognized that they had strong cognitive impairment.

All the patients were treated with HAART, which consisted of a multidrug combination of zidovudine (ZDV), nelfinavir (NFV), and lamivudine (3TC; protease inhibitors). The experiments were conducted with the understanding and consent of each participant, and the ethical committee of the hospital approved the experiments.

The methods of investigation employed in this study were the following: (1) MRS, (2) neurological examination, (3) psychiatric assessment, (4) EEG, and (5) number connection test (NCT, a psychometric test). ¹⁵ These investigations were carried out on all the patients under standardized conditions.

¹H MRS Study Protocol

All imaging and single volume spectroscopy studies were done on a 1.5-Tesla MRI Scanner (Siemens Magnetom Vision, Siemens Medical AG, Erlangen, Germany). The metabolite spectra were acquired using a stimulated echo acquisition mode, short echo time (TE) localization sequence (TE of 20 ms, mixing time of 15 ms, repetition time of 1500 ms, 250 acquisitions), and frequency-selective water suppression. The voxel selection (for measurement positions see below) was based on the need for favorable test conditions. These conditions are essential for screening a large series of patients with a multivoxel protocol in a relatively short time as well as for including areas that are histologically involved in HIV. The voxel size was 8 cm³ for all measurement positions. The following particular voxels were selected:

1. The occipital gray matter (OGM, mid-parietal line). Aside from a minimal proportion of white matter (WM) originating from U-fibers, the voxels contained gray matter exclusively (Figure 1).

2. The parietal WM (PWM).

3. The frontal WM (FWM).

Figure 1.

The measurement locations are shown in different orientations. Metabolites were measured in the frontal white matter, the parietal gray matter, and the occipital white matter.

Finally, the signals were processed by means of zero filing, a Fourier transformation, baseline correction, and a phase correction.

The signal areas were evaluated during magnetic resonance analysis using computer algorithms. The creatine (Cr)-normalized (X/Cr) relationships of the metabolites were determined using the program LCModel. ¹³

Processing of the Spectroscopic Data

The spectral raw data were processed with LCModel, ¹⁶ using the concentration-calibrated in vitro model spectra with N-acetyl-aspartate and N-acetyl-aspartatyl-glutamate (NAA; processed together); Cr and phosphor-Cr (processed together); choline (Cho), phosphocholine, and glycerophosphocholine (processed); myo-inositol (mI); and glutamate (Glu) and glutamine (Gln), which were also processed together and abbreviated as Glx. All of the metabolite concentrations were quantified as ratios to Cr.

Clinical Investigations and Score Value

All the clinical results for the EEG, the NCT, the neurological investigation, and the psychiatric investigation were summarized as a score value. This score value is a measure of the clinical stage of HIV encephalopathy. For the score values, the results of the EEG (decreased frequencies) were given up to 2 points. Similarly, the results of the NCT were rated with values up to 2 points. An unusual neurological or psychiatric test result would be given 1 additional point each.

Patients exhibiting a score of 0 to 1 points were assessed as not showing signs of HIV encephalopathy. Patients with 2 to 3 points were regarded as possibly having encephalopathy, and patients with 4 to 6 score points were regarded as having HIV encephalopathy.

Results

Groups

As shown in Figure 2, the signal intensities for the NAA/Cr ratio, the Cho/Cr ratio, and the mI/Cr ratio were compared for patients without HIV encephalopathy (group 0, 0-1 points), patients with possible encephalopathy (group 1, 2-3 points), and for patients with encephalopathy (group 2, 4-6 points). For the PWM and the OGM, several metabolite ratios changed with an increasing clinical impairment of the patients (score value). In detail, the following changes were found in patients with HIV encephalopathy when compared with HIV-positive patients without HIV encephalopathy:

Figure 2.

Comparison of the spectroscopic results for the different measurement locations: the frontal white matter, the PWM, as well as the occipital gray matter. The metabolite ratios myo-inositol to creatine ratio, N-acetyl-aspartate (including N-acetyl-aspartatyl-glutamate) to creatine ratio, glutamine (including glutamate) to creatine ratio, and choline (including phosphocholine and glycerophosphocholine) to creatine ratio were compared. The graph represents the intensity ratios relative to creatine versus the different patient groups (group 0 HIV-positive without HIV encephalopathy, group 1 HIV-positive with a possible HIV encephalopathy, and group 2 HIV-positive with manifest HIV encephalopathy). Zero indicates HIV-positive patients without encephalopathy, 1 indicates patients with a mild HIV encephalopathy, and 2 indicates patients with severe HIV encephalopathy. The myo-inositol intensity was significantly increased in the PWM. Myo-inositol in the PWM directly correlates with the clinical extent of the HIV encephalopathy. * indicates statistically significant correlation.

1. increased mI/Cr ratios,

2. no statistically significant changes in the Cho/Cr ratios, and

3. slightly decreased NAA/Cr ratios.

The mI/Cr ratios in the PWM and OGM correlated significantly with increasing score values (Table 1 and Figure 2) and increasing clinical impairment.

Table 1.

Statistical Significance of MRS Metabolite Changes at Different Positions.^a

	Cho/Cr	NAA/Cr	mI/Cr	Glx/Cr
PWM	o	o	x	o
OGM	o	o	x	o
FWM	x	o	o	o

Abbreviations: Cho/Cr, choline (including phosphocholine and glycerophosphocholine) to creatine ratio; FWM, frontal white matter; Glx/Cr, glutamine (including glutamate) to creatine ratio; mI/Cr, myo-inositol to creatine ratio; NAA/Cr, N-acetylaspartate (including N-acetyl-aspartatyl-glutamate) to creatine ratio; OGM, occipital gray matter; PWM, parietal white matter.

^a “o” indicates no significant change relative to HIV-patients without encephalopathy; “x” indicates significant change relative to HIV-patients without encephalopathy.

The mI/Cr ratio seemed to be a suitable, early parameter for monitoring the progress of HIV encephalopathy. The main result was the significant correlation of the mI/Cr ratio with the degree of HIV encephalopathy. On the other hand, the Cho signal increased with increasing score in both the cortex and the cerebral medulla, while in the FWM, the Cho signal decreased.

In the results, we found that MRS is suitable as an important supplement for clinical investigations of HIV-positive patients. The HIV-positive patient receives an MRI at regular intervals when a questionable cognitive impairment has been found to clarify and/or exclude in CNS infections. In this study, we found that additional MRS would be useful in additional studies. The MRS investigations performed at regular intervals would be useful for objectively measuring any questionable cognitive impairment. In addition, such an investigation could also aid in choosing the correct therapy by monitoring the patient and optimizing the therapy. ¹⁷ When MRS indicates a cognitive impairment, other CNS medications, such as certain virostatics, should be used. A rapid change in medication can disrupt an ongoing impairment of brain function. ¹⁷ Therefore, the HIV-positive patient would benefit from such an extended therapy. Furthermore, the possibility of objectively documenting the course of the disease becomes apparent. For the individual patient, this would indicate an improvement in his or her medical support. Importantly, early therapy is necessary to effectively treat an HIV-associated dementia (HAD). Therefore, a safe diagnosis of this disease is necessary. Once brain tissue is lost, this tissue cannot be replaced or brought back by any therapy. Therefore, only an early diagnosis can help the patient, and this can be significantly improved with the use of MRS. With an early diagnosis of HAD, the treating physician would be able to change the patient’s medication to stop the progression of encephalopathy. Because the presence of HAD increases mortality, an early and sufficient therapy can increase AIDS survival. ¹⁸ Thus, for the treating physician, MRS serves as a valuable method of diagnosis. The progress evaluation could objectively determine whether and how the brain function is impaired by the HIV infection. Given the use of health services and the otherwise enormous costs that an HIV-positive patient incurs for his or her treatment, the costs of additional MRS are low, even if a conventional MRI is performed. Furthermore, the use of MRS could be recommended in addition to the results of the routine diagnostics of the HIV-positive patient. Studies with a larger number of patients are required to confirm these results. Thus, MRS could replace conventional diagnostics. Furthermore, this could save time and money because conventional diagnostics are very time-consuming. In contrast to previous studies, this study directly compared conventional diagnostics and the new MRS instrument. This provides the clinician with the opportunity to directly compare both the approaches (conventional vs MRS). This is also relevant because the HIV-positive patient is primarily treated by the physicians of internal medicine, dermatologists, and venereologists. However, these physicians are limited in their experience in diagnosing psychiatric and/or neurological diseases, such as HAD. In particular, for these neurologically inexperienced physicians, an objective method would be important for diagnosing HIV-associated cognitive impairments, because these colleagues are frequently strained with the diagnosis of HAD.

Discussion

The ability of a neurologically/psychiatrically inexperienced physician to use MRS for determining the presence of an HIV-associated cognitive impairment in a patient and distinguish HAD from a purely depressive disorder would be beneficial. Therefore, the authors suggest that, in the future, patients should use MRS in the context of an MRI (which is performed in nearly all cases) to clarify the cognitive impairment of the HIV-positive patient. With only a small additional expenditure of time, valuable information regarding the cognitive clinical condition of the patient can be archived for neurologically/psychiatrically inexperienced physicians. Based on these data, the patient can be treated more effectively. An early and objective clarification of HIV-associated impairments and/or encephalopathy is necessary for many reasons, including the following, and should be supported.

Interpretations of the Changes Found

The NAA measured at a chemical shift of 2.01 ppm is widely accepted as an indicator of neurons (perikaria and neuro-axons). ¹⁹ It is present in neurons, predominantly as a reservoir of the glutamate neurotransmitter, in the form of N-acetyl-aspartyl-glutamate ^20,21 part of the NAA measured here. In addition to mI, NAA is found to be one of the most important modulators of osmotic pressure. ²²

The NAA represents a measure of the number of nerve cells. Here, NAA decreased during the course of HIV encephalopathy. However, the decrease was small and found only in the advanced stages of the disease.

Creatine measured at a chemical shift of 3.02 ppm is likely involved in energy metabolism of the brain. As is the common standard, all the other metabolites were divided by the area under the curve of Cr. ^20,21

Cho measured at a chemical shift of 3.20 ppm serves as a marker of cell membrane turnover. It is present in high concentrations in membrane-rich structures such as myelin. It is also a constituent of neurotransmitters (acetylcholine), although at much lower concentrations. ^20,21

As a component of lecithin, Cho is present in the cell membranes of all cells of the CNS. Through remodeling, astrocyte numbers increase; hence, the Cho signal should increase. Several authors have described an increase in the parietal Cho signal. ^23,24 However, this was not seen in the present study. On the other hand, Chang et al ²⁵ reported that the increase in the Cho signal reverses during antiretroviral therapy (ART). The absence of a significant increase in our study could be an indication that the HAART used was effective. Nevertheless, with modern antiretroviral treatments, the changes in the Cho signal may be too small to be detectable.

Myo-inositol measured at 3.54 ppm serves as an indicator of glial cells (eg, astrocytes). As a sugar (pentasaccharide) that is found in normal brain tissue, the majority of the spectroscopically visible, free, intracellular mI serves as one of the most important organic osmolytes. ^22,26 During the periods of osmotic stress, the osmotic balance is preserved by regulating mI transport across the plasma membrane. ²¹ Furthermore, mI is a key precursor of membrane phospholipids such as phosphoinositides. Thus, mI is involved in both cell membrane and myelin-sheet turnover. Increased membrane turnover, as well as damage to myelin, can result in increased free mI. On the other hand, mI-containing phosphoinositides are also an integral part of several signal transduction pathways. ²⁷ However, as neurotransmitters, they are only present at nanomolar concentrations; so, presumably, they are not part of the mI detected here. High levels of mI were found in astrocytes and related to in vitro glial cell activation. Several diseases characterized by astrogliosis and demyelination, such as Alzheimer disease and multiple sclerosis, respectively, show increased mI and mI/Cr ratios in proton nuclear magnetic resonance (¹H-NMR) spectroscopy. An increased mI concentration was also detected in other metabolic and inflammatory diseases of WM. Because astrocytic proliferation and demyelination are common for all these diseases, the role of mI in astrogliosis and cell volume maintenance in reactive astrocytes has been discussed and confirmed previously. ²²

Myelin is a substantial component of the WM. Myelin is formed by oligodendroglia and encloses nerve cell axons. It functions as an electrical insulator for nerve cells, assisting in saltatory information transfer and fast-thought processing. Thus, it is logical that the defects in the myelin wrapping coincide with a slowdown in thought processes. The main clinical results of our study were slowdowns of motor and thought processes in the impaired HIV-positive patients. Assignment into groups 1 and 2 (score of >2) was done according to mental, cognitive, and/or motor slowdown. These clinical findings are supported by the fact that HIV is rarely found in nerve cells but is more often present in astrocytes and microglia. It is possible that these cells are the main targets for HIV in the CNS.

In astrocytomas of patients with a tumor in CNS, Cho, but not mI, has been found to increase. ²⁸ In our study, therefore, it may be difficult to interpret the increase in the mI signal as a sign of the gliotic degradation in the CNS. ^28,29

With respect to the early increase in the mI signal and the decrease in the NAA signal, the results presented here are consistent with some other reports. ^25,26 In those studies, however, correlations with clinical patient data or conventional diagnostic methods were not made.

In 1998, Harrison et al ³⁰ also tried to correlate MRS data with the clinical data, but they were unable to detect mI, which was found to be the most important metabolite here, because they did not use short TEs.

Based on our results, we expect that MRS may be suitable for the early and certain diagnosis of HIV encephalopathy. For the patient, early diagnosis of HIV encephalopathy is decisive because timely and appropriate medication can prevent the advancement of the disease. The importance of preventing ongoing dementias forms the foundation for the future use of available spectroscopy methods, together with modern magnetic resonance tomography scanners, in routine clinical diagnoses of HIV encephalopathy.

As shown here, the extent of the cognitive impairment correlated with the spectroscopic changes. Therefore, based on our data, the mI/Cr ratio in the PWM was the best spectroscopic parameter. The parietal measuring position produced more reliable and stable results with respect to motion artifacts than the frontal measuring position. Based on the described physiological roles of mI, as an osmotic modulator and an indicator of cell membrane collapse, we believe that we have identified an early parameter of cerebral changes within the context of HIV. The early diagnosis of an existent HIV encephalopathy has important implications in providing an early therapy for the patients, due to which the degeneration of existing brain tissue can be prevented. Nevertheless, our results should be further confirmed by long-term studies.

From the clinical point of view, the patient can only be helped effectively with an early and safe diagnosis of HAD. ¹⁷ Because of the robust effectiveness of modern ARTs, there are more long-term survivors of HIV infections. ³¹ Therefore, it is possible that more patients will have HIV-associated cognitive impairments. These patients should be diagnosed and treated appropriately. Because of existing HIV infections and other life circumstances, many HIV-positive patients are depressive and their cognitive impairments are often not recognized because of the progression of additional depression. ³² Therefore, the treating physician is in need of a methodology to help identify patients who may have HAD. According to this study, modern MRS might potentially help the treating physician to identify and adequately treat the correct patients who demonstrate changes in brain structure. Because of the increasing number of HIV-positive patients ⁵ in developing countries, ³³ these patients are often treated by physicians with specialties other than neurology or psychiatry, such as physicians of general practice and internal medicine, dermatologists, and venereologists. In particular, for these physicians, an objective method would be beneficial. The MRS has the potential to help diagnose and identify HIV-associated cognitive impairments in the future. Also, because of a large intersubject variability of different educational levels of the HIV-positive patients, an additional new form of dementia might be difficult to diagnose. ³⁴

Limitations of the Study and ¹H-NMR Spectroscopy at the Moment

The number of patients investigated in this study (N = 14) has been limited and so are the conclusions drawn from it. Furthermore, at the moment, ¹H-NMR spectroscopy is still a very expensive method and can therefore currently not be the method of choice for monitoring HIV therapy.

Especially, MRS is not readily available in developing countries and also in areas where HIV is most prevalent at the moment. In addition, one would need significant MRS expertise to process the data from the machine. Therefore, at the moment, the clinician still needs to augment with clinical skills, rather than use an expensive and resource-intensive machine. However, this might change in the future.

Conclusion

Many neurologically/psychiatrically inexperienced physicians have difficulties in diagnosing a HAD. Therefore, the use of MRS to distinguish HAD from a purely depressive disorder would be beneficial. Cognitive impairment in a patient that is possibly the result of HIV infection (similar to HAD) could be better diagnosed if MRS were available. Therefore, we believe that examining both the MRS and the conventional diagnostics of many HIV-positive patients would be beneficial. With only a small additional amount of time, valuable information can be acquired for the diagnosis of a cognitive impairment in the patient, and based on these data, the patient can receive a superior treatment. The MRS could be helpful for neurologically/psychiatrically inexperienced physicians.