Introduction

Human immunodeficiency virus (HIV) is a member of the Lentivirus genus, part of the Retroviridae family and has neuroinvasive, neurotropic and neurovirulent properties [1, 2]. HIV infection leads to a wide range of clinical features because it may involve almost all systems including the nervous system and the optic nerve [3–5]. The examination of visual evoked potentials (VEPs) provides information about functional changes of the visual pathway to the brain visual cortex. Depending on the visual stimulation used, various subsystems of the visual pathway can be activated. The pattern reversal of a black/white checkerboard activates the primary visual area V1, which is reflected by a dominant positive P100 peak (P-VEPs) [6, 7]. The motion-onset visual evoked potentials (M-VEP) recordable via the activation of the magnocellular system and the dorsal stream of the visual pathway are characterized by the motion-onset-specific negative peak N160 in the extra-striatal temporooccipital and parietal cortices [8]. Axonal pathology in the optic nerve can be recognized by VEPs independently of overt clinical symptoms or detectable morphological changes [9, 10].

Before the introduction of combined antiretroviral therapy (cART), numerous studies reported neuro-ophthalmological and electrophysiological abnormalities, even in the early stages of infection. During the 1990s, prolonged latencies and reduced amplitudes of the P-VEPs and reduced amplitudes of the pattern electroretinogram (PERG) suggested retinal and postretinal visual pathway dysfunction in neurologically asymptomatic HIV-seropositive patients [11–17].

The advent of cART nearly two decades ago resulted in a profound decline in morbidity and mortality and raised the hope for an eradication of CNS complications [18]. The multifocal electroretinogram (mfERG) findings suggested early diffuse dysfunction of the inner retina resulted from severe HIV disease even in the cART era [19]. Surprisingly, relatively few P-VEP studies have reexamined the effect of HIV on CNS function since the widespread use of cART [20]. To our knowledge, no studies have used M-VEPs for the testing of visual motion processing in HIV-seropositive patients, neither before nor during the cART era. Therefore, the goal of the current study was to explore visual function in virally suppressed HIV patients undergoing cART by using pattern-reversal and motion-onset VEPs.

Methods

Patients

Between June 2013 and June 2015, 20 HIV+ patients who had visited the Department of Infectious Diseases (Faculty Hospital in Hradec Kralove in the Czech Republic) were enrolled into the electrophysiological study. Fourteen men (3 heterosexuals and 11 homosexuals) and 6 heterosexual women with serum antibodies to HIV as determined by ELISA and confirmed by Western blot were included. The demographic and clinical characteristics of the included patients are shown in Table 1. Their visual acuity is specified in Fig. 1. All patients had ≥230 × 106 CD4 cells/L of blood at the time of enrollment. The duration of HIV infection was estimated considering the first positive HIV antibody test. It was found to be 0.1–20.7 years (median 3.5 years) at the time of inclusion in the study. The mean age of patients at inclusion was 38 years (2 patients were ≤25 years and 3 subjects were >45 years). The HIV disease staging classification according to the Centers for Disease Control and Prevention (CDC) was determined in all patients (A1–C3) [21]. The CDC categorization of HIV/AIDS is based on the lowest documented CD4 cell count as well as previously diagnosed HIV-related conditions. The International AIDS Society—USA Panel advises the start of treatment in patients with symptomatic HIV disease and patients with CD4 cell counts below 350 × 106 CD4 cells/L of blood or viral load above 50,000–100,000 copies/mL of blood [22]. At inclusion, 6 patients had not undergone any treatment for HIV. Fourteen HIV patients had been taking antiretroviral medication for at least 2 months before participation in the study (median 4.4 years). All of these patients had a combination of different groups of antiretroviral medicines into one complete regimen (protease inhibitor + ritonavir, nucleoside/nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors or integrase inhibitor). The Montreal Cognitive Assessment (MoCA) was used as a screening instrument for a cognitive function evaluation. A score of ≤25 was considered to represent cognitive impairment [23].

Table 1 Demographic and clinical characteristics of the pursued patients
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Fig. 1
figure 1

Monocular pattern-reversal VEPs (40′ and 20′) are plotted in four columns. The subject responses are organized in rows and sorted by age so that the eldest subject is in the last row. The subject numbers correspond to those of Table 1. The green lines indicate the upper borders (M + 2.5 SD) of the latency norm (Table 2) with respect to the age of each subject. Pathological responses are plotted by red lines. Corresponding visual acuity obtained by the Landolt C test is listed in the right-most columns of the figure. Abbreviations are consistent with those of Table 2

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Visual evoked potentials (VEPs)

The VEPs were examined in the Electrophysiological laboratory at Charles University in Prague—Faculty of Medicine in Hradec Kralove. Two types of visual stimuli were used to elicit VEPs (pattern-reversal and motion-onset stimuli) with the aim of testing the function of the two fundamental subsystems of the visual pathway and their cortical projections.

In the pattern-reversal stimulation (P-VEP), a black and white checkerboard (contrast 96% according to Michelson) with check sizes of 40′ and 20′ was reversed at a frequency of 2/s [24].

Motion-onset stimuli (M-VEP) consisted of low contrast (10%) gray concentric circles that randomly expanded/contracted (radial motion). We recorded three variants of M-VEP responses to the onset of radial motion: (1) a stimulus comprising 37° × 28° of the central visual field (FF); (2) a stimulus field outside the central 20° (M20°); and (3) the central 8° of the visual field (C8°). All motion-onset stimuli had the same timing: 200 ms of motion followed by a 1-second interstimulus interval (stationary pattern) [8, 25].

The stimuli were presented on a 21” computer monitor (Vision Master Pro 510, Iiyama, Japan) subtending 37° × 28° of the visual field at a 0.6 m viewing distance. The monitor was driven using a Visual Stimulus Generator 2/5 (CRS Ltd., UK) at a 105-Hz vertical refresh frequency. A mean luminance of 17 cd/m2 was used for all stimuli. The correct fixation of the stimulus field center was monitored via a near-infrared camera. Forty single sweeps were averaged because this number of sweeps in our recording conditions with the Faraday cage provides a good signal-to-noise ratio. Monocular VEPs were recorded from six unipolar derivations using the right ear lobe as a reference. Four derivations from the midline (Oz, Pz, Cz and Fz) and two lateral derivations (Ol and Or that were 5 cm to the left and right from the Oz position, respectively) were used to cover areas with maximum amplitudes for both the P-VEPs and M-VEPs. Latencies and interpeak amplitudes of the P100 peak were always evaluated in the Oz derivation. Because of interindividual differences in the topographies of the M-VEPs [26], latencies and interpeak amplitudes of the N160 motion-onset-specific peak were read from one of the lateral occipital or Pz leads. We selected the lead with the maximum interpeak amplitude.

Individual VEP parameters were compared to laboratory normal values. Norms were derived from a control group of 70 healthy subjects with significant age-dependent VEPs changes [27, 28]. VEP latencies that exceeded the value of the mean + 2.5 standard deviation were considered as pathologically prolonged. The upper limits of the latency norms are shown in Table 2.

Table 2 Upper borders (M + 2.5 SD) of normal VEP latencies from the control group of 70 normal subjects, with respect to their age-dependent changes
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Interocular latency differences over 10 ms in the P100 peak and 20 ms in the N160 peak were also recognized as pathological.

Systematic interocular differences of the VEP amplitudes exceeding approximately 3 µV appeared to be significant for the detection of unilateral pathology. VEP amplitudes in single eyes were not used for pathological detection due to known significant interindividual variability [29].

Statistical analyses

The data were statistically processed with R software version 3.2 [30], using the “nortest”, “psych” and “ggplot2” packages. Because the Anderson–Darling test had rejected the hypothesis of normal data distribution, the Spearman nonparametric test of the relationship between clinical and electrophysiological parameters was used. A relationship was considered to be statistically significant when the probability level (P) was below an alpha level of 0.05. The alpha level was corrected for multiple comparisons by a Holm–Bonferroni adjustment. The Kruskal–Wallis nonparametric test was used for assessment of differences among three groups defined by the clinical severity scale (CDC) in pursued electrophysiological or clinical parameters.

Results

The 20 cooperating patients were divided into two groups: (1) neurologically symptomatic subjects (4/20) and (2) neurologically asymptomatic patients (16/20).

Neurologically symptomatic subjects with cognitive impairment—2 cases

A heterosexual couple with no visual or other neurological complaints and an unclear duration of infection before the incidental detection of HIV scored in the MoCA 19/30 points (female) and 22/30 points (male), below the normal limit (26/30 points). Woman No. 19 displayed prolonged M-VEP latencies in both eyes and normal P-VEP reactions. Man No. 20 had prolonged R20′ P-VEP latencies in both eyes and a M20° M-VEP latency delay in one eye (for details, see Figs. 1, 2).

Fig. 2
figure 2

Monocular motion-onset VEPs are plotted in six columns. The derivation with the shortest latency and the largest amplitude was selected from the lateral occipital and parietal leads. Figure arrangement is similar to Fig. 1. Abbreviations are consistent with those of Table 2

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Neurologically symptomatic subjects with neuro-ophthalmological manifestation—2 cases

Patient No. 1, who was adherent to the cART therapy inconsistently, complained of a few weeks of blurred vision in the right eye. VEP examination showed absent reactions to P-VEP and small M-VEP reactions with prolonged latencies in the right eye (M-VEP latencies: FF 234 ms, M20° 244 ms, C8° no reaction; interpeak amplitudes: FF 4.5 µV, M20° 5.1 µV, C8° 3.5 µV). The left eye showed completely normal reactions for left eye stimulation (P-VEP latencies R40′ 106 ms, R20′ 118 ms; interpeak amplitudes: R40′ 14.8 µV, R20′ 17.1 µV and M-VEP latencies FF 140 ms, M20° 144 ms, C8° 158 ms; interpeak amplitudes: FF 16.1 µV, M20° 13 µV, C8° 6.3 µV). These findings, together with clinical symptoms, suggested the presence of acute optic neuritis. The brain magnetic resonance imaging suspicion of cytomegalovirus (CMV) encephalopathy with right eye retinitis and facial nerve palsy on the right side was confirmed by positive polymerase chain reaction in the cerebrospinal fluid (CSF-PCR) for CMV-DNA.

Patient No. 15 suffered from a headache, diffuse arthralgia and fever. Lyme borreliosis coinfection was detected with a high titer IgG in the blood (ELISA, TestLine, Brno, Czech Republic) and symptoms resolving within 14 days after oral doxycycline therapy. Three months later, the patient without any visual difficulties had an electrophysiological picture of monocular optic nerve involvement with prolonged latencies in the P-VEP and M-VEPs of the right eye with a VEP recovery to normal within 1 year.

Neurologically asymptomatic patients—16 cases

Eleven patients with duration of HIV infection between 0.1 and 20.7 years had VEP reactions within our laboratory limits.

In 5 cases, the VEP evaluation revealed a binocular pathology. Patients with an HIV infection for 2.9–8 years without visual or neurological difficulties displayed prolonged latencies or slurred reactions in M-VEPs in all motion variants. Abnormal M-VEPs were accompanied by P-VEP latency delay in only case No. 17.

Effect of clinical severity (CDC score)

Three subgroups divided by the CDC clinical criteria were defined as follows: group a: A1 + A2 + A3, 10 patients, group b: B1 + B2 + B3, 6 patients, and group c: C1 + C2 + C3, 4 patients. The Kruskal–Wallis nonparametric ANOVA did not show any significant differences among the sensory parameters, the cognitive test and the biochemical markers—see Fig. 3.

Fig. 3
figure 3

The boxplots illustrate medians, interquartile ranges, minimum and maximum CD4 cell counts (top) and HIV copies in plasma (bottom) among three groups defined by CDC clinical score (group a = A1 + A2 + A3, 10 patients, group b = B1 + B2 + B3, 6 patients, and group c = C1 + C2 + C3, 4 patients). The nonparametric Kruskal–Wallis test did not show any intergroup differences as well as differences among other followed parameters (not depicted here)

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Relationship among sensory/cognitive and clinical parameters

The Spearman rank correlation test of relationships between disease duration, CD4, HIV copies in plasma, MoCA and electrophysiological parameters also did not show any significant results. The independence between VEPs latencies of R40′ or expanding FF and CD4 cell counts at the time of inclusion in the study is demonstrated in Fig. 4. The graphs show that visual pathology was not related to the CD4 cell count, which is coded as the size of dots.

Fig. 4
figure 4

A weak relationship between visual pathology and the CD4 cell count is illustrated by the P100 latency of the pattern-reversal R40′ VEP in the upper plot and the N160 latency of the motion-onset FF VEP in the bottom plot. Every point position represents a subject’s left and right eye response, and the size of the point corresponds to the CD4 cell count. Normal VEP findings are located in gray areas, and normal CD4 counts are green colored

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Discussion

Before the widespread use of cART, electrophysiological studies reported optic nerve dysfunction in 1–45% of neurologically asymptomatic HIV-seropositive patients as detected by P-VEPs [11–16].

In the present study, P-VEP pathology was observed in 3 neurologically symptomatic subjects, specifically in two with Lyme and cytomegalovirus infection, as well as in one patient with cognitive impairment. Only 1 of 16 neurologically asymptomatic patients with bilaterally prolonged M-VEP latencies in all motion variants showed concurrent P-VEP R40′ latency delay in one eye.

The P-VEP is regarded as a sensitive marker for the detection of optic neuritis. In HIV+ patients, most cases of optic neuritis are associated with opportunistic infections [3]. However, the advent of cART has reduced the incidence and severity of opportunistic infections in the HIV-infected population [31]. Our study supports the proposition that the initiation of cART therapy for HIV patients has substantially reduced the risk of impaired optic nerve function due to opportunistic infections. In the present study, not a single patient in our group treated with cART had clinical signs or VEP evidence of acute optic neuritis or residual effects after the neuritis except for one patient with Lyme borreliosis coinfection who had a VEP picture of monocular optic nerve involvement with recovery within 1 year after antibiotic treatment. The only patient refusing cART treatment with 280 × 106 CD4 cells/L and a viral load of 310,000 copies/mL suffered from optic neuritis as a manifestation of opportunistic infections. The VEP picture of optic neuritis in our study is characterized by the monocular influence of the P-VEP with the finding of the current involvement of M-VEP. Such a VEP image is also found in patients with optic neuritis in multiple sclerosis or neuroborreliosis [32, 33].

In our study, 4 out of 16 neurologically asymptomatic virally suppressed HIV patients had a prolongation of the motion-onset-specific N160 peak in both eyes with normal P-VEP findings. In 3 out of these 4 patients, the ERG, PERG, mfERG and funduscopic examination was additionally performed and no evidence of the retina damage was found. The fourth patient with the same VEP results was not examined because he committed suicide in the meantime.

Although VEP examination is not specific enough to localize impairment, we may speculate that the likelihood of cortical involvement is greater than that of an optic nerve lesion because the latter is unlikely to cause binocular M-VEP pathology and spared ERGs and P-VEPs as we observed in our patients. Moreover, the N160 peak is generated in the extra-striatal temporooccipital and parietal cortices [34], and a loss of cortical neurons in these regions has been found in asymptomatic HIV-positive subjects in the early stages of infection [35] and in many neuroimaging studies [36–40]. Therefore, we hypothesize that motion-onset VEPs may help to detect early central nervous system involvement in HIV-positive subjects.

The viewpoint of broader cortical impairment is further underlined by two patients with cognitive deficits and pathological VEPs. However, we cannot speculate about the role of HIV infection in their cognitive deficits because their pre-disease states were unknown.

Only one patient suffering from CMV encephalopathy with optic neuritis had a sudden decrease in vision in one eye. No visual complaints in 19 of twenty patients indicate that despite normal visual acuity, the subclinical dysfunction of the visual system may be present in HIV+ patients. In such a situation, VEPs could serve as a sensitive insight into disease development as they have been demonstrated in optic nerve pathology in patients without overt clinical symptoms [10, 12, 41].

For a deeper understanding of the functional changes in the visual system and CNS in HIV patients on cART, we plan to observe the long-term development of VEP findings in a follow-up study.

Conclusion

In the current study, nine of 20 HIV patients displayed electrophysiological abnormalities. We found abnormal P-VEP in 20% of HIV patients and M-VEP pathology in 45% of HIV+ subjects. Impairment of the P-VEP was predominantly observed in neurologically symptomatic subjects.

Prolongation of the M-VEP was detected in patients with neuro-ophthalmological symptoms as well as in neurologically asymptomatic subjects. The functional changes of the visual system detectable with the use of motion-onset VEPs appeared separately from the changes detected by the standard pattern-reversal VEP examination. The similar results of higher motion-onset VEP sensitivity in various CNS pathologies have been previously reported by our laboratory [8, 42, 43].

Limitations

The small group of the patients examined and the wide range of the duration of HIV infection (low homogeneity of the group) certainly represent shortcomings of this study. Unfortunately, the real duration of infection was known for only a small number of patients. Furthermore, the duration of infection was derived from the first positive HIV antibody test in most subjects.

Another shortcoming of the study is the lack of information about the functional changes in the retina and intraorbital portion of the optic nerve in most patients. Optic nerve fundus investigation was not included in this study.