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Wednesday, December 29, 2010

NeuroAIDS: Characteristics and Diagnosis of the Neurological Complications of AIDS

TUGAS PENGAYAAN NEUROLOGI












NeuroAIDS: Characteristics and Diagnosis of the Neurological Complications of AIDS








Dewi Sri Wulandari
0610710031







LABORATORIUM ILMU PENYAKIT SARAF
FAKULTAS KEDOKTERAN
UNVERSITAS BRAWIJAYA
RSU DR. SAIFUL ANWAR
MALANG
2010




NeuroAIDS: Karakteristik dan Diagnosis Komplikasi Neurologis dari AIDS

Abstrak
Komplikasi neurologis dari AIDS (neuroAIDS) termasuk gangguan neurokognitif dan dementia karena AIDS (HIV-associated dementia = HAD). HAD merupakan komplikasi infeksi HIV pda CNS yang paling berpengaruh dan merugikan. Walaupun telah ditemukan perkembangan dalam pengetahuan tentang gejala klinis, patogenesis, dan aspek neurobiologI HAD, hal ini masih merupakan tantangan daam ilmu pengetahuan dan terapi. Pemahaman tentang mekanisme neuroinvasi HIV, proliferasi CNS, dan patogenesis HAD memberikan dasar interpretasi ciri-ciri diagnosis HAD dan bentuk yang lebih ringan, HIV-associated minor cognitive/motor disorder (MCMD). Strategi diagnosis terbaru memiliki keterbatasan yang signifikan, namun diharapkan penggunaan biomarker dapat membantu pasien dan klinisi dalam mempredisikan onset proses penyakit dan mengevaluasi efek terapi baru.

Pendahuluan
HIV adalah retrovirus patogen yang dapat memicu disfungsi neurologi dan neurodegenerasi. Perubahan ini dapat bermanifestasi klinis dengan berbagai cara dalam konteks suatu sindrom yang disebut neuroAIDS yang meliputi, namun tidak terbatas pada, berbagai derajat gangguan neurokognitif, mielopati vakuolar, dan neuropati perifer. HAD mungkin merupakan sindrom neuroAIDS dan neurodegenerasi karena HIV yang paling merugikan. HAD ditandai dengan penurunan progresif fungsi esensial CNS, seperti kognisi, kontrol metabolik, dan perilaku. Pada stadium lanjut HAD, pasien mengalami kesulitan dalam melaksanakan perintah motorik dan intelektual dasar. Pasien yang mengalami gejala yang lebih ringan yang disebut MCMD dapat menjadi pelupa dan tidak dapat menyelesaikan regimen ARV yang ketat dibandingkan dengan pasien normal. Bahkan pasien dengan gangguan neurofisiologi subklinis yang tidak mengalami gangguan fungsional memiliki resiko mortalitas yang lebih tinggi daripada pengidap HIV dengan kognitif yang normal.

Tabel 1. Stadium klinis AIDS dementia complex dalam Memorial Sloan Kettering Cancer Center Scale
Stadium Nama Alternatif Deskripsi
0
0.5

1


2



3



4 Normal
Subclinical

Mild


Moderate



Severe



End stage Pasien dengan fungsi mental dan motorik yang normal.
Pasien tanpa atau dengan gejala minimal atau ekuivokal tanpa gangguan melaksanakaan pekerjaan atau aktivitas sehari-hari. Gejala ringan (reflek snout, gerakan mata dan ekstremitas menurun) dapat muncul. Gait dan kekuatan dalam batas normal.
Pasien dapat melakukan semua aktivitas sehari-hari kecuali aspek yang lebih rumit, namun dengan tanda-tanda ekuivokal atau gangguan fungsional, intelektual atau motorik. Pasien dapat berjalan tanpa bantuan.
Pasien dapat melakukan aktivitas dasar perawatan diri, namun tidak dapat melakukan atau mempertahankan aspek kehidupan yang lebih rumit. Pasien masih dapat berpindah tempat, namun mungkin memerlukan bantuan 1 orang.
Pasien dengan ketidakmampuan intelektual nyata (tidak dapat mengikuti informasi, mempertahankan percakapan kompleks, respon menjadi lambat) dan ketidakmampuan motorik (tidak dapat berjalan tanpa bantuan, memerlukan walker atau bantuan orang lain, biasanya dengan gerakan lengan yang lambat dan janggal.
Pasien hampir dalam kondisi vegetatif dengan komprehensi intelektual, sosial, dan respon yang rudimenter. Terjadi mutisme absolut dengan paraparesis atau paraplegi dan inkontinensia ganda.

Laporan tentang peningkatan skor tes neurofisiologi pada pasien yang diterapi dengan ARV dan penurunan insiden komplikasi CNS pada pasien yang diterapi dengan HAART memberikan harapan bahwa HAD dan MCMD dapat diatasi. Namun, HAART tampaknya hanya mempengaruhi, bukan menghilangkan, efek HIV pada CNS. Hal ini mungkin disebabkan kurang adekuatnya penetrasi berbagai regimen HAART ke CNS, kegagalan HAART untuk mengeliminasi reservoir virus di CNS (misalnya mikroglia parenkim yang berumur panjang), persistensi provirus dalam CNS, atau kegagalan HAART untuk mengurangi inflamasi pada CNS setelah siklus inflamasi yang terus-menerus terpicu.

Namun, beberapa karakteristik klinis HAD/MCMD telah berubah baru-baru ini, terutama karena HAART. Sebelum era HAART, mayoritas pasien yang mengalami HAD pada tahap lanjut AIDS, akan mengalami perburukan progresif sampai meninggal, biasanya dalam 6 bulan. Dengan adanya HAART, waktu interval rata-rata antara diagnosis HAD dan kematian meningkat dari 6 menjadi 44 bulan. Sebagai hasil bertambahnay harapan hidup pasien HIV yang diterapi dengan HAART, peningkatan durasi penyakit dan sindrom yang lebih ringan, prevalensi keseluruhan gangguan neurokognitif meningkat walaupun insiden kasus baru HAD menurun. Apabila neurodegenerasi karena HIV tidak dihilangkan, sebagian besar pasien HIV+ dengan infeksi sistemik yang tidak terkontrol dengan baik dapat mengalami gangguan neurologis. Hal ini selain merugikan pasien secara personal, juga membebankan biaya yang besar bagi masyarakat.

Kami telah mempelajari patogenesis HAD selama 2 dekade terakhir, namun pemahaman lengkap tentang bagaimana HIV mempengaruhi otak masih belum jelas. Salah satu masalah yang dihadapi peneliti dan klinisi adalah pencarian penanda yang menunjukkan perkembangan HAD/MCMD di stadium awal proses degeneratif. Yang dapat digunakan oleh klinisi untuk menegakkan diagnosa penyakit pada stadium awal dan memonitor efek terapi. Walaupun berbagai tes telah disarankan, diagnosa HAD/MCMD hanya berdasarkan presentasi gejala klinis dan adanya confounding/kondisi komorbid dieksklusikan sebagai penyebab utama gangguan.

Review ini merangkum metode diagnosa terbaru untuk HAD/MCMD dan potensi penggunaan biomarker yang dapat membantu peneliti dan klinisi untuk memprediksi onset proses penyakit dan mengevaluasi efek terapi baru. Sebagai dasar pemahaman ciri-ciri diagnosa HAD/MCMD, kami juga membahas mekanisme neuroinvasi HIV, proliferasi CNS dan patogenesis HAD. Kami akan mendiskusikan keuntungan dan kerugian penanda diagnosa terbaru untuk MCMD/HAD.

1. Gejala klinis neuroinvasi HIV dan HAD
Berdasarkan adanya abnormalitas CSF pada pasien infeksi HIV dan penemuan kembali Simian immunodeficiency virus (SIV) dari CSF pada hewan coba, tampak bahwa neuroinvasi HIV terjadi pada awal setelah infeksi dan merupakan ciri infeksi HIV yang terdapat di mana-mana. Namun, gejala klinis yang berkaitan dengan neuroinvasi biasanya ringan dan self-limited, serta tidak diperhatikan oleh pasien. Sebagian besar pasien HIV+ hanya mengalami sedikit atau tidak ada keluhan berkaitan dengan CNS sampai tahap akhir penyakit. Beberapa pasien mengalami meningitis aseptik HIV selama infeksi primer dan serokonversi. Pada kasus yang jarang, hal ini dapat berkembang menjadi HIV ensefalitis yang mematikan. Munculnya gejala neurologis pada infeksi primer berhubungan dengan tingginya RNA HIV di CSF dan prognosis yang buruk. Pemeriksaan CSF pada penyakit otak, walaupun tidak merefleksikan otak secara langsung, merupakan prosedur standar karena merupakan salah satu metode terbaik untuk mendeteksi infeksi penyerta seperti neurosifilis.

HAD adalah diagnosis klinis yang mengacu pada kumpulan tanda dan gejala yang mempengaruhi kognisi, fungsi motoris central, dan tingkah laku pada pasien HIV+ yang tidak mengalami infeksi CNS sekunder atau penjelasan lain dari sindrom ini. Pasien HAD dapat bervariasi jenis dan derajat presentasi gejalanya. Skenario yang umum antara lain:
• Pasien yang datang sendiri dengan keluhan hilangnya memori dan kurangnya koordinasi dengan onset baru.
• Pasien yang dibawa oleh keluarganya karena perilaku aneh atau memburuknya hygiene yang baru terjadi.
• Pasien yang menunjukkan gejala psikiatrik dengan ciri depresi, psikosis, mania, atau agitasi; pasien ini kemudian dirujuk ke neurologi karena gejalanya tidak menghilang setelah terapi psikiatrik.

Baru-baru ini, delirium telah dilaporkan dalam konteks HAD. Kasus lainnya masih melaporkan tidak adanya gejala, namun perburukan kognitif subklinis dapat dideteksi dengan tes neuropsikologis yang dilakukan untuk penelitian atau work up diagnosis klinis, atau penemuan atrofi korteks dan white matter yang terdeteksi pada neuroimaging otak.

Pada awal epidemi AIDS, banyak pasien secara khas mempresentasikan gejala sistemik dan neurologis, yang sekarang disebut AIDS full-blown dan HAD, pada tahap akhir penyakit. Dengan banyaknya data yang terkumpul, menjadi jelas bahwa terdapat spectrum terlibatnya CNS pada pasien infeksi HIV, berkisar dari gangguan kognitif yang sangat ringan sampai sangat parah. Juga dicatat bahwa pada sebagian besar bentuk dementia, pasien tidak mengalami sindrom de novo secara keseluruhan. Di samping itu, gejala klinis muncul setelah habisnya sisa fungsi kognitif otak setelah berbulan-bulan sampai bertahun-tahun. Penurunan kognitif yang berkaitan dengan HIV terjadi secara bertahap dengan kecepatan yang berbeda-beda.

Nosologi gangguan kognitif berkaitan dengan HIV telah berkembang sejak 20 tahun yang lalu. Salah satu skema yang paling awal muncul dan paling banyak digunakan adalah Memorial Sloan Kettering Cancer Center Scale (MSKCC). Pada skema ini, peneliti menggunakan istilah AIDS Dementia Complex (ADC) untuk menggambarkan ciri kognitif, motorik, dan tingkah laku yang tampak pada sindrom ini dan derajat keparahan gejala dari yang paling ringan sampai paling berat. Namun, dengan menggunakan skema ini, secara teoritis mungkin untuk mendiagnosa ADC pada pasien dengan disfungsi motorik dan perilaku, tetapi bukan kognitif. Klasifikasi ini diikuti oleh klasifikasi American Academy of Neurology (AAN), yang memperkenalkan istilah MCMD untuk pasien yang mengalami gangguan ringan yang belum memenuhi kriteria dementia. Banyak peneliti sekarang mengkonsep bahwa pasien yang pertama mengalami subsindrom atau fase subklinis gangguan neurokognitif berkaitan dengan HIV (disebut juga asymptomatic neurocognitive impairment = ANI), seperti yang didiskusikan dalam nosologi baru yang disarankan, ditandai dengan penurunan neuropsikologi tanpa adanya perubahan pada aktivitas sehari-hari pasien (misalnya pasien asimtomatik secara klinis dan defisit neurokognitif hanya ditemukan melalui pemeriksaan neurofisiologis formal). Lebih jauh, sistem klasifikasi ini berfokus pada stadium keparahan gangguan neurokognitif. Sistem ini mengeksklusikan ciri motorik dan perilaku yang digunakan dalam sistem MSKCC dan AAN untuk mengidentifikasi stadium penyakit. Karena alasan ini, istilah HIV-associated mild neurocognitive disorder (MND) digunakan sebagai pengganti MCMD. Sebagai kesimpulan, klasifikasi penyakit neurologis HIV merupakan bidang yang masih berkembang dan ketiga sistem klasifikasi di atas masih digunakan sampai sekarang. Dalam penelitian ini, gangguan kognitif pada pasien ANI secara kasar ekuivalen dengan stadium 0.5 pada skala MSKCC; MND/MCMD dengan stadium 1 MSKCC; dan HAD dengan stadium 2-4 MSKCC.

Pasien dengan MCMD mengalami sedikit penurunan fungsi kognitif dan motorik sentral (tidak memenuhi diagnosis dementia), namun masih dapat bekerja dan merawat diri. Pasien HAD mengalami permasalahan nyata dalam aktivitas sehari-hari dan bergantung pada orang lain dalam melaksanakan tugas kompleks; kadang-kadang pasien dapat menjadi sama sekali tidak mampu karena gangguan kognitifnya. Terdapat kontroversi apakah perubahan motorik sentral dan perilaku, yang termasuk dalam definisi AIDS dementia complex, harus termasuk dalam kriteria staging atau harus dilakukan staging secara terpisah.

Beberapa pasien infeksi HIV juga mengalami kejang, mioklonis, koreoatetosis, atau gangguan gerak hiperkinetik lainnya. Walaupun gejala ini dapat muncul pada HAD/MCMD, hal ini tidak umum dan memerlukan pemeriksaan lebih lanjut untuk infeksi oportunistik CNS atau tumor. Mielopati vakuolar, sindrom medulla spinalis yang mirip dengan disfungsi medulla spinalis akibat kekurangan vitamin B12, dapat terjadi bersamaan dengan HAD/MCMD, namun tidak termasuk bagian dari sindrom dementia.

2. Metode diagnosis terbaru
2.1. Pemeriksaan neurofisiologis
HIV Dementia Score (HDS) adalah alat skiring yang digunakan untuk memeriksa fungsi kognitif pasien suspek HAD. Skor kurang dari sama dengan 10, dari skor maksimal 16, dianggap abnormal. HDS merupakan metode yang sederhana dan mudah dilakukan, serta cocok untuk pasien yang tidak mampu melakukan tes kognitif lengkap. Namun, HDS tidak spesifik untuk HAD, dan kurang bermanfaat untuk pasien dengan tingkat pendidikan rendah atau buta huruf.

Pada stadium awal MCMD, jarang ditemukan kelainan pada pemeriksaan neurologis; apabila ada kelainan yang ditemukan ringan, seperti nistagmus, tremor ringan, sedikit penurunan koordinasi, hiperrefleksi simetris, atau frontal release signs. Pada tahap lanjut penyakit seperti HAD, banyak pasien mengalami penurunan frekuensi berkedip, ekspresi wajah datar, mirip dengan pasien Parkinson. Defisit motorik yang berkaitan dengan HAD dapat mempengaruhi sistem piramidal, menyebabkan kelemahan UMN, meningkatnya tonus, spastisitas, hiperrefleksi, dan melambatnya gait, serta sistem ekstrapiramidal, menyebabkan berkurangnya koordinasi, ataksia, gangguan motorik halus, dan tremor. Nistagmus dapat terjadi dan diukur secara kuantitatif. Mungkin penemuan motorik yang dapat diamati adalah berkurangnya kecepatan gerakan volunter secara umum. Secara keseluruhan, tanda-tanda neurologis seperti abnormalitas motorik ekstrapiramidal (misalnya tremor) dan frontal release signs (misalnya refleks snout), secara statistik lebih banyak terjadi pada pasien HIV positif daripada kontrol HIV negatif. Walaupun tanda-tanda ini dapat berkaitan dengan gangguan neurokognitif, namun bukan termasuk diagnosa HAD. Sebagai tambahan, terdapat beberapa bukti bahwa tanda-tanda ekstrapiramidal lebih jarang terjadi pada wanita HIV positif daripada pria HIV positif pada stadium penyakit yang sama. Oleh karena itu, tanda-tanda tersebut bukan merupakan marker HAD yang reliabel dan dapat rancu dengan Parkinson pada pasien yang lebih tua.

Manifestasi neurofisiologi klasik HAD/MCMD terutama menunjukkan disfungsi sirkuit frontal dan subkortikal otak, sesuai dengan penemuan berbagai penelitian neuropatologi dan neuroimaging. Karena alasan itu, HAD kadang-kadang dimasukkan dalam dementia subkorteks. Perlambatan psikomotor yang menunjukkan defisit pada pengolahan informasi sentral, tampaknya merupakan ciri yang paling awal dan paling umum dari infeksi HIV pada CNS. Perlambatan psikomotor dapat diukur dengan tes neurofisiologis, misalnya waktu reaksi. Sebagai perbandingan dengan kontrol HIV negatif, pasien infeksi HIV lebih lambat, bahkan ketika mempertahankan akurasi respon. Perubahan ini dapat tampak sebelum pasien mengalami gejala neurologis. Beberapa peneliti berpendapat bahwa perlambatan psikomotor merupakan prediktor terbaik HAD dan ensefalitis HIV, walaupun hal ini tidak diterima secara universal. Juga terdapat pertanyaan apakah hasil pemeriksaan neurofisiologis berkaitan dengan faktor biologis.

Baru-baru ini, Shiramizu, dkk., melakukan studi eksplorasi untuk memeriksa baseline HIV DNA dan hubungannya dengan defisit neurofisiologis individu. HIV DNA plasma berhubungan dengan baseline neuropsikologis yang tidak tergantung umur, etnis, IQ, kadar HIV-1 RNA plasma; namun, HIV DNA plasma tidak menggambarkan perubahan yang akan datang pada defisit neuropsikologis. Peneliti menyimpulkan bahwa HIV DNA dan defisit neuropsikologis bervariasi setiap waktu.

Pemeriksaan neurologis merupakan alat dengan teknologi rendah yang relatif murah untuk memeriksa pasien infeksi HIV dengan suspek MCMD/HAD. Pemeriksaan ini sangat membantu dalam mendeteksi lesi otak fokal dan abnormalitas neurologis seperti afasia, yang menyingkirkan diagnosa HAD/MCMD tanpa komplikasi, pasa infeksi oportunistik, stroke, atau penyebab lain gangguan neurokognitif. Namun, interpretasi pemeriksaan neurologis memerlukan pengetahuan medis, psikiatri, neuroanatomi, dan neurofisiologi. Pemeriksa perlu dilatih untuk melakukan dan menginterpretasi pemeriksaan dengan cara yang seragam karena hal ini sangat penting dalam studi klinis. Abnormalitas yang ditemukan bukan merupakan dignosa HAD/MCMD karena tanda-tanda neurologis yang ditemukan tidak spesifik pada penyakit ini.

Pemeriksaan neurologis tidak memprediksikan pasien yang akan mengalami HAD/MCMD. Tidak terdapat bukti bahwa pemeriksaan tersebut berguna dalam mengkuantifikasi respon terhadap HAART atau terapi lain, mungkin karena pemeriksaan ini sulit untuk dikuantifikasi secara keseluruhan. Sebagai kesimpulan, pemeriksaan neurologis merupakan alat yang paling penting untuk diagnosa klinis karena membantu menemukan proses penyakit lain, namun merupakan marker prediktif yang relatif lemah untuk perkembangan HAD/MCMD.

2.2. Neuroimaging
Neuroimaging CNS berperan penting dalam diagnosa dan eksplorasi patogenesis HAD dan memungkinkan klinisi menyingkirkan infeksi oportunistik dan keganasan. Modalitas neuroimaging yang paling sering digunakan CT scan dan MRI otak. CT scan kepala digunakan untuk menemukan kalsifikasi basal ganglia pada pasien infeksi HIV-1. Pada MRI otak, HAD bermanifes sebagai area hiperintens pada gambar T2-weighted pada substansia alba subkortikal. Lesi substansia alba hiperintens T2-weighted ini biasanya tanpa efek massa atau penyerapan kontras, dan berkembang dari abnormalitas pada area kecil menjadi abnormalitas difus pada proses penyakit lanjut. Di samping itu, juga tampak atrofi regio kaudatus selektif, dan atrofi umum otak nonspesifik dengan hilangnya substansia grisea dan pembesaran sulci kortikal dan sistem ventrikel.

Proton MR-spectroscopy (MRS) telah digunakan sebagai alat riset untuk mengeksplor patogenesis HAD, untuk membedakan keterlibatan substansia alba dan grisea, dan untuk mendeteksi keterlibatan CNS pada psien HIV positif yang masih asimtomatis secara neurologis. MRS serial juga digunakan untuk memeriksa efek HAART pada gangguan kognitif yang berkaitan dengan AIDS. Diffusion Tensor Imaging (DTI), yang mengukur difusi dalam substansia alba otak, merupakan teknik neuroimaging advanced yang digunakan untuk mendeteksi abnormalitas substansia alba pada pasien HIV positif dibandingkan dengan pasien normal. Sebuah studi menunjukkan bahwa pasien dengan elevasi konstanta difusi tertinggi dan penurunan anisotropi terbesar mengalami penyakit HIV tahap lanjut, sementara pasien yang mendapat HAART memiliki viral load terendah, dengan anisotropi dan konstanta difusi normal.

MRI fungsional meliputi kumpulan data fungsional dan anatomis yang menunjukkan perubahan aliran darah dan kadar oksigenasi pada vaskuler lokal dan yang berhubungan dengan aktivitas saraf otak. Menggunakan fMRI, peneliti dapat menghubungkan tugas mental yang dilakukan di unit MRI dengan aktivitas otak. fMRI telah digunakan untuk mempelajari pasien dengan HAD dan mengeksplor dasar patofisiologi gangguan kognitif. Teknik imaging eksperimental lainnya, seperti continuous arterial spin labeled (CASL) MRI, single-photon emission computed tomography (SPECT), dan positron emission tomography (PET), telah digunakan untuk mempelajari patogenesis HAD dan efek terapi antivirus. Sebagai kesimpulan, berbagai teknik neuroimaging digunakan sebagai alat riset untuk mempelajari patogenesis HAD lebih lanjut dan membantu klinisi untuk menyingkirkan patologi intrakranial lainnya yang mirip dengan HAD.

3. Mekanisme neuropatogenesis HIV
Untuk mengidentifikasi dan mengembangkan biomarker klinis yang berguna untuk deteksi awai dan prediksi perkembangan HAD, diperlukan pemahaman mekanisme invasi dan proliferasi HIV pada CNS, serta mekanisme patogen mempromosi HAD.
3.1. Neuroinvasi HIV dan proliferasi CNS
3.1.1. Melalui sawar darah-otak
Segera setelah infeksi sistemik, HIV-1 menembus otak. Beberapa mekanisme diusulkan untuk menjelaskan bagaimana HIV menembur sawar darah otak. Hipotesis Trojan horse, yang merupakan postulat yang diterima secara luas, menyatakan bahwa HIV memasuki otak melalui sel CD4+ yang terinfeksi dan monosit yang melalui sawar darah otak. Sekali sel-sel ini mendapatkan akses ke lingkungan CNS, mereka akan menginfeksi sel mikroglia residen CNS lainnya dan astrosit. Monosit dari sirkulasi perifer berdiferensiasi menjadi makrofag saat memasuki CNS dan berperan penting dalam patogenesis HAD. Di antara reseptor kemokin yang diekspresikan oleh sel manusia, ko-reseptor CCR5 adalah yang paling signifikan untuk masuknya HIV-1 ke monosit/makrofag dan mikroglia. Juga terdapat hipotesis bahwa virus bebas dalam darah menembus otak melalui sel endotel mikrovaskular, yang kemudian diikuti dengan infeksi astrosit.

Sel endotel serebral membentuk tight junction pada sawar darah otak, yang dapat dirusak oleh protein Tat (transactivator of transcription) HIV-1 untuk membantu HIV-1 masuk ke CNS. Telah ditunjukkan bahwa Tat mempengaruhi ekspresi protein tight junction sel endotel serebral claudin-1 dan 5, yang meningkatkan permeabilitas sawar darah otak. Walaupun infeksi langsung sel endotel in vivo oleh HIV-1 tidak diketahui, kerusakan sawar darah otak karena protein HIV (misalnya Tat) tidak ekuivokal. Kerusakan tight junction endotel pada sawar darah otak telah tampak pada sample postmortem basal ganglia dan dalam substansia alba pada pasien ensefalitis HIV, mengkonfirmasi prediksi awal. Kerusakan sawar darah otak yang diukur dengan penyerapan kontras magnetic resonance (MR) berhubungan dengan gangguan neurokognitif.

Makrofag yang berasal dari monosit yang terinfeksi HIV-1 juga mempengaruhi integritas sawar darah otak, yang memfasilitasi migrasi transendotelial leukosit yang terinfeksi HIV dari perifer ke CNS. Ricardo-Dukelow, dkk., meneliti proteomic platform yang mengintegrasikan elektroforesis gel dengan spektrometri sequencing peptide untuk mengetahui efek makrofag yang terinfeksi HIV-1 terhadap pola ekspresi protein sel endotel serebral manusia. Peneliti menemukan upregulasi lebih dari 200 protein oleh sel endotel serebral manusia, yang berperan dalam metabolisme, voltage-gated ion channel, heat shock, transportasi, sitoskeleton, regulator, dan calcium binding protein. Penemuan studi ini divalidasi dengan Western blot analysis. Penulis menyimpulkan bahwa makrofag yang berasal dari monosit yang terinfeksi HIV-1 menimbulkan efek yang signifikan terhadap proteom sel endotel serebral manusia dan oleh karena itu mempengaruhi integritas struktural dan fungsional sawar darah otak, dan berpotensi membuka sawar darah otak terhadap influks HIV dan neurotoksin sistemik.

3.1.2. Reservoir HIV pada CNS
Sekali HIV memperoleh akses ke CNS, makrofag otak dan sel mikroglia berperan sebagai reservoir utama HIV-1 dan alat replikasi HIV. Bukti yang diperoleh dari hibridisasi in situ dan studi imunohistokimia menunjukkan bahwa virus memiliki predileksi pada makrofag di daerah perivaskular, terutama sel CD4+. Sebagai tambahan, sel yang berasal dari neuroektoderm, termasuk astrosit, juga dicurigai sebagai reservoir infeksi HIV, dan studi in vitro dan postmortem mendukung hipotesis ini. Infeksi pada sel ini tidak tergantung CD4+ dan reseptor permukaan tambahan telah disarankan sebagai alternatif.

Pada CNS, infeksi kronik astrosit dan mikroglia telah ditegakkan. Astrosit mengekspresikan CXCR4 dan mungkin ko-reseptor HIV-1 lainnya seperti CCR5. Replikasi virus pada astrosit secara umum terbatas, walaupun astrosit dapat menghasilkan virus dalam kondisi tertentu, seperti priming dengan interferon (IFN)-γ dan dapat berperan sebagai reservoir virus. Sebagian besar studi mengindikasikan bahwa neuron dan oligodendroglia tidak terinfeksi secara produktif, namun dapat menghasilkan protein virus.

3.2. Neurodegenerasi dan patogenesis HAD
Dari observasi yang telah dilakukan, secara umum disetujui bahwa HIV tidak merusak CNS melalui mekanisme seperti lisis sel yang diinduksi virus dan hilangnya neuron yang terjadi tanpa adanya infeksi produktif pada neuron oleh HIV-1. Konsep sekarang adalah bahwa infeksi HIV, terutama pada makrofag menyebabkan pelepasan molekul terlarut oleh sel yang terinfeksi virus dan sel yang tidak terinfeksi virus namun teraktivasi.

Disfungsi neuron dan kematian, disebabkan oleh efek neurotoksin terlarut yang dilepaskan oleh makrofag yang terinfeksi virus dan mikroglia, merupakan komponen yang signifikan dalam patogenesis HAD. Situasi menjadi cukup kompleks karena beberapa mediator seperti TNF-, menghasilkan efek toksik dan protektif. Banyak dari neurotoksin ini dapat mengaktifkan nuclear factor kappa B (NF-kB) yang secara luas diekspresikan oleh neuron dan sel nonneuron, serta memiliki efek neuroprotektif kuat setelah terpapar protein HIV-1.

3.2.1. Peranan protein virus
Dalam CNS, makrofag terinfeksi dan sel mikroglia melepaskan protein virus, termasuk protein selubung HIV gp120 dan protein regulator HIV Tat, yang mengganggu homeostasis kalsium neuron. Protein neurotoksik ini dapat menginduksi apoptosis kultur neuron sehingga menyebabkan neuron rentan terhadap eksitotoksisitas dan stres oksidatif. Beberapa protein tersebut dapat merangsang proses degeneratif yang menyebabkan disfungsi neurologis dan kematian neuron, serta merusak integritas sawar darah otak.

Sejumlah protein virus HIV-1 berperan dalam proses neurodegeneratif yang dirangsang oleh ensefalitis HIV. Ensefalitis HIV merupakan proses neuropatologis difus yang cenderung lebih parah pada substansia alba dan subkorteks cerebri, terutama basal ganglia. Secara neuropatologis, ensefalitis HIV ditandai dengan adanya multinucleated giant cell (yang mungkin berasal dari fusi mikroglia atau makrofag yang diinduksi HIV), sel inflamasi kronik perivaskuler, dan nodul mikroglia. Beberapa studi telah menunjukkan adanya asam nukleat spesifik HIV-1, DNA virus, dan protein Nef HIV-1 pada astrosit dalam konteks ensefalitis HIV. Hef adalah determinan utama virulensi lentivirus serta penting dalam replikasi efektif dan viral load yang tinggi. Nef memodulasi sejumlah jalur sinyal dan dipertimbangkan sebagai faktor progresi AIDS; protein ini juga berperan dalam patogenesis HAD.

Baru-baru ini, Lehmann, dkk., mengeksplor lebih lanjut mekanisme interaksi Nef dengan astrosit, yang mengekspresian Nef sekali terinfeksi HIV-1. Dengan protein array, peneliti menemukan bahwa monosit menunjukkan kemotaksis ketika terpapar supernatan kultur sel. Astrosit U25IM6 yang mengekspresikan Nef dan CC chemokine ligan-2/monocyte chemotactic protein-1 (CCL2/MCP1) telah diidentifikasi sebagai faktor yang memediasi fenomena ini. Sebagai tambahan, peneliti menunjukkan bahwa ekspresi CCL2/MCP1 yang diinduksi Nef tergantung pada myristoylation moiety of Nef dan kalmodulin fungsional. Dipercaya bahwa ekspresi CCL2/MCP1 yang diinduksi Nef oleh astrosit memfasilitasi migrasi transendotel monosit yang terinfeksi HIV-1 ke CNS.

Protein virus lain yang berimplikasi pada neurodegenerasi adalah HIV viral regulatory protein (Vpr), protein yang terdapat dalam jumlah banyak pada isolate HIV-1, HIV-2, dan SIV. Vpr adalah protein inti 14 kDa, 96 asam amino yang diisolasi dari CSF pasien dengan HAD dan mungkin terlibat dalam kematian neuron. Vpr menginduksi apoptosis neuron melalui aktivasi kaspase dan telah dilaporkan meningkatkan permeabilitas mitokondria, yang menyebabkan pelepasan sitokrom c dan apoptosis. Untuk mendukung peran Vpr dalam apoptosis sel, sebuah studi menunjukkan bahwa Vpr menarget HAX-1 (protein mitokondria antiapoptosis) dan dislokasi protein dari posisi normal, yang menyebabkan instabilitas membrane mitokondria dan kematian sel.

Untuk menyelidiki lebih jauh peran Vpr dalam perkembangan HAD, Wheeler,dkk., memeriksa keberadaan Vpr dalam jaringan otak, menggunakan sample jaringan dari pasien ensefalitis HIV. Peneliti menunjukkan bahwa Vpr terdapat dalam jumlah terdeteksi pada basal ganglia dan korteks frontal otak yang terinfeksi HIV. Pemberian imunofluoresen ganda menunjukkan bahwa Vpr hanya terdapat di makrofag dan neuron, tidak pada astrosit. Keberadaan Vpr intraneuron mendukung lebih jauh pendapat bahwa protein regulator ini mungin berperan dalam kerusakan neuron pada HAD.

Terpisah dari interaksi destruktif antara Vpr dan sistem mitokondria dalam patogenesis HAD, mikroorganel ini berpartisipasi dalam patogenesis HAD melalui mekanisme lainnya. Menggunakan model kultur neuron korteks tikus, Norman, dkk., meneliti efek nonletal Tat pada sel ini. Peneliti menemukan bahwa Tat merangsang penurunan cepat PH mitokondria internal dan inhibisi kompleks IV rantai transport electron. Pemaparan neuron korteks tikus terhadap Tat juga berkaitan dengan penurunan NAD(P)H dan konsentrasi Ca dalam mitokondria neuron, dengan penurunan hebat dalam aktivitas respirasi mitokondria. Peneliti menyimpulkan bahwa abnorrmalitas respirasi mitokondria setelah paparan terhadap Tat, berkontribusi dalam patogenesis HAD dengan menghambat sinyal neuron.

3.2.2. Peranan Molekul Inflamasi
Selain protein virus, banyak zat proinflamasi dilepaskan oleh makrofag terinfeksi yang teraktivasi dan sel glia, yang memulai reaksi inflamasi dalam CNS. Makrofag yang berasal dari monosit di sistem imun perifer mengikuti entry CNS, merupakan sel imunokompeten residen di CNS. Makrofag yang berasal dari monosit menjadi teraktivasi sebagai respon terhadap berbagai rangsangan. Dipercaya bahwa aktivitas besar-besaran makrofag dan mikroglia oleh HIV-1 protein dan release mediator proinflamasi oleh sel terinfeksi, merangsang perkembangan HAD. Mediator proinflamasi dihasilkan oleh makrofag teraktivasi dan sel mikroglia, termasuk tapi tidak terbatas pada sitokin, enzim seperti metalloproteinase dan kemokin seperti stromal derived factor (SDF) 1 (protein neurotoksik yang menyebabkan neurodegenarasi). Produk ini juga tampak merekrut sel teraktivasi tambahan ke dalam proses inflamasi. Terdapat interaksi antara berbagai molekul toksik yang menimbulkan proses yang dapat berlanjut sendiri tanpa adanya virus.

Dua sitokin utama, TNFα dan interleukin (IL)-1β, mengalami upregulasi pada infeksi HIV-1 dan menginduksi kematian neuron. Efek neurotoksik sitokin proinflamasi tersebut dimediasi melalui sejumlah mekanisme, termasuk peningkatan permeabilitas sawar darah otak, yang memudahkan influks monosit yang terinfeksi HIV, dan meningkatkan efek neurotoksik sitokin proinflamasi lainnya. Sebagai tambahan, TNFα dan IL-1β menginduksi overstimulasi reseptor N-methyl-D-aspartate (NMDA), yang menyebabkan peningkatan kalsium neuron ke tingkat letal. TNFα juga menghambat uptake glutamate sehingga meningkatkan konsentrasi glutamate di sinaps ke level yang berpotensi letal. Peningkatan produksi dan pelepasan TNFα dari monosit yang terinfeksi HIV dimediasi oleh ligan CD40 yang sinergis dengan Tat.

Sitokin neurotoksik yang ketiga yang berperan utama dalam perkembangan HAD adalah IFNγ. IFNγ mengaktivasi jalur JAK/STAT, yang merupakan kunci regulator inflamasi dan sinyal apoptosis. Dengan keberadaan protein HIV gp120 dan Tat, kapasitas IFNγ mangaktivasi JAK/STAT meningkat pesat. Dalam serangkaian penelitian pada neuron dan tikus, Giunta, dkk., menunjukkan bahwa epigallocatechin-3-gallate (EGCG) dalam teh hijau menurunkan neurotoksisitas kerusakan neuron oleh IFNγ yang dirangsang gp120 dan Tat.

3.2.3. Efek obat
Nucleoside reverse transcriptase inhibitors (NRTIs) juga berperan dalam menyebabkan HAD. Studi terbaru oleh Opii, dkk., meneliti peranan NRTI 2′,3′-dideoxycytidine (ddC; zalcitabine) dalam neuropatologi HAD. Peneliti menginkubasi isolat sinaptosom dan mitokondria selama 6 jam dengan CSF yang mengandung zalcitabine dan mendeteksi peningkatan signifikan stress oksidatif pada zalcitabine 40nM yang diukur melalui protein karbonil dan 3-nitrotirosin. Juga ditemukan bahwa zalcitabine 40nM dapat menyebabkan release sitokrom c, menurunkan kadar protein antiapoptosis, dan meningkatkan kadar protein proapoptosis, sehingga meningkatkan potensi apoptosis. Penemuan ini mendukung peranan obat-obatan NRTI, terutama zalcitabin, dalam promosi HAD.

4. Biomarker Laboratorium untuk HAD/MCMD
Viral load plasma dan limfosit CD4+ sering digunakan sebagai marker prognostik respon pengobatan HIV. Namun, CD4+ lebih berguna dalam memeriksa respon sistemik, daripada CNS, terhadap HAART. Viral load plasma juga kurang membantu dalam diagnosis dan prognosis HAD/MCMD, karena tidak merefleksikan viral load CNS. Berkembangnya pemahaman tentang patobiologi HAD mengarahkan studi marker laboratorium yang diharapkan dapat membantu diagnosis HAD/MCMD dan memeriksa respon terapi.

4.1. Imunofenotip selular
Kompartemen perivaskular merupakan target utama masuknya HIV ke CNS. Kapsid dan protein kapsul virus terdeteksi di sel endotel otak dan makrofag perivaskular. Namun, keberadaan makrofag dalam CNS merupakan prediktor yang lebih baik daripada deteksi materi virus berkaitan dengan perburukan neurologis. Peneliti telah mengidentifikasi sejumlah monosit aktif di darah perifer, yang mendahului atau bersamaan dengan perkembangan HAD/MCMD. Sel invasif jaringan ini ditandai dengan aktivasi imunofenotip CD14+/CD16+ dan CD14+/CD19+. Monosit ini bermigrasi melalui sawar darah otak dan menyerang reservoir makrofag perivaskular yang terinfeksi HIV. Makrofag perivaskular yang terinfeksi HIV tidak mengalami apoptosis dan menjadi reservoir HIV jangka panjang di CNS, walaupun tampaknya produksi virus aktif di CNS tetap rendah saat onset AIDS. Sejumlah sel monosit darah perifer juga mengembangkan profil proteomik unik yang tampak sebelum terjadinya gangguan kognitif.

4.2. Marker CSF
Sejak awal riset neuroAIDS, peneliti telah menggunakan CSF untuk mempelajari neuropatogenesis HAD/MCMD. Abnormalitas CSF terjadi segera setelah infeksi HIV dan CSF telah digunakan untuk mengidentifikasi pola temporal yang berbeda selama perjalan penyekit HIV tanpa komplikasi. Sebagai tambahan, analisis CSF merupakan alat diagnosis penting pada neurosifilis, infeksi oportunistik, dan tumor pada pasien AIDS.

Terdapat banyak keuntungan pemeriksaan CSF. CSF dapat diambil melalui lumbal punksi yang merupakan prosedur yang relatif aman dan mudah dibandingkan dengan biopsi otak. Lumbal punksi dapat diulang secara serial sebelum dan sesudah terapi. Proses lumbal punksi dan pemeriksaan CSF lebih murah dibandingkan dengan biopsi otak dan neuroimaging. Secara umum, marker CSF banyak berkaitan dengan penurunan kognitif dibandingkan dengan marker yang sama di darah.

Sebelum adanya HAART, sejumlah parameter CSF dipelajari sebagai marker HAD/MCMD. Terdapat beberapa penemuan nonspesifik seperti pleositosis CSF (peningkatan leukosit), peningkatan sintesis IgG dalam CSF (pengukuran aktivasi imun dan produksi antibodi intratekal), keberadaan oligoclonal band unik pada CSF (masing-masing menunjukkan klon intratekal unik dari sel B), dan bocornya albumin CSF (pengukuran integritas dan permeabilitas sawar darah otak). Pada penelitian tersebut, pengukuran inflamasi pada sawar darah otak dilakukan dengan beberapa metode. Isoelectric focusing (IEF) CSF dan spesimen serum cognate in vivo diperiksa dengan imunohistokimia untuk mendeteksi IgG pada CSF yang tidak terdapat di serum cognate. Hal ini disebabkan adanya klon sel plasma di otak yang tidak terdapat di darah. Klon sel plasma tersebut menghasilkan IgG di otak yang disekresikan ke CSF. Persamaan Tourtellotte mengoreksi difusi protein darah ke otak melalui sawar darah otak, menghitung nilai dinamis produksi IgG bersih dalam otak, mengukur sintesis IgG dalam sawar darah otak.

Kepentingan pemeriksaan CSF didukung hasil penelitian bahwa CSF pasien HIV mengandung satu atau beberapa faktor neurotoksik, seperti peningkatan kadar asam kuinolinik, neurotoksin eksitatori yang merupakan metabolit triptofan dan berperan sebagai agonis reseptor NMDA. Asam kuinolinik dalam jumlah kecil terdapat di CNS dalam kondisi normal, namun konsentrasinya meningkat ketika terjadi peningkatan IFNγ atau sitokin lainnya, termasuk sitokin proinflamasi, pergeseran metabolisme ke jalur kinurenin. Asam kuinolinik di CSF meningkat paralel dengan gangguan neurologis dan menurun setelah terapi antretrovirus. Kadar tinggi berkaitan dengan atrofi selektif bagian otak yang rentan terhadap injuri eksitotoksik. Namun, seperti sebagian besar marker CSF berkaitan dengan aktivasi makrofag dan mikroglia, peningkatan asam kuinolinik tidak spesifik pada HAD. Pemeriksaannya sulit dan mahal, memerlukan spektrometri massa.

Subunit neurofilamen rantai ringan adalah marker sensitif CSF untuk injuri akson dan neurodegenerasi yang meningkat pada pasien dengan AIDS dementia complex. Namun, marker nonspesifik ini juga meningkat pada proses neurodegeneratif lainnya.

4.2.1. Marker Virus
Viral load CSF dilaporkan berkaitan dengan gangguan kognitif, dan umumnya diperiksa melalui deteksi dan pengukuran kuantitatif antigen p24 HIV pada CSF, sebelum perkembangan teknik amplifikasi gen untuk mengukur replikasi aktif virus. Metode lain yang digunakan untuk deteksi HIV termasuk kultur virologis CSF, pengukuran kuantitatif DNA provirus pada sel CSF melalui PCR, dan pengukuran kuantitatif RNA HIV viral load dengan PCR atau teknik amplifikasi asam nukleat berdasarkan urutannya. Kuantifikasi metabolit monoamin di CSF tidak berkaitan dengan stadium HAD.

4.2.2. Marker protein yang dihasilkan host
Beberapa faktor larut CSF yang terlibat dalam inflamasi telah dipelajari sebagai biomarker potensial HAD, misalnya CCL2/MCP1, mediator inflamasi yang meningkat pada HAD, ensefalitis HIV, dan ensefalitis SIV. Namun, CCL2/MCP1 tidak spesifik pada HIV, tetapi juga meningkat pada infeksi sitomegalovirus pada CNS, penyakit yang mirip dengan HAD/MCMD.

β2-Microglobulin, komponen protein molekul MHC kelas I pada permukaan sel dan indeks turnover sel, telah dipelajari sebagai marker progresi penyakit HIV sistemik dan marker HAD/MCMD pada CSF. Dibandingkan dengan marker nonspesifik imunoaktivasi lainnya, β2-Microglobulin cenderung lebih tinggi pada pasien HIV+ yang akan mengalami gangguan neurokognitif, dan lebih tinggi pada pasien HIV yang mengalami gangguan neurokognitif dibandingkan dengan pasien HIV yang tidak terganggu. Marker ini paralel dengan keparahan disfungsi neurokognitif. Kadar β2-Microglobulin menurun setelah terapi antiretroviral efektif. Walaupun dapat diukur dengan relatif murah dengan enzyme immunoassay (EIA), β2-microglobulin mengalami masalah yang sama dengan marker lainnya, yaitu kurang spesifik untuk infeksi HIV, oleh karena itu, infeksi oportunistik harus disingkirkan dulu. Beberapa laporan menyatakan β2-microglobulin bukan marker yang sensitif untuk perburukan neurologis yang berlajut pada pasien yang diterapi HAART.

Neopterin, produk antara metabolisme guanosin trifosfat yang dihasilkan oleh makrofag aktif, merupakan marker lain imunoaktivasi nonspesifik yang selalu dipelajari berhubungan dengan β2-microglobulin. Marker ini dapat diukur dengan teknik laboratorium standar (misalnya EIA dan high-performance liquid chromatography [HPLC]). Seperti β2-microglobulin, peningkatan neopterin CSF bermanfaat untuk memprediksi pasien yang beresiko terkena HAD; namun tidak seperti β2-microglobulin, neopterin tidak kembali ke kadar normal pada CSF pasien yang diterapi HAART. Tidak jelas apakah neopterin relevan secara klinis dan mencerminkan aktivasi imun intratekal yang berlangsung terus. Menariknya, kadar neopterin yang tinggi berkaitan dengan tingginya reactive oxygen species, dan rendahnya antioksidan. Hal ini menunjukkan bahwa neopterin merupakan indikator stres oksidatif, kondisi yang menyertai penyakit neurodegeneratif, termasuk HAD/MCMD. Seperti marker inflamasi lainnya, neopterin kurang spesifik untuk HAD/MCMD dan juga meningkat pada penyakit infeksi dan inflamasi lainnya.

TNFα berkaitan dengan apoptosis pada beberapa kondisi neurodegeneratif, termasuk HAD/MCMD. Glass, dkk., melaporkan hubungan antara peningkatan mRNA TNFα pada jaringan otak postmortem dan riwayat HAD. Peneliti lain menemukan bahwa kadar TNFα CSF pasien HIV yang mengalami dementia lebih tinggi dibandingkan dengan pasien HIV yang tidak mengalami dementia. Pada laporan kasus oleh Gendelman, dkk., pasien HIV-1 seropositif, antiretroviral-naïve, mengalami penurunan kadar TNFα CSF setelah 12 minggu terapi dengan HAART. Namun TNFα CSF jarang terdeteksi pada studi lainnya, dan TNFα tidak spesifik untuk HAD sehingga kegunaannya sebagai biomarker terbatas. Namun deteksi monocyte chemoattractant protein-1 (MCP-1) dan macrophage colony-stimulating factor (M-CSF) di plasma berkaitan dengan waktu terjadinya HAD.

Marker lainnya adalah Fas dan Fas ligan (FasL). Fas adalah reseptor protein yang dapat diekspresikan pada permukaan membrane, atau dalam bentuk terlarut dan bersirkulasi (sFas). FasL juga dikenal sebagai Apo-1 atau CD95, dan merupakan bagian dari superfamili reseptor TNFα. Cross-linking Fas dengan FasL atau activating antibody menginduksi apoptosis sel yang mengekspresikan fas; untuk alasan ini Fas disebut juga death receptor. Ketika sistem apoptosis ini gagal berfungsi, dapat berkembang menjadi keganasan. Peningkatan FasL berkaitan dengan imunoaktivasi kronis. Kadar sFas plasma meningkat pada infeksi HIV dan menurn secara signifikan setelah HAART, namun tidak kembali ke kadar normal fisiologis. sFas dapat diukur pada cairan tubuh dengan teknik EIA. Kadar sFas CSF meningkat pada HAD dibandingkan dengan control yang seronegatif, dan kadarnya menurun dengan terapi antiretroviral.

4.3. Keuntungan dan kerugian marker laboratorium
Terdapat beberapa permasalahan berkaitan dengan penggunaan marker CSF pada studi HAD/MCMD. Pertama, terdapat pertanyaan apakah virus yang terdapat di CSF mewakili virus yang terdapat di parenkim otak. Terdapat banyak sumber HIV di CSF, termasuk produksi lokal dari sel monosit CSF, virus yang melalui sawar darah otak dari sirkulasi sistemik (sawar yang terdapapt pada tight junction sel kuboid di pleksus koroideus), dan virus yang terlepas dari parenkim otak dan mengalir melalui pleksus koroideus ke CSF.

Kedua, terdapat beberapa variasi regional pada neuropatologi dan virology kualitatif dan kuantitatif pada otak. Namun variasi ini tidak jelas karena komposisi CSF yang relative homogen. Efek HAART pada HIV bervariasi antara kompartemen plasma, CSF, dan parenkim otak, selama dan setelah terapi tiap kompartemen dapat merespon dengan kecepatan yang berbeda. Lebih jauh, virus dapat tetap tinggal dalam otak setelah dibersihkan dari CSF.

Akhirnya, sebagian besar marker ini tidak spesifik untuk HIV dan hanya bermanfaat apabila infeksi lain telah disingkirkan.

Sebagai kesimpulan, marker laboratorium darah dan CSF relative murah diperoleh dan dapat diikuti secara serial, namun bukan indikator yang baik untuk perubahan regional yang diinduksi HIV atau kerusakan otak. Banyak dari marker ini tidak spesifik terhadap penyebab aktivasi makrofag dan inflamasi, serta tidak dapat digunakan sebagai indikator keparahan HAD/MCMD; oleh karena itu, marker-marker tersebut perlu digunakan dengan hati-hati.
Table 2 merangkum beberapa tes yang berkaitan dengan diagnosis neuroAIDS.

5. Perubahan metode diagnosis pada era HAART
Sebelum adanya HAART, HAD secara khas terjadi pada pasien dengan CD4+ rendah (di bawah 200 sel/mm3). Namun, dari kelompok ini hanya sedikit yang mengalami HAD/MCMD. Post HAART, rata-rata CD4+ pasien HAD meningkat. Pasien HIV dapat mengalami dementia pada jumalh CD4+ yang tinggi karena walaupun sistem imun mereka telah membaik secara parsial dengan terapi, mereka tidak normal secara imunologis. Di samping itu, mereka terpapar neuroinflamasi selama jangka waktu lama. Jumlah CD4+ ini perlu diobservasi ketat dan merupakan faktor resiko yang signifikan untuk HAD/MCMD.

Sebelum HAART, sebagian besar studi yang mengukur RNA viral load CSF menemukan korelasi positif dengan gangguan neurokognitif. Namun korelasi ini tampaknya tidak lagi dipertahankan. Sevigny, dkk., menyatakan bahwa replikasi virus yang terus-menerus tidak diperlukan untuk berkembangnya dementia. Sekali HIV merangsang produksi abnormal mediator toksik oleh sistem imun, disfungsi neurologis akan terjadi tidak tergantung pada jumlah virus. Observasi ini mengarahkan beberapa peneliti bahwa mungkin saat in terdapat berbagai bentuk HAD. Subtipe yang diusulkan termasuk HAD kronis, di mana kompliansi parsial dan resistensi virus menyebabkan progresi disfungsi neurokognitif yang lambat dan bervariasi, dan HAD inaktif dengan kontrol virus yang baik namun terdapat deficit menetap karena kerusakan yang terjadi sebelum terapi efektif. Sebagai tambahan, peningkatan harapan hidup pasien HIV menyebabkan kondisi komorbid lebih potensial menimbulkan efek pada fungsi kognitif, memungkinkan adanya bentuk hybrid dementia atau HAD transform.

Tidak dapat diasumsikan bahwa marker darah dan CSF berkorelasi dengan penurunan neurokognitif sebelumnya. Kadar β2-microglobulin dan neopterin CSF kurang berarti dalam mengikuti progresi penyakit CNS pada pasien yang diterapi HAART. Apakah perubahan pada HAD mencerminkan kenampakan histologi otak saat kematian belum dapat ditentukan, namun telah dilakukan observasi. Insiden ensealitis HIV dan hubungannya dengan HAD sangat bervariasi. Seperti yang disebutkan sebelumnya, bahkan sebelum HAART, 50% pasien HAD tidak mengalami ensefalitis HIV. Penemuan ini berlawanan dengan studi terakhir bahwa 95% subjek dengan gangguan neurokognitif mengalami ensefalitis HIV, dan sebagai tambahan, 45% subjek dengan neurokognitif normal juga mengalami ensefalitis HIV. Terdapat beberapa faktor yang mempengaruhi perbedaan ini, seperti perbedaan interobserver karena subjektivitas pemeriksaan neuropatologi dan neuropsikiatri. Namun kompleksitas patogenesis HAD juga berperan. Paparan terhadap HAART yang bervariasi potensial meningkatkan kompleksitas secara dramatis.

Terdapat bukti bahwa bahkan pasien yang diterapi secara konsisten mengalami inflamasi dalam tingkat rendah. Dalam sebuah studi kadar neopterin, β2-microglobulin, dan RNA HIV CSF pada pasien asimtomatis yang diterapi HAART diikuti, RNA HIV menjadi tidak terdeteksi dan kadar β2-microglobulin menjadi normal. Namun, bahkan setelah 2 tahun terapi, kadar neopterin, walaupun menurun relatif dibandingkan dengan sebelum terapi, tetap meningkat. Apakah hal ini disebabkan replikasi virus yang tidak terdeteksi belum diketahui. Namun hal ini mengarah pada kemungkinan terjadinya HAD kronik yang lebih ringan namun signifikan. Hal ini mungkin tidak mencerminkan ensefalitis HIV tetapi patologi lainnya.

Anthony, dkk menggunakan sistem analisis gambar untuk mengkuantifikasi mikroglia aktif di otak pasien HIV yang diterapi HAART dan gangguan neurokognitif dibandingkan dengan pre HAART pada pasien ensefalitis HIV dan pasien HIV yang tidak mengalami ensefalitis. Menariknya, hipokampus dan basal ganglia pasien dengan kognitif normal yang diterapi HAART memiliki mikroglia/makrofag yang sama banyak dengan pasien pre HAART dengan atau tanpa ensefalitis HIV, yang diukur dengan CD68+, marker upregulasi dan aktivasi sel mikroglia yang menandai adanya neuroinflamasi yang sedang berlangsung. Hal ini juga menunjukkan ketidakmampuan HAART untuk mengeradikasi efek HIV pada CNS dan menimbulkan pertanyaan tentang implikasi klinis dari fakta ini.

6. Arah diagnosis HAD di masa depan
Batasan-batasan pada tes neuropsikologi menimbulkan permasalahan dalam pemeriksaan pasien HIV dalam taraf internasional. Pengembangan tes yang valid untuk minoritas dan populasi imigran di berbagai negara memberikan tantangan bagi neuropsikologis dan klinisi.

Sebagai tambahan, gejala infeksi HIV saat ini lebih kompleks dibandingkan dengan sebelum adanya HAART. Tampaknya korelasi antara beberapa biomarker dan disfungsi neurokognitif telah melemah. Mekanisme patogenik yang menyebabkan HAD telah berubah dan berbeda antar individu tergantung paparan terhadap protokol antiretrovirus. Namun, interval antara infeksi awal pada CNS dan perkembangan HAD menunjukkan kesempatan intervensi terapi. Seperti yang disebutkan sebelumnya, HAART tidak mencegah atau menyembuhkan HAD. Sekali bergerak, proses patologis yang dirangsang dengan keberadaan virus dan disregulasi imun menjadi berjalan sendiri dan sulit untuk dimodulasi. Karena intervensi profilaksis mahal dan memiliki efek samping, diharapkan adanya marker untuk mengidentifikasi pasien yang beresiko terkena HAD.

Produk yang dihasilkan oleh makrofag teraktivasi, astrosit, dan sel apoptotik sebagai respon terhadap infeksi HIV pada CNS berkontribusi dalam disfungsi otak dan merusak neuron secara langsung. Produk ini dapat menginduksi perubahan gen dan profil ekspresi proteinnya sendiri, yang merusak fungsinya. Saat ini studi tentang faktor terlarut spesifik untuk mendeteksi HIV/AIDS sedang dilakukan. Penelitian ini dilakukan bersamaan dengan teknik imaging in vivo seperti CT dan MRI, serta diarahkan untuk tujuan penting analisis in vivo penyakit otak menggunakan gabungan teknik molekuler dan imaging.

Perkembangan teknis dalam metodologi, seperti imunofluorensen flow cytometri, mengembangkan kuantifikasi dan karakterisasi populasi sel terinfeksi HIV (misalnya CD4+, CD4+CCR5+, CD4+CD25+FoxP3+ regulatory T cells) yang menginfiltrasi sawar darah otak dan menginvasi parenkim otak. Eksplorasi ekspresi marker aktivasi pada populasi sel CD8+ yang terlibat dalam sitolisis sel yang terinfeksi virus penting untuk dilanjutkan. Imunohistokimia dan flow cytometri, serta teknologi molekular, virologis, dan ekspresi gen, merupakan metode laboratorium yang berguna untuk menganalisis sel yang terlibat dalam HAD. Pendekatan baru untuk menginterpretasi data ini, seperti analisis fraksi, akan menjadi standar karakterisasi yang lebih baik tentang gangguan patologis pada bentuk, struktur, dan fungsi populasi sel CNS pada HAD. Riset molekular selanjutnya bertujuan menemukan gangguan DNA koding dan nonkoding pada sel teinfeksi HIV dan sel apoptosis pad parenkim otak pasien HAD. Aktivasi dan patologi seluler berhubungan dengan proses pada bagian nonkoding dan koding genom manusia, dengan konsekuensi signifikan untuk fungsi dan survival sel.

Studi tentang biomarker laboratorium menunjukkan bahwa walaupun CSF bukan merupakan akses yang tepat ke otak, CSF lebih mencerminkan HIV dan aktivitas imunitas daripada darah perifer. Namun, tidak mudah untuk melakukan lumbal punksi dan pemeriksaan CSF berulang pada pasien asimtomatis di luar studi. Idealnya, marker HAD/MCMD seharusnya diperoleh dengan cara yang kurang invasif, misalnya melalui tes tambahan yang dilakukan pada darah yang diambil saat perawatan pasien. Lebih-lebih, pelacakan marker terisolasi pada proses kompleks seperti inflamasi mungkin terbatas karena proses ini mencerminkan jalur umum yang digunakan oleh berbagai proses. Marker CSF/darah yang penting lainnya dapat dihasilkan dari informasi rangkaian asam nukleat spesifik pada virus, serta polimorfisme nukleotida tunggal spesifik dan perubahan genetik yang meningkatkan kerentanan terhadap HAD. Sebagai tambahan, analisis ekspresi gen pada tingkat transkripsi dan translasi menunjukkan metode diagnosis mendatang, dengan pentingnya penggunaan bioinfromatika. Akhirnya, definisi klinis dan nosologi HAD serta gangguan neuropsikiatri berkaitan dengan HIV masih dipelajari. Diharapkan perkembangan baru akan berkontribusi dalam menegakkan tes diagnosis molekular pada setting klinis yang stabil.












































Molecular Diagnosis & Therapy:
1 January 2008 - Volume 12 - Issue 1 - pp 25-43
Neurological Disorders

NeuroAIDS: Characteristics and Diagnosis of the Neurological Complications of AIDS

Minagar, Alireza

Author Information
1 Department of Neurology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana, USA
2 Department of Pathology, USC Keck School of Medicine, Los Angeles, California, USA
3 National Neurological AIDS Bank, Los Angeles, California, USA
4 Departments of Cellular and Molecular Physiology, Louisiana State University Health Sciences Center Shreveport, Shreveport, Louisiana, USA
5 Division of Oral Biology and Medicine, UCLA School of Dentistry, Los Angeles, California, USA
6 Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA
7 Department of Immunology, Tehran University, Medical Sciences, Tehran, Iran
8 Department of Psychiatry and Behavioral Medicine, University of South Florida, College of Medicine, Tampa, Florida, USA
9 Department of Internal Medicine, Division of Infectious Diseases, University of South Florida, College of Medicine, Tampa, Florida, USA

Correspondence: Dr Paul Shapshak, Department of Psychiatry & Behavioral Medicine, University of South Florida, College of Medicine, 3515 East Fletcher Avenue (MDT14), Room 333, Tampa, FL 33613, USA. E-mail: pshapsha@health.usf.edu

Abstract
The neurological complications of AIDS (NeuroAIDS) include neurocognitive impairment and HIV-associated dementia (HAD; also known as AIDS dementia and HIV encephalopathy). HAD is the most significant and devastating central nervous system (CNS) complications associated with HIV infection. Despite recent advances in our knowledge of the clinical features, pathogenesis, and neurobiological aspects of HAD, it remains a formidable scientific and therapeutic challenge. An understanding of the mechanisms of HIV neuroinvasion, CNS proliferation, and HAD pathogenesis provide a basis for the interpretation of the diagnostic features of HAD and its milder form, HIV-associated minor cognitive/motor disorder (MCMD). Current diagnostic strategies are associated with significant limitations, but it is hoped that the use of biomarkers may assist researchers and clinicians in predicting the onset of the disease process and in evaluating the effects of new therapies.

Introduction
Human immunodeficiency virus (HIV) is a pathogenic retrovirus capable of triggering neurological dysfunction and neurodegeneration. These changes can be manifested clinically in several ways in the context of one major syndrome known as 'NeuroAIDS', which includes, but is not limited to, varying degrees of neurocognitive impairment, vacuolar myelopathy, and peripheral neuropathy. HIV-associated dementia (HAD) is perhaps the most devastating component of the NeuroAIDS syndrome and HIV-related neurodegeneration. HAD is characterized by a progressive, disabling decline in essential central nervous system (CNS) functions, such as cognition, motor control, and behavior.[1] In the advanced stages of HAD, the patient has difficulty performing even the most basic intellectual or motor tasks (see table I). Less seriously afflicted patients, who manifest a milder form of HIV-associated impairment known as HIV-associated minor cognitive/motor disorder (MCMD), can become forgetful and are less likely to comply with their stringent antiretroviral regimens than unaffected patients.[2] Even patients with subclinical neuropsychological impairment who report no functional problems may be at increased risk for mortality as compared with cognitively normal HIV-infected individuals.[3]


Reports of improved neuropsychological test scores in patients treated with antiretrovirals, along with decreasing numbers of new CNS complications in patients treated with highly active antiretroviral therapy (HAART), have fostered hope that HAD and MCMD could be eliminated.[4] However, HAART appears to have only modified or attenuated, rather than eliminated, the effects of HIV on the CNS.[5,6] This may be due to the inadequate penetration of many HAART regimens into the CNS,[7] the failure of HAART to eliminate CNS viral reservoirs (such as long-lived parenchymal microglia), the persistence of provirus within the CNS,[8] or the failure of HAART to reduce levels of inflammation within the CNS once a self-perpetuating inflammatory cycle is triggered.
Nonetheless, some of the clinical characteristics of HAD/MCMD have changed in recent years, most likely because of HAART. In the pre-HAART era, the majority of patients developed HAD in the advanced stages of AIDS, and proceeded to progressively deteriorate until death, usually within 6 months. With the advent of HAART, the mean time interval between the diagnosis of HAD and death has increased from 6[10-12] to 44 months.[1,13] As a result of the longer lifespan of HAART-treated HIV patients, the increased duration of the disease, as well as an increase in the milder forms of the syndrome, the overall prevalence of neurocognitive impairment has actually increased[14] even though the incidence of new cases of HAD has declined.[4,15] If HIV-associated neurodegeneration is not eliminated, large numbers of HIV-positive patients with otherwise well controlled systemic infection will eventually become disabled through neurological impairment. Apart from the personal tragedy, this would represent a tremendous cost to society.
We have learned much about the pathogenesis of HAD over the past two decades, yet a complete understanding of the mechanisms by which HIV affects the brain remains elusive. One of the most pressing issues facing researchers and clinicians is the search for markers that indicate the development of HAD/MCMD early in the course of the neurodegenerative process, which could then be used by clinicians to reliably diagnose the disease in its earliest stages and to follow the effects of therapy. Although numerous tests have been proposed to date, the diagnosis of HAD/MCMD continues to be based on presentation of the appropriate clinical features of the syndrome, and after significant confounding or co-morbid conditions have been excluded as the primary cause of impairment.
This review summarizes the current diagnostic methods for HAD/MCMD and the potential use of biomarkers that may assist researchers and clinicians to predict the onset of the disease process and evaluate the effects of new therapies. As a basis for understanding the diagnostic features of HAD/MCMD, we also outline the mechanisms of HIV neuroinvasion, CNS proliferation, and HAD pathogenesis. We discuss the advantages and disadvantages of the currently used diagnostic markers for MCMD/HAD.

1. Clinical Features of HIV Neuroinvasion and HIV-Associated Dementia (HAD)
Based on the presence of cerebrospinal fluid (CSF) abnormalities in HIV-infected patients,[13,16-19] as well as the recovery of simian immunodeficiency virus (SIV) from CSF in animal models,[20,21] it appears that HIV neuroinvasion occurs early after infection and is an almost ubiquitous feature of HIV infection. Nonetheless, the clinical symptoms associated with early neuroinvasion are usually mild and self-limited, and may go unnoticed by the patient. Most HIV-positive individuals have few or no CNS-related complaints until the advanced stages of disease.[7] A few patients develop an HIV 'aseptic meningitis' during the period of primary infection and seroconversion.[22] In rare instances, this will proceed to a fulminating and potentially deadly HIV encephalitis.[23] The presence of neurologic symptoms during primary infection has been associated with higher HIV RNA levels in CSF and also with a poorer prognosis.[10,22] An examination of CSF in brain disease, although not a direct reflection of the brain itself, is standard practice because it is one of the best methods of detecting concurrent infections such as neurosyphilis.
HAD is a clinical diagnosis that refers to a constellation of signs and symptoms affecting cognition, central motor functions, and behavior in an HIV-seropositive individual who has no secondary CNS infections or other explanation for this syndrome.[24] Patients with HAD may vary in the type and degree of clinical symptoms at presentation. Common scenarios include:
* patients who refer themselves because they are concerned about their memory loss or new-onset lack of coordination;
* patients who are brought in by friends or family members because of their new bizarre behavior or deteriorating hygiene;
* patients who present to psychiatrists with features of depression, psychosis, mania, or agitation;[25] these patients are then referred to neurologists when their symptoms fail to resolve after psychiatric treatment.
Recently, delirium has been reported in the context of HAD.[26] Still others may report no symptoms whatsoever, but subclinical cognitive deterioration may be detected by neuropsychological tests ordered as part of a research study or clinical diagnostic work up, or on the adventitious finding of cortical atrophy and white matter disease detected on a neuroimaging study of the brain.
In the early years of the AIDS epidemic, most patients typically presented for medical attention during the very advanced stages of systemic and neurologic disease, with symptoms that would currently be considered full-blown AIDS and HAD. As more data accumulated, it became apparent that there is a full spectrum of CNS involvement in HIV-infected patients, ranging from very mild to extremely severe cognitive impairment (table I). It was also noted that, as in most other forms of dementia, patients did not experience the full syndrome de novo. Rather, clinical symptoms appeared after a wearing down of cerebral/cognitive reserve over a period of months, if not years. HIV-associated cognitive decline proceeds sequentially through these varying levels of impairment, albeit at different rates depending on the individual.
The nosology of HIV-associated neurocognitive disorders has evolved over the past 20 years. One of the earliest, and most widely used, schema is the Memorial Sloan Kettering Cancer Center Scale (MSKCC; see table I). In this schema, the investigators used the term AIDS Dementia Complex (ADC) to describe the cognitive, motor, and behavioral features seen in this syndrome and grade the severity of these symptoms from very mild to very severe. However, using this schema, it is theoretically possible to diagnose ADC in a patient with motor and behavioral, but not cognitive, dysfunction. This classification was followed by the American Academy of Neurology (AAN) classification, which introduced the term MCMD in recognition that many patients had a mild level of impairment that did not qualify as dementia.[27] Many researchers currently conceptualize that patients first develop a subsyndromic or subclinical phase of HIV-associated neurocognitive impairment (also called asymptomatic neurocognitive impairment [ANI]), as discussed in a proposed new nosology,[28] which is characterized by neuropsychologic decline in the absence of notable changes in the patient's activities of daily living (i.e. the patients are clinically asymptomatic and their neurocognitive deficits found only by formal neuropsychological examination).[29] Furthermore, this new classification system focuses solely on staging the severity of HIV-associated neurocognitive impairment. It excludes the motor and/or behavioral features used in the MSKCC and AAN systems to identify the stage of disease. For this reason, the term HIV-associated mild neurocognitive disorder (MND) is used in lieu of MCMD. In summary, the classification of HIV neurological disease is an evolving field, and all three of the classification systems described above are still in use. For our purposes, the cognitive impairment seen in patients with ANI is roughly equivalent to stage 0.5 of the MSKCC scale[30]; MND/MCMD to stage 1 of the MSKCC; and HAD to stages 2-4 of the MSKCC scale.[27]
Patients with MCMD have a minor but noticeable decline in their cognitive and central motor functions (insufficient for a diagnosis of dementia), but are still able to work and care for themselves. Those with HAD have significant problems with activities of daily living and rely on others for assistance to complete tasks; they may eventually become fully disabled due to cognitive impairment.[31] There is controversy as to whether central motor and behavioral changes, which were included in the original definitions of AIDS dementia complex, should be included in the staging criteria or whether they should be staged separately.
Some HIV-infected patients also develop seizures, myoclonus, choreoathetosis, or other hyperkinetic movement disorders.[32] While these symptoms can occur in the setting of HAD/MCMD, they are not common and warrant serious investigation for CNS opportunistic infection(s) or tumor(s). Vacuolar myelopathy, a spinal cord syndrome that resembles the spinal cord dysfunction seen in vitamin B12 deficiency, may also occur simultaneously with HAD/MCMD, but is not considered part of the dementia syndrome.

2. Current Diagnostic Methods
2.1 Neuropsychological Assessment
The HIV Dementia Score (HDS) is a bedside screening tool used to assess cognitive functioning in patients with suspected HAD.[33] A score of 10 points or under, out of a possible 16, is considered to be abnormal. The HDS is simple and inexpensive to administer, and is appropriate for patients who are too ill to complete extended cognitive testing. However, the HDS is not specific for HAD,[34] and it is less useful in patients who are poorly educated or illiterate.[35]
In the early stages of MCMD the neurologic examination is relatively unremarkable; if any abnormalities are apparent they would typically be subtle, such as mildly impaired smooth pursuit eye movements, a slight tremor, mildly decreased coordination, mild symmetrical hyper-reflexia, or frontal release signs. In more advanced disease such as HAD, many patients develop a decreased blink rate[35] and a flat facial expression, similar to that seen in patients with advanced Parkinson's disease.[1] The motor deficits associated with HAD can affect both the pyramidal system, causing an upper-motor neuron weakness, increased tone, spasticity, hyper-reflexia and a slowed gait,[36] and the extrapyramidal system,[32] resulting in decreased coordination, ataxia, impairment of fine motor skills, and tremor. The smooth pursuit movements of the eyes are also notably affected and these abnormalities can be quantitatively measured.[30] Perhaps the most noticeable motor finding is generalized slowing of voluntary movement. Overall, neurologic signs such as extrapyramidal motor abnormalities (e.g. tremor) and frontal release signs (e.g. the 'snout reflex'1), are statistically more common in HIV-positive persons than in HIV-negative controls.[37] Although these signs may be associated with neurocognitive impairment[38,39] they are not diagnostic of HAD. In addition, there is some evidence that these extrapyramidal signs are less common in HIV-positive women than in HIV-positive men at a similar stages of disease.[40] Thus, they are not likely to be reliable markers of HAD and can be clinically confused with Parkinsonism in older patients.
The classical neuropsychological manifestations of HAD/MCMD point primarily to dysfunction of the frontal and subcortical circuits of the brain, as is consistent with the findings of many neuropathologic and neuroimaging studies.[41,42] For that reason, HAD is sometimes classified as a 'subcortical' dementia.[43] Psychomotor slowing, which reflects a deficit in the central processing of information, appears to be among the earliest, and most common, features of HIV infection of the CNS. Psychomotor slowing can be measured by neuropsychological tests such as reaction time. In comparison to HIV-negative controls, HIV-infected subjects are typically slower, even while they maintain the accuracy of their responses.[44] These changes may appear before the patient has developed any neurologic symptoms. Some investigators argue that psychomotor slowing is the best predictor of HAD and HIV encephalitis, although this is not universally accepted. There is also a question as to whether neuropsychologic measures correlate with biologic factors.
Recently, Shiramizu et al.[45] performed an exploratory study to examine baseline HIV DNA and its association with individual neuropsychological deficits. Plasma HIV DNA was significantly associated with baseline neuropsychological deficits independent of age, ethnicity, IQ, and plasma HIV-1 RNA levels; however, plasma HIV DNA did not predict future changes in neuropsychological deficits. The investigators concluded that HIV DNA and neuropsychological deficits co-vary over time.
The neurologic examination is a relatively inexpensive low technology tool for assessing HIV-infected patients with suspected MCMD/HAD. It is extremely helpful in detecting focal brain lesions and neurologic abnormalities such as aphasia, which should immediately point away from a diagnosis of uncomplicated HAD/MCMD and toward an opportunistic infection, stroke, or another cause of neurocognitive decline. However, interpretation of the neurologic exam results requires some knowledge of medicine, psychiatry, neuroanatomy, and neurophysiology. Examiners need to be trained to administer and interpret the examination in a uniform way for it to be useful in clinical studies. An isolated abnormality on examination is not diagnostic for HAD/MCMD, as the neurologic signs found in such patients are not specific to this disease.
The neurologic exam does not predict who will develop HAD/MCMD. There is no evidence that it is useful in quantifying the response to HAART or other therapy, probably because the examination is difficult to quantify at all. In summary, the neurologic exam is an important tool for clinical diagnosis because it helps rule out other disease processes, but is a relatively poor predictive marker for developing HAD/MCMD.

2.2 Neuroimaging
Neuroimaging of the CNS plays a significant role in both the diagnosis and exploration of HAD pathogenesis, and enables clinicians to exclude opportunistic infections and malignancies. The two most commonly used neuroimaging modalities are the CT scan and magnetic resonance imaging (MRI) of the brain. Plain CT scans of the brain commonly reveal calcification of the basal ganglia in HIV-1-infected patients. On brain MRI scans, HAD manifests as ill-defined areas of hyperintense signals on T2-weighted images of the subcortical white matter.[46] These T2-weighted hyperintense white-matter lesions are usually without associated mass effect or contrast enhancement, and evolve from small areas of abnormality to diffuse abnormality in patients with more advanced disease process. Selective atrophy of caudate region, and nonspecific generalized brain atrophy with loss of gray matter and enlargement of the cortical sulci and ventricular system have also been demonstrated.[47]
Proton MR-spectroscopy (MRS) has been used solely as a research tool to explore the pathogenesis of HAD, to differentiate the extent of white matter versus gray matter involvement, and to detect CNS involvement in HIV-seropositive patients who are still neurologically asymptomatic.[48] Serial MRS imaging has also been used to assess the effect(s) of HAART on AIDS-associated cognitive impairment.[48]
Diffusion tensor imaging (DTI), which measures the diffusion properties within brain white matter, is another advanced neuroimaging technique that has been used to detect white matter abnormalities in HIV-seropositive individuals as compared with normal individuals. One study showed that patients with the highest diffusion constant elevations and largest anisotropy decreases had the most advanced HIV disease, while those receiving HAART had the lowest viral load levels, along with normal anisotropy and diffusion constants.[49]
Functional MRI (fMRI) involves the acquisition of functional and anatomical data that demonstrate changes in blood flow and oxygenation levels in the local vasculature and that correlates with neural activity in the brain. Using fMRI, neuroscientists can link a mental task performed in the MRI unit to brain activity across time. fMRI has been used to study patients with HAD and explore the pathophysiologic basis of their cognitive impairment. Other experimental imaging techniques, such as continuous arterial spin labeled (CASL) MRI, single-photon emission computed tomography (SPECT), and positron emission tomography (PET), have been used to study the pathogenesis of HAD and the effects of antiviral treatment.[50-52]
In summary, various neuroimaging techniques are currently being employed as research tools to further dissect HAD pathogenesis and to assist clinicians to exclude other concurrent intracranial pathologies that may imitate HAD.

3. Mechanisms of HIV Neuropathogenesis
In order to identify and develop useful clinical biomarkers for the early detection and/or prediction of the development of HAD, an understanding of the mechanisms of HIV invasion and proliferation in the CNS, and the pathogenic mechanisms promoting HAD, is required.
3.1 HIV Neuroinvasion and CNS Proliferation
3.1.1 Crossing the Blood-Brain Barrier
Shortly after systemic infection, HIV-1 penetrates the brain.[16,17,19,53,54] Several mechanisms have been proposed by which HIV crosses the blood-brain barrier (BBB). The 'Trojan horse' hypothesis is a widely accepted postulate that HIV enters the brain via infected CD4+ T cells and monocytes that cross the BBB (figure 1).[55,56] Once these cells gain access to the CNS environment they infect other resident CNS microglial cells and astrocytes. Monocytes from the peripheral circulation differentiate into macrophages on entry into the CNS, and play a significant role in the pathogenesis of HAD. Among the chemokine receptors expressed by human cells, the CCR5 co-receptor is most significant for HIV-1 entry into monocytes/macrophages and microglia.[57,58] It is also hypothesized that cell-free virus from blood penetrates the brain via, or between, microvascular endothelial cells, after which astrocyte infection follows.[59-61]

Cerebral endothelial cells create the tight junctions of the BBB, which can be altered by the HIV-1 protein Tat (transactivator of transcription) to assist HIV-1 entry into the CNS.[62] It has been demonstrated that Tat alters the expression of the cerebral endothelial cell tight junction proteins claudin-1 and -5, which in turn increases the permeability of the BBB.[62] Although direct infection of endothelial cells in vivo with HIV-1 is unconfirmed, damage to the BBB due to HIV proteins (such as Tat) is unequivocal. Disruption of the endothelial tight junctions of the BBB has been demonstrated in postmortem samples of basal ganglia and deep white matter from patients with HIV encephalitis,[38] confirming prior predictions.[18,19] Disruption of the BBB as measured by post-contrast magnetic resonance (MR) enhancement also correlates with neurocognitive impairment.[63]
HIV-1-infected monocyte-derived macrophages also compromise the integrity of the BBB, which in turn translates into facilitation of transendothelial migration of HIV-infected leukocytes from the periphery into the CNS. Ricardo-Dukelow et al.[64] applied a proteomic platform that integrates difference-gel electrophoresis with tandem mass spectrometry peptide sequencing to determine the effects of HIV-1-infected macrophages on the pattern of protein expression by human cerebral endothelial cells. The investigators observed the upregulation of more than 200 proteins by human cerebral endothelial cells, which included metabolic, voltage-gated ion channel, heat shock, transport, cytoskeletal, regulatory, and calcium binding proteins. The findings of this study were validated by Western blot analysis. The authors concluded that HIV-1-infected monocyte-derived macrophages exert significant effects on the proteome of human cerebral endothelial cells and thereby alter the structural and functional integrity of the BBB, potentially opening the BBB to an influx of HIV and systemic neurotoxins.

3.1.2 Reservoirs of HIV in the CNS
Once HIV gains access to the CNS, brain macrophages and microglial cells serve as the chief reservoirs of HIV-1 and the workhorses of HIV replication.[65,66] Evidence obtained from in situ hybridization and immunohistochemical studies indicate that the virus has a predilection for macrophages localized at the perivascular areas, largely in CD14+ cells.[67-69] In addition, cells of neuroectodermal origin, including astrocytes, are also suspected to be reservoirs of HIV infection, and in vitro as well as postmortem studies support this hypothesis.[70,71] Infection of these cells is CD4-independent[72,73] and additional surface receptors have been suggested as alternatives.[65,74]
In the CNS, chronic infections of astrocytes and microglial cells have been well established.[75] Astrocytes express CXCR4 and possibly other HIV-1 co-receptors such as CCR5.[76] Viral replication by astrocytes is generally restricted,[77] although astrocytes can produce low levels of virus under particular circumstances, such as priming by interferon (IFN)-γ,[78] and may act as reservoirs for the virus.[79] Most studies indicate that neurons and oligodendroglia are not productively infected, although they may produce viral proteins.[80,81]

3.2 Neurodegeneration and HAD Pathogenesis
Given these observations, it is generally agreed that HIV does not damage the CNS by mechanisms such as viral-induced cell lysis, and that neuronal loss occurs in the absence of productive neuronal infection by HIV-1. The present concept is that HIV infection, particularly in macrophages, leads to the release of soluble molecules by both virally infected cells and activated, noninfected cells.
Neuronal dysfunction and death, caused by the effects of soluble neurotoxins released by virally infected macrophages and microglia, are significant components of HAD pathogenesis. The situation is made particularly complex by the fact that some mediators, such as tumor necrosis factor (TNF)-α, exert both toxic and protective effects.[82-84] Many of these neurotoxins can paradoxically activate nuclear factor-kappa B (NF-κB), which is widely expressed by neurons and nonneuronal cells[85-87] and possesses strong neuroprotective effects after toxic insults, including exposure to HIV-1-encoded proteins.[88,89]

3.2.1 Role of Viral Proteins
Within the CNS, infected macrophages and microglial cells release viral proteins including the HIV envelope protein gp120, and the HIV regulatory protein Tat,[90] which disrupt neuronal calcium homeostasis.[91] These neurotoxic proteins can induce apoptosis of cultured neurons and, therefore, render neurons vulnerable to excitotoxicity and oxidative stress.[91] Such substances are believed to trigger neurodegenerative processes responsible for neurologic dysfunction and neuronal death. They can also disrupt the integrity of the BBB, as discussed previously with regard to Tat.[62,92]
A number of HIV-1 viral proteins have been implicated in the neurodegenerative process triggered by HIV encephalitis. HIV encephalitis is a diffuse neuropathologic process which tends to be most severe in cerebral white matter and subcortical cerebral structures, especially basal ganglia. Neuropathologically, HIV encephalitis is characterized by the presence of multinucleated giant cells (which possibly result from HIV-induced fusion of microglia and/or macrophages), perivascular chronic inflammatory cells, and microglial nodules. Several studies have shown the presence of HIV-1-specific nucleic acids,[81,93,94] viral DNA, and HIV-1 Nef protein in astrocytes in the context of HIV encephalitis.[95] Nef is a major determinant of lentivirus virulence and is essential for effective virus replication and high viral load. Nef modulates a number of signaling pathways and is considered a progression factor for AIDS;[96] it has also been investigated as a significant player in the pathogenesis of HAD.[97]
Recently, Lehmann et al.[97] further explored the mechanism(s) of Nef interaction with astrocytes, which constitutively express Nef once infected with HIV-1. Using a protein array, the investigators found that monocytes exhibited chemotaxis when subjected to cell culture supernatants of Nef-expressing astrocytic U251MG cells, and CC chemokine ligand-2/monocyte chemotactic protein-1 (CCL2/MCP1) was identified as the factor mediating this phenomenon. In addition, the investigators demonstrated that Nef-induced CCL2/MCP1 expression depended on the myristoylation moiety of Nef and was dependent on functional calmodulin. It is believed that the Nef-induced CCL2/MCP1 expression by astrocytes facilitates the trans-endothelial migration of HIV-1-infected monocytes into the CNS.[75,98]
Another viral protein implicated in neurodegeneration is HIV viral regulatory protein R (Vpr), a highly conserved protein among HIV-1, HIV-2, and SIV isolates. Vpr is a 14 kDa, 96 amino acid nuclear protein that has been isolated from the CSF of patients with HAD[99] and may be involved in neuronal death.[94] Vpr induces neuronal apoptosis through the activation of caspase 8,[100,101] and has been reported to increase mitochondrial permeability, which in turn leads to release of cytochrome C and apoptosis.[79] Supporting the role of Vpr in cell apoptosis, a study has demonstrated that Vpr targets HAX-1 (an anti-apoptotic mitochondrial protein) and dislocates this protein from its normal position; an event which translates into mitochondrial instability and cell death.[102]
To further investigate the role of Vpr in the development of HAD, Wheeler et al.[103] assessed the presence of Vpr in brain tissue, using a panel of tissue samples from patients with HIV encephalitis. The investigators demonstrated that Vpr was present in detectable quantities in the basal ganglia and frontal cortex of HIV-1-infected brains. Application of double immunofluorescence demonstrated that Vpr was only present in macrophages and neurons, and not in astrocytes. The intraneuronal presence of the Vpr further supports the idea that this regulatory protein may play a role in neuronal damage occurring in the context of HAD.
Apart from the destructive interactions between Vpr and the mitochondrial system in the pathogenesis of HAD, these micro-organelles participate in the pathogenesis of HAD through other mechanism(s).[104] Using a model of cultured rodent cortical neurons, Norman et al.[104] assessed the nonlethal effects of Tat on these cells. The investigators found that Tat elicited a rapid drop in internal mitochondrial pH as well as inhibition of complex IV of the electron transport chain. Exposure of rodent cortical neurons to Tat was also associated with decreased NAD(P)H and calcium concentration within neuronal mitochondria, with subsequent decrease in mitochondrial respiratory activity. The investigators concluded that such mitochondrial respiratory abnormalities, following exposure to Tat, may contribute to the pathogenesis of HAD by impairing neuronal signaling.

3.2.2 Role of Inflammatory Molecules
In addition to viral proteins, numerous pro-inflammatory substances are released, either by activated, infected macrophages or by glial cells, which in turn promote the inflammatory reaction within the CNS. Macrophages, derived from monocytes of the peripheral immune system following CNS entry, are the resident immunocompetent cells of the CNS. Monocyte-derived macrophages become activated in response to various insults. It is believed that the aberrant activation of macrophages and microglia by HIV-1 proteins, as well as the pro-inflammatory mediators released by the infected cells, promotes the development of HAD.[105-107] The pro-inflammatory mediators produced and released by activated macrophages and microglial cells include, but are not limited to, cytokines, enzymes such as metalloproteinases, and chemokines such as stromal-derived factor (SDF) 1 (a neurotoxic protein that has been implicated in neurodegeneration).[108] These products have also been shown to recruit additional activated cells into the inflammatory process.[109] There ensues a complex interplay of various toxic molecules in what may possibly become a self-perpetuating process that requires little or no virus to continue (figure 2).

Two major cytokines, TNFα and interleukin (IL)-1β, are upregulated in HIV-1 infection and induce neuronal death.[110] The neurotoxic effects of these pro-inflammatory cytokines are mediated through a number of mechanisms, including increasing the permeability of the BBB, which in turn allows further influx of HIV-1-infected monocytes, and enhancing the neurotoxic effects of the other pro-inflammatory cytokines. In addition, TNFα and IL-1β induce over-stimulation of the N-methyl-D-aspartate (NMDA) receptors, which results in a lethal increase in neuronal Ca2+ levels. TNFα is also known to inhibit glutamate uptake, thereby increasing the concentration of glutamate in synapses to potentially neurotoxic levels.[102] An increase in TNFα production and release from HIV-infected monocytes is mediated by CD40 ligand (CD40L) synergizing with Tat.[107]
A third significant neurotoxic cytokine with a major role in the development of HAD is IFNγ.[111] IFNγ activates the JAK/STAT pathway, which is a key regulator of inflammation and apoptosis signaling. In the presence of HIV proteins gp120 and Tat, the capacity for IFNγ activation of the JAK/STAT pathway is further enhanced. In a series of experiments on primary neurons and mice, Giunta et al.[112] demonstrated that green tea-derived (-)-epigallocatechin-3-gallate (EGCG) reduced the neurotoxicity of IFNγ-enhanced neuronal damage from gp120 and Tat.

3.2.3 Drug Effects
Nucleoside reverse transcriptase inhibitors (NRTIs) may also have a role in promoting HAD. A recent study by Opii et al.[113] examined the possible role of the NRTI 2′,3′-dideoxycytidine (ddC; zalcitabine) in the neuropathology of HAD. The investigators treated and incubated isolated synaptosomes and mitochondria for 6 hours with CSF-achievable concentrations of zalcitabine and detected significant increase in oxidative stress with zalcitabine 40 nM measured by protein carbonyls and 3-nitrotyrosine. They also observed that zalcitabine at a concentration of 40 nM could cause the release of cytochrome C and, in addition, decrease the levels of anti-apoptotic proteins and increase the levels of pro-apoptotic proteins, thereby increasing the potential for apoptosis. These findings support the possible role of this class of drugs, and particularly zalcitabine, in the promotion of HAD.

4. Laboratory Biomarkers of HAD/HIV-Associated Minor Cognitive Motor Disorder
Plasma viral load and CD4+ lymphocyte counts are commonly used as prognostic markers of treatment response in HIV-infected individuals. However, CD4+ tests have been found to be more useful in assessing the systemic, rather than the CNS, response to HAART. Similarly, the plasma viral load is not helpful as a diagnostic or predictive measure of HAD/MCMD, as it is a poor reflection of the CNS viral load.[114,115] An increased understanding of the pathobiology of HAD has led to the study of a number of putative laboratory markers that, it was hoped, would assist in the diagnosis of HAD/MCMD, and help physicians to track the patient's response to therapy.

4.1 Cellular Immunophenotypes
The cerebral perivascular compartment is one of the first targets of HIV upon its entry to the CNS. Both viral capsid and envelope proteins can be detected in cerebral endothelial cells and perivascular macrophages.[116] However, the presence of activated macrophages within the CNS may serve as a better predictor of neurologic deterioration than does the detection of viral material.[117] Investigators have identified a subset of activated monocytes, found in peripheral blood, that appear to precede or coincide with the development of HAD/MCMD.[118] These tissue-invasive cells are characterized by the activated CD14+/CD16+ and CD14+/CD69+ immunophenotypes.[119] Monocytes such as these migrate across the BBB and refresh the perivascular reservoirs of HIV-infected macrophages. HIV-infected perivascular macrophages do not undergo apoptosis and can serve as a long-lasting reservoir of HIV within the human CNS, although it appears that active virus production in the CNS typically remains low prior to the onset of AIDS.[120] A subset of peripheral blood monocytic cells may also develop a unique proteomic (protein) profile that appears to presage the development of cognitive impairment.[121]

4.2 Cerebrospinal Fluid Markers
Since the earliest days of NeuroAIDS research, investigators have exploited the CSF to study the neuropathogenesis of HAD/MCMD. CSF abnormalities occur shortly after HIV infection,[16-19] and the CSF has been used to attempt to identify distinctive temporal patterns during the natural history of uncomplicated HIV disease.[122] In addition, the analysis of CSF is an essential tool in the diagnosis of neurosyphilis, opportunistic infections, and tumors in patients with AIDS.[123]
The advantages of CSF examination are many. CSF can be removed via lumbar puncture, a relatively safe and easy procedure when compared with a brain biopsy. Lumbar punctures can be repeated serially over time, and before and after the initiation of treatments, which is not feasible with brain biopsies. The process of lumbar puncture and CSF examination is less expensive than brain biopsy or neuroimaging. In general, CSF markers have been found to be more highly correlated with cognitive decline than the same markers in blood.[124]
In the pre-HAART era, a number of CSF laboratory parameters were extensively studied as putative markers of HAD/MCMD. Some very nonspecific CSF abnormalities included CSF pleocytosis (elevated white blood cell count), elevated CSF intra-BBB immunoglobulin (IgG) synthesis (a measure of intrathecal immune activation and antibody production),[16,17,122] the presence of unique CSF oligoclonal bands (each representing unique intrathecal clones of B cells),[10] and CSF albumin leakage (a measure of BBB integrity and permeability).[16,18,19] In those studies, measures of intra-BBB inflammation were made using several methods (see table II). Isoelectric focusing (IEF) of CSF versus cognate serum in vivo specimens was coupled with immunohistochemistry to detect IgG bands in CSF that were absent in the cognate sera. These band are due to plasma cell clones in the brain that are absent in the blood. Similarly, these plasma clones produce IgG in brain that is secreted into the CSF. The Tourtellotte equation corrects for diffusion of blood proteins into the brain across the BBB and calculates a dynamic value for net IgG produced within the brain, and is used to measure intra-BBB IgG synthesis (in units of mg of IgG produced per day).

The importance of CSF examination was bolstered by studies that indicate that CSF from HIV-infected patients contains one or more neurotoxic factors,[145] such as elevated levels of quinolinic acid, an excitatory neurotoxin that is a metabolite of tryptophan and acts as an NMDA receptor agonist.[146] Small amounts of quinolinic acid are normally present in the CNS, but concentrations increase when elevated levels of IFNγ or other cytokines, including pro-infammatory cytokines (e.g. IL-6 and TNFα), shift tryptophan metabolism into the kynurenine pathway. Quinolinic acid in CSF increases in parallel with neurological impairment[146] and decreases after antiretroviral therapy.[147] High levels are associated with selective atrophy in brain regions susceptible to excitotoxic injury.[148] However, like most other CSF markers associated with activated macrophages and microglia, the elevation of quinolinic acid is not specific to HAD. It is also difficult and expensive to assay, requiring mass spectrometry.
The light chain subunit of the neurofilament (NFL) protein is another sensitive CSF marker of axonal injury and neurodegeneration that is elevated in patients with AIDS dementia complex.[149] However, this is a nonspecific marker that is elevated in other neurodegenerative processes as well.

4.2.1 Viral Markers
Viral load in the CSF has been reported to correlate with cognitive impairment, and is commonly assessed by the detection and quantitative measurements of CSF HIV p24 antigen (p24 Ag), prior to the development of gene amplification techniques to measure active viral replication.[17] Other methods used for the detection of HIV include virological culture of CSF,[136,137] quantitative measurement of pro-viral DNA in CSF cells by PCR,[127,131] and quantitative measurements of HIV RNA viral load by PCR[147] or nucleic acid sequence-based amplification[124] techniques. The quantification of monoamine metabolites in CSF has not been shown to correlate with the stage of HAD.[150]

4.2.2 Host-Derived Protein Markers
Several CSF-soluble factors involved in inflammation have also been studied as potential biomarkers of HAD. CCL2/MCP1, for example, is an inflammatory mediator reported to be elevated in HAD, HIV encephalitis, and SIV encephalitis.[151,152] However, CCL2/MCP1 is not specific to HIV; for example, it is also elevated in cytomegalovirus infection of the CNS, a disease that can mimic HAD/MCMD.
β2-Microglobulin, a protein component of major histocompatibility complex (MHC) class I molecules on the surface of cells and an index of cell turnover,[138] has been studied extensively as a marker for the progression of systemic HIV disease,[153] as well as a putative CSF marker of HAD/MCMD. As with other nonspecific markers of immunoactivation, CSF β2-microglobulin tends to be higher in neurologically asymptomatic HIV-positive subjects who will go on to develop neurocognitive impairment,[139] and is higher in neurocognitively impaired HIV-positive patients than in non-impaired HIV-infected individuals. This marker parallels, to some degree, the severity of neurocognitive dysfunction.[138] The levels of CSF β2-microglobulin decreases after effective antiretroviral therapy.[154] Although it can be measured relatively inexpensively by enzyme immunoassay (EIA), β2-microglobulin suffers from the same problems as many other putative markers, in that it is not specific to HIV infection, so opportunistic infection and tumor must be ruled out before it is useful. Some reports indicate β2-microglobulin is not a sensitive marker of continued neurological deterioration in HAART-treated patients.[154]
Neopterin, a byproduct of guanosine triphosphate metabolism produced by activated macrophages,[139] is another marker of nonspecific immunoactivation[155] that has often been studied in conjunction with β2-microglobulin. It can be measured by standard laboratory techniques (e.g. enzyme-linked immunosorbent assay[141] and high-performance liquid chromatography [HPLC]).[156] Like CSF β2-microglobulin, elevated CSF neopterin levels may be useful in predicting who is at risk for HAD;[67] however, unlike β2-microglobulin, neopterin does not reliably return to normal levels in the CSF of all HAART-treated patients.[132,154] It is not clear if this is, in fact, clinically relevant and reflects continued intrathecal immune activation. Interestingly, higher levels of neopterin are associated with higher levels of reactive oxygen species, and lower levels of some anti-oxidants.[155] This suggests that neopterin is also an indicator of oxidative stress, a state that has also been implicated in neurodegenerative diseases including HAD/MCMD.[90] Like certain other inflammatory markers, neopterin lacks specificity for HAD/MCMD and is elevated in other infectious and inflammatory disorders.
TNFα has been associated with apoptosis in a number of neurodegenerative conditions, including HAD/MCMD.[157] Glass et al.[117] reported an association between increased levels of TNFα mRNA in postmortem brain tissue and a history of HAD. Other investigators have found that TNFα was more likely to be detected, and/or TNFα levels were higher in the CSF, of HIV-infected patients with dementia compared with those without dementia.[91] In a case report by Gendelman et al.,[158] an HIV-1-seropositive, antiretroviral-naive patient showed a decline in CSF TNFα levels following 12 weeks of treatment with HAART.[158] However, TNFα in CSF was detected infrequently or not at all in several other studies,[133,159] and TNFα is not specific to HAD, limiting its usefulness as a biomarker. However, monocyte chemoattractant protein-1 (MCP-1) and macrophage colony-stimulating factor (M-CSF) detection in plasma were associated with time to HAD.[134]
Another putative marker is Fas and Fas ligand (FasL).[160] Fas is a receptor protein that can be expressed on cell surface membranes or found in a soluble, circulating form (sFas). FasL is also known as Apo-1 or CD95, and is a member of the TNFα receptor superfamily. The cross-linking of Fas with either FasL or an activating antibody induces apoptosis in Fas-expressing cells; for this reason, Fas is sometimes called a death receptor. When this system of apoptosis fails to function, it may contribute to the development of malignancies. Elevated sFas is also associated with chronic immunoactivation. Plasma levels of sFas are elevated in HIV infection and, while they are significantly reduced after HAART, do not return to normal physiological levels.[161] sFas can be measured in body fluids by an enzyme immunoassay technique. CSF sFas levels are elevated in HAD as compared with HIV-seronegative controls,[160,162] and levels decrease with antiretroviral therapy.[162]

4.3 Advantages and Disadvantages of Laboratory Markers
There are a number of issues associated with the use of most of the CSF markers in the study of HAD/MCMD. Firstly, there are questions as to whether the virus recovered from CSF is truly representative of that within the brain parenchyma. There may be multiple sources for the HIV found within the CSF, including local production from CSF monocytic cells, virus that crosses from the systemic circulation through the blood-CSF barrier (a barrier located at tight junctions of cuboidal cells of the choroid plexus), and virus that is shed from the brain parenchyma and flows via the choroid plexus into the CSF.[163-167] Animal studies give credence to this idea of compartmentalization.[148]
Secondly, there is some regional variation in qualitative and quantitative neuropathology and virology within the brain and much work has been done in this regard.[125,142,168-215] However, this variation is obscured by the relatively homogeneous composition of CSF. The effect of HAART on HIV may vary between the plasma, CSF, and brain parenchymal compartments,[132] and during and after therapy each compartment may respond at a different rate. Furthermore, the virus may remain within the brain after it has been cleared from the CSF.[115,216-218]
Lastly, most of these markers are nonspecific for HIV and are only useful when other infections have been excluded.
In summary, blood and CSF laboratory markers are relatively easy to obtain and can be followed in a serial fashion, but they are not good indicators of regional HIV-induced changes or damage in the brain. Indeed, many of these markers are nonspecific markers of ongoing underlying macrophage activation and inflammation and cannot serve as sole indicators of the severity of HAD/MCMD; therefore, these markers should be used with caution.
Table II provides a synopsis of several tests associated with a diagnosis of NeuroAIDS.

5. Diagnostic Changes in the Era of HAART
In the pre-HAART era, HAD typically developed in patients with low CD4+ cell counts (under 200 cells/mm3).[219] However, even in this group of subjects, only a minority developed HAD/MCMD. Post-HAART, the mean CD4+ count at which HAD develops has increased.[12,220,221] HIV-infected patients may be developing dementia at higher CD4+ counts because even though their immune systems have been partly reconstituted with treatment, they are not immunologically normal. In addition, they are exposed to neuro-inflammation for longer periods. HIV-infected patients treated with HAART may be diagnosed with HAD/MCMD at a time when their CD4+ levels are well above the lowest CD4+ counts they had in the course of their disease. This 'nadir' CD4+ count may be the pertinent observation,[12,220-222] and may be a significant risk factor for HAD/MCMD.[223]
Prior to HAART, the majority of studies that measured CSF RNA viral load found a positive correlation with the presence of neurocognitive impairment.[124,224] However, this correlation seems to no longer hold. Sevigny et al.[114] suggested that ongoing viral replication may not be required for dementia to develop. Once HIV triggers an abnormal production of toxic mediators by the immune system, neurologic dysfunction may progress irrespective of viral levels. These observations have led some investigators to propose that there now may be multiple forms of HAD.[220,224] Suggested subtypes include 'chronic' HAD, in which partial compliance or viral resistance leads to slow, variable progression of neurocognitive dysfunction, and 'inactive' HAD with good viral control but some fixed deficits from damage incurred prior to effective treatment. In addition, the increased longevity of HIV-infected individuals has resulted in more potential for co-morbid conditions to have a significant impact on cognitive functioning, suggesting the possibility of hybrid forms of dementias or 'transformed' HAD.[224]
Similarly, it cannot be assumed that blood and CSF markers that correlated well with neurocognitive decline in the past still do so. CSF levels of β2-microglobulin and neopterin, for instance, are less valuable for following the progression of CNS disease in HAART-treated patients.[114,130,224] Are these apparent changes in HAD reflected in the histologic appearance of the brain at death? This question has not been directly addressed; however, one observation can be made. The incidence of HIV encephalitis and its relationship to HAD has always been variable. As mentioned previously, even prior to HAART, in one study 50% of patients with HAD did not have HIV encephalitis.[132] These findings contrast with a later study by a different group in which 95% of their subjects with any degree of neurocognitive impairment had HIV encephalitis and, additionally, 45% of their neurocognitively normal subjects also had HIV encephalitis.[225] This cohort had some exposure to antiretrovirals but this was not documented in detail. There are mundane factors that may contribute to this disparity, such as the inter-observer differences resulting from the subjectivity of neuropathologic and neuropsychiatric examinations. However, the complexity of the pathogenesis of HAD likely plays a role as well. The variable exposure of different individuals to HAART potentially increases this complexity dramatically.
There is evidence that even in patients receiving consistent therapy there is ongoing, low-level neuroinflammation. In one study, CSF levels of neopterin, β2-microglobulin, and HIV RNA were followed in neurologically asymptomatic patients on HAART.[154] HIV RNA levels became undetectable and β2-microglobulin levels normalized. However, even after 2 years of treatment, neopterin levels, although decreased relative to pre-treatment, remained elevated. Whether this is because of the ongoing undetectable viral replication is unknown, as is the clinical significance. However, it certainly opens the door to the possibility of 'chronic' HAD or of milder, but still significant, impairment. This may not be reflected by the presence of HIV encephalitis but rather some more subtle pathology.
Anthony et al.[226] used an image analysis system to quantify activated microglia in the brains of HIV-infected patients compliant with HAART and neurocognitively intact at death compared with pre-HAART HIV encephalitis-positive and HIV-infected/HIV encephalitis-negative patients. Interestingly, in the hippocampus and basal ganglia the HAART-treated, cognitively normal group had as many activated microglia/macrophages as the pre-HAART patients with or without HIV encephalitis, as measured by CD68 positivity, a marker of upregulation and activation of microglial cells that indicates the presence of ongoing neuro-inflammation. This also points to the inability of HAART to completely eradicate the effects of HIV on the CNS, and raises questions as to the clinical implications of this fact.

6. Future Directions for HAD Diagnostics
Clinicians and researchers can anticipate that they will be confronted by one of the major limitations of neuropsychological testing, that is, the questionable applicability of many currently used standardized tests to the growing numbers of under-educated, differently acculturated, and/or non-English speaking HIV-infected populations in English-speaking countries. This poses a problem for assessing HIV-infected individuals on the international stage, and much work is being done internationally in this regard. This is also a growing problem in the US, because HIV is spreading rapidly in minority communities. These populations differ significantly from those in which the test norms were developed. Developing valid tests and norms for minority and immigrant populations in any country represents a clear challenge for neuropsychologists and clinicians.
Adding to this challenge is the fact that the course of HIV infection is now more complex than in the pre-HAART era. It appears that the correlation between some biomarkers and neurocognitive dysfunction has weakened. It seems likely that the pathogenic mechanisms resulting in HAD may have changed and may differ among individuals depending on their exposure to antiretroviral protocols. However, the interval between initial infection of the CNS and development of overt HAD represents an important window of opportunity for therapeutic intervention. As mentioned previously, HAART neither completely prevents nor entirely reverses HAD. Once set in motion, some pathologic processes triggered initially by the presence of virus and immune dysregulation may become self-sustaining and difficult to modulate. Since any prophylactic interventions are likely to be expensive and have adverse effects, markers that identify people at risk for the development of HAD are desirable.
Products released by activated macrophages, astrocytes, and apoptotic cells in response to HIV-infection in the CNS may contribute to cerebral dysfunction by directly damaging neurons. These products may induce alterations in gene and protein expression profiles in neurons themselves, which are deleterious to their function.[227,228] Studies of specific soluble factors that may assist in the diagnosis of HIV/AIDS,[229] now utilizing cutting edge modern molecular tools specifically in the context of NeuroAIDS, are ongoing. This work is being done in association with in vivo imaging techniques such as CT and MRI,[53,230,231] and is directed to the important goal of in vivo analysis of brain disease using combined molecular and imaging technologies.
Technical advances that expand current methodologies, such as immunofluorescence flow cytometry, will improve the quantification and characterization of HIV-infected cell populations (e.g. CD4+, CD4+CCR5+, CD4+CD25+FoxP3+ regulatory T cells) that infiltrate the BBB and invade the brain parenchyma. It will be important to continue to explore the expression of activation markers in CD8+ T-cell population involved in cytolysis of virally infected cells.[226,227,231,232] Immunohistochemistry and flow cytometry, as well as molecular, virologic, and gene expression technologies, will continue to provide useful laboratory methods to analyze cells involved in HAD. Increasingly, new or improved approaches to interpret these data, such as fractal analysis,[226,228] will become standardized to permit a better characterization of the pathological alterations of form/structure/function in CNS cell populations in HAD. Future molecular research is likely to uncover significant alterations in coding versus noncoding DNA in HIV-infected and apoptotic cells within the brain parenchyma of patients with HAD. Events related to cellular activation and pathology may be associated with processes related to the noncoding as well as coding portions of the human genome, with significant consequences for the function and survival of the cell.
Studies of laboratory biomarkers indicate that even though the CSF is not a precise window to the brain, it better reflects HIV and immune activity than does the peripheral blood. However, it is not feasible to perform widespread lumbar punctures and repeat the CSF testing of clinically asymptomatic patients outside of a research study. Ideally, markers of HAD/MCMD should be obtainable in a less invasive manner, for instance, through additional tests on blood that is drawn in the course of patient care. Moreover, tracking isolated markers of complex processes such as inflammation is possibly of limited use as these reflect common pathways used by many processes. A 'profile' that could be developed of abnormalities may be more specific to HAD. Other potentially important markers in CSF/blood may be derived from specific viral nucleic sequence information (an entire field in itself), as well as specific single nucleotide polymorphisms and genetic changes that increase susceptibility to HAD.[143,144,233] Additionally, analyses of gene expression at the transcriptional and translational levels show promise for future diagnostics, with the added importance of the use of bioinformatics.[135,233-239] Finally, the clinical definitions and nosology of HAD and HIV-associated neuropsychiatric disorders is currently in flux.[28,233] It is hoped that new developments will contribute to the establishment of reliable molecular diagnostic tests in a clinically stable setting.

Acknowledgments
The work was supported by grants to Dr Shapshak (NIH grants DA 14533, DA 12580, and GM 056529), Drs Commins and Singer (NIH grant NS 38841), and Dr Chiappelli (NIH grant DA 07683).
The authors have no conflicts of interest directly relevant to the content of this review.
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HIV Associated Dementia (HAD)/Minor Cognitive Motor Disorder (MCMD)

Key words:
Gangguan kognitif, fungsi motorik sentral, dan perilaku

Keluhan utama:
1. gangguan memori dan koordinasi
2. gangguan perilaku dan penurunan hygiene
3. gangguan psikiatri (depresi, psikosis, mania, agitasi)

Anamnesa dan pemeriksaan fisik:
1. perlambatan gerakan volunter
2. gejala pyramidal
a. parese UMN
b. peningkatan tonus
c. spastisitas
d. hiperrefleksi
e. penurunan gait
3. gejala ekstra pyramidal
a. penurunan koordinasi
b. ataksia
c. gangguan gerakan motorik halus
d. tremor
e. gangguan gerakan mata halus
f. penurunan frekuensi berkedip
g. pendataran ekspresi wajah
4. frontal release sign (misalnya reflek snout)
;
Neuropatofisiologi:
1. disfungsi sirkuit frontal dan subkorteks
2. psikomotor melambat karena deficit pda proses sentral

Neuroimaging:
1. atrofi regio kaudatus
2. atrofi otak umum dengan hilangnya gray matter serta pelebaran sulci dan sistem ventrikel
3. CT scan: kalsifikasi basal ganglia
4. MRI: sinyal hiperintens pada T2-weighted subkorteks white matter

Laboratorium:
1. viral load
2. CD4+ count
3. CSF marker (bukan indikator yang baik untuk perubahan regional yang diinduksi HIV atau kerusakan otak, sebagian besar tidak spesifik terhadap penyebab aktivasi makrofag dan inflamasi, serta tidak dapat digunakan sebagai indikator keparahan HAD/MCMD)

Terapi:
HAART (masih memungkinkan virus tinggal dalam parenkim otak)

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