ABSTRAK
Latar belakang : Cytomegalovirus (CMV) merupakan salah satu infeksi oportunistikpada pasien dengan sindrom immunodefisiensi (AIDS). Gejala klinis dan CT scantidak dapat menegakkan diagnosa definitif ensefalitis CMV. Oleh karena itudiperlukan uji alternatif untuk menegakkan diagnosis infeksi CMV pada pasien HIVdengan infeksi otak. Salah satu uji yang sensitif dan spesifik adalah Real TimePolymerase Chain Reaction (rPCR).Tujuan : Mendapatkan uji deteksi molekular CMV pada pasien HIV dengantersangka infeksi otak.Metode : Penelitian dilakukan dalam 3 tahap. Tahap 1 adalah optimasi konsentrasiprimer, probe, suhu annealing, volume elusi ekstraksi DNA, dan volume cetakan.Tahap 2 adalah uji spesifisitas (reaksi silang) dan uji sensitivitas (ambang batasdeteksi DNA) rPCR dan tahap 3 adalah penerapan uji rPCR yang sudah dioptimasiterhadap sampel plasma, urin, dan LCS.Hasil : Kondisi optimal uji rPCR telah diperoleh dengan konsentrasi primer danprobe 0,1 μM, dengan kondisi suhu reaksi rPCR: aktivasi enzim pada 950C selama 3menit; 45 siklus pada 950C selama 15 detik (denaturasi) dan 560C selama 1 menit(annealing dan ekstensi). Volume elusi ekstraksi DNA yang optimal untuk ketigajenis sampel (LCS, plasma dan urin) adalah 40 μL, dan volume cetakan rPCR untukLCS, plasma, dan urin, masing-masing adalah 5, 4, dan 3 μL. Uji rPCR mampumendeteksi DNA pada 50.000 jumlah kopi/mL dan tidak bereaksi silang denganStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, EBV,HSV,dan VZV. Penerapan ujirPCR pada sampel klinis memberikan hasil negatif pada semua sampel LCS, 72,22%positif pada sampel plasma, dan 72,22% positif pada sampel urin.Kesimpulan: Telah dilakukan optimasi uji rPCR dengan minimal deteksi DNACMV 50.000 jumlah kopi/mL dan tidak bereaksi silang dengan mikroorganisme yangberpotensi menyebabkan positif palsu (false positive).ABSTRACT
Background: Cytomegalovirus (CMV) is one of opportunistic infections in patientswith Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are nottypical, and CT scans can not define encephalitis CMV specifically. Therefore, it isimportant to apply an alternative assay for sensitive and specific detection of CMVinfection in HIV patients with suspected central nervous system (CNS) infections.One of the assays is real time polymerase chain reaction (rPCR).Objective: To obtain a molecular assay for detection of CMV in HIV patients withsuspect CNS infections.Methods: This study was conducted in three phases. The first is optimization ofconcentrations of primers, probe, annealing temperature, final elution of DNAextraction, and volume of PCR template. The second is determinations of sensitivity(minimal detection of DNA) and specificity (cross-reaction) of the optimized rPCR,and the third is application of the rPCR for clinical samples of plasma, urine, andliquor cerebrospinal (LCS).Results: The rPCR reaction showed optimal concentrations of primers and probe at0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing andextension). Final elution of DNA extraction was 40 μL and volume of PCR templatesfor urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimaldetection of DNA at 50,000 copies/mL and was not cross-reacted withStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), HerpesSimplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR forclinical samples showed that the rPCR yielded 72.22% positive for plasma or urine,and negative for all LCS samples.Conclusion: The rPCR has been optimized in this study with minimal DNA detectionat 50,000 copies/mL and was not cross-reacted with other microorganisms that arepotential to cause false positive results.;Background: Cytomegalovirus (CMV) is one of opportunistic infections in patientswith Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are nottypical, and CT scans can not define encephalitis CMV specifically. Therefore, it isimportant to apply an alternative assay for sensitive and specific detection of CMVinfection in HIV patients with suspected central nervous system (CNS) infections.One of the assays is real time polymerase chain reaction (rPCR).Objective: To obtain a molecular assay for detection of CMV in HIV patients withsuspect CNS infections.Methods: This study was conducted in three phases. The first is optimization ofconcentrations of primers, probe, annealing temperature, final elution of DNAextraction, and volume of PCR template. The second is determinations of sensitivity(minimal detection of DNA) and specificity (cross-reaction) of the optimized rPCR,and the third is application of the rPCR for clinical samples of plasma, urine, andliquor cerebrospinal (LCS).Results: The rPCR reaction showed optimal concentrations of primers and probe at0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing andextension). Final elution of DNA extraction was 40 μL and volume of PCR templatesfor urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimaldetection of DNA at 50,000 copies/mL and was not cross-reacted withStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), HerpesSimplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR forclinical samples showed that the rPCR yielded 72.22% positive for plasma or urine,and negative for all LCS samples.Conclusion: The rPCR has been optimized in this study with minimal DNA detectionat 50,000 copies/mL and was not cross-reacted with other microorganisms that arepotential to cause false positive results.;Background: Cytomegalovirus (CMV) is one of opportunistic infections in patientswith Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are nottypical, and CT scans can not define encephalitis CMV specifically. Therefore, it isimportant to apply an alternative assay for sensitive and specific detection of CMVinfection in HIV patients with suspected central nervous system (CNS) infections.One of the assays is real time polymerase chain reaction (rPCR).Objective: To obtain a molecular assay for detection of CMV in HIV patients withsuspect CNS infections.Methods: This study was conducted in three phases. The first is optimization ofconcentrations of primers, probe, annealing temperature, final elution of DNAextraction, and volume of PCR template. The second is determinations of sensitivity(minimal detection of DNA) and specificity (cross-reaction) of the optimized rPCR,and the third is application of the rPCR for clinical samples of plasma, urine, andliquor cerebrospinal (LCS).Results: The rPCR reaction showed optimal concentrations of primers and probe at0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing andextension). Final elution of DNA extraction was 40 μL and volume of PCR templatesfor urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimaldetection of DNA at 50,000 copies/mL and was not cross-reacted withStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), HerpesSimplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR forclinical samples showed that the rPCR yielded 72.22% positive for plasma or urine,and negative for all LCS samples.Conclusion: The rPCR has been optimized in this study with minimal DNA detectionat 50,000 copies/mL and was not cross-reacted with other microorganisms that arepotential to cause false positive results.;Background: Cytomegalovirus (CMV) is one of opportunistic infections in patientswith Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are nottypical, and CT scans can not define encephalitis CMV specifically. Therefore, it isimportant to apply an alternative assay for sensitive and specific detection of CMVinfection in HIV patients with suspected central nervous system (CNS) infections.One of the assays is real time polymerase chain reaction (rPCR).Objective: To obtain a molecular assay for detection of CMV in HIV patients withsuspect CNS infections.Methods: This study was conducted in three phases. The first is optimization ofconcentrations of primers, probe, annealing temperature, final elution of DNAextraction, and volume of PCR template. The second is determinations of sensitivity(minimal detection of DNA) and specificity (cross-reaction) of the optimized rPCR,and the third is application of the rPCR for clinical samples of plasma, urine, andliquor cerebrospinal (LCS).Results: The rPCR reaction showed optimal concentrations of primers and probe at0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing andextension). Final elution of DNA extraction was 40 μL and volume of PCR templatesfor urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimaldetection of DNA at 50,000 copies/mL and was not cross-reacted withStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacteriumtuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), HerpesSimplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR forclinical samples showed that the rPCR yielded 72.22% positive for plasma or urine,and negative for all LCS samples.Conclusion: The rPCR has been optimized in this study with minimal DNA detectionat 50,000 copies/mL and was not cross-reacted with other microorganisms that arepotential to cause false positive results. |
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