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Abstrak :
ABSTRAK
Latar belakang : Cytomegalovirus (CMV) merupakan salah satu infeksi oportunistik pada pasien dengan sindrom immunodefisiensi (AIDS). Gejala klinis dan CT scan tidak dapat menegakkan diagnosa definitif ensefalitis CMV. Oleh karena itu diperlukan uji alternatif untuk menegakkan diagnosis infeksi CMV pada pasien HIV dengan infeksi otak. Salah satu uji yang sensitif dan spesifik adalah Real Time Polymerase Chain Reaction (rPCR). Tujuan : Mendapatkan uji deteksi molekular CMV pada pasien HIV dengan tersangka infeksi otak. Metode : Penelitian dilakukan dalam 3 tahap. Tahap 1 adalah optimasi konsentrasi primer, probe, suhu annealing, volume elusi ekstraksi DNA, dan volume cetakan. Tahap 2 adalah uji spesifisitas (reaksi silang) dan uji sensitivitas (ambang batas deteksi DNA) rPCR dan tahap 3 adalah penerapan uji rPCR yang sudah dioptimasi terhadap sampel plasma, urin, dan LCS. Hasil : Kondisi optimal uji rPCR telah diperoleh dengan konsentrasi primer dan probe 0,1 μM, dengan kondisi suhu reaksi rPCR: aktivasi enzim pada 950C selama 3 menit; 45 siklus pada 950C selama 15 detik (denaturasi) dan 560C selama 1 menit (annealing dan ekstensi). Volume elusi ekstraksi DNA yang optimal untuk ketiga jenis sampel (LCS, plasma dan urin) adalah 40 μL, dan volume cetakan rPCR untuk LCS, plasma, dan urin, masing-masing adalah 5, 4, dan 3 μL. Uji rPCR mampu mendeteksi DNA pada 50.000 jumlah kopi/mL dan tidak bereaksi silang dengan Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Candida spp, Toxoplasma gondii, EBV,HSV,dan VZV. Penerapan uji rPCR 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 DNA CMV 50.000 jumlah kopi/mL dan tidak bereaksi silang dengan mikroorganisme yang berpotensi menyebabkan positif palsu (false positive).ABSTRACT
Background: Cytomegalovirus (CMV) is one of opportunistic infections in patients with Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are not typical, and CT scans can not define encephalitis CMV specifically. Therefore, it is important to apply an alternative assay for sensitive and specific detection of CMV infection 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 with suspect CNS infections. Methods: This study was conducted in three phases. The first is optimization of concentrations of primers, probe, annealing temperature, final elution of DNA extraction, 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, and liquor cerebrospinal (LCS). Results: The rPCR reaction showed optimal concentrations of primers and probe at 0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45 cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing and extension). Final elution of DNA extraction was 40 μL and volume of PCR templates for urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimal detection of DNA at 50,000 copies/mL and was not cross-reacted with Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR for clinical 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 detection at 50,000 copies/mL and was not cross-reacted with other microorganisms that are potential to cause false positive results.;Background: Cytomegalovirus (CMV) is one of opportunistic infections in patients with Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are not typical, and CT scans can not define encephalitis CMV specifically. Therefore, it is important to apply an alternative assay for sensitive and specific detection of CMV infection 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 with suspect CNS infections. Methods: This study was conducted in three phases. The first is optimization of concentrations of primers, probe, annealing temperature, final elution of DNA extraction, 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, and liquor cerebrospinal (LCS). Results: The rPCR reaction showed optimal concentrations of primers and probe at 0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45 cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing and extension). Final elution of DNA extraction was 40 μL and volume of PCR templates for urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimal detection of DNA at 50,000 copies/mL and was not cross-reacted with Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR for clinical 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 detection at 50,000 copies/mL and was not cross-reacted with other microorganisms that are potential to cause false positive results.;Background: Cytomegalovirus (CMV) is one of opportunistic infections in patients with Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are not typical, and CT scans can not define encephalitis CMV specifically. Therefore, it is important to apply an alternative assay for sensitive and specific detection of CMV infection 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 with suspect CNS infections. Methods: This study was conducted in three phases. The first is optimization of concentrations of primers, probe, annealing temperature, final elution of DNA extraction, 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, and liquor cerebrospinal (LCS). Results: The rPCR reaction showed optimal concentrations of primers and probe at 0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45 cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing and extension). Final elution of DNA extraction was 40 μL and volume of PCR templates for urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimal detection of DNA at 50,000 copies/mL and was not cross-reacted with Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR for clinical 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 detection at 50,000 copies/mL and was not cross-reacted with other microorganisms that are potential to cause false positive results.;Background: Cytomegalovirus (CMV) is one of opportunistic infections in patients with Aquired Immunodeficiency Syndrome (AIDS). Clinical manifestations are not typical, and CT scans can not define encephalitis CMV specifically. Therefore, it is important to apply an alternative assay for sensitive and specific detection of CMV infection 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 with suspect CNS infections. Methods: This study was conducted in three phases. The first is optimization of concentrations of primers, probe, annealing temperature, final elution of DNA extraction, 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, and liquor cerebrospinal (LCS). Results: The rPCR reaction showed optimal concentrations of primers and probe at 0.1 μM, with thermal cycler: 950C for 3 min (enzyme activation), followed by 45 cycles of 950C for 15 sec (denaturation) and 560C for 1 min (annealing and extension). Final elution of DNA extraction was 40 μL and volume of PCR templates for urine, plasma, and LCS was 3, 4, and 5 μL, respectively. The rPCR had minimal detection of DNA at 50,000 copies/mL and was not cross-reacted with Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Candida spp, Toxoplasma gondii, Epstein-Bar Virus (EBV), Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV). Application of rPCR for clinical 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 detection at 50,000 copies/mL and was not cross-reacted with other microorganisms that are potential to cause false positive results.

Abstrak :
Latar belakang: Infeksi susunan saraf pusat (SSP) dapat berakibat fatal bagi anak. Salah satu komplikasi infeksi SSP adalah gangguan keseimbangan natrium yang dapat menyebabkan keterlambatan diagnosis, memperberat gejala infeksi SSP, dan berkaitan dengan luaran buruk. Meskipun demikian, masih sedikit penelitian yang berupaya memprediksi gangguan keseimbangan natrium pada anak dengan infeksi SSP. Tujuan: Penelitian ini bertujuan untuk mengidentifikasi dan memprediksi gangguan keseimbangan natrium pada anak dengan infeksi SSP. Metode: Penelitian ini merupakan studi prognostik dengan rancangan penelitian kohort retrospektif di RSCM menggunakan data rekam medis dari Januari 2020-Desember 2023. Subyek yang diteliti ialah anak berusia >1 bulan sampai 18 tahun yang mengalami infeksi SSP. Prediktor yang diteliti adalah penurunan kesadaran saat masuk rumah sakit, dugaan patogen penyebab, sepsis, kelainan struktural kranioserebral sebelumnya, dan kejang sebelum masuk RS. Hasil: Terdapat 76 subyek yang mengalami infeksi SSP. Median usia subyek ialah 1,61 tahun (rentang 0,09-17,14 tahun). Proporsi lelaki dan perempuan hampir sama dengan lelaki sebanyak 39 (51,3%). Jenis infeksi SSP terbanyak ialah meningitis bakterialis (22 pasien, 28,9%). Terdapat 54 episode gangguan keseimbangan natrium pada 48 subyek (63,1%). Etiologi gangguan keseimbangan natrium diketahui pada 13 pasien dengan penyebab terbanyak ialah cerebral salt wasting (CSW) pada empat subyek. Pada analisis multivariat regresi logistik hanya penurunan kesadaran saat masuk rumah sakit yang dapat memprediksi gangguan keseimbangan natrium pada subyek dengan infeksi SSP probable dan terkonfirmasi dengan RR 1,5 (IK 95% 1,033-2,176), nilai p=0,033. Gangguan keseimbangan natrium pada infeksi SSP probable dan terkonfirmasi meningkatkan risiko kematian dengan RR 7,8 (IK 95% 1,074-56,65), nilai p=0,015. Simpulan: Penurunan kesadaran saat masuk rumah sakit merupakan prediktor gangguan keseimbangan natrium pada anak dengan infeksi SSP probable dan terkonfirmasi. Gangguan keseimbangan natrium pada populasi ini dapat meningkatkan risiko kematian secara signifikan. ......Background: Central nervous system (CNS) infection can be fatal for children. One of the complications of CNS infection is impaired sodium balance which can cause delayed diagnosis, aggravate symptoms of CNS infection, and is associated with poor outcomes. However, few studies have attempted to predict sodium balance disturbances in children with CNS infections. Objective: This study aims to identify and predict impaired sodium balance in children with central nervous system infection. Methods: This is a prognostic study with an retrospective cohort design at RSCM using medical record data from January 2020-December 2023. The subjects studied were children aged >1 month to 18 years who had CNS infections. The predictors studied were decreased consciousness at admission, suspected causative pathogen, sepsis, previous craniocerebral structural abnormalities, and seizures before admission. Results: There were 76 subjects with central nervous system infection. The median age of the subjects was 1.61 years (range 0.09-17.14 years). The proportion of males and females was almost equal with males 39 (51.3%). The most common type of CNS infection was bacterial meningitis (22 subjects, 28.9%). There were 54 episodes of sodium balance disorder in 48 subjects (63.1%). The etiology of sodium balance disorders was known in 13 patients with the most common cause was cerebral salt wasting (CSW) in four subjects. In multivariate logistic regression analysis, only decreased consciousness at hospital admission predicted sodium balance disturbance in subjects with probable and confirmed CNS infection with RR 1,5 (95% CI 1,033-2,176), p value=0,033. Impaired sodium balance in probable and confirmed CNS infection increased the risk of death with RR 7,8 (95% CI 1,074-56,65), p value=0,015. Conclusion: Decreased consciousness at hospital admission is a predictor of impaired sodium balance in children with probable and confirmed CNS infection. Impaired sodium balance in this population can significantly increase the risk of death.