Specific Real-time Reverse Transcription Research Articles

Genomic Characterization of SARS-CoV-2 Isolates Obtained from Antalya, Türkiye

As the number of coronavirus diseases-2019 (COVID-19) cases have decreased and measures have started to be implemented at an individual level rather than in the form of social restrictions, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) still maintains its importance and has already taken its place in the spectrum of agents investigated in multiplex molecular test panels for respiratory tract infections in routine diagnostic use. In this study, we aimed to present mutation analysis and clade distribution of whole genome sequences from randomly selected samples that tested positive with SARS-CoV-2 specific real-time reverse transcription polymerase chain reaction (rRT-PCR) test at different periods of the pandemic in our laboratory with a commercial easy-to-use kit designed for next-generation sequencing systems. A total of 84 nasopharyngeal/oropharyngeal swab samples of COVID-19 suspected patients which were sent for routine diagnosis to the medical microbiology laboratory and detected as SARSCoV-2 RNA positive with rRT-PCR were randomly selected from different periods for sequence analysis. Library preparation for sequencing was performed with the commerical EasySeq SARS-CoV-2 RC PCR kit (Nimagen, the Netherlands). The data generated from the Illumina MiSeq system (Illumina Inc, San Diego, CA, USA) were analysed using CLC Genomics Workbench (CLC, Qiagen, Hilden, Germany). Nextstrain clades detected in order of frequency were 21J (Delta) (25%, n= 21), 21L (Omicron) (23.8%, n= 20), 20B (19%, n= 16), 20A (15.5%, n= 13), 21K (Omicron) (11.9%, n= 10), 19A (3.6%, n= 3), and 22B (Omicron) (1.2%, n= 1). Excluding one patient sample which was identified as 22B (Omicron), a total of 2829 common distinct mutations (2076 missense, 551 synonymous, 192 deletions and 10 insertions) were detected. 100 mutations were observed in the non-coding 5' untranslated region (UTR). The majority of the mutations were located in the Spike gene region (1120 mutations), followed by the ORF1a (624 mutations), nucleocapside (315 mutations) and ORF1b (263 mutations) gene regions. Sampling times of the patients were March 2020 (n= 1), April 2020 (n= 11), May 2020 (n= 1), June 2020 (n= 2), July 2020 (n= 3), August 2020 (n= 1), September 2020 (n= 5), November 2020 (n= 2), December 2020 (n= 6), December 2021 (n= 19), January 2022 (n= 11), March 2022 (n= 16), April 2022 (n= 3), and June 2022 (n= 3). As a result, in this study, SARS-CoV-2 variants and mutations in the Mediterranean Region of Türkiye, Antalya province were analyzed in detail. To the best of our knowledge, no SARS-CoV-2 genome analysis study from the pandemic period has been reported in our province. When the sequences from our study were uploaded to the GISAID Instant Audacity system and the related genomes obtained from different countries in the EpiCoV database metadata were examined, the top two countries in terms of similarity and which could be associated with the main entry route of the virus into Türkiye were Germany and the United Kingdom. In today's world, where it is discussed what can be done to be prepared for possible new pandemics based on the COVID-19 pandemic, the importance of being proactive in molecular surveillance studies is indisputable. Developing countries should be supported and encouraged to research new variants and share data in addition to known variants in pandemic control. At this point, we believe that past pandemic data reported from different geographical regions will also be valuable in terms of predicting the circulation network and taking precautions in a possible new pandemic.

Toscana Virus: Ten Years of Diagnostics in Portugal.

Toscana virus (TOSV) is an emerging sandfly-borne virus within the Phlebovirus genus. Although most infections caused by this virus present as asymptomatic or with minimal symptomatology, TOSV may emerge as a febrile disease or sporadic cases of neurological disease such as meningitis or meningoencephalitis. This pathogen is distributed throughout the Mediterranean basin, along with the spatial distribution of its recognized sandfly vector, Phlebotomus perniciosus. Portugal, after Italy, was the second country considered endemic for this virus, with the first case of acquired infection published in 1985. Although little is known about the circulation of this virus in Portugal, the laboratory diagnosis of TOSV is available at the Centre for Vectors and Infectious Diseases Research of the National Institute of Health Dr. Ricardo Jorge (CEVDI/INSA), since 2007. The aim of this study is to report the results of the diagnosis of TOSV at the CEVDI/INSA, between 2009 and 2018. The diagnosis of TOSV in the CEVDI/INSA is included in the arboviruses and vector-borne neurotropic viruses panels or can be performed, when specified, for TOSV only. Direct detection is made in cerebrospinal fluid samples and is available for TOSV by specific real-time reverse transcription polymerase chain reaction followed by conventional real-time reverse transcription polymerase chain reaction for sequencing purposes, if positive. For indirect diagnosis, performed in serum samples, an in-house immunofluorescence assay for the detection of IgM and IgG antibodies against TOSV is used. A commercial immunofluorescence assay consisting in a mosaic of four phleboviruses is also available, in which, in addition to TOSV, antibody detection for sandfly fever Naples virus, sandfly fever Sicilian virus and sandfly fever Cyprus virus can be done. All diagnostic tests requested by clinicians to the CEVDI/INSA for arboviruses, neurotropic viruses and/or TOSV between January 2009 and December 2018, were included in this study. During the study period, the CEVDI/INSA received samples from 608 patients with diagnostic requests for TOSV. Five acute TOSV infections and one acute sandfly fever Sicilian virus infection were confirmed in serum samples. Three other patients had serological evidence of previous contact with the virus. Two of the six patients with acute infection developed febrile syndrome, and the other four presented with neurological disease: meningitis (n = 2), meningoencephalitis (n = 1) and severe depression of consciousness (n =1). These infections were most likely acquired in the districts of Faro (3), Lisbon (2) and Setúbal (1). In Portugal, the number of laboratory diagnostic requests for TOSV is low when compared to the numbers of requests for other less prevalent vector-borne viruses. The Faro district presented the highest number of TOSV-specific diagnostic requests which seems to indicate a higher level of recognition by clinicians in that region. Febrile syndrome and neurological disease were the clinical manifestations that were present in acute cases. In this study, in addition to the Faro district, recent infections were also detected in the districts of Lisbon and Setúbal. It is probable that TOSV may be distributed throughout the mainland territory since its main vector is present from north to south. In 2017, the sandfly fever Sicilian virus was associated for the first time with human disease in our country, thus alerting to the circulation of this phlebovirus. Even though the number of identified cases in Portugal is low, TOSV circulates and causes disease in our country. The diagnosis of this and other phleboviruses should not be neglected in the differential diagnosis of febrile syndrome and viral meningitis and meningoencephalitis, especially during the warmer months, when the vector's activity is higher.

Variability of cycle threshold values in an external quality assessment scheme for detection of the SARS-CoV-2 virus genome by RT-PCR.

The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (Ct) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. Ct values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (Ct values) were assessed. A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting Ct values for a total of 92 different protocols. In all three positive samples, the means of the Ct values for the E-, N-, S-, RdRp-, and ORF1ab-genes varied by less than two cycles. However, 7.7% of reported results deviated by more than ±4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report Ct values along with a qualitative result. This study highlights the limitations of interpreting Ct values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of Ct values with respect to the target gene.

Building the National SARS-CoV-2 Laboratory Diagnostic Capacity in Taiwan.

Through early and proactive laboratory testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes novel coronavirus 2019, Taiwan has demonstrated an efficient and rapid control response to contain the outbreak. Two days after the World Health Organization announced the complete viral genome sequence, the national laboratory of the Taiwan Centers for Disease Control developed a specific real-time reverse transcription polymerase chain reaction (real-time RT-PCR) test for SARS-CoV-2. The national laboratory network was further strengthened through the recruitment of medical centers and regional hospitals distributed throughout most geographical regions of the country. Ultimately, a network of 60 laboratories with a capacity of 7,342 real-time RT-PCR tests per day was established. Between January 14 and August 5, 2020, a total of 158,772 tests were conducted, corresponding to 120,487 cases. Test results were obtained within 24 hours, enabling an efficient and rapid control response.

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2.

BackgroundHighly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid.MethodsA rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection.ResultsThis assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.

Human pegivirus persistence in human blood virome after allogeneic haematopoietic stem-cell transplantation

ObjectivesBecause commensal viruses are defined by the immunologic tolerance afforded to them, any immunomodulation, such as is received during haematopoietic stem-cell transplantation, may shift the demarcation between innocuous viral resident and disease-causing pathogen. MethodsWe analysed by deep-sequencing the plasma virome of 40 allogeneic haematopoietic stem-cell transplantation patients 1 month after transplantation. Because human pegivirus (HPgV) was highly prevalent, we performed a 1-year screening of 122 plasma samples by specific real-time reverse transcription PCR assay. We used the log-rank test and the Gray test to assess association with outcomes, and the Mann-Whitney test and multivariable linear regression model to assess association with T-cell reconstitution. ResultsPolyomaviruses (PyV) (20/40 patients), anelloviruses (16/40), pegiviruses (14/40) and herpesviruses (14/40) were most frequently identified, including ten cytomegalovirus; three Epstein-Barr virus; two herpes simplex virus type 1; one human herpesvirus 6b and one human herpesvirus 7; 18 Merkel cell–PyV; two BK-PyV; three PyV-6; and one JC-PyV. Papillomavirus and adenovirus were identified in 11 and two patients, respectively. The HPgV specific real-time reverse transcription PCR screening identified 51 of 122 positive samples, high virus loads and persistent infections up to 1 year after transplantation. Comparison between patients with or without HPgV infection at time of transplantation did not reveal a significant difference in infections, engraftment, survival, graft vs. host disease, relapse or immune reconstitution. ConclusionsThe blood virome after allogeneic haematopoietic stem-cell transplantation includes several DNA viruses, notably herpesviruses and PyV. Among RNA viruses, HPgV is highly prevalent and persists for several months, and it thus may deserve special attention in further research on immune reconstitution.

Evaluation of 793/B-like and Mass-like vaccine strain kinetics in experimental and field conditions by real-time RT-PCR quantification.

Infectious bronchitis virus (IBV) is a great economic burden both for productive losses and costs of the control strategies. Many different vaccination protocols are applied in the same region and even in consecutive cycles on the same farm in order to find the perfect balance between costs and benefits. In Northern Italy, the usual second vaccination is more and more often moved up to the chick's first d of life. The second strain administration together with the common Mass priming by spray at the hatchery allows saving money and time and reducing animal stress. The present work compared the different vaccine strains (Mass-like or B48, and 1/96) kinetics both in field conditions and in a 21-day-long experimental trial in broilers, monitoring the viral replication by upper respiratory tract swabbing and vaccine specific real time reverse transcription PCR (RT-PCR) quantification. In both field and experimental conditions, titers for all the vaccines showed an increasing trend in the first 2 wk and then a decrease, though still remaining detectable during the whole monitored period. IBV field strain and avian Metapneumovirus (aMPV) presence also was also investigated by RT-PCR and sequencing, and by multiplex real-time RT-PCR, respectively, revealing a consistency in the pathogen introduction timing at around 30 d, in correspondence with the vaccine titer's main decrease. These findings suggest the need for an accurate knowledge of live vaccine kinetics, whose replication can compete with the other pathogen one, providing additional protection to be added to what is conferred by the adaptive immune response.

SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes

BackgroundThe monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures.ResultsWe developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes.ConclusionsBeing a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.

Astrovirus MLB2, a New Gastroenteric Virus Associated with Meningitis and Disseminated Infection.

Next-generation sequencing has identified novel astroviruses for which a pathogenic role is not clearly defined. We identified astrovirus MLB2 infection in an immunocompetent case-patient and an immunocompromised patient who experienced diverse clinical manifestations, notably, meningitis and disseminated infection. The initial case-patient was identified by next-generation sequencing, which revealed astrovirus MLB2 RNA in cerebrospinal fluid, plasma, urine, and anal swab specimens. We then used specific real-time reverse transcription PCR to screen 943 fecal and 424 cerebrospinal fluid samples from hospitalized patients and identified a second case of meningitis, with positive results for the agent in the patient's feces and plasma. This screening revealed 5 additional positive fecal samples: 1 from an infant with acute diarrhea and 4 from children who had received transplants. Our findings demonstrate that astrovirus MLB2, which is highly prevalent in feces, can disseminate outside the digestive tract and is an unrecognized cause of central nervous system infection.

Reverse transcription loop-mediated isothermal amplification for rapid and quantitative assay of covert mortality nodavirus in shrimp.

A disease known as covert mortality disease has become an increasing problem in the shrimp farming industry in recent years in China and several countries of Southeast Asia, leading to serious losses in production. Litopenaeus vannamei (also known as Pacific white shrimp) is affected by this disease that leads to a range of clinical symptoms including hepatopancreas atrophy and necrosis, soft shell, slow growth, and abdominal muscle whitening and necrosis in the acute stage of disease. A new nodavirus, termed covert mortality nodavirus (CMNV), has been shown to be the etiological agent. In this study, we report a sensitive and specific real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and quantitative detection of CMNV. The optimal conditions for this newly developed RT-LAMP reaction were found to be 6mM MgCl2 and 1.6mM dNTPs, an incubation temperature of 65°C and a reaction time of 50min. The analytical sensitivity of the RT-LAMP assay was estimated to be 6.3pg total RNA of CMNV-infected shrimp and 27 copies of the target plasmid. The diagnostic sensitivity and specificity of the newly developed assay versus the standard nested reverse transcription PCR (RT-PCR) assay was 96.4% and 94.4%, respectively. The reaction products were detected by visual inspection after staining with an in-tube DNA fluorescent dye, a measure taken to eliminate the risk of contamination. The quantitative RT-LAMP assay for CMNV showed high correlation coefficient (r2=0.9953) when the initial templates were above 1000 copies, however the correlation coefficient decreased when the initial templates were lower than 1000 copies. Test of viral load in shrimp indicated that the viral loads varied from 1.5×102 to 6.7×106 copies per mg of cephalothorax tissue. Thus, the CMNV RT-LAMP assay is a sensitive and specific new tool for the field detection and quantification of CMNV in the diagnosis and surveillance of covert mortality disease.

Validación de una técnica de RT-PCR en tiempo real para la detección del virus de fiebre aftosa y evaluación de su desempeño en la infección aguda

A specific real time reverse transcription polymerase chain reaction (RT-PCRrt) for the detection of foot-and-mouth disease virus was validated using the LightCycler thermocycler 2.0 and its reagents as recommended by the World Organization for Animal Health and was assessed for the detection of the virus in acute infection of cattle experimentally vaccinated and challenged with virus A Argentina/2001 or A24 Cruzeiro. The technique proved to be robust, showing coefficients of variation lower than 4% for different ARN extractions, days or repetitions and was able to detect up to 0,4 TCID 50%, and/or up to 100 RNA molecules. In probang samples, diagnostic sensitivity was 93.1 (95% CI 86.5–96.6) and diagnostic specificity 100 (95% CI 96.3-100). The results of the challenge in vaccinated or multivaccinated bovines showed that although there were high levels of clinical protection in the vaccinated group, FMDV could be detected in all challenged groups. However, detection was 100 times lower in immunized animals.

Development of a Usutu virus specific real-time reverse transcription PCR assay based on sequenced strains from Africa and Europe

Usutu virus (USUV) has been isolated in several African and European countries mainly from mosquitoes and birds. However, previous benign and two recent severe cases of human infections point out the need of a tool for the identification of USUV in human samples. A published real-time reverse transcription (RT) PCR assay for the detection of USUV in human blood or cerebrospinal fluid does not take into account the genetic variability of USUV in different geographic regions. Therefore, this article presents a quantitative real-time RT-PCR assay based on sequences from Europe and Africa. Primers and probe were designed in conserved regions among USUV strains that differed from closely related flaviviruses. The specificity of the assay was investigated by testing 16 other flaviviruses circulating in Africa. The sensitivity was determined by testing serial dilutions of virus and RNA standard. Intra- and inter-assay coefficients of variation were evaluated by 10 reactions in a same and in different assays, respectively. The assay provides high analytical specificity for USUV and detection limits of 1.2pfu/reaction for virus dilutions in L-15 medium or human serum and 60 copies/reaction for the RNA standard. The assay needs to be evaluated in a clinical context and integrated in standard diagnosis of flaviviral diseases.

Development and evaluation of a novel TaqMan fluorescence probe-based real-time reverse transcriptase polymerase chain reaction assay for detection and quantification of West Nile virus

In order to improve and accelerate the detection of West Nile virus (WNV), a rapid and specific real-time reverse transcription polymerase chain reaction (rtRT-PCR) was established. Primers and probe were designed according to the conservative sequence of capsid protein gene of WNV. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of rtRT-PCR. The amplifying curve showed that this method could successfully amplify 101 copies/μl WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. The assay system showed high reproducibility with coefficient of variation (CV) < 2%. The detection of WNV can be completed within 2 to 3 h. By detecting cDNA samples (n = 55) with rtRT-PCR and the conventional PCR assay, the established rtRT-PCR showed 96.36% (37 + 16/55) coincidence rate with the conventional PCR. All the results showed that the newly established rtRT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV. Key words: West Nile virus, capsid protein gene, real-time RT-PCR.

Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus

Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) > or = 93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.

Detection and identification of human metapneumovirus by real time reverse transcription PCR

To develop a rapid, sensitive and specific real time reverse transcription PCR for detecting and identifying human metapneumovirus. The Hmpv-L gene of human metapneumovirus was chosen as target gene, the primers and TaqMan probe were designed, and the PCR reaction was optimized systematically. The total RNA was extracted from respiratory specimens, and reverse transcription was performed through random primer. The cDNA was detected by using real time PCR. The specificity, sensitivity and reproducibility of real time PCR were estimated. The real time PCR was applied to detect 180 clinical respiratory specimens. The human metapneumovirus can be detected using real time reverse transcription PCR accurately and quickly, and the sensitivity was 1 copy/microl. The coefficient of variation of intra-assay and inter-assay was less than 5%. Among those 180 specimens, 28 (15.56%) were positive for human metapneumovirus, the clinical diagnoses for these 28 patients were pneumonia (15.60%, 17/109) and bronchiolitis (15.49%, 11/71). 21 positive specimens were from patients under 2 years of age, and 6 positive specimens were from patients between 2 and 5 years of age, only 1 positive specimens was from patients over 5 years. It is demonstrated that real time reverse transcription PCR is a reliable, accurate and feasible assay for human metapneumovirus, which has become one of the most important pathogens induced acute respiratory infections in pediatric patients.

Feasibility and clinical significance of real-time quantitative RT-PCR assay of PML-RARalpha fusion transcript in patients with acute promyelocytic leukemia.

To study the relationship between the expression level of the PML-RARalpha fusion transcripts and the clinical status and efficiency of the therapy in acute promyelocytic leukemia (APL) patients, we applied a very sensitive and specific real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) system to quantify the dose of PML-RARalpha fusion transcripts in a series of APL patients at distinct disease stages. A total of 31 APL patients (19 males and 12 females; aged from 8 to 74 years) from eight hospitals in Shanghai were analysed. Real-time Quantitative RT-PCR was used to measure the normalized dose (DoseN) of PML-RARalpha fusion transcripts. A wide range of PML-RARalpha DoseN above 1 x 10(3) was noted in 25 newly diagnosed patients. PML-RARalpha DoseN was significantly decreased after remission induction with ATRA, ATRA/chemotherapy or As2O3 and further reduced after consolidation. The fact that all patients with long disease free survival had a constantly low PML-RARalpha DoseN below 2 x 10(2) and a higher level predicted impending relapse suggests that this value could serve as a 'threshold' for molecular remission. PML-RARalpha DoseN was also of prognostic value in a group of relapsed patients, since good response to As2O3 reinduction was accompanied by a remarkable reduction of fusion transcript level, whereas patients with high PML-RARalpha Dose(N) after the second CR tended to relapse again rapidly. These results confirm that real-time RT-PCR assay for PML-RARalpha transcripts in APL patients is useful in reflecting leukemic burden, assessing response to treatment and indicating the ultimate clinical outcome or curability of disease.