Real-time Nucleic Acid Sequence-based Amplification Research Articles

Prevalence of Candida albicans in High-Risk Human Papillomavirus-Positive Women: A Study in Diyarbakır Province, Turkey.

The human papillomavirus (HPV) is a significant public health concern due to its association with the development of cervical cancer. Although inflammation caused by Candida spp. has been shown to facilitate oncogenesis, the interactions between HPV and Candida spp. remain unclear. This study aimed to determine the prevalence and genotype distribution of HR-HPV infection HR-HPV-positiveCandida albicans in HR-HPV-positive individuals in Diyarbakır province in Turkey. Cervical samples were taken from 350 participants aged 20-69 years who applied to Diyarbakır Gazi Yaşargil Training and Research Hospital, Gynecology and Obstetrics Clinic. For detection of HPV presence and HR-HPV genotyping, PCR/direct cycle sequencing was used. E6/E7 mRNA expression of HPV-16, -18, -31, -33, and -45 was determined by type-specific real-time NASBA assay (NucliSENS EasyQ(®)HPV v1.1). The presence of Candida albicans in cervical specimens of HR-HPV-positive women was investigated by RAPD-PCR and culture methods. Results. Of the 350 women who participated in the study, 24% were HPV positive and 10.5% were found to be HR-HPV positive. HR-HPV positivity was most frequently detected in the age range of 40-49 years. Among HR-HPV-positive women, C. albicans was found in 59.4%. Conclusion. The most frequent HR-HPV genotype was HPV16, followed by HPV31. Of women who tested positive for HR-HPV, C. albicans was discovered in 59.4%. C. albicans infection may occur when the immune system is weakened or the balance of the vaginal flora is disturbed, increasing tissue damage in the vaginal area and the risk of carcinogenesis of HR-HPV. Therefore, knowing the presence of Candida infection in HR-HPV-positive women is essential to plan the treatment and prevent the risk of secondary disease.

Molecular Diagnostic Tools and Malaria Elimination: A Review on Solutions at Hand, Challenges Ahead and Breakthroughs Needed

Malaria is a serious public health problem worldwide. Globally concerted efforts are underway to control and eliminate it. Despite recent slowdown, substantial achievements have been recorded in the last 20 years. However, its eradication requires successful elimination of all <i>Plasmodium</i> parasites among symptomatic, asymptomatic, and sub-microscopic infections. This review is aimed at assessing the role of molecular diagnostic tools in malaria elimination. Quality assured malaria diagnosis is fundamental to control and elimination of malaria. High-throughput molecular diagnostic tools are important for the diagnosis, and monitoring of interventions to mitigate malaria. Molecular techniques such as real-time PCR, LAMP, nPCR, RT-PCR, multiplex-PCR, NASBA, and CLIP-PCR have been instrumental for malaria control and elimination. They enabled the detection and identification of symptomatic, asymptomatic, and sub-microscopic parasitemia. They are also important in the discovery, and development of drugs. Despite their tremendous contribution and immense potential, they are not readily available in malaria-endemic settings, fail to detect hypnozoites and infectious gametocytes as well as not sufficiently optimized for fieldwork. Those challenges might delay malaria elimination thereby threatening the quest to reach the goal of a malaria-free world by 2050. Therefore, we need novel tools fit for field application and for detecting hypnozoites, infectious gametocytes, and in vitro analysis of <i>Plasmodium vivax</i>.

Evaluation of real-time NASBA assay for the detection of SARS-CoV-2 compared with real-time PCR.

Purpose In January 2020, the COVID-19 pandemic started and has severely affected all countries around the world. The clinical symptoms alone are not sufficient for a proper diagnosis. Thus, molecular tests are required. Various institutes and researchers developed real-time PCR-based methods for the detection of the virus. However, the method needs expensive equipment. In the present study, we developed a real-time NASBA assay for the detection of SARS-CoV-2.MethodsPrimers and molecular beacon probes for RdRp and N genes were designed. In silico analysis showed that primers and the probes were specific for SARS-CoV-2. The standard samples with known copy numbers of the virus were tested using the NASBA assay and an FDA-approved real-time PCR kit. A series of standard samples were prepared and tested. Clinical sensitivity, precision analysis, and clinical assessment of the assay were performed.ResultsThe limit of detection of the assay was 200 copies/mL. The clinical sensitivity of the assay was 97.64%. The intra-assay and inter-assay for both N and RdRp genes were less than 5% and 10%, respectively. Clinical assessment of the assay showed that the positive agreement rate and negative agreement rate of the assays were determined to be 97.64% and 100%, respectively.ConclusionsThe results of the present study show that the developed real-time NASBA is a sensitive and specific method for the detection of SARS-CoV-2 and is comparable with real-time PCR. NASBA is an isothermal signal amplification method, and if stand-alone fluorescent readers are available, the real-time NASBA can be used without the need for expensive thermocyclers. In addition compared to other isothermal methods like LAMP, the primer design is straightforward. Thus, real-time NASBA could be a suitable method for inexpensive SARS-CoV-2 detection.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11845-022-03046-2.

A duplex real-time NASBA assay targeting a serotype-specific gene for rapid detection of viable Salmonella Paratyphi C in retail foods of animal origin.

Salmonella enterica serovar Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were identified by comparative genomics for Salmonella Paratyphi C, SPC_0871, SPC_0872, and SPC_0908. Based on the SPC_0908 and xcd genes for testing Salmonella spp., we developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with a molecular beacon approach for the simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference from natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 cfu/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in foods of animal origin.

Rapid and simultaneous detection of Japanese encephalitis virus by real-time nucleic acid sequence-based amplification

Japaneses encephalitis (JE) is most common zoonoses caused by Japanese encephalitis virus (JEV) with a high mortality and disability rate. To take timely preventive and control measures, early and rapid detection of JE RNA is necessary. But due to characteristic brief and low viraemia, JE RNA detection remains challenging. In this study, a real-time nucleic acid sequence-based amplification (RT-NASBA) was developed for rapid and simultaneous detection of JEV. Four pairs of primer were designed using a multiple genome alignment of all JEV strains from GenBank. NASBA assay established and optimal reaction conditions were confirmed by using primers and probe on ns1 gene of JEV. The specificity and sensitivity of the assay were compared with RT-PCR by using serial RNA and virus cultivation dilutions. The results showed that JEV RT-NASBA assay was established, and robust signals could be observed in 10 min with high specificity. The limit of dectetion of RT-NASBA was 6 copies per reaction. The assay was thus 100 to 1, 000 times more sensitive than RT-PCR. The cross-reaction was performed with other porcine pathogens, and negative amplification results indicated the high specificity of this method. The novel JEV RT-NASBA assay could be used as an efficient molecular biology tool to diagnose JEV, which would facilitate the surveillance of reproductive failure disease in swine and would be beneficial for public health security.

Development of a Quantitative Real-time Nucleic Acid Sequence based Amplification (NASBA) Assay for Early Detection of Apple scar skin viroid.

An assay for detecting Apple scar skin viroid (ASSVd) was developed based on nucleic acid sequence based amplification (NASBA) in combination with real-time detection during the amplification process using molecular beacon. The ASSVd specific primers for amplification of the viroid RNA and molecular beacon for detecting the viroid were designed based on highly conserved regions of several ASSVd sequences including Korean isolate. The assay had a detection range of 1 × 104 to 1 × 1012 ASSVd RNA copies/μl with reproducibility and precision. Following the construction of standard curves based on time to positive (TTP) value for the serial dilutions ranging from 1 × 107 to 1 × 1012 copies of the recombinant plasmid, a standard regression line was constructed by plotting the TTP values versus the logarithm of the starting ASSVd RNA copy number of 10-fold dilutions each. Compared to the established RT-PCR methods, our method was more sensitive for detecting ASSVd. The real-time quantitative NASBA method will be fast, sensitive, and reliable for routine diagnosis and selection of viroid-free stock materials. Furthermore, real-time quantitative NASBA may be especially useful for detecting low levels in apple trees with early viroid-infection stage and for monitoring the influence on tree growth.

MODERN METHODS OF DIAGNOSTICS AND TREATMENT OF CHLAMYDIA INFECTION

The review reflects the current understanding of the etiology, pathogenesis, and treatment of chlamydia. The advantages of new methods for the diagnosis of chlamydial infection using methods such as real-time polymerase chain reaction (PCR-RV) and real-time NASBA transcription amplification (NASBA-Real-Time) are described. Comparison with older methods, such as bacteriological, immunomorphological and immunological, has been made; diagnostic and treatment algorithms for chlamydial infection in pregnant women have been compiled.

The development and application of reagents kit for detection RNA of the causative agents of melioidosis and glanders by transcription-based amplification (NASBA) in real-time.

The polymorphism of clinical manifestations of melioidosis and glanders and their high mortality require improvement of diagnostics for detection of this agents. The perspectivity of development of transcription-based amplification real-time NASBA diagnostic kits is determined by high analytical sensitivity and the opportunity to perfom the verification of the results of other methods for pathogenic Burkholderia species detection. The fragment of 23S rRNA gene was selected as the target for development of real-time NASBA kit. The high specificity of the constructed oligonucleotides was confirmed during the analysis of wide range of heterological strains of microorganisms and sequencing of amplified fragments of 23S rRNA gene. The analytical sensitivity of the developed kit allowed to detect Burkholderia pseudomallei and Burkholderia mallei in concentration of 1×101 microbial cells per ml. The high functional characteristics of developed kit as well as the possibility to use it in case of appereance of discordant result during the detection of pathogenic Burkholderia species were demonstrated while studying biological samples.

A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters

The presence of Escherichia coli in environmental waters is considered as evidence of faecal contamination and is therefore commonly used as an indicator in both water quality and food safety analysis. The long period of time between sample collection and obtaining results from existing culture based methods means that contamination events may already impact public health by the time they are detected. The adoption of molecular based methods for E. coli could significantly reduce the time to detection. A new quantitative real-time PCR (qPCR) assay was developed to detect the ybbW gene sequence, which was found to be 100% exclusive and inclusive (specific and sensitive) for E. coli and directly compared for its ability to quantify E. coli in environmental waters against colony counts, quantitative real-time NASBA (qNASBA) targeting clpB and qPCR targeting uidA. Of the 87 E. coli strains tested, 100% were found to be ybbW positive, 94.2% were culture positive, 100% were clpB positive and 98.9% were uidA positive. The qPCR assays had a linear range of quantification over several orders of magnitude, and had high amplification efficiencies when using single isolates as a template. This compared favourably with qNASBA which showed poor linearity and amplification efficiency. When the assays were applied to environmental water samples, qNASBA was unable to reliably quantify E. coli while both qPCR assays were capable of predicting E. coli concentrations in environmental waters. This study highlights the inability of qNASBA targeting mRNA to quantify E. coli in environmental waters, and presents the first E. coli qPCR assay with 100% target exclusivity. The application of a highly exclusive and inclusive qPCR assay has the potential to allow water quality managers to reliably and rapidly detect and quantify E. coli and therefore take appropriate measures to reduce the risk to public health posed by faecal contamination.

HPV types and E6/E7 mRNA expression in cervical samples from Turkish women with abnormal cytology in Ankara, Turkey.

Human papillomaviruses have been established as a risk factor for invasive carcinoma of the uterine cervix. HPV E6/E7 oncogene expression has recently emerged as a promising biomarker to determine the risk for progression to high-grade cervical lesions. The aim of this study was to evaluate HPV mRNA and DNA detection in samples with abnormal cytology. Cervical specimens were obtained at the Department of Obstetrics and Gynecology via cervical brushes during January-October 2011. Liquid-based cytology slides were evaluated according to the 2001 Bethesda System. Cytology specimens from a total of 81 women with abnormal cytology were included. Real-time PCR and NASBA assays were performed to detect HPV DNA and E6/E7 mRNA, respectively. HPV DNA was identified in 73 samples (90.1%). HPV E6/E7 mRNA expression was observed in 45 samples (55.6%). A statistically significant difference was observed among cytological diagnosis groups. In 25 patients, a biopsy was performed during the follow-up. HPV DNA was detected in all of these patients. HPV E6/E7 expression was present only in CIN I-III diagnosed patients. The E6/E7 mRNA test is a robust indicator of cytological atypia and correlates better with progressive lesions than DNA assays.

Effectiveness of spiramycin for treatment of experimental toxoplasmosis in rats by the Real-time NASBA method

Effectiveness of spiramycin for treatment of experimental toxoplasmosis in rats by the Real-time NASBA method

Real-time nucleic acid sequence-based amplification (NASBA) assay targeting MIC1 for detection of Cryptosporidium parvum and Cryptosporidium hominis oocysts

Both Cryptosporidium parvum and Cryptosporidium hominis are often associated with cryptosporidiosis in humans, but whereas humans are the main host for C. hominis, C. parvum is zoonotic and able to infect a variety of species. The oocyst transmission stages of both species of parasites are morphologically identical and molecular techniques, usually polymerase chain reaction (PCR), are required to distinguish between oocysts detected by standard methods in environmental samples, such as water. In this study, we developed two primer sets for real-time nucleic acid sequence-based amplification (NASBA), targeting the MIC1 transcript in C. parvum (CpMIC1) and C. hominis (ChMIC1). Using these primer sets, we were not only able to detect low numbers of C. parvum and C. hominis oocysts (down to 5 oocysts in 10 μl, and down to 1 oocyst using diluted RNA samples), but also distinguish between them. One of the primer sets targeted an exon only occurring in CpMIC1, thereby providing a tool for distinguishing C. parvum from other Cryptosporidium species. Although mRNA has been suggested as a tool for assessing viability of Cryptosporidium oocysts, as it is short-lived and may have high transcription, this NASBA assay detected MIC1 mRNA in inactivated oocysts. RNA within the oocysts seems to be protected from degradation, even when the oocysts have been killed by heating or freeze-thawing. Thus, our approach detects both viable and non-viable oocysts, and RNA does not seem to be a suitable marker for assessing oocyst viability.

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis

Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans.

A handheld sensor assay for the identification of grouper as a safeguard against seafood mislabeling fraud

Increases in international trade and global seafood consumption, along with fluctuations in the supply of different seafood species, have resulted in fraudulent product mislabeling. Grouper species, due to their high demand and varied commercial availability, are common targets for mislabeling by exploiting inefficient inspection practices. Compounding this problem is the fact that there are currently 64 species of fish from eleven different genera allowed to be labeled “grouper” per U.S. Food and Drug Administration guidelines. This wide diversity makes it difficult for regulators to discern legally salable groupers from restricted species. To obviate taxonomic misidentification when relying on external phenotypic characteristics, regulatory agencies are now employing genetic authentication methods which typically offer species-level resolution. However, standard genetic methods such as DNA barcoding require technical expertise and long turnover times, and the required instrumentation is not amenable for on-site analysis of seafood. To obviate some of these limitations, we have developed a handheld genetic sensor that employs a real-time nucleic acid sequence-based amplification assay (RT-NASBA) previously devised in our lab, for the analysis of fish tissue in the field. The base RT-NASBA assay was validated using a lab-based, benchtop RNA purification method as well as non-portable, commercial RT-NASBA analyzer. Described herein, is an uncomplicated method for purifying RNA from fish tissue in the field, which had similar efficiency to the benchtop method demonstrated through direct comparisons. We have also demonstrated that the field sensor is only slightly less sensitive than the benchtop instrument, and could discern 80.3% of groupers (no target sequence available for three species) on the 2014 FDA Seafood List from potential impostors. The complete field assay requires fewer than 80 min for completion and can be performed outside of the lab in its entirety.

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study.

BackgroundQuantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA.ResultsThe reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance.ConclusionsBayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0402-2) contains supplementary material, which is available to authorized users.

Comparative detection of rotavirus RNA by conventional RT-PCR, TaqMan RT-PCR and real-time nucleic acid sequence-based amplification

Rotavirus is one of the major viral pathogens leading to diarrhea. Diagnosis has been conducted by either traditional cultural, serological methods or molecular biology techniques, which include RT-PCR and nucleic acid sequence-based amplification (NASBA). However, their differences regarding accuracy and sensitivity remain unknown. In this study, an in-house conventional RT-PCR assay and more importantly, an in-house real-time NASBA (RT-NASBA) were established, and compared with a commercial TaqMan RT-PCR assay. The results showed that all of these methods were able to detect and distinguish rotavirus from other diarrhea viruses with a 100% concordance rate during the course of an evaluation on 20 clinical stool samples. However, RT-NASBA was much quicker than the other two methods. More importantly, the limit of detection of RT-NASBA could reach seven copies per reaction and was one to two logs lower than that of conventional RT-PCR and TaqMan RT-PCR. These results indicate that this in-house assay was more sensitive, and thus could be used as an efficient diagnosis tool for rotavirus. To the best of our knowledge, this is the first direct comparison among three different assays for the detection of rotavirus. These findings would provide implication for the rational selection of diagnosis tool for rotavirus.

Performance of HPV E6/E7 mRNA RT-qPCR for screening and diagnosis of cervical cancer with ThinPrep® Pap test samples

Recent research has shown that oncogenic human papillomavirus (HPV) DNA, which is currently used in the screening and diagnosis of cervical cancer, can be detected not only in high-grade cervical lesions, but also in low-grade cervical lesions and normal tissues. For this reason, HPV tests targeting the E6 and E7 mRNA of five oncogenic HPV strains (HPV genotypes 16, 18, 31, 33, and 45), which are known to be responsible for the oncogenesis of cervical cancer, have been commercialized using a real-time nucleic acid sequence based amplification (NASBA) assay. Previous data has shown that the real-time NASBA assay has higher clinical specificity than HPV DNA testing (97.1% vs. 53.7%). However, the sensitivity of the real-time NASBA assay was lower than that of HPV DNA testing (41.1% vs. 100%). Despite the fact that there are more than 16 oncogenic HPV genotypes known to cause cervical cancer (HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 69), the commercialized real-time NASBA kit was designed to detect only five genotypes (16, 18, 31, 33, and 45). Therefore, in the present study, CervicGen HPV RT-qDX (Optipharm), a commercial diagnostic kit targeting a HPV E6/E7 mRNA based on RT-qPCR assay was evaluated with RNA extracted from ThinPrep® Pap samples, and the results were compared to real-time NASBA data. The sensitivity and specificity of the RT-qPCR assay were 91% and 98.6%, respectively, for the detection of cervical intraepithelial neoplasia CIN2+ high-grade cervical lesions. Therefore, the CervicGen HPV RT-qDX assay showed a significantly higher sensitivity (91.1%) compared to the real-time NASBA assay (41.1%). In normal cytohistology cases, the specificity was 98.6% and 53.7% for HPV mRNA RT-qPCR and HPV DNA testing, respectively. These results demonstrate that HPV mRNA RT-qPCR better reflects clinical diagnosis. In conclusion, it is suggested that HPV mRNA RT-qPCR overcomes the shortcomings of lower specificity seen in the DNA assay and the lower sensitivity of the commercialized HPV mRNA real-time NASBA assay when testing from ThinPrep® Pap samples.

Human papillomavirus genotype distribution and E6/E7 oncogene expression in Turkish women with cervical cytological findings.

Infection with certain human papillomavirus (HPV) genotypes is the most important risk factor related with cervical cancer. The objective of the present study was to investigate the prevalence of HPV infection, the distribution of HPV genotypes and HPV E6/E7 oncogene mRNA expression in Turkish women with different cervical cytological findings in Mersin province, Southern Turkey. A total of 476 cytological samples belonging to women with normal and abnormal cervical Pap smears were enrolled in the study. For the detection and genotyping assay, a PCR/direct cycle sequencing approach was used. E6/E7 mRNA expression of HPV-16, 18, 31, 33, and 45 was determined by type-specific real-time NASBA assay (NucliSENS EasyQ(®)HPV v1.1). Of the 476 samples, 106 (22.3%) were found to be positive for HPV DNA by PCR. The presence of HPV was significantly more common (p<0.001) in HSIL (6/8, 75%) when compared with LSIL (6/14, 42.9%), ASC-US (22/74, 29.7%) and normal cytology (72/380, 18.9%). The most prevalent genotypes were, in descending order of frequency, HPV genotype 66 (22.6%), 16 (20.8%), 6 (14.2%), 31 (11.3%), 53 (5.7%), and 83 (4.7%). HPV E6/E7 oncogene mRNA positivity (12/476, 2.5%) was lower than DNA positivity (38/476, 7.9%). Our data present a wide distribution of HPV genotypes in the analyzed population. HPV genotypes 66, 16, 6, 31, 53 and 83 were the predominant types and most of them were potential carcinogenic types. Because of the differences between HPV E6/E7 mRNA and DNA positivity, further studies are required to test the role of mRNA testing in the triage of women with abnormal cervical cytology or follow up of HPV DNA positive and cytology negative. These epidemiological data will be important to determine the future impact of vaccination on HPV infected women in our region.

Molecular Methods in Microbiology and their Clinical Application

Conventional microbiology is an inexpensive but protracted diagnostic method and there are certain limitations associated with it. Molecular methods have been increasingly incorporated in laboratories, particularly for the detection and characterization of isolates and for the diagnosis of diseases due to fastidious, slow growing, nonviable or non-cultivable organisms which cannot be detected by conventional culture techniques. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. These tests provide timely results which are useful for high-quality patient care at a reasonable cost and are associated with improvement in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for micro-organisms detection, genotyping and quantification. Molecular methods are rapid and highly sensitive and specific. This review will focus on various molecular techniques which are performed in clinical microbiology laboratories and their clinical applications and therefore help in management of infectious diseases.

Comparison of the detection of HPV-16, 18, 31, 33, and 45 by type-specific DNA- and E6/E7 mRNA-based assays of HPV DNA positive women with abnormal Pap smears

This study compares the type-specific human papillomavirus (HPV) DNA test with E6/E7 mRNA detection assay because of their importance in cervical cancer screening programs. A total of 105 women with positive high-risk Hybrid Capture 2 or Abbott RealTime High Risk HPV screening test and an abnormal cervical Pap smear were enrolled in the study. HPV typing was performed by multiplex real-time PCR (HPV High Risk Typing Real-TM test). HPV-16, 18, 31, 33, and 45 E6/E7 mRNAs were determined by type-specific real-time NASBA assay (NucliSENS EasyQ® HPV v1.1). Infections caused by HPV-16, 18, 31, 33, and 45 types increased with severity of cervical cytology (p=0.008). Global positivity of five HPV E6/E7 mRNAs was lower than DNA positivity within women with atypical squamous cells of undetermined significance (p=0.016; p=0.008). High agreement of the tests was found in the groups of women with low-grade (p=1.000; p=0.063) and high-grade squamous intraepithelial lesion (p=0.250; p=0.125). Type-specific agreement of both diagnostic approaches was high regardless of cytology. Based on the found differences between HPV-16, 18, 31, 33, and 45 E6/E7 mRNA and DNA positivity, further study is needed to test the role of mRNA testing in the triage of women with atypical squamous cells of undetermined significance in Pap smear.