In-house Polymerase Chain Reaction Assay Research Articles

A novel nonsense variant in RHAG underlies a Nordic Rhnull phenotype.

The extremely rare Rhnull phenotype is characterized by the absence of all Rh antigens on erythrocytes. It is divided into the regulator and amorph types based on the underlying genetic background. The more common regulator type depends on critical variants silencing RHAG, which encodes RhAG glycoprotein, necessary for RhD/RhCE expression. Rhnull cells have altered expression of glycophorin B and LW glycoprotein. Four unrelated Rhnull individuals were investigated. Serological testing was performed according to standard blood bank practice. RHD/RHCE and S/s allele-specific Polymerase chain reaction (PCR) genotyping was done on genomic DNA using in-house PCR assays. RHAG, and in some cases also RHD/RHCE, were sequenced. Initial s phenotyping results triggered additional serological investigation. Anti-Rh29 was identified in all four individuals. Extended typing with anti-S and anti-s showed that the three samples predicted to type as s+ failed to react with 2 of 5 anti-s. Sequence analysis of all 10 RHAG exons and the immediate intron/exon boundaries revealed a single nucleotide variant in the 3'-end of intron 6, c.946 -2a>g in all samples. RHD/RHCE showed no alterations. A novel Nordic Rhnull allele was identified. In addition, it was shown that s+ Rhnull red blood cells are not only U- but also have qualitative changes in their s antigen expression.

Detection of human papillomavirus in fresh and dried urine through an automated system for cervical cancer screening in low- and middle-income countries.

The majority of cervical cancer cases and associated deaths occur in low- and middle-income countries (LMICs), where sociocultural barriers, poor access to prevention and care, and technical and practical difficulties hinder screening coverage improvement. Using urine specimens for human papillomaviruses (HPV) molecular screening through automated testing platforms can help to overcome these problems. We evaluated the high-risk (HR) HPV detection performance of the Xpert® HPV test on GeneXpert® System (Cepheid), on fresh and dried urine (Dried Urine Spot [DUS]) samples as compared to an in-house polymerase chain reaction (PCR) genotyping assay. Forty-five concentrated urine samples collected from women with known cytological and HPV infection status, determined through in-house PCR and genotyping assays, were tested "as is" and as DUS with the Xpert® HPV test. This system detected HR-HPV in 86.4% of fresh and in 77.3% of dried urine samples collected from HPV+ women, correctly identifying HR-HPV infection in 100% of women with low- and high-grade lesions. High concordance (91.4%, k = 0.82) was found between PCR test and Xpert® HPV Test from urine. Urine-based Xpert® HPV test seems to be a suitable screening test for detection of HR-HPV infections associated with low- and high-grade lesions requiring follow-up monitoring or treatment. This methodology, relying on noninvasively collected samples and on available rapid testing platforms, could facilitate large, at-scale screening programs, particularly in LMICs and rural areas, thus reducing adverse outcomes of HPV infection and facilitating achievement of the WHO cervical cancer elimination goal.

Comparative Evaluation of Luminex xTAG® Gastrointestinal Pathogen Panel and Direct-From-Stool Real-Time PCR for Detection of C. difficile Toxin tcdB in Stool Samples from a Pediatric Population.

Detection of Clostridioides difficile toxins in patients with gastroenteritis has increasingly been accomplished through the use of enteric multiplex syndromic panels. Comparisons of the performance of these panels to both direct-from-stool (DFS) and culture-enriched stools followed by polymerase chain reaction (PCR) methods in pediatric populations are limited. Here, we compare the performance of the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) to our DFS in-house real-time PCR (DFS RT-PCR) assay for the detection of C. difficile toxin gene, tcdB, using 2641 stool specimens collected from children enrolled in the Alberta Provincial Pediatric EnTeric Infection Team (APPETITE) study in Alberta, Canada. We used culture enrichment followed by in-house RT-PCR to resolve discordant results between the two assays. We found excellent agreement (k = 0.89) between the GPP and our DFS RT-PCR assay: the positive percent agreement between the two assays was 97%, and the negative percent agreement was 99%. GPP, a multi-analyte platform can easily be implemented into a routine diagnostic laboratory for detecting enteric pathogens including C. difficile.

Melting Curve-based Assay as an Alternative Technique for the Accurate Detection of SARS-CoV-2.

Background:Early and cost-effective diagnosis and monitoring of the infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critically important to anticipate and control the disease. We aimed to set up a SYBR Green-based one-step real-time polymerase chain reaction (PCR) as a lower-cost alternative method to detect the virus.Materials and Methods:An in-house SYBR Green-based PCR assay targeting the envelope (E) and RNA-dependent RNA polymerase (RdRp) genes, was set up to diagnose the infection, and was compared with the reference probe-based PCR method.Results:When the commercial probe-based assay was considered as the reference method, SYBR Green-based PCR had a slightly lower sensitivity (81.98% and 86.25% for E and RdRp targets, respectively) and a good specificity (100% and 94.44% for E and RdRp targets, respectively). For both gene targets, three different melting temperature (Tm) patterns were found in the PCRs of the nasopharyngeal/oropharyngeal swab samples, but no size polymorphism was seen in agarose gel electrophoresis.Conclusion:Further studies to improvement of the assay are needed to make it an inexpensive and reliable tool for the diagnosis of COVID-19.

Diagnosis of Bacterial Meningitis and AMR Profile Using Molecular and Immunological Techniques

Background: Bacterial meningitis (BM) is severe complication of central nervous system (CNS) and is often associated with high mortality and morbidity rates if not timely diagnosed and treated. Current diagnostic tools for BM and drug resistance suffer from lack of sensitivity due to paucibacillary nature of Cerebrospinal fluid (CSF).
 Objectives: The objective of the study is to develop rapid and efficacious immunological tools (Enzyme linked immunosorbent assay and Lateral flow) for diagnosis of BM in hospital settings. In addition, in-house molecular assays will be developed for diagnosis of extended spectrum beta lactamases and Carbapenems drug resistance in community and hospital acquired BM infection
 Methodology: A prospective observational study will be carried out in patients admitted in IPD awards of CIIMS, Nagpur. Diagnosis of BM will be done using conventional gold standards and by in-house designed nested polymerase chain reaction (PCR) for 8 etiological pathogens. An in-house ELISA assay will be standardized and optimized based on culture filtrates antigen isolated from clinical isolates of etiological pathogens associated with BM for immunological diagnosis. For diagnosis of drug resistance, in-house conventional PCR assay targeting ESBL (TEM, SHV and CTX-M) and Carbapenems (NDM, OXA-48, VIM) resistance genes will be standardized and optimized. The sensitivity and specificity of developed test will be determined by standard ROC curve using medical software. The diagnostic accuracy will be determined based on concordance with gold standards.
 Expected results: A simple, rapid ELISA assay will be developed for etiological diagnosis BM in hospital settings. In addition, in house molecular assay will be developed for rapid identification of ESBL and Carbapenems drug resistance in BM cases.
 Conclusion: A simple, rapid, efficacious immunological and molecular tools for diagnosis of BM and drug resistance will be developed for improved management of cases in hospital settings as governance of bacterial meningitis in India is poor.

Lyme neuroborreliosis in Swedish children\u2014PCR as a complementary diagnostic method for detection of Borrelia burgdorferi sensu lato in cerebrospinal fluid

The aim of this study was to evaluate polymerase chain reaction (PCR) as a diagnostic method for the detection of Borrelia burgdorferi s.l. in CSF of Swedish children with LNB. This study was performed retrospectively on CSF and serum samples collected from children evaluated for LNB (n = 233) and controls with other specific neurological disorders (n = 59) in a Swedish Lyme endemic area. For anti-Borrelia antibody index, the IDEIA Lyme Neuroborreliosis kit (Oxoid) was used. Two in-house real-time PCR assays targeting the 16S rRNA gene were evaluated (TaqMan® and LUX™). Among patients classified as LNB cases (n = 102), five children (5%) were Borrelia PCR-positive in CSF with the TaqMan® assay. In the Non-LNB group (n = 131), one patient was Borrelia PCR positive with the TaqMan® assay. Among controls (n = 59), all CSF samples were PCR negative. When amplifying and sequencing ospA, we found B. garinii (n = 2), B. afzelii (n = 2), B. bavariensis (n = 1), and one untypable (n = 1). With the LUX™ technology, all CSF samples were PCR negative. The TaqMan® assay could detect only few cases (n = 6) of B. burgdorferi s.l. in CSF among children with LNB and the sensitivity was very low (5%). However, using larger CSF volumes and centrifugation of samples, the PCR technique could still be useful as a complementary diagnostic method when evaluating LNB. Furthermore, detection of spirochete DNA in clinical matrices, including CSF, is the method of choice for studying epidemiological aspects of LNB, a tick-borne emerging disease.

Standardization of an in-house multiplex real-time polymerase chain reaction for the simultaneous detection of Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex Virus 1 and 2, and Treponema pallidum infection among pregnant women.

An in-house multiplex real-time polymerase chain reaction (PCR) was developed in two cocktails for the identification of six Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and Treponema pallidum (syphilis) (TORCH-S) agents, which causes congenital infection among pregnant women. Standardization and validation of an in-house multiplex real-time PCR assay for the detection of TORCH-S infection. This study was conducted from February 2017 to February 2019. Primers specific for T. gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and T. pallidum were designed using Primer3 software (https://bioinfo.ut.ee/primer3-0.4.0/). The primer sequences obtained were subjected to BLAST analysis using BLAST database. Synthetic DNA was obtained to use as positive control templates for all the six TORCH-S agents. The lower limit of the detection was performed using plasmid construct for each virus serially diluted from 10-1 to 10-9. An in-house multiplex real-time PCR was standardized and validated in two cocktails for TORCH-S agents, cocktail-1 (HSV1, rubella, and T. gondii), and cocktail-2 (HSV2, CMV, and T. pallidum). The lower limit of the detection for HSV1, rubella, and Toxoplasma were 60.7 copies/10 μl input, 76.4 copies/10 μl input, and 34.4 copies/10 μl input and for HSV2, CMV, and T. pallidum were 80.8 copies/10 μl input, 166 copies/10 μl input, and 43.7 copies/10 μl input, respectively. TORCH-S infection is one of the significant reasons for irregular pregnant outcomes. It is absolutely important to screen TORCH-S infection for women who had the histories of abnormal pregnancies to prevent birth defects and perinatal complications. This multiplex real-time PCR assay provides a rapid, sensitive, and specific technique to detect these six TORCH-S agents.

Real-time PCR assay for detecting Lactobacillus plantarum group using species/subspecies-specific genes identified by comparative genomics

The Lactobacillus plantarum group is composed of four closely related species or subspecies. To clarify the L. plantarum group classification and accurately provide health benefits of probiotics, it is important to distinguish them correctly. In this study, species- or subspecies-specific genes were identified to distinguish the L. plantarum group, and a real-time polymerase chain reaction (PCR) assay was developed to accurately detect them. Specific genes, such as protein-coding genes present in all strains of target species or subspecies but not in other bacterial strains, were explored from 70 genome sequences using comparative genomics. As a result, genes encoding a transporter, major facilitator family protein, and hypothetical proteins were confirmed as specific genes. Specificity of primers was evaluated with 55 lactic acid bacteria, and all primers showed 100% specificity. Probiotic products and fermented food samples were used to validate the developed real-time PCR assay. Of the probiotic products, one product was found to contain L. pentosus, even though it was labeled to contain L. plantarum. Using our in-house developed real-time PCR assay, the products labeled as L. plantarum were identified at the subspecies level, and the L. plantarum group species or subspecies in fermented food samples were successfully qualified and quantified.

DETECTION OF CANINE PARVOVIRUS TYPE 2 BY A COMMERCIALLY AVAILABLE IN-HOUSE PCR

Canine parvovirus type 2 (CPV-2) is an important pathogen that causes anorexia, vomiting, acute gastroenteritis, and bloody diarrhea. CPV-2 was divided into three variants (2a–2c) based on residue 426 of the VP2 protein. Recently, a novel Asian CPV-2c isolate was more prevalent in the Asian continent. The diagnostic tools of CPV-2 are characterized into traditional (such as immunochromatography test) and molecular methods based on their pathogen detection mechanisms. However, the low sensitivity of the immunochromatographic tests is supported by their simplicity and rapid ability to provide results for CPV-2. Surprisingly, some immunochromatography tests showed low sensitivity for the detection of novel CPV-2c compared to a real-time polymerase chain reaction (PCR) assay. Sixty rectal swabs were collected from naturally infected dogs with CPV-2 at the Animal Disease Diagnostic Center, National Pingtung University of Science and Technology, Taiwan, from 2016 to 2019. A novel in-house QubeMDx PCR system was used for diagnosis of three CPV-2 variants and compared to real-time PCR diagnosis. This diagnostic assay detected all of CPV-2 variants within 30 min and the sensitivity was identical to a real-time PCR assay using clinical samples. This study provides evidence that this novel, commercially available in-house PCR assay (QubeMDx PCR system) provides useful, simple, sensitive, reliable, and rapid diagnosis of three CPV-2 variants in veterinary clinics.

A Seven-Year Evaluation of Viral Central Nervous System Infections

Identification of viral agents causing central nervous system (CNS) infections increased by the application of nucleic acid tests. In this study, the results of polymerase chain reaction (PCR) for viral agents were evaluated in cerebrospinal fluid (CSF) samples taken from patients with CNS infection. CSF samples taken from 1185 patients between 2010 and 2017 were tested for the presence of Herpes simplex virus (HSV) type 1 and 2, Varicella Zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus ve enterovirus by PCR in Dokuz Eylul University Hospital. Tests were performed according to the clinicians' orders and results were evaluated retrospectively. The number of tests performed were 1038 for HSV, 882 for adenovirus, 865 for enterovirus, 496 for VZV, 100 for EBV and 92 for CMV. Commercial tests were used for EBV, CMV (Artus QS-RGQ Kits, Qiagen, Germany) and enterovirus (GeneXpert, Cepheid, USA) while the other viruses (HSV, VZV, adenovirus) were tested by in-house real-time PCR assays. Ninety-one CSF (7.7%) samples were positive. The mean age was 13 (<1 to 76 years) while median was seven. The most frequently detected pathogens were enterovirus (63/91, 69%) and HSV-1 (14/91,15%). The number of patients positive for adenovirus, VZV, EBV and CMV were five, four, three and two, respectively. In one patient, both enterovirus (Ct: 29.5) and EBV (Ct: 38.53) were positive. The number of positive samples were increased in summer months. Enterovirus RNA positive patients (n= 60/63, 95.2%) were ≤ 18 years old while 29% were younger than one year of age. Enterovirus positive samples peaked in 2012 and 2014 and detected mainly in summer (60.3%) and autumn (20.6%) months. VZV was mostly detected in patients greater than 65 years of age. Mean Ct of the positive reactions was 31.87 ± 3.5 (22.88-40.32). The lowest and the highest Ct values were detected in HSV-1 assay. The mean Ct value of enterovirus assay (30.4; 25.7-35.9) was lower than the other pathogens' values. In the seven-year period, 7.7% of the1185 patients' CSF samples were positive for viral nucleic acids. As expected, enteroviruses were the most common pathogens in children and detected mainly in summer-autumn period. Syndromic approach in CNS infections could increase the viral pathogen detection.

Identification of FLT3 and NPM1 Mutations in Patients with Acute Myeloid Leukaemia.

Objective:The most frequent acquired molecular abnormalities and important prognostic indicators in patients with Acute Myeloid Leukaemia (AML) are fms-like tyrosine kinase-3 gene (FLT3) and nucleophosmin-1 (NPM1) mutations. Our study aims to develop a cost effective and comprehensive in-house conventional PCR method for detection of FLT3-ITD, FLT3-D835 and NPM1 mutations and to evaluate the frequency of these mutations in patients with cytogenetically normal (CN) AML in our population. Methods:A total of 199 samples from AML patients (95 women, 104 men) were included in the study. Mutation analyses were performed using polymerase chain reaction (PCR) and gene sequencing. Result:Sixty-eight patients were positive for the mutations. FLT3-ITD mutations were detected in 32 patients (16.1%), followed by FLT3-D835 in 5 (2.5%) and NPM1 in 54 (27.1%). Double mutations of NPM1 and FLT3-ITD were detected in 23 cases (11.6%). Assays validation were performed using Sanger sequencing and showed 100% concordance with in house method. Conclusion:The optimized in-house PCR assays for the detection of FLT3-ITD, FLT3-D835 and NPM1 mutations in AML patients were robust, less labour intensive and cost effective. These assays can be used as diagnostic tools for mutation detection in AML patients since identification of these mutations are important for prognostication and optimization of patient care.

Implementation and evaluation of the Presto combined qualitative real-time assay for Chlamydia trachomatis and Neisseria gonorrhoeae in Rwanda.

BackgroundThe Presto combined qualitative real-time assay for Chlamydia trachomatis and Neisseria gonorrhoeae (Presto CT/NG PCR assay) is appealing for developing countries, because it can be used with multiple DNA extraction methods and polymerase chain reaction (PCR) platforms.ObjectivesThe objective of the study was to implement and evaluate the Presto CT/NG PCR assay at the National Reference Laboratory (NRL) in Kigali, Rwanda, where no real-time PCR assays for the detection of C. trachomatis or N. gonorrhoeae were available.MethodsThe Presto CT/NG PCR assay was first evaluated at the Institute of Tropical Medicine (ITM) in Antwerp, Belgium. Next, NRL laboratory technicians were trained to use the assay on their ABI PRISM 7500 real-time PCR instrument and their competencies were assessed prior to trial initiation. During the trial, endocervical swabs were tested at the NRL, with bi-monthly external quality control testing monitored by the ITM. The final NRL results were evaluated against extended gold standard testing at the ITM, consisting of the Abbott m2000 RealTime System with confirmation of positive results by an in-house real-time PCR assay for C. trachomatis or N. gonorrhoeae.ResultsOf the 192 samples analysed using the Presto assay at the NRL, 16 samples tested positive for C. trachomatis and 17 tested positive for N. gonorrhoeae; four of these were infected with both. The sensitivity and specificity of the Presto assay were 93.3% (95% confidence interval [CI]: 68.1% – 99.8%) and 99.4% (95% CI: 96.8% – 100%) for C. trachomatis and 100% (95% CI: 76.8% – 100%) and 98.8% (95% CI: 95.8% – 99.9%) for N. gonorrhoeae.ConclusionC. trachomatis and N. gonorrhoeae testing with the Presto assay was feasible in Kigali, Rwanda, and good performance was achieved.KeywordsqPCR; Chlamydia trachomatis; Neisseria gonorrhoeae.

The short-chain dehydrogenases/reductases (SDR) gene: A new specific target for rapid detection of Mycobacterium tuberculosis complex by modified comparative genomic analysis

BackgroundEarly detection of tuberculosis is one of the crucial steps for TB control. Although, the sensitivity of conventional methods like Lowenstein Jensen (LJ) culture and direct staining is quite low, molecular techniques like polymerase chain reaction (PCR) are more sensitive and be considered as useful tools for rapid detection of tuberculosis. Various genes like IS6110 and mpb64 have been used as target for detection of M. tuberculosis, but more research is needed to find the most specific targets.The short-chain dehydrogenases/reductases family (SDR) is one of a very large family of NAD- or NADP-dependent oxidoreductase enzymes which is present in all M. tuberculosis strains. The large part of SDR sequences in tuberculosis is completely conserved and different from non-tuberculosis mycobacterium. The aim of the study was to develop an in-house PCR assay using the SDR target for rapid detection of M. tuberculosis from clinical specimens. MethodM. tuberculosis-specific sequences were found using modified genome comparison method and the primers were designed by the Primer Premier 5.0 software. A PCR assay was developed targeting the nucleotide sequences within the SDR gene. A total of 50 cultivated specimens and 120 clinical specimens were evaluated by PCR. ResultsThe clinical evaluation of SDR PCR assay showed high specificity (100%) and high sensitivity (88.5%). The analytical sensitivity was 10 fg of template DNA which is theoretically equivalent to 2 copy of genomic DNA per microliter. The SDR is a new specific target of M. tuberculosis and no cross-reactivity was observed to non-tuberculosis mycobacterium and other pathogenic bacteria. ConclusionsBased on our results, the SDR gene can be considered as a useful target for detection of M. tuberculosis complex from clinical specimens.

Health service impact of testing for respiratory pathogens using cartridge-based multiplex array versus molecular batch testing

There is increasing demand for access to rapid microbiological testing, with a view to improving clinical outcomes. The possibility of rapid testing has been facilitated by development of cartridge-based random access molecular technologies that are now widely available. Whether the expense of cartridge-based assays is justified in terms of clinical or laboratory cost savings is controversial. This prospective study evaluated the impact of the Biofire FilmArray Respiratory Panel (‘FilmArray’), a cartridge-based random access molecular test, compared with standard batched molecular testing using an ‘in-house’ respiratory polymerase chain reaction (PCR) on laboratory and health service outcomes for adult patients at a tertiary-level adult hospital in Melbourne, Australia.Laboratory result turnaround time was significantly reduced with the FilmArray (median 4.4 h) compared to a standard validated in-house respiratory PCR assay (median 21.6 h, p < 0.0001) and there was a significant increase in diagnostic yield with the Filmarray (71/124, 57.3%) compared to in-house PCR (79/200; 39.5%; p = 0.002). Despite improved result turnaround time and increased diagnostic yield from testing, there was no corresponding reduction in hospital length of stay or use of isolation beds.Although cartridge-based molecular testing reduced turnaround time to result for respiratory pathogen testing, it did not impact on health service outcomes such as hospital length of stay. Further work is warranted to determine whether cartridge-based tests at the point of care can improve clinical and health service impacts.

Prevalence of Mycoplasma genitalium and mutations associated with macrolide and fluoroquinolone resistance in Finland.

The aim was to examine the prevalence of Mycoplasma genitalium and to determine the prevalence of mutations leading to resistance to macrolides and fluoroquinolones in a sexually transmitted infection clinic setting in Finland, and as a service evaluation, to validate the performance of a commercial Aptima® Mycoplasma genitalium assay. Urogenital samples were studied for M. genitalium with an automated commercial Aptima® Mycoplasma genitalium assay on the Panther® system (Hologic), and with an in-house real-time polymerase chain reaction (PCR) (mgpB). Positive specimens were further studied for mutations associated with macrolide resistance within the 23S rRNA gene and the known quinolone resistance-determining regions within genes gyrA, gyrB and parC. Altogether 17/303 (5.6%) of samples contained M. genitalium by either test. Two of the samples positive by the Aptima assay were not detected by the in-house PCR assay, although the internal control (beta-globin gene) was amplified. The Aptima assay gave an invalid result for five samples, all of which were negative by the in-house PCR. Mutations resulting in macrolide resistance were detected in 30.8% of M. genitalium-positive specimens. Prevalence of M. genitalium infections in the specimens tested is similar to that in other parts of Europe, 5.6%. The Aptima® Mycoplasma genitalium assay detected slightly more positives than the in-house PCR assay. Mutations resulting in macrolide resistance were common in M. genitalium and detection of these mutations is recommended in diagnostic laboratories to assist in selection of treatment.

The Very Low Frequency of Epstein-Barr JC and BK Viruses DNA in Colorectal Cancer Tissues in Shiraz, Southwest Iran.

Viruses including Epstein-Barr virus (EBV), JCV and BKV have been reported to be associated with some cancers. The association of these viruses with colorectal cancers remains controversial. Our objective was to investigate their infections association with adenocarcinoma and adenomatous polyps of the colon. Totally, 210 paraffin-embedded tissue specimens encompassing 70 colorectal adenocarcinoma, 70 colorectal adenomatous and 70 colorectal normal tissues were included. The total DNA was extracted, then qualified samples introduced to polymerase chain reaction (PCR). The EBV, JCV and BKV genome sequences were detected using specific primers by 3 different in-house PCR assays. Out of 210 subjects, 98 cases were female and the rest were male. The mean age of the participants was 52 ± 1.64 years. EBV and JCV DNA was detected just in one (1.42%) out of seventy adenocarcinoma colorectal tissues. All adenomatous polyp and normal colorectal tissues were negative for EBV and JCV DNA sequences. Moreover, all the patients and healthy subjects were negative for BKV DNA sequences. The results suggested that EBV and JCV genomes were not detectable in the colorectal tissue of patients with colorectal cancer in our population. Hence, BKV might not be necessitated for the development of colorectal cancer. The findings merit more investigations.

Development of a real-time PCR assay and comparison to CHROMagarTM STEC to screen for Shiga toxin-producing Escherichia coli in stool, Cape Town, South Africa.

IntroductionShiga toxin-producing Escherichia coli (STEC) is an emerging infectious pathogen which could lead to haemolytic uremic syndrome. Even though previous studies have compared the performance of CHROMagarTMSTEC to real-time polymerase chain reaction (PCR) in Europe, no study has been done to assess its performance on African isolates.ObjectivesThis project aimed to validate and test an in-house-developed duplex real-time PCR and use it as a reference standard to determine the performance of CHROMagarTMSTEC on African isolates from diarrhoeic stool samples.MethodsThis study evaluated STEC diagnostic technology on African isolates. An in-house-developed duplex real-time PCR assay for detection of stx1 and stx2 was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of CHROMagarTMSTEC. Real-time PCR was used to screen for stx in tryptic soy broth and the suspected STEC isolates, while conventional PCR was used to detect the other virulence genes possessed by the isolates.ResultsThe real-time PCR limit of detection was 5.3 target copies/μL of broth. The mean melting temperature on melt-curve analysis for detection of stx1 was 58.2 °C and for stx2 was 65.3 °C. Of 226 specimens screened, real-time PCR detected stx in 14 specimens (6.2%, 95% confidence interval = 3.43% – 10.18%). The sensitivity, specificity, negative predictive value and positive predictive value of the CHROMagarTMSTEC were 33.3%, 77.4%, 95.3% and 11.3%.ConclusionsThe in-house developed real-time PCR assay is a sensitive and specific option for laboratory detection of STEC as compared to CHROMagarTMSTEC in this setting.

Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting.

Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting.

Comparison of a new commercial real-time PCR assay (RealCycler PJIR kit, Progenie Molecular) to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections

The atypical fungus Pneumocystis jirovecii ( P. jirovecii ) is the causative agent of a life threatening pneumonia [ Pneumocystis pneumonia (PCP)] in immunosuppressed patients. PCP diagnosis is essentially based on the detection of the fungus in pulmonary specimens using microscopic examination and polymerase chain reaction (PCR). For the past 20 years, the use of PCR assays has improved the sensitivity of P. jirovecii detection. The main objective of the present study was to compare the efficiency of the RealCycler PJIR kit (Progenie Molecular; pmPCR), to an in-house PCR assay (ihPCR), both assays targeting the mitochondrial large subunit ribosomal rRNA gene. Thirty-four patients were retrospectively enrolled in the study. Fourteen patients were initially diagnosed with PCP, 15 were considered to be pulmonary colonized by the fungus, and five were negative for P. jirovecii detection (negative control group). Archival DNA specimens (extracted from the patient's pulmonary specimens) were examined with ihPCR and pmPCR on the same day using the same real-time PCR instrument (Applied Biosystems 7500 Real-Time PCR system). The ihPCR assay was performed as described elsewhere by Meliani et al. and Totet et al. [1,2] . The pmPCR assay was performed according to the manufacturer's recommendations. Concordant results were obtained with the two PCR assays for all DNA specimens but one (33/34). Thus, the concordance rate between the two methods was evaluated at 97% (Cohen's kappa, 0.905). The discrepant result concerned one specimen from a colonized patient previously known to have a cycle threshold (Ct) value ≥ 35 using ihPCR. This specimen was confirmed to be positive with ihPCR and was negative with pmPCR. Taking into account Ct values for positive samples, the correlation factor between the two PCR methods has been evaluated at 0.97 ( R 2 = 0.9481). Taking into account the Cohen's kappa > 0.8, the RealCycler PJIR kit (Progenie Molecular) appears to be an efficient technique to detect P. jirovecii in patients with PCP and in patients colonized by P. jirovecii .

Diarrheagenic pathogens in adults attending a hospital in Singapore.

BackgroundSingapore’s diarrhoeal notification system is based on specific pathogens. Official data may thus be skewed towards notifiable diseases. Limited information is available on the profiles of aetiological agents responsible for acute gastroenteritis (AGE) cases, especially among the adult population. To understand the frequency and distribution of potential causative agents of diarrheal disease in Singapore, we screened adults’ stool samples collected from a large public hospital.MethodsThe stool samples were screened for 18 diarrheagenic pathogens using a combination of commercial multiplex polymerase chain reaction (PCR), in-house singleplex PCR and immunochromatographic assays. One hundred adult faecal samples that were collected from October 2013 to January 2014 for routine diagnostic purposes and submitted for culture at Tan Tock Seng Hospital, Singapore were used.ResultsPathogens were detected in 32 % of the samples. The predominant organisms encountered were norovirus genogroup II (11 %), Aeromonas spp. (9 %) and Campylobacter spp. (5 %). One sample was positive for both verocytotoxigenic E. coli (VTEC) and E. coli O157:H7. Two other samples were positive for VTEC only, and one other sample was positive for E. coli O157:H7 only. Astrovirus, C. perfringens, Shigella spp. and toxigenic C. difficile were each detected in 2 % of the samples. Cryptosporidium parvum, Giardia lamblia, group A rotavirus, Salmonella spp. and Vibrio spp. were each detected in 1 % of the samples. No L. monocytogenes, Y. enterocolitica, enteric adenovirus, or norovirus genogroup I were detected.ConclusionOur preliminary findings suggest that pathogens causing non-notifiable diseases might have contributed considerably to the adult hospitalised AGE cases. However, as the samples were from an adult hospital, the data obtained may not be representative of the whole community. Thus, a larger study to collect clinical samples and risk exposure data from primary healthcare clinics and children hospital is planned for, to gain a more holistic perspective on the epidemiology of AGE in Singapore. A larger study may also offer valuable insights for improving the approach of microbiological surveillance of food, as well as strategizing inspection efforts along the food supply chain by public health authorities.