Monoplex Polymerase Chain Reaction Research Articles

Antimicrobial resistance profiling of coagulase negative staphylococci isolated from bovine mastitis

Mastitis plays a crucial role in the economics of dairy industry by deteriorating the quality and quantity of milk produced, as well as endangering the animal health and welfare. The objective of this study was to investigate the antimicrobial resistance (AMR) profile of coagulase negative staphylococci (CNS) isolated from bovine mastitis. Microbiological evaluation by morphological, cultural and biochemical characterisation as well as by monoplex polymerase chain reaction (PCR) of the 83 quarter milk samples revealed that CNS were the most predominant bacteria (32.53%). It was also concluded that most of the CNS were resistant to multiple antibiotics. The phenotypic and genotypic AMR profiling was done using in vitro disc diffusion assay and PCR, respectively, to identify the resistance pattern towards penicillin, methicillin, tetracycline and enrofloxacin. The results depicted a significant difference between the phenotypic and genotypic resistance of CNS against penicillin, methicillin and tetracycline. This outcome on the interaction of phenotypic and genotypic AMR profiling is intriguing and opens a huge scope for future studies on the transcriptomic and proteomic aspects of drug resistance. A better knowledge of the AMR profile guides the dairy producers in developing suitable timely intervention strategies for the economic management of mastitis, which in turn helps in tackling AMR and reduces the threat of its zoonotic transmission.

A comprehensive study of Helicobacter pylori infection: molecular analysis, antibacterial susceptibility, and histopathological examination.

Helicobacter pylori is a pathogen associated with gastroduodenal diseases. This study aimed; (i) to investigate H. pylori presence by invasive tests in adult dyspeptic patients, (ii) to determine antibiotic susceptibility and genotypic characteristics of the H. pylori isolates, and (iii) to investigate the relationship between the H. pylori genotypes and the histopathological findings. In this cross-sectional study, gastric biopsy samples from 208 adult dyspeptic patients were used for culture, tissue Polymerase Chain Reaction (PCR), and histopathological analysis. Antibiotic susceptibility of the H. pylori isolates was analyzed by gradient method. Analysis of the virulence genes was performed by monoplex PCR. Genetic profiles (from A to H) were created based on the virulence genes presence. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) was used for the genotyping of the H. pylori isolates. The mean age of the patients was 46 (± 15) years and 128 (61.5%) of them were female. H. pylori positivity was detected by culture, tissue PCR and histopathological examination in 59 (28.4%), 114 (54.8%) and 81 (38.9%) patients, respectively. The overall prevalence of H. pylori was found to be 63% (131/208). All H. pylori isolates were susceptible to tetracycline and amoxicillin. The resistance rates for metronidazole, clarithromycin, levofloxacin, and rifampicin were 67.2%, 27.9%, 34.4% and 13.11%, respectively. Multi drug resistance (MDR) was detected at the rate of 45.9% (28/61). While the most common virulence gene was cagA (93.44%), the least common was vacAm1 (23%). The predominant genetic profile was profile A (47.5%). ERIC-PCR results revealed a total of 26 different patterns. A high prevalence of H. pylori was detected in adult dyspeptic patients as in developing countries. It was observed significant genotypic heterogeneity and virulence gene diversity within the isolates. A considerable resistance rate detected against antibiotics such as clarithromycin, metronidazole, and levofloxacin, which are frequently used in the eradication of H. pylori, should be taken into consideration when creating regional empirical treatment regimens.

Detection of pneumococcal carriage among under five healthy children with multiple co-colonizing serotypes with impact of Pneumococcal conjugate vaccine.

Background: The distribution of serotypes causing invasive pneumococcal diseases are diverse, limiting the proportion of IPD cases pneumococcal conjugate vaccine can prevent. More studies are need to estimate the rate of pneumococcal acquisition and serotype replacement following introduction of pneumococcal conjugate vaccine. Methods:The study was conducted in the department of microbiology of a tertiary hospital. Nasopharyngeal swabs were collected among 200 under five children from Pediatric outpatient department. Streptococcus pneumoniae were isolated and identified by culture, Gram staining, biochemical test and polymerase chain reaction (PCR). An initial screening of the nasopharyngeal swabs were done by the primer cpsA to identify the pneumococci by monoplex PCR. Then primers that are serotype specific were used for serotyping by multiplex PCR with positive and negative control. Results: Out of 200 nasopharyngeal swabs, 67 (33.50%) were positive by culture and 92 (46%) were positive by PCR for S. pneumoniae. Out of 200 children, 90 (45%) were received PCV and 110 (55%) were not vaccinated. Among vaccinated children, 3 (12%) S. pneumoniae were detected in fully vaccinated children and 19 (29.23%) S. pneumoniae were detected in partially vaccinated children by culture. In case of PCR, 4 (16%) S. pneumoniae were detected in fully vaccinated children and 25 (38.46%) S. pneumoniae were detected in partially vaccinated children. Among not vaccinated children, 45 (40.90%) and 63 (57.27%) S. pneumoniae were detected by culture and PCR respectively .The predominant serotypes were 34F, 35B, 6A, 6B, 14, 23F, 3, 19A, 19F, 4, 18C, 7F as carriage strains. Conclusion. Children represent a consistent population of pneumococcal-naive individuals. So, we found detection rate of S. pneumoniae and serotypes inchildren as carrier was relatively more in non-vaccinated children.

Molecular detection of metallo-β-lactamase genes in carbapenem-resistant isolates of Pseudomonas aeruginosa recovered from patients in Al-Diwaniyah province, Iraq

Introduction: The World Health Organization(WHO) has rated Carbapenems Resistant Pseudomonas aeruginosafrom toppriority pathogens to research and develop new antibiotics.Objectives: The current work intended to detect the presence of the metallo-β-lactamase genes in Pseudomonasaeruginosaisolates cultivated from some healthcare centers in Al-Diwaniyah City, Iraq.Materials and methods: After induction of specific traditional cultivation and identification methods for 630 samples (244 burnswabs, 163 sputa, 115 urine samples, and 108 ear swabs), a monoplex polymerase chain reaction (MPCR) method aiming at 8metallo-β-lactamases (MBLs) genes was followed. Partial-gene sequencing of the MBLs was also performed to understand someof the evolution history of those isolates.Results: The P. aeruginosa was identified in 100 isolates. MBL-related MPCR results showed the presence of blaVIM, blaSPM,blaGIM, blaNDM, blaAIM,blaSIM, and blaDIM genes in 16 (66.6%), and 14 (14.3%), 13 (54.1%), 1 (36.5%), 9 (37.5%), 8 (33.3%), 2(8.3%), respectively, of the isolates. No detection of the blaIMP was observed. The phylogenic study demonstrated high matchingrates, 99%, with global strains from the NCBI-related database.Conclusion: To the best of our awareness, the current work is the first in Iraq identifying metallo-β-lactamase genes inPseudomonasaeruginosa isolates.

Respiratory Viruses and Atypical Bacteria Co-Infection in Children with Acute Respiratory Infection.

BACKGROUND:Acute respiratory infections (ARI) are one of the prevalent pediatric diseases. Coinfections of respiratory viruses and atypical bacterial respiratory pathogens are common.AIM:This study aimed to determine the prevalence of co-infection between respiratory pathogens including viruses, bacteria and atypical bacteria in a sample of Egyptian children presenting with symptoms of acute respiratory tract infection.METHODS:This one-year prospective cohort study conducted in Abo El Rish Pediatric Hospital, Cairo University over one year included children presenting with symptoms of acute respiratory infection. Enrolled children were subjected to nasopharyngeal swabs or throat swabs and then processed to detect viral, bacterial and atypical bacterial causative agents by culture), retrotranscription polymerase, Monoplex polymerase chain reaction (PCR) and Multiplex PCR.RESULTS:Viral etiological agents were detected in 20 cases (20.8%), while 76 patients (79.2%) had no definite viral aetiology. The most abundant virus detected was Rhinovirus in 36 (27.3%), followed by 21 (15.9%) were positive for RSV, 12 (9.1%) were positive for HMPV, 6 (4.5%) were positive for adenovirus and 3 (2.3%) were positive for influenza B. For Atypical bacterial causes Mycoplasma were positive for 9 (6.8%) cases and one case was positive for Bordetella parapertussis. Viral and atypical bacteria Co infection were detected in 14 (10.6%) of cases.CONCLUSION:These results suggest that coinfection with bacteria or atypical bacteria in children with acute respiratory tract infection is common and this co-infection can induce serious illness. The multiplex reverse-transcriptase polymerase chain reaction should become an essential tool for epidemiological studies and can fill the gap between clinical presentation and definitive diagnosis.

Development of Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Detection of Influenza A, B and Adenoviruses

Millions of people in developing countries lose their lives due to acute respiratory infections, such as Influenza A & B and Adeno viruses. Given the importance of rapid identification of the virus, in this study the researchers attempted to design a method that enables detection of influenza A, B, and adenoviruses, quickly and simultaneously. The Multiplex RT PCR method was the preferred method for the detection of influenza A, B, and adenoviruses in clinical specimens because it is rapid, sensitive, specific, and more cost-effective than alternative methods. After collecting samples from patients with respiratory disease, virus genome was extracted, then Monoplex PCR was used on positive samples and Multiplex RT-PCR on clinical specimens. Finally, by comparing the bands of these samples, the type of virus in the clinical samples was determined. Performing Multiplex RT-PCR on 50 samples of respiratory tract led to following results; flu A: 12.5%, fluB: 50%, adeno: 27.5%, negative: 7.5%, and 2.5% contamination. Reverse transcription-multiplex Polymerase Chain Reaction (PCR) technique, a rapid diagnostic tool, has potential for high-throughput testing. This method has a significant advantage, which provides simultaneous amplification of numerous viruses in a single reaction. This study concentrates on multiplex molecular technologies and their clinical application for the detection and quantification of respiratory pathogens. The improvement in diagnostic testing for viral respiratory pathogens effects patient management, and leads to more cost-effective delivery of care. It limits unnecessary antibiotic use and improves clinical management by use of suitable treatment.

Prevalence of TEM, SHV, and CTX-M genes of extended-spectrum β-lactamase-producing Escherichia coli strains isolated from urinary tract infections in adults.

Urinary tract infections (UTIs) are one of the major sources of widespread infectious diseases in the community as well as in the hospitals which increase the cause of morbidity and mortality. Prevalence of extended-spectrum-β-lactamase (ESBL)-producing uropathogenic E. coli isolates has been found to be increased rapidly across the world. The present study was undertaken to find out the frequency of bla TEM, bla CTX-M, and bla SHV genes among E. coli isolates from UTI and detect their sensitivity pattern. A total of 112 non-repeated E. coli isolates obtained from urine samples of UTI diagnosed patients were included in this study. Antibiotic susceptibility test was done by disc diffusion method. Seventy seven (68.75%) isolates were MDR and tested for ESBL. ESBL-positive isolates were screened for bla TEM, bla CTX-M, and bla SHV genes by monoplex PCR (polymerase chain reaction). Among 46 ESBL-producing E. coli isolates, 8.69% harboured all the three bla genes. The bla TEM was the predominant (93.47%) gene followed by bla CTX-M (82.6%) and bla SHV (4.34%). It can be concluded that the prevalence of MDR (multidrug resistance) ESBL-producing E. coli appears to be high and the highest identified gene was bla TEM. The knowledge of resistance pattern can help physician's select suitable empirical antibiotic regimens, so that antibiotics showing high-resistance pattern can be avoided.

Antibiotic resistance profile and random amplification typing of β-lactamase-producing Enterobacteriaceae from the local area of Al-Taif and nearby cities in Saudi Arabia

Background: The emergence of extended-spectrum ?-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae constitutes a serious problem because of the transfer of resistance genes from one organism to another. Objectives: To screen the antibiotic susceptibility of E. coli and K. pneumoniae from inpatients at King Abdul-Aziz Hospital, Al-Taif, Saudi Arabia and to detect common ESBL genes, and random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) genotyping of the Enterobacteriaceae. Methods: We analyzed 43 bacterial strains isolated from clinical samples of sporadic cases for their reactivity to different antibiotics. Strains that showed resistance by monoplex PCR were screened for ESBL TEM- and CTXM-1-, -2-, and -9-encoding genes. Random amplifications were used for typing the resistant strains. Results: The majority of the ESBL containing strains were sensitive to meropenem, amikacin, gentamicin, and tigecycline. PCR detection using ESBL gene-specific primers showed that 17 of 43 strains harbored genes for ESBL TEM, CTX-M-1, or CTX-M-2. RAPD typing revealed marked variation among the ESBL-producing E.coli in relation to each other. Conclusions: Considerably high incidence of ESBL-producing bacteria was present patients from the local area of Al-Taif and nearby cities in Saudi Arabia with TEM and CTX-M subtypes being the most commonly detected variants. There was evidence of a polymorphic genetic pattern among ESBL-producing bacteria. Keywords: Antibiotic resistance, E.coli, Klebsiella pneumoniae, RAPD typing, Saudi Arabia

Molecular Detection of Methicillin-Resistant Staphylococcus aureus by Non-Protein Coding RNA-Mediated Monoplex Polymerase Chain Reaction.

Non-protein coding RNA (npcRNA) is a functional RNA molecule that is not translated into a protein. Bacterial npcRNAs are structurally diversified molecules, typically 50–200 nucleotides in length. They play a crucial physiological role in cellular networking, including stress responses, replication and bacterial virulence. In this study, by using an identified npcRNA gene (Sau-02) in Methicillin-resistant Staphylococcus aureus (MRSA), we identified the Gram-positive bacteria S. aureus. A Sau-02-mediated monoplex Polymerase Chain Reaction (PCR) assay was designed that displayed high sensitivity and specificity. Fourteen different bacteria and 18 S. aureus strains were tested, and the results showed that the Sau-02 gene is specific to S. aureus. The detection limit was tested against genomic DNA from MRSA and was found to be ~10 genome copies. Further, the detection was extended to whole-cell MRSA detection, and we reached the detection limit with two bacteria. The monoplex PCR assay demonstrated in this study is a novel detection method that can replicate other npcRNA-mediated detection assays.

Phenotypic and Molecular Characterization of Plasmid- Encoded Extended Spectrum Beta-Lactamases Produced by Escherichia coli and Klebsiella spp from Lahore, Pakistan

Purpose: To investigate the distribution of plasmid-encoded extended spectrum beta-lacatamases (ESBLs) in Lahore, Pakistan using different phenotypic and molecular methods.Methods: Escherichia coli and Klebsiella spp were obtained over a period of nineteen months (June 2007 to December 2008). Both were tested by the double disk synergy test, combined disk test and Epsiometer-test (E-test) to evaluate their ability to detect ESBLs. The genotypes of ESBLs were analyzed by monoplex polymerase chain reaction (PCR), multiplex PCR, DNA sequencing and isoelectric focusing.Results: 662 E. coli and 153 Klebsiella spp were analyzed. Among these isolates, 39.3 % E. coli and 26.1 % Klebsiella spp were positive for extended spectrum beta-lactamases (ESBLs).71.9 % E. coli and 79.6 % Klebsiella spp showed minimum inhibitory concentration (MIC) in the range > 32/0.064 = 500 μl/mL for cetatzidime/cetatzidime + clavulanic acid, while 66.5 % E. coli and 69.1 % Klebsiella spp revealed MIC in the range of > 16/0.016 = 1000 μl/mL for cefotaxime/cefotaxime + clavulanic acid. Antibiotic susceptibility testing revealed that imipemem, meropenem and tazocine were the most effective in the management of such infections. The most frequent genotype of ESBL was OXA (19.2 %) for E. coli and SHV (92.5 %) for Klebsiella spp. The highest genotypic combination found was the combination of TEM/OXA (44.2 %) for E. coli.Conclusion: The resistance of E. coli and Klebsiella spp-producing ESBLs in Pakistan is a serious issue, and TEM, OXA and SHV type ESBL were the most common genotypes. Some isolates produced two or three genotypes at a time. Multiplex PCR of ESBL may help in early detection as well as phenotypic antibiotic therapy of these infections.Keywords: Beta-lactamases, Escherichia coli, Klebsiella spp, Antibiotic susceptibility, Plasmidencoded, Structural genes, Imipemem, Meropenem, Tazocine

Investigation of multidrug-resistant fatal colisepticaemia in weanling pigs.

Escherichia coli is usually a benign commensal of the gut microflora. However, when E. coli acquires virulence genes it can multiply rapidly and cause disease through colonisation of the intestinal mucosa. Escherichia coli can become a significant pathogen in young pigs. We report an investigation of fatal colisepticaemia in weanling pigs from emerging farms where piglets and weaners were diarrhoeic and the mortality rate ranged between 15% and 70% in each litter. Faecal and tissue samples were processed for histopathology, bacteriology and molecular biology (multiplex and monoplex polymerase chain reaction) and we recovered enteroaggregative multidrug-resistant E. coli producing EAST-1 enterotoxin. An association between poor housing conditions and the observed cases was established and future management programmes were recommended to reduce the impact of such pathogens. Enteroaggregative E. coli is becoming a major problem in the pig industry. It therefore becomes necessary to establish the full impact of E. coli on the South African pig industry and to determine the geographic extent of the problem.

Single step detection of HIV-1 proviral DNA and housekeeping β-actin gene from dried blood spots by a monoplex polymerase chain reaction

There is a growing need for developing a simple, rapid, reliable and cost effective method for detection of HIV-1 for early diagnosis of the infection especially in developing countries and in resource limited settings. A method for simultaneous detection of the HIV-1 p17 gene and the house keeping human β-actin gene from dried blood spots (DBS) by a monoplex polymerase chain reaction (PCR) is described. Genomic DNA was extracted from 40 HIV-1 positive and 40 HIV-1 negative DBS and used as templates to amplify both the HIV-1 p17 and β-actin genes simultaneously under the same cycling condition by a single round PCR. This method of detection of HIV-1 may provide a simple, rapid and cost effective alternative in resource limited settings; however, it would require testing a larger number of samples before widespread use.

Performance of a novel microarray multiplex PCR for the detection of 23 respiratory pathogens (SYMP-ARI study)

Symptoms of acute febrile respiratory tract infection are often unspecific, but the rapid identification of pathogens allows optimised patient management. The objective of this study was to evaluate a novel multiplex polymerase chain reaction (PCR) suspension microarray which detects 19 viral and four atypical bacterial targets. A comprehensive set of sensitive monoplex real-time PCR assays was used for each pathogen as the gold standard. A panel of archived as well as 300 prospectively collected clinical samples was analysed by both methods. At least one target was detected in 165/300 (55 %) samples by monoplex PCR and in 140/300 (46 %) samples by multiplex PCR, respectively. The positivity rate was significantly higher in paediatric patients compared to adults [126/154 (82 %) vs. 39/146 (27 %) by monoplex and 114/154 (74 %) vs. 26/146 (18 %) by multiplex PCR, respectively]. Among all samples, 17/300 (5.6 %) were positive for atypical bacteria by monoplex and 8/300 (2.6 %) by multiplex PCR, respectively. Multiple detections were recorded in 35/300 (11.6 %) samples by monoplex and 26/300 (8.7 %) by multiplex PCR. For the most common pathogens, the sensitivity ranged from 57 to 93 % and the specificity ranged from 95 to 100 %. The overall concordance between both methods was 77 % [95 % confidence interval (CI) 72–81 %]. False-negative results by multiplex PCR were mainly due to the low target concentration. Compared to monoplex PCR, the novel microarray assay proved its principle but displayed overall lower sensitivities, potentially restricting its use to paediatric patients. For some targets, only small numbers of positive samples were available, requiring larger studies to firmly assess the sensitivity and specificity.

Evaluation of Seeplex® STD6 ACE Detection kit for the diagnosis of six bacterial sexually transmitted infections

Traditionally, the diagnosis of bacterial sexually transmitted infection (STI) has been dependent on the isolation of the causative pathogens by culturing endocervical or urethral swab specimens on selective media. While such procedures typically provide excellent diagnostic accuracy, they are often time-consuming and expensive. A multiplex polymerase chain reaction (PCR) assay, based on a semi-automated detection system, was evaluated for the detection of six STI causative organisms. The Seeplex(®) STD6 ACE (auto-capillary electrophoresis) Detection assay employed six pairs of dual priming oligonucleotide (DPO™) primers specifically targeted to unique genes of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Mycoplasma hominis, and Trichomonas vaginalis. A total of 739 specimens (304 cervical swabs and 435 urine samples) collected for 4 months were tested, and results were compared to those obtained with a combined monoplex PCR. The concordance between the multiplex PCR and monoplex PCR assay was 100% for both sensitivity and specificity. We also tested for the presence of two pathogenic bacteria (C. trachomatis and N. gonorrhoeae) and compared the results obtained with the multiplex PCR and BD ProbeTec duplex strand displacement amplification (SDA). The results of the multiplex PCR and duplex SDA were 99.7% concordant for C. trachomatis and 100% concordant for N. gonorrhoeae. The multiplex PCR assay using the Seeplex(®) STD6 ACE Detection kit proved to be a novel cost-effective and fast diagnostic tool with high sensitivity and specificity for the simultaneous detection of six STI pathogens.

Monoplex LightCycler Polymerase Chain Reaction Quantitation of the HER2 Gene for Quality Assurance of HER2/neu Testing by Immunohistochemistry and Fluorescence In Situ Hybridization

Assessment of HER2 by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is a standard practice for breast carcinomas. Testing is associated with a 20% disagreement between laboratories. The College of American Pathologists (CAP) guidelines for HER2 testing include validation of HER2 test methods by achieving 95% concordance with another validated method. Our laboratory requires IHC 3+ FISH nonamplified specimens to undergo retesting by polymerase chain reaction (PCR). A random sample of IHC 2+ cases are routinely tested by PCR. We found this practice useful for resolving discrepancies in HER2 testing. At clinician request, seventy-nine 3+ and one hundred forty-eight 2+ cases were tested by FISH. In 22 cases, IHC was 3+ but FISH was nonamplified. These 22 cases underwent HER2 LightCycler monoplex polymerase chain reaction (MPCR) testing. Seventeen 2+ nonamplified cases were tested by MPCR. Twenty-one 3+, FISH nonamplified cases were found to be MPCR nonamplified. One IHC 3+, FISH nonamplified case was MPCR amplified. Seventeen 2+, FISH nonamplified cases were MPCR nonamplified. In all but one case, FISH and MPCR were concordant. American Society of Clinical Oncology/CAP guidelines propose validation of testing procedures by showing 95% concordance with a validated test for positive and negative assays. Specific actions are not recommended to resolve discordances between tests. Our laboratory uses 3 different modalities for HER2 testing. We have found that our 2 methods for testing gene amplification status show a higher degree of concordance between themselves than either did with IHC. Review of the 3+ IHC nonamplified cases showed them to have a dark, granular circumferential staining pattern.

Quantitation of RHD by real‐time polymerase chain reaction for determination of RHD zygosity and RHD mosaicism/chimerism: an evaluation of four quantitative methods

Determination of RHD zygosity of the spouse is crucial in preconception counseling of families with history of hemolytic disease of the fetus and newborn caused by anti-D. RHD zygosity can be determined by quantitative real-time polymerase chain reaction (PCR) basically by determining RHD dosage, and this feature is relevant in investigating RHD mosaicism and chimerism. Monoplex and duplex real-time PCRs, uncalibrated and calibrated, were tested. RHD zygosity was determined for 72 samples and compared with serology-based prediction of RHD zygosity. Additionally, a range of constructed RHD dosages were compared with determined RHD dosages. Finally three samples, Days 1, 90, and 120, from a patient with loss of D were included. All setups enabled differentiation between hemi- and homozygous samples. Results from calibrated methods had the smallest variation and enabled differentiation at the mean +/- 3SD. Incongruity between determined and predicted RHD zygosity was found in three samples. PCR-sequence-specific priming specific for the hybrid downstream Rhesus box was performed on those samples confirming the real-time PCR results. Determined RHD dosage equaled constructed RHD dosage in both calibrated real-time PCRs. Patient samples showed RHD with a mean of 1, 5, and 8 percent relative to GAPDH in all samples. Duplex calibrated real-time PCR was a robust and exact method for determination of RHD zygosity and applicable in evaluation of RHD mosaicism and chimerism. This was illustrated with patient samples, where this assay revealed for the first time signs of mosaicism in both expression of D antigens and in RHD dosage.

Evaluation of HER-2/neu Gene Status in Osteosarcoma by Fluorescence In Situ Hybridization and Multiplex and Monoplex Polymerase Chain Reactions

Previous reports suggest that the human epidermal growth factor 2 (HER-2/neu) receptor may be overexpressed in osteosarcoma. To determine whether osteosarcomas have amplifications of the HER-2/neu gene. We studied a series of osteosarcomas by fluorescence in situ hybridization (FISH) and by 2 real-time polymerase chain reaction assays that measure the amount of HER-2/neu DNA relative to a control gene. The HER-2/ neu monoplex and multiplex assays were capable of identifying those cases of breast cancer that were known to overexpress HER-2/neu as assessed by FISH. We initially studied 21 cases of osteosarcoma by FISH analysis (using a technique that included a probe for chromosome 17), 11 of which had their HER-2/neu gene amplification status previously reported. None of these osteosarcoma cases showed HER-2/neu amplification by our FISH analysis and subsequent quantitative (multiplex) polymerase chain reaction. Apparent expression of HER-2/neu protein was observed in several of the cases but the immunoreactivity was localized to the cytoplasm and was not membranous in character. An additional 35 osteosarcoma specimens were subjected to monoplex polymerase chain reaction analysis, and amplifiable DNA was recovered from 19 specimens (54%). None of these samples had HER-2/neu amplification by monoplex PCR analysis and only one case had membranous immunoreactivity graded as 1+. Although a small subset of osteosarcomas had weak noncircumferential membranous immunoreactivity for HER-2/neu protein, no osteosarcomas demonstrated positive (2+ or 3+) immunoreactivity for HER-2/ neu protein and none showed HER-2/neu gene amplification by either FISH or polymerase chain reaction.

Correlation of HER2 Gene Amplification with Immunohistochemistry in Breast Cancer as Determined by a Novel Monoplex Polymerase Chain Reaction Assay

Amplification of the HER2 oncogene in breast cancer identifies patients who are likely to respond to anti-HER2 mAb therapy. Current clinical practice dictates that all breast cancers first undergo HER2 screening by IHC. Strongly positive (3+ on a 0-to-3+ scale) IHC cases are considered as HER2-amplified tumors and are not evaluated further because of the strong correlation between HER2 gene amplification as measured by FISH and 3+ IHC. This strong correlation has recently been questioned, and some data suggest that over 50% of 3+ IHC HER2 immunostains may not be due to HER2 gene amplification. To help resolve this discrepancy, the authors developed a quantitative PCR assay for HER2. Quantitative PCR was used to determine the amount of HER2 DNA relative to a control gene, IF2 (eukaryotic translation initiation factor, 2p11.1-q11.1). The PCR assay is performed on genomic DNA isolated from paraffin-embedded breast cancer tissue. The PCR assay developed is a monoplex assay in which the HER2 and IF2 PCRs are performed in separate cuvettes. Cases of HER2 FISH amplified breast cancer and HER2 FISH nonamplified breast cancer were chosen for study by monoplex HER2 PCR. HER2 overexpression was evaluated by IHC. Twenty-two cases of HER2-positive and 22 cases of HER2-negative breast cancer, as determined by FISH, were assayed for HER2 by PCR and IHC. Sixteen of the 44 cases were interpreted as 3+ IHC. All 16 showed HER2 amplification by PCR and 15 showed HER2 amplification by FISH. One FISH negative case was found to be HER2 amplified by PCR and showed 3+ IHC stain, suggesting the FISH result in this case was underinterpreted. Two FISH positive cases were found to be negative by PCR and negative in IHC as well, suggesting the FISH result in these cases was overinterpreted. The authors conclude that 3+ IHC membrane staining correctly identifies neoplasms showing HER2 gene amplification. Monoplex HER2 PCR may offer significant advantages over both IHC and FISH for HER2 testing in breast cancer.

Characterization of the D18S535 STR Locus in Northern Ontario European and Aboriginal Populations for Forensic Purposes

Genetic characterization of one European and three aboriginal populations from northern Ontario was undertaken to assess the utility of the D18S535 short tandem repeat locus (STR) as a genetic marker for forensic DNA typing in the region. The D18S535 locus was amplified using monoplex polymerase chain reaction (PCR), separated by denaturing polyacrylamide gel electrophoresis (PAGE), and visualized using the silver-stain detection method. The generated population data demonstrated that the D18S535 locus is highly polymorphic with a heterozygosity of > or = 0.75. The exact test showed violations of Hardy-Weinberg equilibrium in two of the aboriginal populations. Pairwise comparisons of allele-frequency distributions indicated that the four northern Ontario populations were significantly different from each other. This test also revealed that the northern Ontario populations differed significantly from ten European populations (from Germany, Spain, and Croatia) and one population from South America (from Argentina). Forensic parameters showed that the D18S535 locus is highly discriminating (power of discrimination > or = 0.85, chance of exclusion > or = 0.51); however, the lack of Hardy-Weinberg equilibrium in some of the populations must be taken into account in the application of these results to northern Ontario forensic casework.

Multiplex PCR for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila in community-acquired pneumonia

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila. Oligonucleotide primers for the amplification of the DNA of these three organisms were optimized for use in combination in the same reaction. PCR products were detected by the Micro-Chip Electrophoresis Analysis System. Clinical samples were obtained from 208 community-acquired pneumonia (CAP) patients who were participants in a multicenter CAP surveillance study performed at seven medical schools and their affiliate hospitals in Japan. No significant differences in the sensitivity of each primer set were observed when tested in both the multiplex and monoplex PCR assays. Our multiplex PCR was able to reliably detect 10 copies/100 μl of each of the three pathogen DNAs. Of the panel of 208 samples, 14 of 15 C. pneumoniae, 10 of 10 M. pneumoniae, eight of eight L. pneumophila and 165 of 176 negative samples were correctly identified. Eleven cases who were the multiplex PCR positive and conventional method negative were observed. The PCR findings were of possible significance in at least four of these patients. Our multiplex PCR assay could potentially be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical usefulness.