Duplex PCR Assay Research Articles

Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a ‘perfect’ agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.

A new duplex PCR assay for the rapid screening of mating-type idiomorphs of pathogenic Sporothrix species

Sporothrix schenckii and allied species are thermodimorphic fungi widely distributed in nature which causes human and animal sporotrichosis, the most common subcutaneous mycosis globally. Sporotrichosis is acquired after a traumatic inoculation of soil or plant material contaminated with Sporothrix propagules or through bites and scratches from diseased cats. In Ascomycota, the master regulators of sex are MAT genes that lie in a single mating-type locus, in Sporothrix these are determined by two nonhomologous alleles, MAT1-1 and MAT1-2. We assessed the whole-genome sequences of medically relevant Sporothrix to develop a single-tube duplex PCR assay to screen S. brasiliensis, S. schenckii, S. globosa, and S. luriei idiomorphs (MAT1-1 or MAT1-2) and understand the distribution and incidence of mating-type strains from natural populations. Using our duplex PCR assay, a 673 bp amplicon (α-box protein) was consistently amplified from all MAT1-1 isolates, while a 291 bp fragment was only amplified from the isolates harboring MAT1-2 (HMG box). Molecular evidence suggests heterothallism (self-sterility) as the unique mating strategy among the species evaluated. The mating-type identity of 93 isolates revealed a nearly equal distribution (1:1 ratio) of mating type alleles within species but deviating between different outbreak areas. Remarkably, for S. brasiliensis in Rio de Janeiro, we report an overwhelming occurrence of MAT1-2 (1:13 ratio; χ2 = 10.286, P = 0.0013) opposing the high prevalence MAT1-1 in the Rio Grande do Sul (10:1 ratio; χ2 = 7.364, P = 0.0067). Therefore, the population structure of Sporothrix species refers from paucity to regular cycles of sexual recombination in most of the studied regions. Our PCR-based mating-type diagnostic assay is proposed here as an important marker to track the geographical expansion during the long-lasting outbreak of cat-transmitted sporotrichosis driven by S. brasiliensis.

Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates

BackgroundCampylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates.MethodsA total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping.ResultsOut of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates.ConclusionSignificant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

Two new rapid PCR-based methods for identification of Acinetobacter baumannii isolated from clinical samples

The accurate identification of Acinetobacter spp. is challenging due to their high phenotypic and biochemical similarities. Because clinical relevance and antibiotic susceptibility are significantly different among different genomic species of Acinetobacter, the exact identification of A. baumannii is necessary and it can help us prevent inappropriate antibiotic use and inferior clinical care. This project employed a sequence-specific PCR assay for the rpoB region in A. baumannii to distinguish it from non-Acinetobacter baumannii Acinetobacter species. Moreover, a duplex PCR assay was used to detect blaOXA-51-like and gluconolactonase genes as a second identification method. In this study, 210 isolates of Acinetobacter spp. were considered and identified by PCR-sequencing of rpoB gene as a reference test. PCR-sequencing of rpoB revealed that 179 isolates were A. baumannii and 31 were non- A. baumannii Acinetobacter strains. PCR amplification targeting the rpoB gene as the first method, detected 182 isolates of A. baumannii, while duplex PCR assay confirmed 163 isolates as A. baumannii. Data analysis indicated that the sensitivities of sequence-specific PCR of the rpoB gene and duplex PCR assay were 100% and 91.06%, respectively, while specificities were 91.18% and 100%, respectively. Given the data, it was revealed that these two methods showed a reasonable potential for the accurate identification of A. baumannnii from non- A. baumannii species. Sequence-specific PCR assay for the rpoB gene and duplex PCR assay for blaOXA-51-like and gluconolactonase genes are rapid, reliable and cost-effective methods which can be used in clinical laboratories for the accurate identification of A. baumannii.

A New Duplex PCR-Assay for the Detection and Identification of Paracoccidioides Species.

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the P. brasiliensis complex and P. lutzii in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for Paracoccidioides species. Primers PbraCx-F and PbraCx-R targeting P. brasiliensis DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting P. lutzii DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 Paracoccidioides revealed 100% specificity (AUC = 1.000, 95%CI 0.972–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the P. brasiliensis complex (n = 7) or P. lutzii (n = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient’s organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to P. brasiliensis (S1) and P. lutzii in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the P. brasiliensis complex and P. lutzii, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas.

The presence and genetic characteristics of porcine circovirus 3 from pigs in Southern and Central provinces of Vietnam

Porcine circovirus type 3 (PCV3) is an emerging circovirus species that has recently been reported in different countries around the world, suggesting a widespread circulation. This study was carried out in order to investigate the presence and further genetic characteristics of PCV3 from swine herds in Southern and Central provinces of Vietnam. A duplex PCR assay for rapid detection of PCV3 in pigs was established with a pair of specific primers designed between rep and cap gene segment to amplify full-length ORF2 and another set of primers binding to COX1gene serving as an internal amplification control (IAC). The resulting duplex PCR was used to examine PCV3 presence in 94tissue and serum samples. Subsequently, PCV3 was detected in 10 out of 94 cases (10.6%). The infection rate in sows (14.3%) was higher than that in grower pigs (7.7%). Regarding nucleotide sequence comparison, 10 ORF2 genes were selected for nucleotide sequencing and their alignment showed 97.2% - 99.5% homology. According to the phylogenetic analysis and sequence alignment of cap gene, all the sequences were clustered into group PCV3a,including 9 strains of sub-group PCV3a1 and only one strain of subgroup PCV3a2. These findings indicated that the PCV3a group is circulating in swine farms in Vietnam. This study provides better insights into epidemiology of this pathogen in the national swine industry.

Duplex real-time PCR combined with melting curve analysis for rapid detection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Financial loss and health risk caused by the substitution with Salmo salar and Oncorhynchus mykiss have been widely reported around the world, highlighting the necessity to establish rapid and accurate methods for species identification. The aim of the present study was to develop a novel method for rapid identification of S. salar and O. mykiss based on duplex PCR assay (conventional and fluorescent). Specifically, after sequence alignment, specific primers for S. salar and O. mykiss based on the COⅠ (cytochrome oxidase I) and cyt b (cytochrome b) genes were designed and the specificity was verified against 23 common fish species. Clear and distinguishable bands of the expected sizes of 108 bp, and 207 bp, as well as the melting peaks around 81.6 ℃ and 84.7 ℃, were observed for S. salar, and O. mykiss, respectively. Moreover, the feasibility of melting curve analysis for detecting the adulteration of different amounts of O. mykiss with S.salar has also be confirmed. Therefore, the novel assay in the present study allows a fast and accurate identification of S. salar and O. mykiss in processed fish products.

Evaluation of the performance of an in-house duplex PCR assay targeting the IS6110 and rpoB genes for tuberculosis diagnosis in Cameroon

BackgroundTuberculosis (TB) remains a major public health concern in many low-income countries accounting for approximately two-thirds of deaths in people living with human immunodeficiency virus (HIV) infection. With prompt, accurate and appropriate treatment, almost all TB disease can be cured. The present study was to evaluate the diagnostic performance of an in-house duplex PCR (D-PCR) using IS1610 and rpoB specific primers in sputum samples from TB suspected patients.MethodsA hospital-based cross-sectional study was conducted at the Limbe and Buea Regional Hospitals of the South West Region of Cameroon from June 2016 to April 2017. Sputum samples, decontaminated with hypertonic saline/sodium hydroxide solution were centrifuged and pellets processed for smear microscopy, culture and DNA extraction. Suspected inhibition was resolved by serial dilution of genomic DNA. Results were compared to culture as gold standard as well as a Composite Reference Standard (CRS).ResultsA total of 129 participants aged between 5 to 82 years were enrolled in to the study. The median age of the participants was 37 years (interquartile range, IQR: 27–50 years), with 54.3% being male. Forty-seven samples (36.4%) were positive by direct sputum microscopy, 49 (38%) by microscopy after concentration, 51 (39.5%) by culture and 62 (40.1%) by D-PCR. PCR inhibition was resolved in 85.7% (18/21) of the samples that had inhibition. The overall sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios and area under the curve AUC) of the D-PCR was 93.5, 94, 94, 94%, 15.6, 0.005 and 89.0% respectively using CRS as reference. The sensitivities of D-PCR observed among different sample categories were 95.7, 87.5 and 87.5% for smear-and culture-positives, smear-negative/culture-positive, and clinically diagnosed cases respectively.ConclusionIS1610 and rpoB duplex PCR using relatively cheap decontamination and DNA extraction methods in addition to simple serial dilutions to resolve PCR inhibition shows high sensitivity in the diagnosis of paucibacillary tuberculosis.

Diagnostic Challenges and Prospects Associated With Zoonotic Tuberculosis of Central Nervous System

Introduction: The diagnosis of Tuberculous Meningitis (TBM) has remained a challenge due to its insidious onset and the failure of conventional diagnostic tests. The present study aimed to identify the mycobacterial pathogen in the CSF of patients with TBM and a poor prognosis. Methods: We retrospectively recruited 224 TBM and 34 non-TBM patients admitted to the Central India Institute of Medical Sciences, Nagpur, India, in 2014. The CSF samples of these patients were subjected to a duplex PCR assay for the species-specific identification of the causative pathogen. Results: M. bovis and infection with M.tuberculosis were detected in 7% (18) and 32.9% (85) of the patients, respectively. Moreover, 14% (36) of the study samples were culture positive; however, the mycobacterial pathogens could not be differentiated to the species level. Conclusion: The present study findings emphasized the potentially vital importance of M. bovis identification for appropriate patient management. The obtained data also demonstrated the persistent significance of M. bovis, as a zoonotic pathogen.

TaqMan-MGB probe quantitative PCR assays to genotype and quantify three mtDNA mutations of Leber hereditary optic neuropathy

Leber hereditary optic neuropathy (LHON) is a degenerative disease of the optic nerve associated with one of three mitochondrial DNA (mtDNA) m.3460G>A, m.11778G>A and m.14484T>C mutations. Although several procedures are available to genotype these mutations, quantitative approaches with rapid, low-cost and easy to handle advantages for three LHON mtDNA mutations are rarely reported. Here, we firstly developed a “one-step” tetra-primer amplification-refractory mutation system (T-ARMS) PCR for qualitative genotyping of three LHON mtDNA mutations. Subsequently, we established single, duplex and triplex TaqMan MGB probe-based fluorescence quantitative PCR (qPCR) assays to perform both qualitative and quantitative analyses of three LHON mtDNA mutations. Standard curves based on tenfold diluted plasmid standard exhibited high specificity and sensitivity, stable repeatability and reliable detectable ability of TaqMan probe qPCR assays without cross-reactivity upon probes combination. Moreover, by comparing with SYBR Green qPCR, we further validated the feasibility of the triplex-probe qPCR assay for the quantitative detection of mtDNA copy number in blood samples. In conclusion, our study describes a rapid, low-cost, easy to-handle, and high-throughput TaqMan-MGB probe qPCR assay to perform both qualitative and quantitative analysis of three primary LHON mtDNA mutations, offering a promising approach for genetic screening and testing of LHON mutations.

Characterization of isolates of Xanthomonas arboricola pv. corylina, the causal agent of bacterial blight, from Oregon hazelnut orchards

Bacterial blight (Xanthomonas arboricola pv. corylina (Xac)) of hazelnut (Corylus avellana L.) was described first in Oregon in 1915 and is now recognized as a damaging disease of young hazelnut trees worldwide. Thousands of hectares of new hazelnut cultivars that are resistant to eastern filbert blight (Anisogramma anomala) are being planted in the Willamette Valley of Oregon, where 99% of the US hazelnut crop is grown. Reports of bacterial blight on young hazelnut trees have increased but information about the causal pathogen is limited. Isolates were recovered from tissues with bacterial blight symptoms that were then characterized for their ability to grow on semi-selective media, their nutrient utilization profiles using Biolog GN2, quinate metabolism, copper tolerance, hypersensitive response on tobacco, and pathogenicity on hazelnut. Additionally, isolates were identified with a duplex PCR assay (ftsX and qumA), 16S rRNA sequence, and multi-locus sequence analysis (MLSA) using rpoD and gyrB. Pathogenic isolates were identified as Xac using morphological, biochemical, molecular, and host assays. Using MLSA, Xac isolates from Oregon separated into two clades, one clade with the type strain and a second clade previously described using isolates from Europe. The phylogenetic diversity of Xac observed in other countries also is present in Oregon.

Diagnostic Challenges and Prospects Associated With Zoonotic Tuberculosis of Central Nervous System.

Introduction:The diagnosis of Tuberculous Meningitis (TBM) has remained a challenge due to its insidious onset and the failure of conventional diagnostic tests. The present study aimed to identify the mycobacterial pathogen in the CSF of patients with TBM and a poor prognosis.Methods:We retrospectively recruited 224 TBM and 34 non-TBM patients admitted to the Central India Institute of Medical Sciences, Nagpur, India, in 2014. The CSF samples of these patients were subjected to a duplex PCR assay for the species-specific identification of the causative pathogen.Results:M. bovis and infection with M.tuberculosis were detected in 7% (18) and 32.9% (85) of the patients, respectively. Moreover, 14% (36) of the study samples were culture positive; however, the mycobacterial pathogens could not be differentiated to the species level.Conclusion:The present study findings emphasized the potentially vital importance of M. bovis identification for appropriate patient management. The obtained data also demonstrated the persistent significance of M. bovis, as a zoonotic pathogen.

Comparative haemato-biochemical alteration in theileriosis and babesiosis detected by duplex PCR in cattle

Blood samples from suspected crossbred cows (327) were examined microscopically as well as confirmed by in-house standardized duplex PCR assay. Out of 327 samples, 107 (32.72%) and 17 (5.19%) samples were positive for T. annulata and B. bigemina respectively by microscopy. When the samples were screened by duplex PCR, 130 (39.75%) and 27 (8.25%) animals had single infection with T. annulata (Group I) and B. bigemina (Group II), respectively. Duplex PCR was able to detect 11% of mixed infections (Group III) compared to 2.75% by microscopy. Haemato-biochemical profile of infected animals (30 for each group) were studied and compared with each other and normal healthy group (Group IV, n=10). The infected group showed significantly decreased levels of TEC, Hb and PCV, red blood cell indices than healthy control animals indicating microcytic hypochromic anaemia. Marked thrombocytopenia was also observed in affected animals. Serum biochemistry of infected cows revealed significantly higher values of AST and low levels of blood glucose, calcium, total protein, albumin as compared to healthy animals. Group I and Group II were further sub-divided into three sub-groups based on severity of infection as latent, subclinical and clinical. No significant difference among biochemical parameters was observed between subgroups of diseased animals but there was significant decline in hematological parameters, viz. haemaoglobin, PCV and TEC. Haemato-biochemical changes were more severe in B. bigemina infected group in contrast to other groups and anaemia is becoming more severe as the disease progress due to extensive intravascular haemolysis.

A duplex PCR assay for the simultaneous detection and differentiation of Muscovy duck parvovirus and goose parvovirus

Both Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) can cause high mortality and morbidity in Muscovy ducklings. MDPVs and GPVs share high nucleotide identity, which can cause errors during differential diagnosis. In this study, the NS genes of both MDPVs and GPVs were chosen for the design of specific primers after genetic comparison. Only three primers (GF1, MF1 and MGR1) were designed for the duplex PCR assay: GF1 is specific for GPV only; MF1 is specific for MDPV only; and MGR1 is highly conserved for both MDPV and GPV. After a series of optimization experiments, the duplex PCR assay amplified a 161-bp fragment specifically for GPV, a 1197-bp fragment specifically for MDPV, and two fragments (161-bp and 1197-bp) for both GPV and MDPV. The lowest detection limit was 103 copies/μl. No amplification was obtained using nucleic acids from other pathogens (including DAdV-A, DuCV, DEV, GHPV, R.A., E. coli., P.M. and S.S.) occurring in Muscovy ducks. Application of the duplex PCR assay in field samples showed that even one-day-old Muscovy ducklings were both MDPV-positive and GPV-positive. In conclusion, a duplex PCR assay for the simultaneous detection and differentiation of MDPV and GPV was established using only three highly specific primers. Our finding suggested that country-wide vaccination with MDPV and GPV vaccines in waterfowls are necessary.

Development of a rapid-viability PCR method for detection of Clostridioides difficile spores from environmental samples

Clostridioides difficile is a common pathogen that is well known to survive for extended periods of time on environmental healthcare surfaces from fecal contamination. During epidemiological investigations of healthcare-associated infections, it is important to be able to detect whether or not there are viable spores of C. difficile on surfaces. Current methods to detect C. difficile can take up to 7 days for culture and in the case of detection by PCR, viability of the spores cannot be ascertained. Prevention of C. difficile infection in healthcare settings includes adequate cleaning and disinfection of environmental surfaces which increases the likelihood of detecting dead organisms from an environmental sample during an investigation. In this study, we were able to adapt a rapid-viability PCR (RV-PCR) method, first developed for detection of viable Bacillus anthracis spores, for the detection of viable C. difficile spores. RV-PCR uses the change in cycle threshold after incubation to confirm the presence of live organisms. Using this modified method we were able to detect viable C. difficile after 22 h of anaerobic incubation in Cycloserine Cefoxitin Fructose Broth (CCFB). This method also used bead beating combined with the Maxwell 16 Casework kit for DNA extraction and purification and a real-time duplex PCR assay for toxin B and cdd3 genes to confirm the identity of the C. difficile spores. Spiked environmental sponge-wipes with and without added organic load were tested to determine the limit of detection (LOD). The LOD from spiked environmental sponge-wipe samples was 104 spores/mL but after incubation initial spore levels of 101 spores/mL were detected. Use of this method would greatly decrease the amount of time required to detect viable C. difficile spores; incubation of samples is only required for germination (22 h or less) instead of colony formation, which can take up to 7 days. In addition, PCR can then quickly confirm or deny the identity of the organism at the same time it would confirm viability. The presence of viable C. difficile spores could be detected at very low levels within 28 h total compared to the 2 to 10-day process that would be needed for culture, identification and toxin detection.

Duplex PCR Assay for Identification of Tissue of Cattle and Buffalo Origin Targeting Mitochondrial Cytochrome b Gene

The aim of this study was to develop PCR assay for simultaneous detection of tissues from cattle and buffalo in a single PCR reaction. Species-specific primers for cattle and buffalo species were designed through homology comparisons and alignment of partial sequences of Cyt.b gene from common food animals. The conditions for species-specific PCR amplification were optimized in terms of quantity and concentration of various components for PCR mix and annealing temperature for each set of designed species-specific primer pairs. The assay revealed the amplification of genomic DNA from cattle to amplicon size of 655 bp, whereas DNA template from buffalo to amplicon length of 249 bp. Simultaneous amplification of DNA templates from cattle and buffalo was observed in duplex PCR assay. Thus, the developed assay could identify the tissues of cattle and buffalo origin, detecting even both the species together and being advantageous over species-specific PCR.

Progressive multifocal leukoencephalopathy in Zambia is caused by JC virus with prototype regulatory region

There are only few documented cases of progressive multifocal leukoencephalopathy (PML) in Africa. Whether this is caused by a lack of JC virus (JCV) spread or alteration in the JCV genome is unknown. We characterized the clinical presentation, laboratory findings, and JCV regulatory region (RR) pattern of the first documented PML cases in Zambia as well as JCV seroprevalence among HIV+ and HIV- Zambians. We identified PML patients with positive JCV DNA PCR in their cerebrospinal fluid (CSF) among subjects enrolled in an ongoing tuberculous meningitis study from 2014 to 2016 in Lusaka. JCV regulatory region was further characterized by duplex PCR in patients' urine and CSF. Of 440 HIV+ patients, 14 (3%) had detectable JCV DNA in their CSF (age 18-50; CD4+ T cells counts 15-155 × 106/μl) vs 0/60 HIV- patients. The main clinical manifestations included altered mental status and impaired consciousness consistent with advanced PML. While prototype JCV was identified by duplex PCR assay in the CSF samples of all 14 PML patients, only archetype JCV was detected in their urine. All PML Zambian patients tested were seropositive for JCV compared to 46% in a control group of HIV+ and HIV- Zambian patients without PML. PML occurs among HIV-infected individuals in Zambia and is caused by CNS infection with prototype JCV, while archetype JCV strains are present in their urine. JCV seroprevalence is comparable in Zambia and the USA, and PML should be included in the differential diagnosis of immunosuppressed individuals presenting with neurological dysfunction in Zambia.

A duplex PCR assay for the simultaneous quantification of Bacteroides HF183 and crAssphage CPQ_056 marker genes in untreated sewage and stormwater

The HF183 marker gene, derived from the 16S rRNA gene of Bacteroides dorei, has been widely used to identify sewage pollution in environmental waters. CrAssphages are recently discovered DNA bacteriophages that are highly abundant in untreated sewage and have shown promises for tracking sewage contamination in environmental waters. In this paper, we report the development of a duplex quantitative PCR (qPCR) assay for simultaneous quantification of HF183 and crAssphage CPQ_056 marker genes in untreated sewage and sewage impacted stormwater. Same primer and probe sequences were used in the duplex qPCR assay as used in published simplex qPCR assays. The performance characteristics of the duplex qPCR assay were similar to its simplex counterparts. We validated the performance of the duplex assay in a collaborative laboratory study with the aim to evaluate reproducibility, sensitivity and concordance for field study. The concordance values between the simplex vs. duplex qPCR assays for HF183 and crAssphage CPQ_056 marker genes ranged from 96.7 to 100% and the mean concentrations of HF183 and CPQ_056 in environmental water samples were remarkably similar or in some cases slightly greater for the duplex qPCR assay suggesting the reliability of this assay for monitoring HF183 and CPQ_056 simultaneously. The newly developed duplex qPCR assay will be a valuable addition to the MST toolbox for sewage pollution monitoring and would allow rapid and comparative sample analysis.

Fast and efficient symbiotic gene-based duplex PCR approach for the preliminary selection of legume root nodule bacteria

The isolation of root nodule bacteria (RNB) usually lead to obtainment of several non-rhizobia, mainly those fast-growing bacteria. The plant-authentication experiments to select the nodulating bacteria are time consuming and can be ruined in case of nodulation of negative controls. To speed up this step in rhizobiology research a fast, simple and efficient duplex PCR assay for the amplification of nodC and nifH genes in was developed aiming to separate rhizobia and non-rhizobia in RNB culture collections. The method was optimized with 43 reference strains and was applied to a new culture collection for validation. Considering all bacteria, 106 out of 109 nodulating rhizobia were positive for both genes. Among non-nodulating, 48 out of 48 were nodC-negative. The results demonstrated the efficiency of this new method that can be completed in a day, to save time and money in RNB isolation projects, fulfilling Koch's postulate, and separating rhizobia from non-rhizobial bacteria.

Comparison of an in-house real-time duplex PCR assay with commercial HOLOGIC\xae APTIMA assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urine and extra-genital specimens

BackgroundExtra-genital Neisseria gonorrhoeae and Chlamydia trachomatis infections are mostly asymptomatic, and important reservoir sites of infection as they often go undetected and may be more difficult to eradicate with recommended therapeutic regimens. Commercial nucleic acid amplification tests (NAATs) have not received regulatory approval for the detection of N. gonorrhoeae and C. trachomatis in extra-genital specimens. The HOLOGIC® APTIMA Combo2 assay for N. gonorrhoeae and C. trachomatis has performed well in evaluations using extra-genital specimens.MethodsWe assessed the performance of an in-house real-time duplex PCR assay for the detection of N. gonorrhoeae and C. trachomatis in urine and extra-genital specimens using the HOLOGIC® APTIMA assays as gold standard comparators. Urine, oropharyngeal and ano-rectal specimens were collected from each of 200 men-who-have-sex-with-men (MSM) between December 2011 and July 2012.ResultsFor N. gonorrhoeae detection, the in-house PCR assay showed 98.5–100% correlation agreement with the APTIMA assays, depending on specimen type. Sensitivity for N. gonorrhoeae detection was 82.4% for ano-rectal specimens, 83.3% for oropharyngeal specimens, and 85.7% for urine; and specificity was 100% with all specimen types. The positive predictive value (PPV) for N. gonorrhoeae detection was 100% and the negative predictive value (NPV) varied with sample type, ranging from 98.5–99.5%. For C. trachomatis detection, correlation between the assays was 100% for all specimen types. The sensitivity, specificity, PPV and NPV of the in-house PCR assay was 100% for C. trachomatis detection, irrespective of specimen type.ConclusionThe in-house duplex real-time PCR assay showed acceptable performance characteristics in comparison with the APTIMA® assays for the detection of extra-genital N. gonorrhoeae and C. trachomatis.