HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples ...and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/10⁶ leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r = 0.900, P < 0.0001). A total of 3,002 specimens from 1,135 infants were tested. The specificity of HIV-DNA and HIV-RNA assays was 100%. HIV-1 infection was diagnosed in nine infants before age 60 days. HIV-DNA levels were low, underlining the need for sensitive assays when highly active antiretroviral therapy (HAART) has been given. The performances of this HIV-DNA assay showed that it is adapted to early diagnosis in children. The results were equivalent to those of HIV-RNA assay. HIV-DNA may be used even in masked primary infection in newborns whose mothers have received HAART. J. Med. Virol. 81:217-223, 2009.
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.
•Comparative genomics revealed novel specific genes to L. plantarum subspecies.•Specific primers for L. plantarum group species showed 100% specificity.•Real-time PCR quantitatively detected L. plantarum group species in food samples.•This real-time PCR is an accurate and simple method to detect L. plantarum group.
Sampling of microbial biomass is crucial for understanding and controlling remediation processes ongoing at contaminated sites in general, particularly when molecular genetic analyses are employed.
...In this study, fiber-based carriers with a nanofiber layer were developed and tested as a method to sample microbial biomass in groundwater for molecular genetic analysis. Nanofiber carriers, varying in the shape and the linear density of nanofibers, were examined throughout a 27-month monitoring period in groundwater contaminated with benzene, toluene, ethylbenzene and xylene isomers (BTEX), and chlorinated ethenes. The effect of carrier shape and nanofiber layer density on the microbial surface colonization and composition of the microbial biofilm was determined using real-time PCR and next-generation sequencing (NGS) analysis. Differences in microbial community composition between nanofiber carriers, groundwater, and soil samples were also analyzed to assess the applicability of carriers for biomass sampling at contaminated sites.
The nanofiber carriers showed their applicability as a sampling tool, particularly because of their easy manipulation that facilitates DNA isolation. The majority of taxa (Proteobacteria, Firmicutes, and Bacteroidetes) present on the carrier surfaces were also detected in the groundwater. Moreover, the microbial community on all nanofiber carriers reflected the changes in the chemical composition of groundwater.
Although the carrier characteristics (shape, nanofiber layer) did not substantially influence the microbial community on the carrier surface, the circular and planar carriers with a nanofiber layer displayed faster microbial surface colonization. However, the circular carrier was the most suitable for biomass sampling in groundwater because of its high contact area and because it does not require pre-treatment prior to DNA extraction.
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•nanofiber carriers examined during 27-month monitoring at a contaminated site•Carriers with nanofiber layer exhibited faster microbial surface colonization.•circular carriers chosen as most suitable for sampling of microflora in groundwater•Carriers ensure faster sample collection and processing prior to DNA extraction.•Microflora on carriers reflected changes in groundwater at a contaminated site.
ABSTRACT
Rapid identification of the causative pathogens of central nervous system infections is essential for providing appropriate management and improving patient outcomes. The performance of ...QIAstat-Dx Meningitis/Encephalitis (ME) Panel—a multiplex PCR testing platform—in detecting pathogens implicated in meningitis and/or encephalitis was evaluated using BioFire FilmArray ME Panel as a comparator method. This multicenter study analyzed 585 retrospective residual cerebrospinal fluid specimens and 367 contrived specimens. The QIAstat-Dx ME Panel showed positive percent agreement (PPA) values of 100% for
Neisseria meningitidis
,
Streptococcus agalactiae
,
Escherichia coli
K1,
Listeria monocytogenes
, and
Cryptococcus gattii
/
neoformans
on clinical samples compared to the BioFire FilmArray ME Panel. The PPA values observed for
Haemophilus influenzae
and
Streptococcus pneumoniae
were 80% and 88.24%, respectively. Negative percent agreement (NPA) values were >99.0% for each of the six bacterial targets and one fungal target tested with clinical samples. One viral target, herpes simplex virus 1, exhibited a PPA value of 100.0%, while the remaining viral targets—human parechovirus, herpes simplex virus 2, human herpes virus 6, and varicella zoster virus—were >90.0%, with the exception of enterovirus, which had a PPA value of 77.8%. The QIAstat-Dx ME Panel detected five true-positive and four true-negative cases compared to BioFire FilmArray ME Panel. The NPA values for all viral pathogens were >99.0%. Overall, the QIAstat-Dx ME Panel showed comparable performance to the BioFire FilmArray ME Panel as a rapid diagnostic tool for community-acquired meningitis and encephalitis.
Introduction.
The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a ...semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis.
Hypothesis/Gap Statement.
FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination.
Aim.
The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures.
Methodology.
A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens:
Shigella
spp.
, Salmonella
spp.
, Campylobacter
spp.,
Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum
spp.
, Dientamoeba fragilis
, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography.
Results.
A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested:
Shigella
spp./enteroinvasive
Escherichia coli
(EIEC) (
P
<0.05),
Salmonella
spp. (
P
<0.05) and
Campylobacter
spp. (
P
<0.05). Marked differences were also observed for the parasites
G. intestinalis
,
Cryptosporidium
spp. and
D. fragilis
.
Conclusion.
These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.
Cistus laurifolius is widely used in folk medicine in Anatolia for the treatment of many ailments. The leaves of the plant are used in the form of tea in the treatment of hemorrhoids in the Western ...Black Sea Region and Central Anatolia.
It was aimed at evaluating the anti-hemorrhoidal effects of C. laurifolus leaves in croton oil-induced hemorrhoid model in rats.
The methanolic and aqueous extracts of C. laurifolius were tested for in vivo anti-hemorrhoidal efficacy using an experimental hemorrhoid model, followed by histological and biochemical analysis. Hemorrhoid was created by using croton oil on the anal region of the rats. TNF-α and VEGF mRNA expression levels were assessed using real-time PCR detections. The extract was also tested for anti-inflammatory properties, which are based on the suppression of an increase in capillary permeability caused by acetic acid. LC-QTOF-MS and RP-HPLC were used for the phytochemical analysis.
In comparison to the control, histological and biochemical assessment showed that the methanolic extract of C. laurifolius is particularly effective against hemorrhoids. The same extract group's TNF-α mRNA expression was found to be the lowest. Additionally, the methanolic extract showed a strong inhibitory effect on the increase in capillary permeability resulted on by acetic acid. Three phenolic compounds were discovered in the extracts by phytochemical analyses, while more than eighteen compounds were found by LC-QTOF-MS analysis. Five of these compounds are phenolic acid derivatives, and flavonoids constitute the majority of the group.
This is the first evidence from the research that C. laurifolius possesses strong anti-inflammatory and anti-hemorrhoidal properties.
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Tuberculosis (TB) remains a major health problem worldwide. Control of TB requires rapid, accurate diagnosis of active disease. However, extrapulmonary TB is very difficult to diagnose because the ...clinical specimens have very low bacterial loads. Several molecular methods involving direct detection of the
Mycobacterium tuberculosis
complex (MTBC) have emerged in recent years.
Tuberculosis (TB) remains a major health problem worldwide. Control of TB requires rapid, accurate diagnosis of active disease. However, extrapulmonary TB is very difficult to diagnose because the clinical specimens have very low bacterial loads. Several molecular methods involving direct detection of the
Mycobacterium tuberculosis
complex (MTBC) have emerged in recent years. Real-time PCR amplification simultaneously combines the amplification and detection of candidate sequences by using fluorescent probes (mainly TaqMan or Molecular Beacons) in automated systems. The multiplex real-time PCR-short assay is performed using locked nucleic acid (LNA) probes (length, 8 to 9 nucleotides) in combination with CodUNG to detect multiple pathogens in clinical samples. In this study, we evaluated the performance of this novel multiplex assay for detecting the MTBC in comparison with that of the conventional culture-based method. The multiplex real-time PCR-short
TUB
assay targets two genes,
whiB3
(redox-responsive transcriptional regulator) and
pstS1
(phosphate-specific transporter), yielding limits of detection (LOD) of 10 copies and 100 copies, respectively, and amplification efficiencies of 92% and 99.7%, respectively. A total of 94 extrapulmonary samples and pulmonary samples with low mycobacterial loads (all smear negative; 75 MTBC culture positive) were analyzed using the test, yielding an overall sensitivity of 88% and a specificity of 95%. For pleural fluid and tissues/biopsy specimens, the sensitivity was 83% and 85%, respectively. In summary, this technique could be implemented in routine clinical microbiology testing to reduce the overall turnaround time for MTBC detection and may therefore be a useful tool for the diagnosis of extrapulmonary tuberculosis and diagnosis using pulmonary samples with low mycobacterial loads.
Human T cell lymphotropic virus (HTLV) is the caustive agent of two main conditions i. e., the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and the adult T-cell ...leukemia/lymphoma (ATLL). HTLV diagnosis is based on serological and molecular approaches; however, an accurate and validated method is still needed. The objective of this study was to establish a rapid and sensitive molecular test to confirm and discriminate HTLV 1/2 types. The test validation was performed as a multicentric study involving HTLV confirmation centers throughout Brazil. Proviral DNA was extracted from whole blood and the amplification was performed using in-house designed primer and probe sets targeting the
pol
genomic region. An internal control to validate the extraction and amplification was also included. The limit of detection (LoD) of the assay was four copies/reaction for HTLV-1 and 10.9 copies/reaction for HTLV-2. The diagnostic sensitivity of the platform was 94.6% for HTLV-1, 78.6% for HTLV-2, and the specificity was 100% for both viruses. Cross-reactions of the test with human viruses including HAV, HBV, HCV, HIV-1/2, and parvovirus B19 were not observed. During the multicentric validation, the test was used to screen a total of 692 blood samples obtained from previously confirmed HTLV-positive individuals. From these, 91.1% tested positive being concordant with the previously obtained results. In conclusion, our duoplex-RT-PCR-HTLV1 /2 presented adequate efficiency for HTLV-1/2 differentiation showing high sensitivity and specificity. Therefore, it can be a suitable tool for confirmation of suspected and inconclusive HTLV cases, prenatal and pre-transplant diagnosis, in Brazil and in other countries HTLV-endemic countries.
Quantitative real-time polymerase chain reaction (QRT-PCR) has become one of the most widely used methods for gene expression analysis. However, the expression profile of a target gene may be ...misinterpreted due to unstable expression of the reference genes under different experimental conditions. Thus, a systematic evaluation of these reference genes is necessary before experiments are performed. In this study, 10 putative reference genes were chosen for identifying expression stability using geNorm, NormFinder, and BestKeeper statistical algorithms in 12 different cucumber sample pools, including those from different plant tissues and from plants treated with hormones and abiotic stresses.
EF1α and
UBI-ep exhibited the most stable expression across all of the tested cucumber samples. In different tissues, in addition to expression of
EF1α and
UBI-ep, the expression of
TUA was also stable and was considered as an appropriate reference gene. Evaluation of samples treated with different hormones revealed that
TUA and
UBI-ep were the most stably expressed genes. However, for abiotic stress treatments, only
EF1α showed a relatively stable expression level. In conclusion,
TUA, UBI-ep, and
EF1α will be particularly helpful for reliable QRT-PCR data normalization in these types of samples. This study also provides guidelines for selecting different reference genes under different conditions.
ObjectivesA major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA ...quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices.DesignReference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/−PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB.ResultsHirt and −PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA.ConclusionsWe present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.
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