Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Preuve par l'ADN: la génétique au service de la justice

[Sommaire] Introduction. - Concepts biochimiques de base. - Concepts génétiques de base. - Quelques méthodes d'analyse de base en biologie. - Analyses génétiques - La caisse à outils de base. - Analyses génétiques - La caisse à outils spécialisée. - Les traces biologiques dans leur utilisation pratique. - Tests indicatifs et recherche des traces. - Quelques éléments d'évaluation probabiliste. - Au-delà du laboratoire. - Glossaire. - Index

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture

Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.

Malaria in the state of Rio de Janeiro, Brazil, an Atlantic Forest area: an assessment using the health surveillance service

The lethality of malaria in the extra-Amazonian region is more than 70 times higher than in Amazonia itself. Recently, several studies have shown that autochthonous malaria is not a rare event in the Brazilian southeastern states in the Atlantic Forest biome. Information about autochthonous malaria in the state of Rio de Janeiro (RJ) is scarce. This study aims to assess malaria cases reported to the Health Surveillance System of the State of Rio de Janeiro between 2000-2010. An average of 90 cases per year had parasitological malaria confirmation by thick smear. The number of malaria notifications due to Plasmodium falciparum increased over time. Imported cases reported during the period studied were spread among 51% of the municipalities (counties) of the state. Only 35 cases (4.3%) were autochthonous, which represents an average of 3.8 new cases per year. Eleven municipalities reported autochthonous cases; within these, six could be characterised as areas of residual or new foci of malaria from the Atlantic Forest system. The other 28 municipalities could become receptive for transmission reintroduction. Cases occurred during all periods of the year, but 62.9% of cases were in the first semester of each year. Assessing vulnerability and receptivity conditions and vector ecology is imperative to establish the real risk of malaria reintroduction in RJ.

Detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection

Viruses are the major contributors to the morbidity and mortality of upper and lower acute respiratory infections (ARIs) for all age groups. The aim of this study was to determine the frequencies for a large range of respiratory viruses using a sensitive molecular detection technique in specimens from outpatients of all ages with ARIs. Nasopharyngeal aspirates were obtained from 162 individuals between August 2007-August 2009. Twenty-three pathogenic respiratory agents, 18 respiratory viruses and five bacteria were investigated using multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay (IIF). Through IIF, 33 (20.4%) specimens with respiratory virus were recognised, with influenza virus representing over half of the positive samples. Through a multiplex real-time RT-PCR assay, 88 (54.3%) positive samples were detected; the most prevalent respiratory viral pathogens were influenza, human rhinovirus and respiratory syncytial virus (RSV). Six cases of viral co-detection were observed, mainly involving RSV. The use of multiplex real-time RT-PCR increased the viral detection by 33.9% and revealed a larger number of respiratory viruses implicated in ARI cases, including the most recently described respiratory viruses [human bocavirus, human metapneumovirus, influenza A (H1N1) pdm09 virus, human coronavirus (HCoV) NL63 and HCoV HKU1].

Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

Accuracy of mucocutaneous leishmaniasis diagnosis using polymerase chain reaction: systematic literature review and meta-analysis

The diagnosis of mucocutaneous leishmaniasis (MCL) is hampered by the absence of a gold standard. An accurate diagnosis is essential because of the high toxicity of the medications for the disease. This study aimed to assess the ability of polymerase chain reaction (PCR) to identify MCL and to compare these results with clinical research recently published by the authors. A systematic literature review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA Statement was performed using comprehensive search criteria and communication with the authors. A meta-analysis considering the estimates of the univariate and bivariate models was performed. Specificity near 100% was common among the papers. The primary reason for accuracy differences was sensitivity. The meta-analysis, which was only possible for PCR samples of lesion fragments, revealed a sensitivity of 71% [95% confidence interval (CI) = 0.59; 0.81] and a specificity of 93% (95% CI = 0.83; 0.98) in the bivariate model. The search for measures that could increase the sensitivity of PCR should be encouraged. The quality of the collected material and the optimisation of the amplification of genetic material should be prioritised.

Molecular diagnosis of strongyloidiasis in tropical areas: a comparison of conventional and real-time polymerase chain reaction with parasitological methods

This study aimed to evaluate the use of conventional polymerase chain reaction (cPCR) and real-time quantitative PCR (qPCR) in the diagnosis of human strongyloidiasis from stool samples in tropical areas. Stool samples were collected from individuals and were determined to be positive for Strongyloides stercoralis (group I), negative for S. stercoralis (group II) and positive for other enteroparasite species (group III). DNA specific to S. stercoralis was found in 76.7% of group I samples by cPCR and in 90% of group I samples by qPCR. The results show that molecular methods can be used as alternative tools for detecting S. stercoralis in human stool samples in tropical areas.

Accès aux soins d'urgence: organisation et utilité de la chaîne des secours [Access to prehospital medical care: organization and utility of the chain of survival].

In order to be effective, access to prehospital care must be integrated into a system described as "the chain of survival". This system is composed of 5 essential phases: 1) basic help by witnesses; 2) call for help; 3) basic life support; 4) professional rescue and transport to the appropriate institution and 5) access to emergency ward and hospital management. Each phase is characterized by a specific organization, dedicated skills and means in order to increase the level of care brought to the patient. This article describes the organization, the utility and the specificity of the chain of survival allowing access to prehospital medical care in the western part of Switzerland.

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

Markov chain montecarlo method applied to rounding zeros of compositional data: first approach

As stated in Aitchison (1986), a proper study of relative variation in a compositional data set should be based on logratios, and dealing with logratios excludes dealing with zeros. Nevertheless, it is clear that zero observations might be present in real data sets, either because the corresponding part is completelyabsent –essential zeros– or because it is below detection limit –rounded zeros. Because the second kind of zeros is usually understood as “a trace too small to measure”, it seems reasonable to replace them by a suitable small value, and this has been the traditional approach. As stated, e.g. by Tauber (1999) and byMartín-Fernández, Barceló-Vidal, and Pawlowsky-Glahn (2000), the principal problem in compositional data analysis is related to rounded zeros. One should be careful to use a replacement strategy that does not seriously distort the general structure of the data. In particular, the covariance structure of the involvedparts –and thus the metric properties– should be preserved, as otherwise further analysis on subpopulations could be misleading. Following this point of view, a non-parametric imputation method isintroduced in Martín-Fernández, Barceló-Vidal, and Pawlowsky-Glahn (2000). This method is analyzed in depth by Martín-Fernández, Barceló-Vidal, and Pawlowsky-Glahn (2003) where it is shown that thetheoretical drawbacks of the additive zero replacement method proposed in Aitchison (1986) can be overcome using a new multiplicative approach on the non-zero parts of a composition. The new approachhas reasonable properties from a compositional point of view. In particular, it is “natural” in the sense thatit recovers the “true” composition if replacement values are identical to the missing values, and it is coherent with the basic operations on the simplex. This coherence implies that the covariance structure of subcompositions with no zeros is preserved. As a generalization of the multiplicative replacement, in thesame paper a substitution method for missing values on compositional data sets is introduced

A sensitive, specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivaxandPlasmodium falciparum infection in field-collected anophelines

We describe a simple method for detection of Plasmodium vivaxand Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed withPlasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochromeb-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.