<i>Plasmodium</i>DNA Contamination between Blood Smears during Giemsa Staining and Microscopic Examination

Aubouy, Agnès; Carme, Bernard

doi:10.1086/424529

Cited by 13 publications

(11 citation statements)

References 4 publications

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“…Though it is likely that the infections detected only by the assay were, in fact, "submicroscopic", they may also be the result of poorly-sensitive microscopy. Moreover, microscopy can be an imperfect reference standard used for malaria diagnosis, and this study did not have microscopy quality control [29,30]. Additionally, the pfldh PCR assay, though simple and easy to perform, detects only P. falciparum infections.…”

Section: Discussionmentioning

confidence: 99%

Comparison of real-time PCR and microscopy for malaria parasite detection in Malawian pregnant women

Rantala

Taylor

Trottman

et al. 2010

Malar J

100

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BackgroundNew diagnostic tools for malaria are required owing to the changing epidemiology of malaria, particularly among pregnant women in sub-Saharan Africa. Real-time PCR assays targeting Plasmodium falciparum lactate dehydrogenase (pfldh) gene may facilitate the identification of a high proportion of pregnant women with a P. falciparum parasitaemia below the threshold of microscopy. These molecular methods will enable further studies on the effects of these submicroscopic infections on maternal health and birth outcomes.MethodsThe pfldh real-time PCR assay and conventional microscopy were compared for the detection of P. falciparum from dried blood spots and blood smears collected from the peripheral blood of 475 Malawian women at delivery. A cycle threshold (Ct) of the real-time PCR was determined optimizing the sensitivity and specificity of the pfldh PCR assay compared to microscopy. A real-time PCR species-specific assay was applied to identify the contribution to malaria infections of three Plasmodium species (P. falciparum P. ovale and P. malariae) in 44 discordant smear and pfldh PCR assay results.ResultsOf the 475 women, P. falciparum was detected in 11 (2.3%) by microscopy and in 51 (10.7%) by real-time PCR; compared to microscopy, the sensitivity of real-time PCR was 90.9% and the specificity 91.2%. If a Ct value of 38 was used as a cut-off, specificity improved to 94.6% with no change in sensitivity. The real-time PCR species-specific assay detected P. falciparum alone in all but four samples: two samples were mixed infections with P. falciparum and P. malariae, one was a pure P. malariae infection and one was a pfldh PCR assay-positive/species-specific assay-negative sample. Of three P. malariae infections detected by microscopy, only one was confirmed by the species-specific assay.ConclusionsAlthough microscopy remains the most appropriate method for clinical malaria diagnosis in field settings, molecular diagnostics such as real-time PCR offer a more reliable means to detect malaria parasites, particularly at low levels. Determination of the possible contribution of these submicroscopic infections to poor birth outcomes and maternal health is critical. For future studies to investigate these effects, this pfldh real-time PCR assay offers a reliable detection method.

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Section: Discussionmentioning

confidence: 99%

Comparison of real-time PCR and microscopy for malaria parasite detection in Malawian pregnant women

Rantala

Taylor

Trottman

et al. 2010

Malar J

100

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“…A number of methods were tried (20 smears per method) to remove the blood from the slides for DNA extraction: use of a scalpel (Aubouy & Carme, 2004) or slide scraper (Sykes et al, 2008), as previously described, and use of a sterile swab moistened in PBS firmly rubbed over the entirety of the slide. Use of the scalpel or slide scraper made it difficult to remove all of the blood from the slide and concern regarding laboratory contamination arose due to blood smear material becoming airborne during removal, so an optimized method using a swab moistened in PBS was used for further studies.…”

Section: Methodsmentioning

confidence: 99%

Investigation of human haemotropic Mycoplasma infections using a novel generic haemoplasma qPCR assay on blood samples and blood smears

Tasker

Peters

Mumford

et al. 2010

Journal of Medical Microbiology

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The aim of this study was to develop quantitative real-time (q)PCR assays to detect all known haemoplasma species, and a human housekeeping gene in order to demonstrate both successful DNA extraction from clinical samples and to test for sample inhibition, and to apply these qPCRs to human blood samples and blood smears. Sensitive and specific generic haemoplasma qPCR assays were developed to amplify haemoplasma species, as well as human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal amplification control. An optimized technique for extracting DNA from stained blood smears was also developed. These methods were applied to anonymized blood samples obtained from 100 human immunodeficiency virus (HIV)-infected South Africans and 920 UK patients undergoing haematological examination, and to 15 blood smears recruited from previous studies describing human haemoplasmosis. Human GAPDH levels were acceptable in all but three of the blood samples and all but two of the blood smears. The latter could have arisen due to DNA degradation due to the old age (over 35 years) of these smears. Haemoplasma infection was found in one HIV-infected South African, but the species could not be characterized due to the very low levels of DNA present. This report describes novel extraction and qPCR methodologies for haemoplasma screening. Previously reported human haemoplasmosis based on cytological diagnosis alone should be viewed with caution.

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“…However, microscopic diagnosis using staining techniques, for instance Gram staining or Giemsa staining to search for bacteria or parasites, respectively, does not usually allow the long-term conservation of samples. 9,10 Moreover, while genomic DNA has been recovered efficiently from untreated glass slides, 11 recycling such glass slides for the DNA extraction is as toxic as some other fixatives. The combination of alcohol and acid (in particular acetic acid), denatures proteins and DNA.…”

Section: Smear Slide Systems That Have Been Used For Other Microorganmentioning

confidence: 99%

Exploring tuberculosis by molecular tests on DNA isolated from smear microscopy slides

Rakotosamimanana

Rabodoarivelo

Palomino

et al. 2017

International Journal of Infectious Diseases

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Tuberculosis (TB) is an infectious disease of global public health importance caused by Mycobacterium tuberculosis complex. The disease has worsened with the emergence of multidrug-resistant (MDR)-TB strains. The timely diagnosis and treatment of TB remains a key public health priority, and laboratories have a critical role in the rapid and accurate detection of TB and drug resistance. Molecular assays based on nucleic acid amplification techniques have been developed for the rapid, sensitive, and specific diagnosis of TB, with the ability to determine the drug sensitivity status. These molecular techniques are now available or are being implemented in developing countries. However, traditional microscopy and culture methods cannot yet be replaced; the molecular assays can be applied in parallel with these tests for the diagnosis of TB or for drug susceptibility testing. Performing such molecular tests is often restricted by constraints with regard to sputum sample storage and safe transportation from remote health centres to central laboratories. Since smear slides are performed routinely for the diagnosis of TB in most TB diagnostic laboratories, they are readily available and could be the ideal tool to transport sputum for further molecular tests. The aim of this review was to provide a comprehensive survey on the use of smear slides for both TB diagnosis and the molecular test approach. Based on the literature, stained smear microscopy slides can be a safe system for the transportation of sputum specimens from remote health centres to reference TB laboratories for further molecular TB or MDR-TB detection, and could help in the rapid diagnosis and therefore timely management of TB patients.

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PlasmodiumDNA Contamination between Blood Smears during Giemsa Staining and Microscopic Examination

Cited by 13 publications

References 4 publications

Comparison of real-time PCR and microscopy for malaria parasite detection in Malawian pregnant women

Comparison of real-time PCR and microscopy for malaria parasite detection in Malawian pregnant women

Investigation of human haemotropic Mycoplasma infections using a novel generic haemoplasma qPCR assay on blood samples and blood smears

Exploring tuberculosis by molecular tests on DNA isolated from smear microscopy slides

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