Detection of Leishmania in Immunocompromised Patients Using Peripheral Blood Spots on Filter Paper and the Polymerase Chain Reaction

Campino, Lenea; Cortes, Sofia; Pires, Renato; Oskam, Linda; Abranches, P.

doi:10.1007/s100960050503

Cited by 24 publications

(28 citation statements)

References 11 publications

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“…Data exist regarding its use in peripheral blood and bone marrow samples, with sensitivities ranging from 73% to 100% and specificity close to 100% for the diagnosis of the initial VL episode, mainly in HIV-infected patients. [7][8][9][10][11][12][13][14][15][16][17][18] However, its value as a useful tool for monitoring VL in HIVinfected patients remains to be proven. Various published studies seem to correlate the presence of a high parasite load level in peripheral blood measured by PCR after an initial episode treated and cured with a higher risk of future clinical relapse (Table 1).…”

Section: Introductionmentioning

confidence: 99%

Ultrasensitive Real-Time PCR for the Clinical Management of Visceral Leishmaniasis in HIV-Infected Patients

Molina¹,

Fisa²,

Riera³

et al. 2013

The American Society of Tropical Medicine and Hygiene

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Abstract. Molecular methods have been proposed as an alternative tool for the diagnosis of visceral leishmaniasis (VL), but no data are available regarding use for monitoring clinical outcome. A prospective cohort study of human immunodeficiency virus-(HIV) and VL-coinfected patients was conducted in a university-affiliated hospital in Barcelona, Spain. Leishmania parasite load was monitored using a real-time polymerase chain reaction (PCR) at baseline and every 3 months. Cutoff values for PCR were determined using receiver operating characteristic (ROC) curves. Overall, 37 episodes were analyzed, and 25 of these episodes were considered as relapsing episodes. A significant decrease of parasite load measured 3 months after treatment could predict the clinical evolution of VL. A parasite load over 0.9 parasites/mL measured 12 months after treatment could predicts relapse with a sensitivity of 100% and a specificity of 90.9%. Monitoring parasite load by an ultrasensitive quantitative Leishmania PCR is useful to predict the risk of relapse after a VL episode in HIV-infected patients.

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Section: Introductionmentioning

confidence: 99%

Ultrasensitive Real-Time PCR for the Clinical Management of Visceral Leishmaniasis in HIV-Infected Patients

Molina¹,

Fisa²,

Riera³

et al. 2013

The American Society of Tropical Medicine and Hygiene

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“…The material dried on filter paper is easily stored and transported, without the need for refrigeration. 3,6,11,13,15,16 Typically, such samples are eluted from the sample paper, and nucleic acid is subsequently isolated from the eluate for use in PCR. Furthermore, some filter-paper products include reagents that lyse cell membranes and stabilize nucleic acids, allowing material to be easily washed and deposited directly into the PCR reaction.…”

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confidence: 99%

“…2,13 To access the biological material for PCR, the material dried on filter paper is commonly excised by using a hole punch. 3,4,6,9,12,13,16,17 This method allows for expedient processing of samples and provides a standard volume of filter paper. However, the use of a single piece of equipment for multiple samples makes the method vulnerable to carryover of nucleic acid between samples.…”

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confidence: 99%

“…Sequentially, ''material'' was excised from an unused filter paper (precut control) from a previously unopened pack, from filter paper with blood from the infected corella sample (known positive sample), and from 5 unused filter papers (contamination controls [1][2][3][4][5]. The sequence of filter paper excisions is illustrated in Fig.…”

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confidence: 99%

“…In method 4 (bleach, tap water, ethanol), no detectable amplification was obtained with PCR of the precut control (Fig. 4, lane 1 Many studies used hole punches to excise material dried on filter paper [2][3][4]7,9,[12][13][14]16,18,20 ; however, only a few reports mention any protocols for detecting possible carryover or detection of false positives in filter-paperbased PCR methods. 9,11 Furthermore, many published studies overlooked any consideration for the possibility of such contamination.…”

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confidence: 99%

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Elimination of False-Positive Polymerase Chain Reaction Results Resulting from Hole Punch Carryover Contamination

et al. 2008

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Abstract. The collection of biological material (e.g., blood) directly onto filter paper for subsequent use in laboratory assays such as polymerase chain reaction (PCR), has become a common practice. Dried cells or fluid on the paper can be readily rehydrated and retrieved into a standard volume of an appropriate elution buffer but introduces a dilution factor to the sample. The use of a common cutting instrument for excising a standard-sized piece of paper that contains the material also introduces the potential for transferring biological material from one sample to subsequent samples, causing false-positive results by PCR. In the present study, filter-paper-collected blood that contained beak and feather disease virus was used to determine if viral DNA could be transferred between samples by a hole punch used to excise sequential filter papers. It was determined that false-positive results could be obtained at least 13 times after a positive sample. Subsequently, the efficacy of 4 methods of hole punch disinfection, flaming, VirkonS, bleach, and a bleachethanol combination, was assessed. The only effective and practical method to destroy DNA was a method where the hole punch was agitated in commercial bleach, rinsed in water, the water was displaced with 100% ethanol and air-dried. This method was simple, cheap, and relatively rapid, and allowed for the use of a single hole punch for a series of samples, without carryover contamination and consequent false-positive results.

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Development and application of 'simple' diagnostic tools for visceral leishmaniasis

Schallig

et al. 2001

Med Microbiol Immunol

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The diagnosis of visceral leishmaniasis is difficult. Due to the limitations of direct methods to detect parasites, indirect immunological methods are widely employed. The simple affordable and sensitive/specific direct agglutination test (DAT) is perhaps the most important diagnostic tool under field conditions. A significant improvement of this test is the use of a freeze-dried antigen, which is heat-stable and has a long shelf-live even under harsh conditions. The performance of this antigen in DAT has been evaluated using samples collected in East Africa. The results of these studies are presented. The detection of Leishmania infection in HIV-co-infected patients is difficult. The combination of DAT-PCR may be useful for the detection of parasite infection in these patients. Finally, we present data to show that the DAT based on the freeze-dried antigen can also be used for the detection of anti-Leishmania antibodies in dogs.

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Detection of Leishmania in Immunocompromised Patients Using Peripheral Blood Spots on Filter Paper and the Polymerase Chain Reaction

Cited by 24 publications

References 11 publications

Ultrasensitive Real-Time PCR for the Clinical Management of Visceral Leishmaniasis in HIV-Infected Patients

Ultrasensitive Real-Time PCR for the Clinical Management of Visceral Leishmaniasis in HIV-Infected Patients

Elimination of False-Positive Polymerase Chain Reaction Results Resulting from Hole Punch Carryover Contamination

Development and application of 'simple' diagnostic tools for visceral leishmaniasis

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