Development of a PCR Assay Followed by Nonradioactive Hybridization Using Oligonucleotides Covalently Bound to CovaLink NH Microwells for Detection of Four Plasmodium Species in Blood Samples from Humans

Abstract

We developed and evaluated a PCR-based assay to detect four Plasmodium species in 79 blood samples from 56 travelers returning from areas where malaria is endemic. DNA amplification targeting a small region of the 18S rRNA gene was performed with Plasmodium genus-specific primers. The biotinylated PCR products were then identified by PCR-colorimetric Covalink NH microwell plate hybridization (CMPH) using species-specific phosphorylated probes covalently bound to a pretreated polystyrene surface. The results from PCR-CMPH showed high specificity, and for 47 of the 56 patients (84%), microscopy and PCR-CMPH results were in agreement. Discordant results were reevaluated with microscopy examination, other molecular methods, and DNA sequencing. Except for one patient, discrepancies were resolved in favor of PCR-CMPH: three mixed infections were detected, four species identification errors were corrected, and two negative results were shown to be positive. Our results indicate that PCR-CMPH is a simple, rapid, and specific method for malaria diagnosis. It employs stable reagents and inexpensive equipment, making it suitable for routine epidemiological use.

PCR-based methods to the diagnosis of imported malaria

“…Concerning mixed infections, practically 100 % of the microscopy results were false, either by excess (diagnostic of a mixed infection when there was a single species) or by omission (reverse situation). Plasmodium falciparum identification appeared to be more reliable, with no misdiagnoses found in eight studies (Morassin et al, 2002;de Monbrison et al, 2003;Calderaro et al, 2004;Ndao et al, 2004;Perandin et al, 2004;Whiley et al, 2004;Machouart et al, 2006;Vo et al, 2007), a 1.7 to 2.8 % misdiagnosis rate in three studies (Rougemont et al, 2004;Rubio et al, 1999;Perandin et al, 2003) and 8.4 % of misdiagnoses in one study (Di Santi et al, 2004). However, the high specificity of PCR methods demonstrated the other side of the coin, namely the intrinsic risk of these tests being unable to detect new molecular variants.…”

“…Concerning Plasmodium species identification, literature analysis reports frequent and sometimes substantial discrepancies between the results from microscopy and those from PCR. From the last 12 published studies that have compared the performance of thin blood smear examination and PCR, 17.1 % (range: 1.5-39) of 923 species identifications by microscopy were corrected after PCR checking (Rubio et al, 1999;Morassin et al, 2002;de Monbrison et al, 2003;Perandin et al, 2003;Calderaro et al, 2004;Di Santi et al, 2004;Ndao et al, 2004;Perandin et al, 2004;Rougemont et al, 2004;Whiley et al, 2004;Machouart et al, 2006;Vo et al, 2007). The rate of misdiagnosis varied from 20 % to 50 % for P. malariae, P. ovale or P. vivax (Morassin et al, 2002;de Monbrison et al, 2003;Perandin et al, 2003;Calderaro et al, 2004;Di Santi et al, 2004;Ndao et al, 2004;Perandin et al, 2004;Rougemont et al, 2004).…”

“…All the patients positive by microscopy were also positive by PCR, whatever the Plasmodium species. When compared to the set of the requests, the gain of sensitivity was 4.4 % (range: from 0 to + 11 %) (Rubio et al, 1999;Filisetti et al, 2002;Morassin et al, 2002;Richardson et al, 2002;de Monbrison et al, 2003;Padley et al, 2003;Patsoula et al, 2003;Perandin et al, 2003;Calderaro et al, 2004;Fabre et al, 2004;Perandin et al, 2004;Rougemont et al, 2004;Whiley et al, 2004;Machouart et al, 2006;Vo et al, 2007). This improvement in sensitivity affected mainly falciparum malaria.…”

Molecular Methods for Diagnosis of Malaria

“…A study titled "Development of a PCR assay followed by nonradioactive hybridization using oligonucleotides covalently bound to Covalink NH microwell for detection of four plasmodium species in blood samples from humans" published in journal of clinical microbiology, Sept. 2006 conducted by M. Machouart et al 42 . The authors developed and evaluated a PCR-based assay to detect four Plasmodium species in 79 blood samples from 56 travelers returning from areas where malaria is endemic (for some patients, several blood samples were collected during the first days of the hospitalization period) and presenting suggestive clinical features were collected in parasitology-mycology laboratories of hospitals in Nancy and Tourcoing (France).…”