Improvement of a PCR test to diagnose infection by Mansonella ozzardi Adequação da técnica da PCR para diagnóstico de infecção de Mansonella ozzardi

Introduction: Mansonelliasis is caused by Mansonella ozzardi. It is widespread in the Amazon region, with a high prevalence. The common exam of thick blood smears stained with Giemsa shows low efficacy levels and has been an obstacle to diagnosing individuals with low blood parasitemia. Methods: In order to increase diagnosis efficacy, the PCR technique was improved. Results and Conclusions: PCR demonstrated the best performance, with sensitivity and negative predictive values (NPV) of 100%, followed by blood filtration through membrane filters, which showed a sensitivity of 88.9% and a NPV of 84.6%, when compared to thick blood smears.

The microfilariae of Mansonella ozzardi (Nematode, Onchocercidae) is the etiological agent of the mansonelliasis.The parasite has a geographic distribution limited to the Americas and it is found from Mexico through Argentina, except Chile, Uruguay, and Paraguay 1 .This filariasis was first reported in Brazil in the City of Manaus in the State of Amazonas 2 and people infected by M. ozzardi were later identified along the Solimoes, Purus and Negro Rivers 3 .These findings reinforce many studies that have warned that M. ozzardi was widely distributed in the State of Amazonas 4 , Brazil; however studies concerning this filariasis remain scarce [5][6][7] .
Mansonella ozzardi is transmitted by insects of the families Ceratopogonidae and Simuliidae (Diptera).Up to now, only the Simuliidaes have been identified as vectors in Brazil 8,9 .Infection symptomatology has been widely discussed and according to Batista et al 10 , people suffering from mansonelliasis present moderate fever, leg coldness, joint aches, dizziness and headaches.Recently, new symptomatology has been attributed to this filariasis, which is characterized by ocular lesions and white rings on the cornea that may eventually cause blindness 11 .
Typically, microscopic examination of a thick blood smear (TBS) stained with Giemsa, has been the main method used in epidemiological assays.Despite the low efficacy of TBS, particularly for patients with low microfilaremia, it is more economically feasible, promptly and easily obtained, and also permits identification of the parasite species even in locations where intermingled infections by other filariidaes occur 12 .More laborious and effective methods are feasible, such as Knott's concentration method (blood lysis by 2% formalin) 12,13 , and by filtering the blood through a polycarbonate membrane filter (PMF) 12 .The PMF is considered the gold-standard.Morales-Rojas et al first suggested polymerase chain reaction (PCR) as an effective method to detect M. ozzardi 14 .
This study aimed to standardize the PCR for diagnosing M. ozzardi and compare its effectiveness in relation to the PMF, which is considered the gold-standard for diagnosing the disease compared to the traditional TBS method.
Forty-seven patients infected by M. ozzardi were randomly selected from 232 individuals diagnosed by PMF (Poretics Corporation, Livermore, USA).These individuals lived alongside the rivers in the municipality of Labrea, State of Amazonas, Brazil (07 o 15'S 64 o 51'W), an area with high prevalence of mansonelliasis.The TBS method was used according to the following procedure: blood was collected from the digital pulp (equivalent to 0.06mL) using disposable lancets and then prepared on microscope slides and left to dry at room temperature.Next, the slides were washed with distilled water, fixed with methanol, stained with Giemsa and dried off.The slides were then subjected to microscopic examination using optical objectives of 10x and 40x by two professionals in a blind controlled trial.PMF was conducted by filtering 1mL of venous blood previously diluted with saline 0.9% through a polycarbonate membrane filter of 3-micrometer pore diameter size (Poretics Corporation -Livermore-USA) 12 .
The PCR method was based on the detection of the GenBank AF228564 DNA fragment from M. ozzardi which comprises the partial sequence of the 18S ribosomal RNA gene, the internal transcribed spacer 1, the complete sequence of the 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS2) and the partial sequence of the 28S ribosomal RNA gene 13 .The forward primer used was 5'GAAAGAAGAAGGATTTTTACT3' on the ITS2 and the reverse primer used was 5'CTTTTCCTCCGCTTAATTATA3' on the 28S ribosomal DNA.These genes are commonly used for species identification on molecular bases since they comprise highly conserved sequences next to highly variable ones.The primers were verified using the NCBI primer design on-line tool based on the Primer3 15 software and showed no unintended DNA products.Genomic DNA was extracted from the samples according to the instructions of the Quiamp DNA Blood Kit (Qiagen®).Blood samples of newborns from the city of Porto Velho were taken as negative control.The eluted DNA were maintained in microtubules and stored at -20ºC.The following procedure was used to amplify the DNA sequence: the samples were maintained A) at a temperature of 94°C for 4min (1 cycle); B) at 94°C for 45sec; at 55°C for 30sec; 72°C for 45sec (35 cycles); C) 72°C for 5min (1 cycle), and D) 4°C for 1min (final time).The amplification product up to 312bp was viewed on 10% polyacrylamide gel with bromide staining (Figure 1).Both the PMF and PCR methods were compared to the common method for M. ozzardi diagnosis (TBS).In order to compare the effectiveness of the methods, the following parameters were considered: sensitivity, specificity, negative predictive value (NPV), and Cohen's kappa coefficient (Tables 1 and 2).The OpenEpi® software was used for statistical analysis, with a confidence interval (CI) of 95%.The project was submitted to the Research Ethics Committee of the São Lucas College, Porto Velho and was approved under the registry number 344/09.
Of the 29 individuals who presented negative by TBS, 62.1% were identified as positive by PMF, while among the 18 who were positive by the TBS method, 88.9% showed the same results by PMF.On the other hand, TBS diagnosed 2 cases that were not diagnosed by PMF (false-negatives), despite its greater sensitivity.Regarding the PCR technique, 89.6% of those who presented negative by TBS were positive by PCR, while 100% of the positive cases showed the same results by PCR.Thus, the PCR technique showed the greatest sensitivity and NPV, followed by PMF, when compared to TBS.However, both these methods showed low specificity and low kappa coefficient compared to TBS.
Despite the encouraging results, the cost of PCR is approximately US$8.60 per test, while PMF has a significantly lower cost, US$0.80 (author's personal information).The difficulties of performing PCR under field conditions must also be taken into account, since this is an important limiting factor in its application.Traditionally, studies concerning the prevalence of the M. ozzardi indicate its low prevalence rate in young individuals [6][7][8] , which is probably due to their low microfilaria concentration.The advent of PCR for mansonelliasis diagnosis, which shows 100% sensitivity, may help correct this bias by providing new epidemiological information on parasitic infections and by promoting the determination of higher prevalence rates.

TABLE 1 -Effectiveness parameters of the polycarbonate membrane filtering method and the thick blood smear exam.
TBS: thick blood smear exam, PMF: polycarbonate membrane filtering method.*confidence interval.

TABLE 2 -Effectiveness parameters of the polymerase chain reaction and the thick blood smear exam.
TBS: thick blood smear exam, PCR: polymerase chain reaction, *confidence interval.