2. Iran J Arthropod-Borne Dis, 2011, 5(1): 7–12 SR Naddaf et al.: Comparison of PCR-Based … 7 Original Article Comparison of PCR-Based Diagnosis with Centrifuged- Based Enrichment Method for Detection of Borrelia persica in Animal Blood Samples *SR Naddaf1, M Kishdehi2, MR Siavashi1 1Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran 2Department of Microbiology, Lahijan Azad University, Gilan, Iran (Received 12 Oct 2010; accepted 24 May 2011) Abstract Background: The mainstay of diagnosis of relapsing fever (RF) is demonstration of the spirochetes in Giemsa-stained thick blood smears, but during non fever periods the bacteria are very scanty and rarely detected in blood smears by mi- croscopy. This study is aimed to evaluate the sensitivity of different methods developed for detection of low-grade spi- rochetemia. Methods: Animal blood samples with low degrees of spirochetemia were tested with two PCRs and a nested PCR targeting flaB, GlpQ, and rrs genes. Also, a centrifuged-based enrichment method and Giemsa staining were per- formed on blood samples with various degrees of spirochetemia. Results: The flaB-PCR and nested rrs-PCR turned positive with various degrees of spirochetemia including the blood samples that turned negative with dark-field microscopy. The GlpQ-PCR was positive as far as at least one spirochete was seen in 5-10 microscopic fields. The sensitivity of GlpQ-PCR increased when DNA from Buffy Coat Layer (BCL) was used as template. The centrifuged-based enrichment method turned positive with as low concentra- tion as 50 bacteria/ml blood, while Giemsa thick staining detected bacteria with concentrations ≥ 25000 bacteria/ml. Conclusion: Centrifuged-based enrichment method appeared as much as 500-fold more sensitive than thick smears, which makes it even superior to some PCR assays. Due to simplicity and minimal laboratory requirements, this method can be considered a valuable tool for diagnosis of RF in rural health centers. Keywords: Borrelia persica, Relapsing fever, Diagnosis, PCR, Enrichment method Introduction Relapsing fever (RF) is an infectious dis- ease with a sudden onset of high fever; it is caused by several species of bacteria belong- ing to the genus Borrelia and, as its name in- dicates, is characterized by the occurrence of one or more spells of fever after the subsi- dence of the primary febrile attack (Burgdorfer 1976). The presence of massive amounts of spirochetes during fever peaks makes diagno- sis of the infection an easy practice with dark-field microscopy or Giemsa staining me- thod (Assous and Wilamowski 2009). How- ever, between the peaks and in milder infec- tions these methods are often negative due to a low number of bacteria in blood stream, making the infection under diagnosed. PCR assays that target different genes including 16S ribosomal RNA (rrs), Flag-ellin (flaB), and Glyc- erophosphodiester phosphodiesterase (GlpQ) were successfully used for detection of spiro- chetes in blood and Ornithodoros tick vectors (Ras et al. 1996, Assous et al. 2006, Halperin et al. 2006, Nordstrand et al. 2007, Oshaghi et al. 2010). Species-specific PCR and RCR-RFLP method were also developed for diagnosis of some Borrelia species (Assous and Wilamowski *Corresponding author: Dr Saied Reza Naddaf, E-mail: snaddaf_2001@yahoo.com Iran J Arthropod-Borne Dis, 2011, 5(1): 7–12 SR Naddaf et al.: Comparison of PCR-Based … 8 2009, Oshaghi et al. 2010). However, the method is costly, labor intensive and requires well-equipped laboratories. The ELISA based on product of GlpQ gene can discriminate between RF and Lyme borreliosis but cannot differentiate between an active and past RF infection (Schwan et al. 1996). Quantitative buffy coat (QBC) analysis of blood samples showed to be a reliable for diagnosis of RF when the spirochetes are low in numbers, but it relies on florescent microscopy, the equipment rarely available in rural health centers (van Dam et al. 1999, Cobey et al. 2001). Recently, a novel centrifugation-based method with mini- mal laboratory requirements showed very promising, detecting concentrations less than 10 bacteria/ml blood (Larsson and Bergstrom 2008). In this study we compare the sensitivity of several PCR assays that amplify DNA sequences of three different loci including rrs, flaB, and GlpQ with the centrifugation-based enrichment method and Giemsa staining to detect bacteria in animal blood samples. Materials and methods Borrelia Borrelia persica was isolated from Orni- thodoros tholozani ticks, collected from Ardebil Province, and maintained in guinea pigs through serial passages for seven successive years. Blood samples Adult guinea pigs were inoculated intrap- eritoneally with 0.5 ml of B. persica infected blood preserved in -70º C with 50% glycerol. From day three, daily amount of 500-600µ l of blood was taken from animals’ heart using insulin syringes and bacteria count was deter- mined using dark-field microscopy. Blood col- lection continued for eight days, until no spi- rochete was detectable in the blood samples with dark-field microscopy. The intensity of in- fection in blood samples was obtained by count- ing the spirochetes using a Neubauer haemo- cytometer. When animals were negative for two successive days (days nine and ten) large amounts of blood was collected from their hearts and amounts of 10 ml were examined for pres- ence of spirochetes using centrifuged-based en- richment method. Also, Buffy Coat Layer (BCL) was obtained from 3 ml of the same blood sam- ples using the lymphocyte®-H kit (Cedarlane, Netherland). We also prepared fifteen serial di- lutions from an infected guinea pig blood sample containing 25×104 spirochetes/ml blood, with so- dium citrate-anticoagulated blood from healthy individuals (Table 3). Giemsa-stained thin and thick smears were prepared in triplicate from all dilutions and the rest of blood samples were examined for presence of spirochetes using centrifuged-based enrichment method. Centrifuged-based enrichment method The method basically comprised two cen- trifugation steps; the blood samples were first centrifuged at 500 x g for 5 min, the plasma were recovered to new tubes and second cen- trifugation was performed at 5000 x g for 10 min. The supernatants were decanted, and the pellets were resuspended in the few remaining micro- liters of plasma. The suspensions were smeared onto a glass slides and air dried for 10 min. The smears were fixed by heating over a flame fol- lowed by a 30 s dip in methanol. The slides were stained with Giemsa and examined for re- covered spirochetes at 1000X magnification. DNA extraction method and PCR Amounts of 200µ l of blood samples and 500 µ l of buffy coats were subjected to DNA extraction, using the Miniprep DNA extrac- tion Kit (Kiagen, Germany) according to manu- facturer's recommendations. Detection of B. persica by PCR was per- formed through amplification of three different genes including rrs, flaB, and GlpQ. The flaB and GlpQ genes were amplified using the prim- ers and thermocycler programs outlined by other authors (Assous et al. 2006, Halperin et al. 2006). The 25µl reactions contained 20 pmol of each primer, 1.7mM MgCl2, 10mM Tris-HCl, Iran J Arthropod-Borne Dis, 2011, 5(1): 7–12 SR Naddaf et al.: Comparison of PCR-Based … 9 50 mM KCl, 200M of dNTPs, 1U of Taq and 3µl of DNA. Amplification of rrs was performed using the nested PCR technique as described previously (Brahim et al. 2005, Nordstrand et al. 2007). The list of primers, target genes, and expected band sizes are reflected in Table 1. Table 1. List of primers used in this study Primer Sequence (5’ to 3’) Target gene Expected band size 128F cag aac ata cct tag aag ctc aag c GlpQ 212 bp 340r gtg att tga ttt ctg cta atg tg BOR1 taa tac gtc agc cat aaa tgc flaB 750 bp BOR2 gct ctt tga tca gttatc att c Fd3 aga gtt tga tcc tgg ctt ag rrs 1st round 613 bp 595R ctt gca tat ccg cct act ca Fd4 ggc tta gaa cta acg ctg gca g 2nd round 527 bp 500R ctg ctg gca cgt aat tag cc Results PCR The GlpQ-PCR did not yield the expected 212bp band with low sprirochetemia blood sam- ples i.e. those that were negative with dark-field microscopy (Table 1, Days 9 and 10). However, the DNA from BCL of the same blood samples was successfully amplified using the same re- agents and PCR conditions. The flaB-PCR and nested rrs-PCR were positive with various degrees of spirochetemia including those that were negative by dark-field microscopy. The details of PCR assays are shown in Table 2. Centrifugation-based enrichment method Centrifugation-based enrichment recovered 890 and 357 spirochetes form two 10 ml of blood samples that were negative with dark-field microscopy examination (Table 2). We could recover 25 spirochetes on the glass slides by microscopy when as low as about 250 bacteria (a concentration equivalent to 50 bacteria/ml) were spiked into 5ml blood samples. No spirochete was recovered with concentration below 25 bacteria in ml of blood (Table 3). Geimsa-stained smears analysis Geimsa-stained thin smears were consis- tently positive with samples containing ≥105 spi- rochetes/ml blood, but only two of the three smears at concentrations 6.26×104 spirochetes/ ml and one of three smears at concentrations 5×104 spirochetes/ml were positive. The Giemsa- stained thick smears were all positive at ≥ 5×104 spirochetes/ml, but only two of the three smears at concentrations 4×104 and 3×103 spirochetes/ ml, and one of the three smears at concentration 25×103 spirochetes/ml were positive (Table 2). Table 2. Details of blood samples and other blood products used for PCR assays Samples Days after inoculation Dark field microscopy Spirochetes in µl of blood Type and amount of sample GLPQ PCR flaB PCR Nested rrsPCR Spirochetes recovered by CEM 1 3 10p ƒ 2750 Blood/200 µl + + + NP 2 4 20p ƒ 6750 Blood/200 µl + + + NP 3 5 1p5 ƒ 375 Blood/200 µl + + + NP 4 6 1p ƒ 1500 Blood/200 µl + + + NP 5 7 1p10 ƒ 250 Blood/200 µl + + + NP 6 8 1p5ƒ 250 Blood/200 µl + + + NP 7 9 Neg. Not seen Blood/200 µl Neg. + + NP 8 10 Neg. Not seen Blood/200 µl Neg. + + 870 and 375 9 9 Neg. Not seen BCL/500 µl + NP NP NP 10 10 Neg. Not seen BCL/500 µl + NP NP NP BCL = Buffy Coat Layer, NP = Not Performed, CBE = Centrifuged-based Enrichment Method 10pƒ=10 spirochetes in one microscopic field, 20pƒ= 20 spirochetes in one microscopic field, 1p5ƒ= 1spirochete in 5 microscopic fields, 1pƒ= 1 spirochetes in one microscopic field, 1p10ƒ=1spirochete in 10 microscopic fields, Neg.= negative, no spirochetes was seen in 30 microscopic fields. Iran J Arthropod-Borne Dis, 2011, 5(1): 7–12 SR Naddaf et al.: Comparison of PCR-Based … 10 Table 3. Details of blood samples used for Giemsa-stain analysis and centrifuged-based enrichment method Sample code Amounts of blood (ml) Number of bacteria spiked into Giemsa- stained thin smears Giemsa- stained thick smears Number of Recovered bacteria Ratio of spirochetes (Recovered/spiked into) 1 5.00 250000 + + + + + + NP NA 2 5.00 150000 + + + + + + NP NA 3 6.00 125000 + + + + + + 970 1/128 4 5.00 100000 + + + + + + NP NA 5 5.00 62500 + + ─ + + + NP NA 6 5.00 50000 + ─ ─ + + + NP NA 7 5.00 40000 ─ ─ ─ + + ─ NP NA 8 5.00 30000 ─ ─ ─ + + ─ NP NA 9 5.20 25000 ─ ─ ─ + ─ ─ 255 1/98 10 5.00 20000 ─ ─ ─ ─ ─ ─ NP NA 11 5.10 12500 ─ ─ ─ ─ ─ ─ 132 1/94 12 5.00 6250 ─ ─ ─ ─ ─ ─ 52 1/120 13 5.00 1250 ─ ─ ─ ─ ─ ─ 31 1/40 14 5.00 625 ─ ─ ─ ─ ─ ─ 25 1/25 15 5.00 250 ─ ─ ─ ─ ─ ─ 25 1/10 16 5.00 125 ─ ─ ─ ─ ─ ─ 0 0 NP= Not performed, NA= Non applicable Discussion Tick-borne Relapsing fever (TBRF) is one of the prevalent bacterial diseases in dif- ferent parts of the world (Karimi 1981, Bar- maki et al. 2010). The disease in Iran is caused primarily by Borrelia persica, which is trans- mitted by Ornithodoros tholozani ticks. Other Borrelia species including B. microtii, B. laty- schevii, and B. baltazardi have also been re- ported from Iran (Karimi et al. 1979, Karimi 1981). From 1997 to 2006, a total of cases 1415 were reported from the entire country, some from areas out of O. tholozani distribu- tion includ hormozgan and Fars Provinces (Masoumi Asl et al. 2009). Most of the RF cases in Hormozgan Province were detected during attempts for diagnosis of malaria para- site in Giemsa-stained blood smears from feb- rile patients. Since, thick smears commonly turn positive during fever peaks that are associated with massive spirochetemia; it is posssible that a large number of RF cases that refer to hos- pitals and health care centers during non-feb- rile periods remain underdiagnosed. The inabil- ities of microscopic analysis to detect spiro- chetes in blood have been demonstrated by some authors (Assous et al. 2006, Halperin et al. 2006, Nordstrand et al. 2007). PCR- based diagnosis offered a new approach to this problem. Our results showed that the flaB- PCR could detect Borrelia flagellin DNA in blood samples with low grade bacteremia that are commonly negative by dark-field micros- copy (Table 2). The nested rrs-PCR also showed very sensitive and was positive with various degrees of bacteremia (Table 2); how- ever it was very vulnerable to cross-contami- nation and led to false positive results when positive controls were included in assays. Di- lution of first round products with distilled water with 1:10 ratio reduced the false positivity re- sults by 90%. The GlpQ-PCR was not posi- tive with low grade spirochetemia blood sam- ples i.e. those that turned negative by dark- field microscopy. However, the DNA from BCL of the same blood samples yielded the expected band with the same PCR protocol (Table 2). Accumulation of spirochetes in BCL was already documented by other authors (van Iran J Arthropod-Borne Dis, 2011, 5(1): 7–12 SR Naddaf et al.: Comparison of PCR-Based … 11 Dam et al. 1999, Cobey et al. 2001). Thus, extraction of DNA from BCL increases the chance of detecting infection with PCR, par- ticularly during non febrile periods. We could recover 870 and 357 spirochetes by centri- fuged–based enrichment method from two 10 ml of guinea pig blood samples that were negative by dark-field microscopy and GlpQ- PCR. In our study, the sensitivity of the cen- trifuged–based enrichment method improved as number of spirochetes spiked into the blood samples decreased; the highest ratio (recovered/ spiked into) was obtained with 250 spirochetes/ 5ml of blood (Table 3). Since the sensitivity of thick smears, the routine method for de- tection of RF agents, was around 25000 spi- rochetes in ml of blood (Table 2), the centri- fuged–based enrichment method could be as much as 500 times more sensitive than thick smear analysis. In conclusion, PCR particu- larly when DNA is extracted from BCL is a useful tool for diagnosis of RF cases that can- not be diagnoses by microscopic analysis. However, the method is commonly available in big hospitals and well-equipped laborato- ries. Centrifuged based enrichment method showed a high sensitivity and even appeared to be superior to GlpQ-PCR. Regarding the fact that it requires the equipments that are commonly available in small laboratories, this method is more feasible for RF diagnostics in underprivileged rural health centers. Acknowledgements We are very grateful to Dr N Piazak for his technical assistance and Dr M Assmar for his guidance throughout the project. 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