Parasites, Hosts and Diseases

"nested PCR"

Brief Communication

Detection of trichomonads in induced sputum from asthma patients in Korea: Myung-hee Yi, Myungjun Kim, Jun Ho Choi, Yoon Hee Cho, Hyun Kyung Oh, Jung-Won Park, Ju Yeong Kim; Parasites Hosts Dis 2025;63(3):254-257.
Published online August 20, 2025; DOI: https://doi.org/10.3347/PHD.25028

Abstract
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Trichomonads are flagellated protozoa that have occasionally been detected in the human respiratory tract, although detection rates have often been underestimated. We applied a nested PCR assay targeting the 18S rRNA gene of trichomonads to induced sputum from asthma patients to determine the prevalence of Trichomonas. Induced sputum was collected from 41 adults with asthma and analyzed through nested PCR using broad-range trichomonad primers and DNA sequencing for species identification. Nested PCR detected trichomonad DNA in 10 of the 41 (24.4%) samples. Sequencing and phylogenetic analysis revealed Trichomonas tenax in 8 cases and Tetratrichomonas sp. in 2 cases. These findings indicate that trichomonads can be present in the lower airways of patients with asthma, warranting further investigation into their clinical relevance.

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Original Articles

Malaria Prevalence in a Low Transmission Area, Jazan District of Southwestern Saudi Arabia: Yousry Hawash, Khadiga Ismail, Khalaf Alsharif, Walaa Alsanie; Korean J Parasitol 2019;57(3):233-242.
Published online June 30, 2019; DOI: https://doi.org/10.3347/kjp.2019.57.3.233

Abstract
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Detailed description of malaria in low transmission areas is crucial for elimination. The current study aimed to provide a comprehensive description for malaria transmission in Jazan, a low transmission district, southwestern Saudi Arabia. Patients at a tertiary care hospital were recruited in our study between August 2016 and September 2018. Malaria diagnosis was performed through a species-specific nested polymerase chain reaction (nested PCR), microscopy and Paramax-3^TM rapid detection test (RDT). Malaria was detected in 30 patients by the PCR, with point prevalence of 10.9%. Of these malaria infections, 80% was imported, 26.6% was asymptomatic and 23.3% was sub-microscopic. Malaria was reported throughout the year, with February/March and September/October peaks. Infection was significantly more in males than in females (P=0.01). Likewise, infections were detected more in febrile than in non-febrile patients (P=0.01). Adult aged 15-24 years, fever and travel were identified as high-risk factors. Malaria was primarily attributed to Plasmodium falciparum mono-infections, followed by P. vivax mono-infections and lastly to falciparum/vivax mixed infections accounting 76.6%, 16.6%, and 6.6% of PCR-confirmed malaria cases, respectively. The nested PCR was superior to the smear microscopy (sensitivity 76.6%; specificity 100%) and the RDT (sensitivity 83.3%, specificity 94.2%). The overall percent agreement between microscopy and the RDT was 92.7% (kappa=0.63). High proportion of imported malaria including sub-microscopic and sub-patent cases were described. We suggest that incorporation of molecular tool into the conventional malaria diagnosis is beneficial in Jazan district.

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An Alternative Method for Extracting Plasmodium DNA from EDTA Whole Blood for Malaria Diagnosis: Krongkaew Seesui, Kanokwan Imtawil, Phimphakon Chanetmahun, Porntip Laummaunwai, Thidarut Boonmars; Korean J Parasitol 2018;56(1):25-32.
Published online February 28, 2018; DOI: https://doi.org/10.3347/kjp.2018.56.1.25

Abstract
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Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris?EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/μl for P. falciparum and 35.2 parasites/μl for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/μl for P. falciparum and 1.4 parasites/μl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.

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Molecular Identification of Cryptosporidium Species from Pet Snakes in Thailand: Benjarat Yimming, Khampee Pattanatanang, Pornchai Sanyathitiseree, Tawin Inpankaew, Ketsarin Kamyingkird, Nongnuch Pinyopanuwat, Wissanuwat Chimnoi, Jumnongjit Phasuk; Korean J Parasitol 2016;54(4):423-429.
Published online August 31, 2016; DOI: https://doi.org/10.3347/kjp.2016.54.4.423

Abstract
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Cryptosporidium is an important pathogen causing gastrointestinal disease in snakes and is distributed worldwide. The main
objective
s of this study were to detect and identify Cryptosporidium species in captive snakes from exotic pet shops and snake farms in Thailand. In total, 165 fecal samples were examined from 8 snake species, boa constrictor (Boa constrictor constrictor), corn snake (Elaphe guttata), ball python (Python regius), milk snake (Lampropeltis triangulum), king snake (Lampropeltis getula), rock python (Python sebae), rainbow boa (Epicrates cenchria), and carpet python (Morelia spilota). Cryptosporidium oocysts were examined using the dimethyl sulfoxide (DMSO)-modified acid-fast staining and a molecular method based on nested-PCR, PCR-RFLP analysis, and sequencing amplification of the SSU rRNA gene. DMSO-modified acid-fast staining revealed the presence of Cryptosporidium oocysts in 12 out of 165 (7.3%) samples, whereas PCR produced positive results in 40 (24.2%) samples. Molecular characterization indicated the presence of Cryptosporidium parvum (mouse genotype) as the most common species in 24 samples (60%) from 5 species of snake followed by Cryptosporidium serpentis in 9 samples (22.5%) from 2 species of snake and Cryptosporidium muris in 3 samples (7.5%) from P. regius.

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Yijun Chai, Haifeng Liu, Lei Deng, Bo Bi, Jingxin Yao, Xingtao Yang, Zhijun Zhong, Hualin Fu, Liuhong Shen, Ziyao Zhou, Yi Geng, Guangneng Peng
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International Journal for Parasitology: Parasites and Wildlife.2019; 10: 274. CrossRef
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Hemozoin Pigment: An Important Tool for Low Parasitemic Malarial Diagnosis: Sarita Mohapatra, Arnab Ghosh, Ruchi Singh, Dhirendra Pratap Singh, Bhawna Sharma, Jyotish Chandra Samantaray, Manorama Deb, Rajni Gaind; Korean J Parasitol 2016;54(4):393-397.
Published online August 31, 2016; DOI: https://doi.org/10.3347/kjp.2016.54.4.393

Abstract
PDF

Low parasitemic condition in malaria remains a diagnostic challenge; as the available diagnostic methods failed to detect. Currently, hemozoin (Hz) pigment is gaining attention in the diagnosis of malaria. The major drawback is ease of detection of Hz in routine practice. A pilot study was conducted to evaluate the role of Hz pigment and to compare the performance of quantitative buffy coat assay (QBC) and PCR in such conditions. Clinically suspected cases of malaria were examined by both Giemsa stain and immunochromatographic test (ICT). Samples positive by ICT and negative by Giemsa stain were further examined by nested PCR targeting 18S rRNA and QBC for the presence of malaria parasites and pigments. Thirty blood samples fulfilled the inclusion criteria out of which 23 were Plasmodium vivax (Pv), 4 Plasmodium falciparum (Pf), and 3 mixed (Pv and Pf) by immunochromatographic test. Twenty-one out of 30 (70%) were positive by nested PCR in comparison to 25/30 (83%) by QBC. Samples containing both malaria parasites and Hz pigment by QBC completely showed concordance with the PCR result. However, 61% of total samples containing only Hz pigment were observed positive by PCR. Hz pigment remains an important tool for malaria diagnosis. Identification of leukocytes containing pigments by QBC not only indicates recent malarial infections but also puts light on severity of the disease. QBC assay is a rapid, highly sensitive, and cost-effective method to detect malaria parasites and Hz pigment especially in low parasitemic conditions.

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Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings: Myat Htut Nyunt, Myat Phone Kyaw, Kyaw Zin Thant, Thinzer Shein, Soe Soe Han, Ni Ni Zaw, Jin-Hee Han, Seong-Kyun Lee, Fauzi Muh, Jung-Yeon Kim, Shin-Hyeong Cho, Sang-Eun Lee, Eun-Jeong Yang, Chulhun L. Chang, Eun-Taek Han; Korean J Parasitol 2016;54(3):253-259.
Published online June 30, 2016; DOI: https://doi.org/10.3347/kjp.2016.54.3.253

Abstract
PDF

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 ?l in 1 sample, was optimal, and the parasite density as low as 2 p/?l for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.

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Yunning Zhong, Ping Xu, Siming Zhong, Juan Ding
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Estimation on local transmission of malaria by serological approach under low transmission setting in Myanmar
Myat Htut Nyunt, Than Naing Soe, Thinzar Shein, Ni Ni Zaw, Soe Soe Han, Fauzi Muh, Seong-Kyun Lee, Jin-Hee Han, Ji-Hoon Park, Kwon-Soo Ha, Won Sun Park, Seok-Ho Hong, Myat Phone Kyaw, Eun-Taek Han
Malaria Journal.2018;[Epub] CrossRef
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Eun-Taek Han, Jong-Seok Lee, Jae-Hun Cheong, Chulhun L. Chang, Myat Htut Nyunt, Wah Wah Aung, Yi Yi Kyaw, Kyaw Zin Thant
Laboratory Medicine Online.2017; 7(3): 94. CrossRef
Optimization and evaluation of the qPCR-based pooling strategy DEP-pooling in dairy production for the detection of Listeria monocytogenes
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Food Control.2017; 82: 298. CrossRef

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Brief Communication

Prevalence of Dirofilaria immitis Infection in Stray Cats by Nested PCR in Korea: Hyung-Jin Park, Sang-Eun Lee, Won-Ja Lee, Jung-Hyun Oh, Easwaran Maheswaran, Kyoung-Won Seo, Kun-Ho Song; Korean J Parasitol 2014;52(6):691-694.
Published online December 23, 2014; DOI: https://doi.org/10.3347/kjp.2014.52.6.691

Abstract
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The purpose of this study was to conduct a survey of Dirofilaria immitis infection among stray cats in Korea using nested PCR. We included 235 stray cats (121 females and 114 males) and evaluated each for the presence of feline heartworm infection. Blood samples were collected from 135 cats in Daejeon, 50 cats in Seoul, and 50 cats from Gyeonggi-do (Province). Of the 235 DNA samples, 14 (6.0%) were positive for D. immitis. The prevalence of infection in male cats (8/114, 7.0%) tended to be higher than that in female cats (6/121, 5.0%), but the difference was not statistically significant. In each location, 8, 2, and 4 cats were positive for infection, respectively, based on DNA testing. No significant differences in the prevalence were observed among the geographic regions, although the rate of infection was higher in Gyeonggi-do (8.0%) than Daejeon (5.9%) and Seoul (4.0%). We submitted 7 of the 14 D. immitis DNA-positive samples for sequencing analysis. All samples corresponded to partial D. immitis cytochrome c oxidase subunit I gene sequences with 99% homology to the D. immitis sequence deposited in GenBank (accession no. FN391553). To the best of our knowledge, this is the first survey using nested PCR to analyze the prevalence of D. immitis in stray cats in Korea.

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Evidence of Dirofilaria immitis in Felids in North-Eastern Italy
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Prevalence of infection with Dirofilaria immitis in cats in Townsville, Australia
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Seroprevalence of Dirofilaria immitis in Cats from Liaoning Province, Northeastern China
Honglie Hou, Lili Cao, Wenzhi Ren, Dansheng Wang, He Ding, Juan You, Xinhua Yao, Hang Dong, Yanbing Guo, Shuxian Yuan, Xichen Zhang, Pengtao Gong
The Korean Journal of Parasitology.2017; 55(6): 673. CrossRef
Toxoplasma gondii, Dirofilaria immitis, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections in stray and pet cats (Felis catus) in northwest China: co-infections and risk factors
Wei Cong, Qing-Feng Meng, Radu Blaga, Isabelle Villena, Xing-Quan Zhu, Ai-Dong Qian
Parasitology Research.2016; 115(1): 217. CrossRef

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Case Report

A Case of Plasmodium ovale Malaria Imported from West Africa: Yunjung Kang, Jinyoung Yang; Korean J Parasitol 2013;51(2):213-218.
Published online April 25, 2013; DOI: https://doi.org/10.3347/kjp.2013.51.2.213

Abstract
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Malaria is a parasitic infection caused by Plasmodium species. Most of the imported malaria in Korea are due to Plasmodium vivax and Plasmodium falciparum, and Plasmodium ovale infections are very rare. Here, we report a case of a 24-year-old American woman who acquired P. ovale while staying in Ghana, West Africa for 5 months in 2010. The patient was diagnosed with P. ovale malaria based on a Wright-Giemsa stained peripheral blood smear, Plasmodium genus-specific real-time PCR, Plasmodium species-specific nested PCR, and sequencing targeting 18S rRNA gene. The strain identified had a very long incubation period of 19-24 months. Blood donors who have malaria with a very long incubation period could be a potential danger for propagating malaria. Therefore, we should identify imported P. ovale infections not only by morphological findings but also by molecular methods for preventing propagation and appropriate treatment.

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The Korean Journal of Parasitology.2020; 58(1): 61. CrossRef
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The Korean Journal of Parasitology.2013; 51(5): 557. CrossRef

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Brief Communications

Detection of Ocular Toxoplasma gondii Infection in Chronic Irregular Recurrent Uveitis by PCR: Sang-Eun Lee, Sung-Hee Hong, Seong-Ho Lee, Young-Il Jeong, Su Jin Lim, Oh Woong Kwon, Sun Hyun Kim, Young Sung You, Shin-Hyeong Cho, Won-Ja Lee; Korean J Parasitol 2012;50(3):229-231.
Published online August 13, 2012; DOI: https://doi.org/10.3347/kjp.2012.50.3.229

Abstract
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Toxoplasma gondii is a zoonotic parasite resulting in human infections and one of the infectious pathogens leading to uveitis and retinochoroiditis. The present study was performed to assess T. gondii infection in 20 ocular patients with chronic irregular recurrent uveitis (20 aqueous humor and 20 peripheral blood samples) using PCR. All samples were analyzed by nested PCR targeting a specific B1 gene of T. gondii. The PCR-positive rate was 25% (5/20), including 5% (1) in blood samples, 25% (5) in aqueous humor samples, and 5% (1) in both sample types. A molecular screening test for T. gondii infection in ocular patients with common clinical findings of an unclear retinal margin and an inflammatory membrane over the retina, as seen by fundus examination, may be helpful for early diagnosis and treatment.

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Wei Cong, Si-Yang Huang, Dong-Hui Zhou, Xiao-Xuan Zhang, Nian-Zhang Zhang, Quan Zhao, Xing-Quan Zhu
The Korean Journal of Parasitology.2013; 51(3): 363. CrossRef
Serologic Survey of Toxoplasmosis in Seoul and Jeju-do, and a Brief Review of Its Seroprevalence in Korea
Hyemi Lim, Sang-Eun Lee, Bong-Kwang Jung, Min-Ki Kim, Mi Youn Lee, Ho-Woo Nam, Jong-Gyun Shin, Cheong-Ha Yun, Han-Ik Cho, Eun-Hee Shin, Jong-Yil Chai
The Korean Journal of Parasitology.2012; 50(4): 287. CrossRef

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Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum: Jae-Ran Yu, Soo-Ung Lee, Woo-Yoon Park; Korean J Parasitol 2009;47(3):293-297.
Published online August 28, 2009; DOI: https://doi.org/10.3347/kjp.2009.47.3.293

Abstract
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Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from 10³ to 10⁴ oocysts, and the nested PCR method was able to detect 10⁰ to 10² oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.

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Original Articles

Imported Malaria in United Arab Emirates: Evaluation of a New DNA Extraction Technique Using Nested PCR: Doaa M. Sultan, Marwa M. Khalil, Ahmed S. Abdouh, Wafaa F. Doleh, Abdul Aziz M. Al Muthanna; Korean J Parasitol 2009;47(3):227-233.
Published online August 28, 2009; DOI: https://doi.org/10.3347/kjp.2009.47.3.227

Abstract
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Local malaria transmission in the United Arab Emirates (UAE) came to an end in 1997. Nevertheless, UAE has been subjected to substantial importation of malaria cases from abroad, concerning both UAE nationals and immigrants from malarious countries with a total number of 2,119 cases in 2007. To evaluate a new DNA extraction technique using nested PCR, blood samples were collected from 132 individuals who presented to Infectious Diseases Department in Rashid Hospital, Dubai, and Central Department of Malaria Control with fever and persistent headache. Giemsa-stained blood films and ELISA test for malaria antibodies were carried out for detection of Plasmodium infection. Plasmodium infections were identified with the genus-specific primer set and species differentiation using nested PCR. A rapid procedure for diagnosis of malaria infections directly from dried blood spots using for the first time DNA extract from FTA Elute cards was evaluated in contrast to extraction techniques using FTA classic cards and rapid boiling technique. Our new simple technique for DNA extraction using FTA Elute cards was very sensitive giving a sensitivity of 100% compared to 94% using FTA classic cards and 62% in the rapid boiling technique. No complex preparation of blood samples was required prior to the amplification. The production cost of DNA isolation in our PCR assay was much less in comparable to that of other DNA extraction protocols. The nested PCR detected plasmodial infection and could differentiate P. falciparum from P. vivax, and also detected the mixed infection.

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Muneaki Hashimoto, Mika Bando, Jun-ichi Kido, Kazumichi Yokota, Toshihiro Mita, Kazuaki Kajimoto, Masatoshi Kataoka
Parasitology International.2019; 73: 101941. CrossRef
Genetic diversity and transmissibility of imported Plasmodium vivax in Qatar and three countries of origin
Mohammed H. Abdelraheem, Devendra Bansal, Mohammed A. Idris, Moawia M. Mukhtar, Muzamil M. Abdel Hamid, Zainb S. Imam, Sisay Getachew, Rakesh Sehgal, Hargobinder Kaur, Amal H. Gadalla, Salam Al-Hamidhi, Zainab Al-Hashami, Ali Al-Jabri, Ali A. Sultan, Hamz
Scientific Reports.2018;[Epub] CrossRef
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Abbas Ali Eskandarian, Sara Moradi, Saeed Abedi
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David Bell, Alessandra E. Fleurent, Michael C. Hegg, John D. Boomgard, Caitlin C. McConnico
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E. C. Oriero, J. Jacobs, J.-P. Van Geertruyden, D. Nwakanma, U. D'Alessandro
Journal of Antimicrobial Chemotherapy.2015; 70(1): 2. CrossRef
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Salama Al-Hamidhi, Mohammed AK Mahdy, Zainab Al-Hashami, Hissa Al-Farsi, Abdulsalam M Al-mekhlafi, Mohamed A Idris, Albano Beja-Pereira, Hamza A Babiker
Malaria Journal.2013;[Epub] CrossRef
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Sepideh Seghatoleslami, Lars Ohlsson, Kristina Hamberg, Peter Carlsson, Dan Ericson, Lennart Ljunggren
American Journal of Molecular Biology.2013; 03(03): 148. CrossRef
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Infection, Genetics and Evolution.2012; 12(6): 1253. CrossRef

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Detection of Plasmodium vivax by Nested PCR and Real-Time PCR: Genc, Ahmet , Eroglu, Fadime , Koltas, Ismail Soner; Korean J Parasitol 2010;48(2):99-103.; DOI: https://doi.org/10.3347/kjp.2010.48.2.99

Abstract
PDF

Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.

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