Nested multiplex PCR for identification and detection of human Plasmodium species including Plasmodium knowlesi

Objective To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria. Methods In this study the nested multiplex malaria PCR was redesigned, targeting the 18S rRNA gene, to identify the fifth human Plasmodium species, Plasmodium knowlesi, together with the other human Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives. Results The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms. The technique showed high sensitivity (100%) and specificity (96%) when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos III and a published real-time PCR malaria assay. Conclusions The technique designed is an economical, sensitive and specific alternative to current diagnosis methods. Furthermore, the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.

Nested multiplex PCR for identification and detection of human Plasmodium species including Plasmodium knowlesi

OBJECTIVE@#To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria.@*METHODS@#In this study the nested multiplex malaria PCR was redesigned, targeting the 18S rRNA gene, to identify the fifth human Plasmodium species, Plasmodium knowlesi, together with the other human Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives.@*RESULTS@#The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms. The technique showed high sensitivity (100%) and specificity (96%) when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos Ⅲ and a published real-time PCR malaria assay.@*CONCLUSIONS@#The technique designed is an economical, sensitive and specific alternative to current diagnosis methods. Furthermore, the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

Molecular screening of Plasmodium infections among migrant workers in Thailand.

Background & objective: A cross-sectional study was conducted to determine the prevalence of Plasmodium infections among migrant workers in Thailand. Methods: A total of 241 migrants at Kanchanaburi, Pathumthani and Nakornpathom provinces of Thailand were recruited in our surveillance. Blood samples were examined for human malaria parasites by using microscopy and semi-nested multiplex PCR (SnM-PCR). Results: Laboratory diagnosis revealed 6.2% total positive rate. As compared to microscopy (26.7%), SnM-PCR was more sensitive (93.3%) for malaria. Plasmodium falciparum was predominant than P. vivax (53% : 40%, respectively). The majority of positive cases were from Myanmar workers who had low parasitaemia and without symptoms. The highest prevalence (13.7%) was found among migrant workers from Kanchanaburi province in western Thailand. Conclusion: These findings indicate risk of malaria transmission from migrant workers. Malaria surveillance should be included in the health-screening program for migrants in Thailand to manage this health risk.

Epidemiological Surveillance of P and G Genotypes of Group A Rotavirus Detected from Diarrheic Patients in Daejeon Region

During 3 years surveillance (January 2001 through December 2003) for acute gastroenteritis in human in Daejeon region, 432 out of 4,869 stool samples were selected as rotavirus-positive specimens by means of antigen-capture enzyme-linked immunosorbent assay (ELISA). The P (VP4) and G (VP7) genotypes for 432 stool samples were investigated by reverse transcription polymerase chain reaction (RT-PCR) and nested multiplex PCR. The most prevalent P subtype was P[8] (44.9%), followed by P[4] (25.7%) and P[6] (17.1%). No cases for P[10] and P[9] subtypes were found through the study. In G subtyping, G1 (53.2%) was the most frequently found G type, followed by G2 (23.1%), G3 (9.5%), G4 (6.7%), and G9 (0.9%). The order of detection rates for G2, G3 and G4 was variable by years. The most common G- and P- type combination found in this study was G1P[8] (33.1%), followed by G2P[4] (20.4%), G1P[6] (10.0%), G3P[8] (7.2%) and G4P[6] (4.2%). The mixed types of G and P were observed most frequently in P[8] (1.4%) and G1 (3.2%), respectively. This is the first molecular epidemiological study for Group A rotavirus in Daejeon region. The results might be useful data for evaluating the epidemiological status of rotaviral diarrhea in the region.

A Comparison of Nested Multiplex-Polymerase Chain Reaction with Indirect Immunofluorescence for Detection and Typing of Herpes Simplex Virus / 대한진단검사의학회지

BACKGROUND: Herpes simplex virus type 1 (HSV-1) is linked specifically to oral and lip infections while HSV-2 is involved in genital infections. We evaluated a recently reported nested-multiplex ploymerase chain reaction (NM-PCR) for the detection and typing of HSV and compared the results with indirect immunofluorescence (IF) after cell culture. METHODS: One hundred thirty three specimens were received from patients suspected of having clinical HSV infections. HSV was cultured by the shell vial method and stained with type specific monoclonal antibodies. NM-PCR was performed using crude samples. RESULTS: HSV was detected in 45 (33.8%) and 46 (34.6%) of the 133 specimens by IF and NMPCR, respectively. All of the HSV IF positive specimens were also positive by NM-PCR. Typing by the two methods concurred in all but two of the 45 specimens; the two specimens were typed as HSV-1 and HSN-2, respectivey, by IF, and both as HSV-1 and HSV-2 by NM-PCR. CONCLUSIONS: Our results suggest that NM-PCR is a rapid and sensitive method for the detection and typing of HSV.

Evaluation of Nested Multiplex PCR in the Diagnosis of Malaria Infection / 대한진단검사의학회지

BACKGROUND: A peripheral blood smear has been the gold standard method for the diagnosis of malaria infection. Recently, many other methods have been introduced, although having inferior sensitivity and specificity to peripheral blood smears. We evaluated Neodin malaria PCR kit and its applicability in clinical settings. METHODS: Samples from seventy patients who visited Korea University hospital were used for evaluation. DNA from EDTA blood was tested in nested multiplex PCR and 470 bp for Plasmodium vivax or 340 bp for Plasmodium falciparum was confirmed after electrophoresis. The detection limit was determined by dilution of malaria positive blood with normal blood. RESULTS: Thirty-five cases of P. vivax and 10 cases of P. falciparum were noted. Except for a case of falciparum malaria, all positive cases were consistent with the peripheral blood smear results. Detection limit was 3.6 parasite/microL. CONCLUSIONS: Neodin malaria nested multiplex PCR has high sensitivity and the ability for species discrimination and may be available in the diagnosis of malaria infection.