Gabriella A. Farcas

Evaluation of the RealArt Malaria LC Real-Time PCR Assay for Malaria Diagnosis

ABSTRACT PCR-based methods have advantages over traditional microscopic methods for the diagnosis of malaria, especially in cases of low parasitemia and mixed infections. However, current PCR-based assays are often labor-intensive and not readily quantifiable and have the potential for contamination due to a requirement for postamplification sample handling. Real-time PCR can address these limitations. This study evaluated the performance characteristics of a commercial malaria real-time PCR assay (RealArt Malaria LC Assay; Artus GmbH, Hamburg, Germany) on the LightCycler platform for the detection of malaria parasites in 259 febrile returned travelers. Compared to nested PCR as the reference standard, the real-time assay had a sensitivity of 99.5%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.6% for the detection of malaria. Our results indicate that the RealArt assay is a rapid (<45 min), sensitive, and specific method for the detection of malaria in returned travelers.

Fatal Severe Acute Respiratory Syndrome Is Associated with Multiorgan Involvement by Coronavirus

Abstract Severe acute respiratory syndrome (SARS) is characterized by pulmonary compromise; however, patients often have evidence of other organ dysfunction that may reflect extrapulmonary dissemination of SARS coronavirus (SARS-CoV). We report on the distribution and viral load of SARS-CoV in multiple organ samples from patients who died of SARS during the Toronto outbreak. SARS-CoV was detected in lung (100%), bowel (73%), liver (41%), and kidney (38%) in 19 patients who died of SARS, with the highest viral loads observed in lung (1.0 × 1010 copies/g) and bowel (2.7 × 109 copies/g). Fatal SARS was associated with multiorgan viral dissemination in a distribution that has implications for disease manifestation, viral shedding, and transmission.

Severe Acute Respiratory Syndrome–associated Coronavirus in Lung Tissue

Efforts to contain severe acute respiratory syndrome (SARS) have been limited by the lack of a standardized, sensitive, and specific test for SARS-associated coronavirus (CoV). We used a standardized reverse transcription-polymerase chain reaction assay to detect SARS-CoV in lung samples obtained from well-characterized patients who died of SARS and from those who died of other reasons. SARS-CoV was detected in all 22 postmortem lung tissues (to 109 viral copies/g) from 11 patients with probable SARS but was not detected in any of the 23 lung control samples (sample analysis was blinded). The sensitivity and specificity (95% confidence interval) were 100% (84.6% to 100%) and 100% (85.1% to 100%), respectively. Viral loads were significantly associated with a shorter course of illness but not with the use of ribavirin or steroids. CoV was consistently identified in the lungs of all patients who died of SARS but not in control patients, supporting a primary role for CoV in deaths.

Real-Time Polymerase Chain Reaction Assay for the Rapid Detection and Characterization of Chloroquine-Resistant Plasmodium falciparum Malaria in Returned Travelers

BACKGROUND Imported drug-resistant malaria is a growing problem in industrialized countries. Rapid and accurate diagnosis is essential to prevent malaria-associated mortality in returned travelers. However, outside of a limited number of specialized centers, the microscopic diagnosis of malaria is slow, unreliable, and provides little information about drug resistance. Molecular diagnostics have the potential to overcome these limitations. OBJECTIVE We developed and evaluated a rapid, real-time polymerase chain reaction (PCR) assay to detect Plasmodium falciparum malaria and chloroquine (CQ)-resistance determinants in returned travelers who are febrile. METHODS A real-time PCR assay based on detection of the K76T mutation in PfCRT (K76T) of P. falciparum was developed on a LightCycler platform (Roche). The performance characteristics of the real-time assay were compared with those of the nested PCR-restriction fragment-length polymorphism (RFLP) and the sequence analyses of samples obtained from 200 febrile returned travelers, who included 125 infected with P. falciparum (48 of whom were infected CQ-susceptible [K76] and 77 of whom were CQ-resistant [T76] P. falciparum), 22 infected with Plasmodium vivax, 10 infected with Plasmodium ovale, 3 infected with Plasmodium malariae malaria, and 40 infected with other febrile syndromes. All patient samples were coded, and all analyses were performed blindly. RESULTS The real-time PCR assay detected multiple pfcrt haplotypes associated with CQ resistance in geographically diverse malaria isolates acquired by travelers. Compared with nested-PCR RFLP (the reference standard), the real-time assay was 100% sensitive and 96.2% specific for detection of the P. falciparum K76T mutation. CONCLUSION This assay is rapid, sensitive, and specific for the detection and characterization of CQ-resistant P. falciparum malaria in returned travelers. This assay is automated, standardized, and suitable for routine use in clinical diagnostic laboratories.

Real-Time PCR Assay for Rapid Detection and Analysis of PfCRT Haplotypes of Chloroquine-Resistant Plasmodium falciparum Isolates from India

ABSTRACT Chloroquine-resistant Plasmodium falciparum (CRPF) malaria isolates in Southeast Asia and sub-Saharan Africa share the same Plasmodium falciparum chloroquine resistance transporter (PfCRT) haplotype (CVIET; amino acids 72 to 76). It is believed that CRPF malaria emerged in Southeast Asia and spread to sub-Saharan Africa via the Indian subcontinent. Based on this assumption, we hypothesized that CRPF isolates in India should possess the same drug resistance haplotype (PfCRT haplotype CVIET) as P. falciparum isolates in Southeast Asia and Africa and that the prevalence of CRPF may be higher and more widespread in India than appreciated. To test this postulate, we utilized a standardized real-time PCR assay to assess the prevalence and distribution of PfCRT haplotypes in P. falciparum isolates (n = 406) collected from Western, Central, and Eastern states in India and compared them to isolates from South America and Africa. Based on the proportion of isolates possessing the molecular marker K76T, the prevalence of chloroquine resistance was high in all five regions of India studied (91%), as well as in Uganda (98%) and Suriname (100%). All isolates from Suriname contained the chloroquine-resistant SVMNT haplotype typical of South American isolates, and 98% of isolates from Uganda possessed the chloroquine-resistant CVIET haplotype characteristic of Southeast Asian/African strains. However, of 246 P. falciparum isolates from across India that contained the molecular marker for chloroquine resistance, 81% contained the SVMNT haplotype. In conclusion, the prevalence of CRPF malaria was high in geographically dispersed regions of India, and the primary haplotype observed, SVMNT, did not support a presumed geographic spread from contiguous Southeast Asia.

Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses

Emerging infectious diseases have caused a global effort for development of fast and accurate detection techniques. The rapidly mutating nature of viruses presents a major difficulty, highlighting the need for specific detection of genetically diverse strains. One such infectious agent is SARS-associated coronavirus (SARS-CoV), which emerged in 2003. This study aimed to develop a real-time RT-PCR detection assay specific for SARS-CoV, taking into account its intrinsic polymorphic nature due to genetic drift and recombination and the possibility of continuous and multiple introductions of genetically non-identical strains into the human population, by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV S, E, M and N genes. These were applied in simple, reproducible duplex and multiplex real-time PCR assays on 25 post-mortem samples and constructed RNA controls, and they demonstrated high target detection ability and specificity. This assay can readily be adapted for detection of other emerging and rapidly mutating pathogens.