High rate of detection of mixed infections of Plasmodium vivax and Plasmodium falciparum in South-East of Iran, using nested PCR

Sistan and Baluchestan province, South-East of Iran, has been reported as an endemic area of malaria [Sadrizadeh B. Malaria in the world, in the eastern Mediterranean region and in Iran: Review article. WHO/EMRO Report 2001: 1-13.]. The main objective of this research was to perform rapid and correct diagnoses of malaria infection. Blood specimens were collected from 140 suspected volunteers. The Giemsa-stained slides examination and nested PCR for amplification of the Plasmodium small subunit ribosomal genes (ssrRNA) were utilized. The results demonstrated 118 out of 140 cases (84.3%) positive for malaria parasites, including 60.7%, 20.7% and 2.9% as having Plasmodium vivax (P.v), Plasmodium falciparum (P.f) and mixed infections (P.v+P.f), respectively by microscopy. The nested PCR detected malaria parasites in 134 samples (94.3%), consisting of 51.4% P.v, 12.6% P.f and 29.3% mixed infections. The PCR analysis detected 37 cases of mixed infections more than that of the routine microscopy. These results suggested that there are a considerable number of cases with mixed infections in the study area that mainly remain undiagnosed by microscopy. It is also concluded that the nested PCR is a suitable complement to microscopy for accurate specific diagnosis of malaria species in field.     

PCR detection of lizard malaria parasites: prevalence of Plasmodium infections with low-level parasitemia differs by site and season

Plasmodium-specific polymerase chain reaction (PCR) primers allowed detection of infections with very low-level parasitemia for 3 species of malaria parasites infecting Anolis lizards at 2 Caribbean sites, Puerto Rico and Saba, Netherlands Antilles. A verification study, using a single-tube nested PCR to eliminate contamination, showed that infections as low as 1 parasite per millions of erythrocytes could be detected by amplifying a 673 bp fragment of the cytochrome b gene. Very low-level parasitemia infections, subpatent under the microscope, were common in A. sabanus on Saba sites, with no significant seasonal difference (31% of infections appearing uninfected by microscopic examination in summer were found infected by PCR, 38% in winter). At the Puerto Rico site, the subpatent infections were also common in A. gundlachi, but were more prevalent in winter (53%) than in summer (17%). A similar high frequency of subpatent infections is known from studies on human and bird malaria, but a previous PCR-based study on a temperate lizard malaria system found few such low-level infections. Differences in the prevalence of subpatent infections by site and season suggest transmission biology may select for distinct life history strategies by the parasite.     

Detection of cytomegalovirus in infant cerebrospinal fluid by conventional PCR, nested PCR and PCR-Digene

The possibility of amplification of human cytomegalovirus (HCMV) DNA in cerebrospinal fluid (CSF) for the diagnosis of HCMV central nervous system (CNS) infection in infants was studied. Single-step PCR, nested PCR and PCR-Digene were used to assay CSF specimens from 37 patients. Criteria for patient inclusion in the study were: 1. clinical manifestations suggesting CMV neuroinfection such as seizures, hypertonia, hypotonia, intracranial calcification, microcephaly, chorioretinitis; 2. any of the following symptoms: anaemia, hepetomegaly, prolonged cholestatic jaundice, or hepatitis, splenomegaly, thrombocytopenia, intrauterine hypotrophy; 3. serologic presentation, and/or positive results for CMV infection obtained by single-step PCR and PCR-Digene in urine and/or blood. PCR-Digene results were positive in 6 CSF samples. Four CSF samples were positive by nested PCR and 1 CSF sample by single step PCR. We found that the double PCR was about ten or more times more sensitive than single PCR and the PCR-Digene was only three times more sensitive than nested-PCR. The results were correlated with serology. Thirty-three out of 37 examined patients were seropositive (ELISA IgG); ELISA IgM gave positive results in 9 patients. In control studies, cells infected with other members of the herpes virus family were negative with these methods, which suggest that amplification combined with primers from the IE and the L-region of CMV is specific. In conclusion, nested-PCR seems to be the best method for early diagnosis of CMV infection in CSF due to an absence of false positive results and its high specificity and sensitivity.     

Search for malaria parasites by PCR and Southern blot in patients with imported malaria in Italy

The present study evaluates the sensitivity, specificity and usefulness of a PCR method with Southern blot hybridization to detect malaria parasites in blood samples from subjects with a suspect clinical diagnosis of malaria imported to Italy. Plasmodia were detected by PCR using a genus-specific primer-set corresponding to the sequences common to P. falciparum, P. vivax, P. malariae and P. ovale, as described by Arai (Arai et al., Nucleosides Nucleotides, 1994, 13, 1363-1364) and Kimura (Kimura et al., Journal of Clinical Microbiology, 1995, 33, 2342-2346). In addition, four distinct tandemly repetitive species-specific probes, described by Kawai (Kawai et al., Analytical Biochimestry, 1993, 209, 63-69), were synthesized to specifically detect the four malaria parasites species by Southern blot hybridization. Fifteen blood samples from 12 patients (7 with malaria) were tested and the genus-specific PCR method showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy, in detecting malaria parasites in the tested blood samples. Fourteen samples (nine were positive and five negative by PCR) were confirmed by Southern blot, whereas only one P. vivax positive sample was not hybridized with the species-specific probes. We conclude that this PCR method with Southern blot hybridization may be useful in detecting malaria parasites in patients with malaria imported to Italy.     

Detection of Dekkera-Brettanomyces strains in sherry by a nested PCR method.

Brettanomyces sp. and its ascosporogenous sexual state, Dekkera sp., have been well documented as spoilage microorganisms, usually associated with barrel-aged red wines. In this report, we describe the genetic characterization, on the basis of DNA content per cell, electrophoretic karyotyping, and mitochondrial DNA restriction patterns, of a Dekkera yeast strain isolated from sherries and of a number of other Brettanomyces and Dekkera strains. By using a genomic DNA fragment of the isolated Dekkera strain, we developed a two-step PCR method which directs the specific amplification of target DNA from this strain and from other Brettanomyces-Dekkera strains. The method efficiently amplified the target DNA from intact cells, obviating DNA isolation, and yielded a detection limit of fewer than 10 yeast cells in contaminated samples of sherry.     

采用套式PCR检测水库产毒微囊藻

根据所测定的微囊藻毒素合成酶mcyB基因的部分核苷酸序列,设计并筛选出两对特异性引物,用于产毒微囊藻的套式PCR检测。套式PCR针对毒素基因的检测结果与ELISA针对微囊藻毒素的检测结果相一致,但灵敏度更高。套式PCR的检测下限达1—10个微囊藻细胞/反应。采用套式PCR对广东12个主要供水水库的247份水样进行了产毒微囊藻检测,共检出阳性水样82份,阳性率为33.2%。这些阳性水样分布于除深圳水库以外的其他11个水库;其中汤溪水库水样套式PCR检出阳性率最高,达67.4%,其水样一步PCR的检出阳性率亦达25.6%,值得引起水文部门重视,并进行进一步跟踪监测。     

Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification.

A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river water were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.     

Detection and monitoring of virus infections by real-time PCR

The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.     

Detection of different developmental stages of malaria parasites by non-radioactive DNAin situ hybridization

Journal of Molecular Histology - A highly sensitive non-radioactive DNAin situ hybridization procedure is described that enables detection and unequivocal identification of various developmental...     

Detection of Legionella pneumophila in water samples by species-specific real-time and nested PCR assays

AIMS: Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. METHODS AND RESULTS: The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-microl reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. CONCLUSIONS: When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.     

DNA from pre-erythrocytic stage malaria parasites is detectable by PCR in the faeces and blood of hosts

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be “sub-microscopic” blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.     

Mixed-genotype infections of malaria parasites: within-host dynamics and transmission success of competing clones.

Mixed-genotype infections of microparasites are common, but almost nothing is known about how competitive interactions within hosts affect the subsequent transmission success of individual genotypes. We investigated changes in the composition of mixed-genotype infections of the rodent malaria Plasmodium chabaudi clones CR and ER by monoclonal antibody analysis of the asexual infection in mice, and by PCR amplification of clone-specific alleles in oocysts sampled from mosquitoes which had fed on these mice. Mixed-clone infections were initiated with a 9:1 ratio of the two clones, with ER as the minority in the first experiment and CR as the minority in the second experiment. When beginning as the majority, clones achieved parasite densities in mice comparable to those achieved in control (single-clone) infections. When they began as the minority, clones were suppressed to less than 10% of control parasitaemias during the early part of the infections. However, in mosquitoes, the frequency of the initially rare clone was substantially greater than it was in mice at the start of the infection or four days prior to the feed. In both experiments, the minority clone in the inocula produced as many, or more, oocysts than it did as a single-clone infection. These experiments show that asexual dominance during most of the infection is poorly correlated to transmission probability, and therefore that the assumption that within-host population size correlates to transmission probability may not be warranted. They also raise the fundamental question of why transmission rates of individual genotypes are often higher from mixed than single-clone infections.     

Evaluation of nested PCR in detection of Helicobacter pylori targeting a highly conserved gene: HSP60

Objective: To comparatively evaluate a new nested set of primers designed for the detection of Helicobacter pylori targeting a highly conserved heat shock protein gene (Hsp60). Methods: A total of 60 subjects having peptic ulcer diseases were tested for the detection of H. pylori using rapid urease test (RUT), histology, culture, and polymerase chain reaction (PCR) in their antral biopsy specimens. A newly designed Hsp60 gene‐based primer set was evaluated against commonly used PCR primers for detection of H. pylori. Results: Forty‐six of the 60 study subjects were found positive for culture isolation and all the 46 culture‐positive specimens were also positive with Hsp60 gene PCR. Of the 46 culture‐positive specimens, 44 were positive for 16S rRNA gene, ureC gene, RUT, and histology whereas only 29 were positive with ureA gene PCR. Of the 14 culture‐negative subjects, 10 were positive with 16S rRNA gene, 4 were positive with ureC (glmM) gene PCR, and 2 were positive with RUT and 1 was positive on histology. Conclusion: This study shows that nested amplification targeting Hsp60 gene is the most sensitive and specific with LR⁺ and LR^? values of ∝ and 0, respectively, when compared with the other three PCR methods. Also, HSP60 gene‐specific nested protocol was the most appropriate for detection of H. pylori in clinical specimens. This is particularly valuable because it can be used as a noninvasive method for detecting H. pylori infection in young children and also, in follow‐up studies with peptic ulcer patients, on samples like feces and saliva.     

Detection of different developmental stages of malaria parasites by non-radioactive DNAin situ hybridization

Summary A highly sensitive non-radioactive DNAin situ hybridization procedure is described that enables detection and unequivocal identification of various developmental stages of human and rodent malaria parasites. Using biotinylated species-specific DNA probes, erythrocytic parasites can be specifically stained in blood smears. Similarly exoerythrocytic stages can be visualized in cell culture and in sections of paraffin-embedded liver. In blood smears, the hybridization procedure provides a rapid detection of (low) parasitemia and species-determination for experienced microscopists at 100 to 400x magnification. Moreover, the procedure can be applied even after previous Giemsa staining of the preparation, enabling revision of patient smears which were difficult to read after routine Giemsa staining.     

Co‐infections by malaria parasites decrease feather growth but not feather quality in house martin

During moult, stressors such as malaria and related haemosporidian parasites (e.g. Plasmodium and Haemoproteus) could affect the growth rate and quality of feathers, which in turn may compromise future reproduction and survival. Recent advances in molecular methods to study parasites have revealed that co‐infections with multiple parasites are frequent in bird–malaria parasite systems. However, there is no study of the consequences of co‐infections on the moult of birds. In house martins Delichon urbica captured and studied at a breeding site in Europe during 11 yr, we measured the quality and the growth rate of tail feathers moulted in the African winter quarters in parallel with the infection status of blood parasites that are also transmitted on the wintering ground. Here we tested if the infection with two haemosporidian parasite lineages has more negative effects than a single lineage infection. We found that birds with haemosporidian infection had lower body condition. We also found that birds co‐infected with two haemosporidian lineages had the lowest inferred growth rate of their tail feathers as compared with uninfected and single infected individuals, but co‐infections had no effect on feather quality. In addition, feather quality was negatively correlated with feather growth rate, suggesting that these two traits are traded‐off against each other. We encourage the study of haemosporidian parasite infection as potential mechanism driving this trade‐off in wild populations of birds.     

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad‐selectivity channel known as the plasmodial surface anion channel, increased Ca⁺⁺ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca⁺⁺ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N‐hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca⁺⁺ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca⁺⁺ permeability, suggesting involvement of parasite‐encoded proteins trafficked to the host membrane. A high‐throughput chemical screen identified the first Ca⁺⁺ transport inhibitors active against Plasmodium‐infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca⁺⁺] is consistent with parasite killing specifically via action on one or more Ca⁺⁺ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca⁺⁺ transport and may be starting points for new antimalarial drugs.