Semi-nested Multiplex Polymerase Chain Reaction Research Articles

Increased peripartum mortality associated with maternal subclinical malaria in Mozambique

BackgroundPlasmodium falciparum infection in pregnant women in sub-Saharan Africa is often asymptomatic. As these forms of malaria are often submicroscopic and difficult to diagnose by conventional methods (microscopy and/or rapid diagnostic test), diagnosis requires the use of molecular techniques such as polymerase chain reaction (PCR). This study analyses the prevalence of subclinical malaria and its association with adverse maternal and neonatal outcomes, a topic that has been scarcely evaluated in the literature.MethodsA cross-sectional study was conducted using semi-nested multiplex PCR to assess the presence of P. falciparum in placental and peripheral blood of 232 parturient pregnant women at the Hospital Provincial de Tete, Mozambique between March 2017 and May 2019. Multivariate regressions were performed to assess the associations of maternal subclinical malaria with several maternal and neonatal outcomes after controlling for the presence of preeclampsia/eclampsia (PE/E) and HIV infection, as well as for other maternal and pregnancy characteristics.ResultsIn total, 17.2% (n = 40) of the women studied had positive PCR for P. falciparum (7 in placental blood only, 3 in peripheral blood only). We found a significant association between subclinical malaria and a higher peripartum mortality risk, which persisted after controlling for maternal comorbidity and maternal and pregnancy characteristics (adjusted odds ratio: 3.50 [1.11–10.97]). In addition, PE/E and HIV infections were also significantly associated with several adverse maternal and neonatal outcomes.ConclusionThis study demonstrated the association of subclinical malaria, as well as of PE/E and HIV, in pregnant women with adverse maternal and neonatal outcomes. Therefore, molecular methods may be sensitive tools to identify asymptomatic infections that can reduce the impact on peripartum mortality and their contribution to sustained transmission of the parasite in endemic countries.

Molecular Epidemiology of Rotavirus Strains in Under Five Children.

To determine the prevalence and genotype distribution of rotaviruses in children presenting to authors' hospital in Middle Black Sea region of Turkey. The results may supply important information about vaccine studies in Turkey. Rotavirus antigen was detected by latex agglutination test and rotavirus RNA was detected by RT-PCR test. On the other hand, rotavirus positive samples were genotyped by semi-nested multiplex polymerase chain reaction. The highest rate of rotavirus positivity (46.9%) was observed among children in the 13 to 24 mo age group. All the positive-strains were in G1-4, G8-9, P [4], P [8], and P [9] genotypes. The most common G and P combination in present study was G9P[8] (n=24, 28.9%). The present results indicated that the most prevalent genotypes were G1, G9, P8, G9P[8] and G1P[8] in authors' region. Rotavirus vaccines used in this region must include mainly these genotypes.

Molecular Detection of Plasmodium malariae/Plasmodium brasilianum in Non-Human Primates in Captivity in Costa Rica.

One hundred and fifty-two blood samples of non-human primates of thirteen rescue centers in Costa Rica were analyzed to determine the presence of species of Plasmodium using thick blood smears, semi-nested multiplex polymerase chain reaction (SnM-PCR) for species differentiation, cloning and sequencing for confirmation. Using thick blood smears, two samples were determined to contain the Plasmodium malariae parasite, with SnM-PCR, a total of five (3.3%) samples were positive to P. malariae, cloning and sequencing confirmed both smear samples as P. malariae. One sample amplified a larger and conserved region of 18S rDNA for the genus Plasmodium and sequencing confirmed the results obtained microscopically and through SnM-PCR tests. Sequencing and construction of a phylogenetic tree of this sample revealed that the P. malariae/P. brasilianum parasite (GenBank KU999995) found in a howler monkey (Alouatta palliata) is identical to that recently reported in humans in Costa Rica. The SnM-PCR detected P. malariae/P. brasilianum parasite in different non-human primate species in captivity and in various regions of the southern Atlantic and Pacific coast of Costa Rica. The similarity of the sequences of parasites found in humans and a monkey suggests that monkeys may be acting as reservoirs of P.malariae/P. brasilianum, for which reason it is important, to include them in control and eradication programs.

Absence of asymptomatic cases of malaria in a historically endemic indigenous locality of the Department of Caaguazú, Paraguay: moving toward elimination.

Paraguay was among the 16 countries that reported zero indigenous malaria cases in 2014. A cross-sectional observational descriptive study was performed in 100 adults from Santa Teresa, Paraguay. Parasite detection was carried out using seminested multiplex polymerase chain reaction (PCR) and microscopy. Among the participants, 44% were female and 56% were male, and 89% had a malaria history. No parasites were detected with either of the methods. There were no asymptomatic cases in Santa Teresa, and this finding is very promising. A longitudinal study should be performed to confirm that there are no asymptomatic cases in this locality.

Prevalence and genotype distribution of rotaviruses in children with gastroenteritis in Rize province.

Determination of the distribution of rotavirus genotypes is essential for understanding the epidemiology of this virus responsible for nearly half a million of deaths in patients with gastroenteritis worldwide. In the present study, we aimed to genotype the rotavirus strains isolated from diarrheal stool samples in children under 5 years old. A total of 1297 fecal samples were collected, and rotavirus antigen was detected in 73 of these samples. Antigen-positive samples were transferred to the Public Health Agency of Turkey, Molecular Microbiology Research Laboratory, and were tested for determination of genotypes G and P using semi-nested multiplex polymerase chain reaction method performed with consensus- and genotype-specific primers. Twelve specimens were found to be negative for rotavirus in genotyping method. All the positive-strains were in G1-4, G8-9, P(4), P(8), and P(9) genotypes. The most frequent GP genotype combinations were found to be G9P(8) in 21 strains (34.4%), G2P(4) in 14 strains (23.0%), and G1P(8) in 12 strains (19.7%). We found 10 distinct genotypes amongst a total of 61 strains. Among the strains isolated and genotyped in our study, 90.2% (55/61) and 67.2% (41/61) have already been included in the two existing commercial vaccines. In conclusion, these findings implicate the necessity of development of region-specific vaccines after evaluation of the local genotype distribution. Further studies on the large number of rotavirus strains would contribute to this process.

A Modified Semi-Nested Multiplex Malaria PCR (SnM-PCR) for the Identification of the Five Human Plasmodium Species Occurring in Southeast Asia

We have modified an existing semi-nested multiplex polymerase chain reaction (PCR) by adding one Plasmodium knowlesi-specific nested PCR, and validated the latter against laboratory and clinical samples. This new method has the advantage of being relatively affordable in low resource settings while identifying the five human Plasmodium species with a three-step PCR.

Prevalence and Molecular Characterization of Dengue Viruses Serotypes in 2010 Epidemic

During August to November 2010, a dengue (DEN) outbreak occurred, and several cities of Pakistan were affected. In this study, serum samples from 317 patients, based on clinical symptoms and blood test, were included from Lahore, Sheikhpura and Gujranwala cities for further diagnosis of different genotypes of DEN virus. The samples were categorized into 2 groups: group 1 containing patients with dengue fever (DF) without hemorrhagic manifestations (DF) (n=228) and group 2 involving patients having DF with hemorrhagic manifestations (dengue hemorrhagic fever) (n=89). The samples found positive for DEN in reverse transcriptase polymerase chain reaction (PCR) by targeting capsid and premembrane genes containing fragment of size ~2.5kb for groups 1 and 2 were found to be 83.33% and 91.01%, respectively; however, the detection limit was quite low by immunoglobulin M antibody capture enzyme linked immunosorbent assay in case of both groups and was found to be 34.70% and 68.53%, respectively. The 16.67% and 8.99% samples of groups 1 and 2, respectively, were found negative in reverse transcriptase PCR analysis. In case of group 1 and group 2, by adopting seminested multiplex PCR strategy, predominant genotype was DEN virus type 2 with prevalence rates of 45.17% and 65.16%, respectively. DEN virus type 1 was the second most prevailing genotype in the population with prevalence rates of 18.85% and 19.10% for groups 1 and 2, respectively.

Comparison of microscopical examination and semi-nested multiplex polymerase chain reaction in diagnosis of Plasmodium Falciparm and P. vivax

We compared light microscopy examination and a semi-nested multiplex PCR (SnM-PCR) assay in endemic areas of the Islamic Republic of Iran. A total of 68 individuals with malaria-positive and suspected malaria symptoms were included in the study. Giemsa-stained thick blood films were examined under a light microscope for malaria parasites in 100 and 200 fields. DNA was extracted from blood samples and SnM-PCR based on the amplification of the small sub-unit ribosomal RNA (ssrRNA) gene sequences was applied. Microscopical examination showed that 48.5% (33.8% P. vivax and 14.7% P.falciparum) and 50% (35.3% P. vivax and 14.7% P. falciparum) of the samples were positive in 100 and 200 fields respectively. SnM-PCR showed the same results as the 200 field microscopy.

Monitoring of failure of chloroquine treatment for Plasmodium vivax using polymerase chain reaction in Sanliurfa province, Turkey

Malaria is a complex disease that varies widely in epidemiology and clinical manifestation in the southeastern part of Turkey. In many regions of the world, chloroquine (CQ) has been the standard treatment for Plasmodium vivax. However, the resistance of the Plasmodium species to antimalarial drugs has become an increasing problem and a concern worldwide. Our target was to determine the Plasmodium species in the southeast region of Turkey and the therapeutic efficacy of CQ used in the treatment of malaria. Blood samples were collected from 180 patients infected with malaria before and after CQ treatment and were subjected to DNA isolation. The isolated DNA was amplified by a seminested multiplex polymerase chain reaction (SnM-PCR) including primers selected on Plasmodium small subunit ribosomal RNA (ssrRNA) genes for identification of the malaria species. The SnM-PCR results showed that only P. vivax exists in this province. It was also determined that there is a therapeutic failure to CQ in 9.5% of patients. These were the second report on identification of P. vivax and the third report on determination of the therapeutic failure in patients who used CQ to cure human malaria in the southeastern region of Turkey. Our results demonstrate that the SnM-PCR is a sensitive, specific, and a rapid tool for the differentiation of malaria species.

Malaria diagnosis by NOW ICT and expert microscopy in comparison with multiplex polymerase chain reaction in febrile returned travellers

Malaria is the most potentially dangerous imported parasitic disease in Spain [1, 2] and other European countries [3], and laboratories must be prepared round the clock to diagnose the disease quickly to start treatment and avoid complications [4]. In recent years, a second generation of rapid diagnostic tests combining the detection of a soluble Plasmodium antigen, the histidine-rich protein II (HRP-2) and aldolase, an antigen common to all species of Plasmodium, has been marketed and proved to be useful [5]. As a recent meta-analysis showed that HRP-2-based tests are superior to the lactate dehydrogenase (LDH) tests in diagnosis of malaria in nonimmune returned travellers [6], we decided to perform a multicentric study to determine the performance of a HRP-2-based diagnostic kit (NOW ICT) and expert microscopy in comparison with multiplex polymerase chain reaction (PCR) as the gold standard, with a target population mainly composed of febrile semi- and nonimmune and travellers returned from Africa. Eligible for the study were all febrile patients (>=38°C),presenting in emergency rooms or ambulatory consults of five hospitals of the Community of Madrid (Spain) who had visited or migrated from an endemic area for malaria in the previous year, to whom the physician in charge requested a malaria test to the laboratory. Patients were excluded if they had received at least one dose of an antimalarial drug in the preceding 4 weeks. Whole-blood samples were collected on ethylenediamine tetraacetic acid (EDTA) tubes for microscopy (thick and thin films stained with Giemsa stain [7]), PCR and HRP-2 detection. The procedure of the NOW ICT (detection of PfHPR2 for P. falciparum and aldolase, a common antigen to the four species of malaria) was performed by laboratory technicians as recommended by the manufacturer [5]. Slides were examined by one experienced microscopist for at least 500 oil fields (x 1,000) before reporting them as negative. Parasite count was rated as very low ( 50,000). The malaria PCR was carried out in the Parasitology Department of the National Microbiology Center following the seminested multiplex malaria PCR method [8] without knowledge of the results of previous thick film and rapid tests. True positives were defined taking multiplex PCR as the gold standard. Sensitivities and specificities and positive (PPV) and negative (NPV) predictive values with 95% confidence interval (CI) were calculated for expert microscopy and NOW ICT.

Plasmodiumdiversity in non-malaria individuals from the Bioko Island in Equatorial Guinea (West Central-Africa)

BackgroundIn this paper we analyse the Plasmodium sp. prevalence in three villages with different isolation status on the island of Bioko (Equatorial Guinea) where malaria is a hyper-endemic disease. We also describe the genetic diversity of P. falciparum, using several plasmodia proteins as markers which show a high degree of polymorphism (MSP-1 and MSP-2). The results obtained from three different populations are compared in order to establish the impact of human movements and interventions.MethodsPlasmodium sp. were analysed in three villages on Bioko Island (Equatorial Guinea), one of which (Southern) is isolated by geographical barriers. The semi-nested multiplex polymerase chain reaction (PCR) technique was used to determine the prevalence of the four human plasmodia species. The genotyping and frequency of P. falciparum populations were determined by PCR assay target polymorphism regions of the merozoite surface proteins 1 and 2 genes (MSP-1 and MSP-2).ResultsThe data obtained show that there are no differences in plasmodia population flow between the Northwest and Eastern regions as regards the prevalence of the different Plasmodium species. The Southern population, on the other hand, shows a minor presence of P. malariae and a higher prevalence of P. ovale, suggesting some kind of transmission isolated from the other two. The P. falciparum genotyping in the different regions points to a considerable allelic diversity in the parasite population on Bioko Island, although this is somewhat higher in the Southern region than the others. There was a correlation between parasitaemia levels and the age of the individual with the multiplicity of infection (MOI).ConclusionResults could be explained by the selection of particular MSP alleles. This would tend to limit diversity in the parasite population and leading up to the extinction of rare alleles. On the other hand, the parasite population in the isolated village has less outside influence and the diversity of P. falciparum is maintained higher. The knowledge of parasite populations and their relationships is necessary to study their implications for control intervention.

Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semi-nested multiplex malaria PCR (SnM-PCR)

A simplified protocol for the identification of Plasmodium species by semi-nested multiplex polymerase chain reaction (SnM-PCR) in human blood samples is compared with microscopical examination of thin and thick blood films in 2 field trials in Côte d'Ivoire and Cameroon. Also, dried blood spots or liquid blood collected from Dutch soldiers returning from Goma, Zaire ( n = 141), Angola ( n = 40), and from Marechaussee (Dutch border police) returning from various parts of the world ( n = 161) were examined, together with miscellaneous other material obtained from laboratories and hospitals. The method is based on features of the small subunit nuclear ribosomal ribonucleic acid (RNA) gene ( ssrDNA), a multicopy gene which possesses both highly conserved domains and domains characteristic for each of the 4 human malaria parasites. The first reaction of the SnM-PCR includes a universal reverse primer with 2 forward primers specific for Plasmodium and mammals, respectively. The mammalian-specific primer was included as a positive control to distinguish uninfected cases from simple PCR failures. The second PCR reaction includes a Plasmodium-specific forward primer plus species-specific reverse primers for P. vivax, P. ovale, P. falciparum and P. malariae. The technique worked better with samples collected in the field as dried blood spots on filter paper and heparinized blood rather than with frozen pelleted blood; it was more sensitive and more specific than the standard microscopical examination.

Identification of equine herpesviruses 1 and 4 by polymerase chain reaction.

To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease. Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR. Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.