Gene-based PCR Assay Research Articles

The Low Genetic Diversity of Dientamoeba fragilis Isolates in Southwest Turkey and Analysis of Clinical Findings.

Dientamoeba fragilis (D. fragilis) is a common intestinal protozoan with a global distribution. In the present study, we aimed to determine genetic diversity of D. fragilis isolates with multilocus sequence typing (MLST) in the southwest of Turkey and analyse the clinical findings. The study included faecal samples from 200 individuals in Aydin, Turkey. The positivity of D. fragilis was determined with 18S rRNA gene-based PCR assay. Six nested-PCR reactions were set to amplify partial D. fragilis housekeeping genes in the positive samples. The sequences were aligned with the references from GenBank to detect nucleotide polymorphisms and haplotypes. Additionally, the clinical findings and demographic characteristics of patients were statistically analysed between D. fragilis-infected and non-infected cases. The positivity of D. fragilis was 16% (32 out of 200 cases) with 18S rRNA based-PCR, and all were classified as "genotype 1". The analysis of six MLST loci revealed different haplotypes only at one locus; the remaining five loci exhibited no polymorphisms. The haplotypes in the present study were identical to at least one previously reported reference, except the locus "large subunit of RNA polymerase II" locus. There were no significant differences in any of the clinical findings or demographic characteristics between the infected and non-infected groups. Our study revealed a low genetic diversity of D. fragilis isolates from Turkey, like other countries including Italy, Denmark, the UK, Australia, and Brazil. The high degree of sequence similarity in housekeeping genes indicated the clonal distribution of D. fragilis.

The use of 18S rRNA for identification of the first record of Tadpole Shrimp Lepidurus apus (Linnaeus, 1758) from Jordan

Abstract. Abushattal S, Alnasarat H, Alnaimat SM, Eid E. 2024. The use of 18S rRNA for identification of the first record of Tadpole Shrimp Lepidurus apus (Linnaeus, 1758) from Jordan. Biodiversitas 25: 1223-1229. The tadpole shrimps are living fossils that survive in temporary water pools and are considered benthic organisms that swim and feed on tiny organisms. Two genera of tadpole shrimps exist worldwide: Triops and Lepidurus, which share similar ecological niches and habitats but never coincide. The occurrence of the tadpole shrimp Lepidurus apus in Jordan was confirmed for the first time using the 18S rRNA Gene-Based PCR Assay. The experimental approach encompassed the amplification of biological specimens utilizing a universal amplicon measuring approximately 1,500 base pairs (bp), specifically targeting the 18S ribosomal RNA (rRNA) gene. Subsequently, the acquired sequences underwent scrutiny through BlastN search against the GenBank database for validation and comparative analysis. Genetic analysis was needed to define the species accurately, especially with tadpole shrimp's generally high morphological variability. This species was recorded in a temporary freshwater pool in the desert of Al-Jafr in Ma’an governorate in southern Jordan. The DNA analysis showed a 100% identical sequence of the 18S rRNA gene with Lepidurus apus sequences. This record is important due to the ecological role of the tadpole shrimps as filter feeders, consuming algae, bacteria, and other small particles of organic matter, and as a food source for various fish, birds, and other aquatic animals. In addition, it will pave the way for future research to identify its economic importance, such as its role in mosquito control and its potential role in aquaculture. Finally, tadpole shrimp are sensitive to environmental changes and could be used to indicate climatic changes and habitat degradation. This is mainly due to their sensitivity to changes in water quality, including temperature, pH, and chemical composition, and their niche requirements for survival and reproduction. In addition, monitoring tadpole shrimp distribution and comparing it with historical records can reveal patterns of climate-induced range shifts.

Development of a novel invA gene-based real-time PCR assay for the detection of Salmonella in food

Development of a novel invA gene-based real-time PCR assay for the detection of Salmonella in food

Molecular characterization and antimicrobial susceptibility of Corynebacterium pseudotuberculosis isolated from skin abscesses of native Korean goats (Capra hircus coreanae).

This study aimed to investigate the molecular characterization and antimicrobial susceptibility of Corynebacterium pseudotuberculosis from skin abscesses of Korean native black goats (KNBG, Capra hircus coreanae) in South Korea. A total of 83 isolates were recovered from skin abscesses of KNBG. Of these isolates, 74 isolates were identified as C. pseudotuberculosis by phospholipase D (PLD) gene-based PCR assay. Each of the isolates possessed all 18 virulence genes (FagA, FagB, FagC, FagD, SigE, SpaC, SodC, PknG, NanH, OppA, OppB, OppC, OppD, OppF, CopC, NrdH and CpaE). The genetic diversity of C. pseudotuberculosis isolates was assessed by the phylogenetic analysis using the concatenated sequences (3073 bp) of five housekeeping genes (fusA, dnaK, infB, groeL1 and leuA) for investigating their genetic diversity. In the results, the isolates belonged to three groups: group 1 (67 isolates), group 2 (one isolate) and group 3 (six isolates) within biovar ovis. However, the groups exhibited low genetic diversity (0.20%-0.41%). In the antimicrobial susceptibility test, most isolates were susceptible to tetracycline, vancomycin, chloramphenicol, ciprofloxacin, erythromycin, enrofloxacin, cefoxitin, ampicillin, gentamycin, cephalothin and doxycycline, whereas they were not susceptible to cefotaxime, trimethoprim and streptomycin. This results suggest the involvement of relatively few clones of C. pseudotuberculosis in Korea. Further, present isolates can threaten public health due to potentially virulent strains with all 18 virulence genes and non-susceptible strains to clinically important antibiotics (CIA) and highly important antibiotics. This study is the first to investigate the genetic diversity and potential pathogenicity of C. pseudotuberculosis biovar ovis isolates from skin abscesses of KBNG in South Korea, and could provide useful information in controlling its infections.

Adherence of Helicobacter pylori to Opisthorchis viverrini gut epithelium and the tegument mediated via L-fucose binding adhesin.

Recent reports implicate both the liver fluke Opisthorchis viverrini as a reservoir of Helicobacter pylori within the human gastrointestinal tract and H. pylori in the pathogenesis of opisthorchiasis-associated cholangiocarcinoma. We postulated that adherence of bacterial ligands to host receptors initiates colonization of the live fluke by H. pylori and here we aimed to assess the molecular interaction between O. viverrini and H. pylori by investigating host receptors for H. pylori in the fluke. Several known receptors of H. pylori including Lewis B, sialyl-Lewis X, Toll-like receptor 4 and L-fucose were detected immunohistochemically and histochemically by focusing analysis on the gut epithelium and tegument of the adult stage of the fluke. The frequency of detection of Lewis B, sialyl-Lewis X, TLR4 and L-fucose in 100 individual worms was 3, 3, 19 and 70%, respectively. Detection of H. pylori by a diagnostic ureA gene-based PCR assay revealed the presence of H. pylori in individual O. viverrini worms in 41 of 49 (79%) worms examined. In addition, numbers of bacteria decreased in a dose- and time-dependent fashion following exposure to fucosidase. These findings suggested that L-fucose represents a tractable receptor for H. pylori that can mediate bacterial colonization of the gut of O. viverrini.

Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.

Antifungal effect of essential oil and different extracts obtained from Nepeta meyeri on Botrytis cinerea

The study concerns the antifungal effect of the aqueous and methanolic extracts, and the essential oil obtained from the aerial parts of Nepeta meyeri Benth. on Botrytis cinerea Pers. The fungus has been isolated from the infected plants of common grape Karaerik (Vitis vinifera L.) cultivating in vineyards in Üzümlü district, Erzincan (Turkey), and was cultured on potato dextrose agar medium in Petri dishes after the identification by 18S rRNA gene-based PCR assay. The concentrations of extracts from N. meyeri in Petri dishes were 2%, 5% and 10% (w/v) for aqueous extract (AE); 500, 1000 and 1500 ppm (v/v) for methanolic extract (ME), and 0.6, 0.8 and 1 µL for essential oil (EO). After the treatments, mycelial growth, spore germination, and germ tube elongation were determined. Sterile distilled water at the same ratios was used for the control treatment. Thirty-six different compounds were identified in the EO of N. meyeri by GC/MS. The highest antifungal activity has been registered for EO of N. meyeri. The inhibition rates in 1 µL/Petri of the EO were 80.72%, 18%, 38.15% on mycelial growth, spore germination and germ tube elongation, respectively. However, AE and ME of N. meyeri showed diverse effects on the studied parameters of B. cinerea. It is suggested that the favourable concentration of EO from N. meyeri can contribute to the prevention of B. cinerea infection (grey mould) which causes disease in vineyards.

Molecular Detection of Brucella melitensis Associated Reproductive Failure in Native Sheep Flock: Potential Public Health Pathogen from Tropical Region of Tamil Nadu

Brucellosis remains a global burden since the last century. In India, both animal and human brucella infections drastically increase every year. Outbreaks of 40 ewe’s abortion were investigated in native sheep’s from the tropical region of Tamil Nadu. Aborted maternal and fetal tissues were initially subjected to Stamps Modified Ziehl Neelsen (S-ZN) staining method. The DNA was extracted and screened for Brucella genome by Brucella cell surface salt extractable proteins (bcsp31) gene based PCR assay. Further, PCR positive samples were subjected for AMOS PCR for B. abortus, B. ovis, B. melitensis and B. suis identification and differentiation. S-ZN staining of aborted tissues revealed pink, coccobacilli organisms giving presumptive identification of Brucella sp. Molecular detection of brucellosis by bcsp31 gene based PCR in placental tissues and fetal abomasal contents of both the animals yielded a specific amplicon of 223 bp confirming it as Brucella genus infection. Molecular detection and differentiation by AMOS PCR revealed a specific amplicon of 731 bp indicating the involvement of B. meletensis infection in the native free-ranging sheep flock. It is a potent public health pathogen, highly essential to be monitored for the strategic control of brucellosis.

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay.

Background:The genus Acanthamoeba is a free-living opportunistic protozoan parasite, which widely distributed in soil and fresh water. Acanthamoeba keratitis, which causes a sight-threating infection of the cornea, is going to rise in Iran and worldwide. The aim of this study was to compare direct microscopy, culture and PCR for detection of Acanthamoeba spp. in clinical samples and to determine the genotypes of Acanthamoeba spp. by sequencing 18SrRNA gene.Methods:Among patients clinically suspected to AK referred to a tertiary ophthalmology center at Mashhad, northeastern Iran. During 2017-18, twenty corneal scrapes specimens obtained. The samples were divided into three parts, subjected to direct microscopic examination, culture onto non-nutrient agar and PCR technique. Sensitivity, specificity, accuracy and likelihood ratio were evaluated.Results:Among 20 persons clinically suspected to amoebic keratitis, 13(69.2%) patients definitely diagnosed as Acanthamoeba keratitis. Wearing contact lens, eye trauma due to foreign particle and swimming in fresh water were the main predisposing factors. Most of patients suffered from pain and photophobia. Corneal ring infiltration and epithelial defect were common clinical sings. Direct examination had the lowest sensitivity and sensitivity of both Nelson-PCR and JDP-PCR methods were equal and highest. In addition, the results of sequencing identified that all strains belonged to T4 genotype.Conclusion:Amoebic keratitis is a sporadic parasitic keratitis, which is mainly seen in contact lens user in Mashhad. PCR based on 18S ribosomal DNA with JDP primers is a reliable and highly sensitive method for diagnosis of Acanthamoeba keratitis in clinically suspected cases.

Detection of Psychrophilic Clostridium spp. in Fecal Samples from Cattle of Different Ages Sampled at the Slaughterhouse Level

Clostridium estertheticum and C. estertheticum-like spp. are obligate anaerobic psychrophiles causing "blown pack" spoilage of chilled vacuum-packed meat. The present study aimed at detecting and isolating these spoilage bacteria in fecal samples of cattle of different ages at the slaughterhouse level. One hundred two swab fecal samples were obtained and enriched anaerobically in prereduced peptone-yeast-glucose-starch (PYGS) medium for 3 weeks at 4°C and then screened for C. estertheticum and C. estertheticum-like spp. by using a 16S rRNA gene-based real-time PCR (RT-PCR) assay. The RT-PCR-positive samples were further enriched for 3 weeks in prereduced PYGS medium and then subjected to an ethanol (50%, v/v) and lysozyme (4 mg/mL) treatment. Isolation was carried out anaerobically on Columbia agar with 5% defibrinated sheep blood at 4°C for 3 weeks. Isolated strains were identified morphologically and by the 16S rRNA gene. Forty (39%) of 102 samples were RT-PCR positive. The frequency of positive samples was the following: 9 (45%) of 20 in calves (aged ≤160 days), 23 (43%) of 54 in young cattle (aged 161 to 1,000 days), and 8 (29%) of 28 in cows or bulls (aged >1,000 days). Six strains were isolated from 6 of 40 RT-PCR-positive samples. Of these, five were from the calves (n = 1) and young cattle (n = 4). The six isolates were identified as C. estertheticum (n = 1), Clostridium frigoriphilum (n = 1), and C. estertheticum-like spp. (n = 4). The present findings confirm that feces of cattle are an important source of psychrophilic Clostridium spp. The fecal carriage among livestock animals at slaughter is strongly correlated with the risk of carcass contamination. Therefore, the maintenance of slaughter hygiene is of central importance.

Development of a real-time PCR assay for detection of African swine fever virus with an endogenous internal control.

Real-time PCR assays are highly sensitive, specific and rapid techniques for the identification of ASF virus (ASFV) (Section 3.8, OIE Terrestrial Manual, 2019). Although an ASFV p72 gene-based real-time PCR assay (a.k.a. the Zsak assay) (Journal of Clinical Microbiology, 2005, 43, 112) has been widely used for ASFV detection, several more ASFV whole genome sequences have become available in the 15years since the design of the Zsak assay. In this study, we developed a new ASFV p72 gene-based real-time PCR after analysis of all currently available sequences of the p72 gene and multiplexed the new assay with a modified Zsak assay aiming to have a broader coverage of ASFV strain/isolates. To reduce false-negative detections, porcine house-keeping gene, beta actin (ACTB), was applied as an internal control. Eight ACTB sequences from the GenBank and 61 partial ACTB sequences generated in this study, and 1,012 p72 sequences from the GenBank and 23 p72 sequences generated at FADDL, were used for ACTB and ASFV primer and probe designs, respectively, to ensure broader host and ASFV coverage. Multiplexing ACTB in the reaction did not affect ASFV amplification. The multiplex assay was evaluated for strain/isolate coverage, sensitivity and specificity. The in silico analysis showed high ASFV strain/isolate coverage: 98.4% (978/994) of all p72 sequences currently available. The limit of detection (LOD) was 6 plasmid copies or 0.1-1 TCID50 /ml of ASFV isolates per reaction. Only targeted ASFV isolates and the viruses in the positive clinical samples were detected, indicating that the assay is highly specific (100% specificity). The test results of 26 ASFV isolates with different country origins showed that this newly developed multiplex assay performed better than the Zsak assay that has been widely accepted and used worldwide, indicating that it may be used as an alternative assay for ASFV detection.

First detection of Theileria parva in cattle from Cameroon in the absence of the main tick vector Rhipicephalus appendiculatus.

A major risk factor for the spread of livestock diseases and their vectors is the uncontrolled transboundary movement of live animals for trade and grazing. Such movements constrain effective control of tick‐transmitted pathogens, including Theileria parva. Only limited studies have been undertaken to identify ticks and tick‐borne diseases (TTBDs) affecting cattle in central African countries, including Cameroon. We hereby report the collection of baseline data on the prevalence of T. parva in Cameroon through a countrywide cross‐sectional survey, conducted in 2016, involving collection of blood samples from cattle from 63 sites across the five agro‐ecological zones (AEZs) of the country. ELISA‐based surveillance of infected cattle was performed on 479 randomly selected samples and revealed specific antibodies to T. parva in 22.7% and T. mutans in 41.1% of cattle. Screening of 1,340 representative DNA samples for the presence of T. parva identified 25 (1.86%) positives using a p104 antigen gene‐based nested PCR assay. The positives were distributed across agro‐ecological zones I, II, III and V. None of the p104 positive cattle exhibited clinical symptoms of East Coast fever (ECF). Using reverse line blot (RLB), 58 (4.3%) and 1,139 (85%) of the samples reacted with the T. parva and T. mutans oligonucleotide probes, respectively. This represents the first report of T. parva from Cameroon. Surprisingly, no Rhipicephalus appendiculatus ticks, the main vector of T. parva, were identified in a parallel study involving comprehensive morphological and molecular survey of tick species present in the country. Only two of the 25 p104 positive cattle were PCR‐positive for the CD8+ T‐cell target schizont‐expressed antigen gene Tp1. Cloning and sequencing of Tp1 amplicons revealed sequence identity with the reference T. parva Muguga. This new finding raises serious concerns of a potential spread of ECF into the central African region.

Babesia (Theileria) equi genotype A among Indian equine population.

Equine piroplasmosis, caused by Babesia (Theileria) equi, is well reported from many parts of India. However, literature regarding its prevalence from semi arid India is limited. Alongside, there is complete absence of information about genetic characterization of B.(T.) equi and the associated genotypes from India. In the present study, the prevalence of B.(T.) equi was studied from semi arid India using 18S ribosomal gene based PCR assay. An overall prevalence rate of 10.46% was recorded. PCR was more sensitive and specific in comparison with blood smears. The found isolates were sequenced. These sequences were aligned and submitted into NCBI. All the isolates showed 100% homology with each other and represented a single haplotype. When compared with other isolates of B.(T.) equi across India and globe, using Genetool and Mega 6 softwares, they showed 99.5-99.2% and 95.7-97.0% homologies, respectively. A unique substitution of G by A at 206 nucleotide position was reported in all found isolates in comparison to earlier reported isolates from India. The studied isolates were found to be phylogenetically closer to isolates from USA and Trinidad than to other parts of the world. All the Indian isolates belonged to genotype A of B.(T.) equi.

Molecular characterization of Cryptosporidium species in sheep and goats in Anhui Province and neighboring provinces

To investigate the prevalence and molecular features of Cryptosporidium in sheep and goats from Anhui Province and neighboring provinces. A total 832 and 781 fresh fecal samples were collected from seven large-scale sheep farms and ten large-scale goat farms in Anhui Province and neighboring provinces of Henan, Jiangsu and Shandong. The prevalence and species of Cryptosporidium were investigated in the fecal samples from the sheep and goats in the study areas using nested PCR assay based on the Cryptosporidium-specific SSU rDNA gene, and the subgenotypes of C. parvum and C. ubiquitum were characterized by amplification and sequencing of the 60 kDa glycoprotein (gp60) gene. The overall prevalence of Cryptosporidium was 5.8% (48/832) in sheep and 8.7% (68/781) in goats in Anhui Province and neighboring provinces, respectively. The SSU rDNA gene-based PCR assay identified C. xiaoi and C. ubiquitum in sheep and C. parvum in goats, and subtyping revealed that all C. ubiquitum subgenotypes belonged to XIIa subtype 2 and C. parvum subgenotypes belonged to IIdA19G1. The identification of zoonotic C. ubiquitum XIIa subtype 2 and C. parvum subtype IIdA19G1 suggests that sheep and goats may serve as a potential source for human Cryptosporidium infections.

Occurrence and genetic diversity of hemoplasmas in beef cattle from the Brazilian Pantanal, an endemic area for bovine trypanosomiasis in South America

Hemotropic mycoplasmas (hemoplasmas) are Gram-negative bacteria that parasitize the erythrocyte surface of a wide variety of mammals. The present study aimed at investigating the occurrence of hemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. Additionally, the objective of this study was to characterize molecularly the genotypes of the found hemoplasmas. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in Corumbá, Nhecolândia sub-region, Mato Grosso do Sul, in Midwest Brazil. Blood samples underwent DNA extraction and standard 16S rRNA gene-based PCR assays for hemoplasmas. The sequences obtained were submitted to phylogenetic inferences, distance analysis, and genotype diversity. The Indirect Enzyme-Linked Immunoabsorbent Assay (iELISA) indicated the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity of said agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive for hemoplasmas in cPCR. The phylogenetic analysis of the obtained sequences confirmed the presence of 'Candidatus Mycoplasma haemobos' and Mycoplasma wenyonii DNA in 0.5% (2/400) and 1.75% (7/400) animals, respectively. Five genotypes of M. wenyonii and one of 'Candidatus M. haemobos' were detected among the sequenced amplicons. The present study showed low molecular occurrence of haemoplasmas in beef cattle sampled in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis. Despite of the conservation of the 16S rRNA gene, there was considerable diversity of hemoplasma genotypes infecting the sampled beef cattle.

Epidemiological survey of Anaplasma marginale in cattle and buffalo in Sri Lanka.

Bovine anaplasmosis caused by Anaplasma marginale represents a serious threat to cattle farming worldwide, especially in the tropics and subtropics. In the present study, archived DNA samples from the blood of cattle (n=437) in the Nuwara Eliya, Galle, Ampara, Polonnaruwa, and Jaffna districts and buffalo (n=327) in the Galle, Polonnaruwa, Mannar, and Mullaitivu districts in Sri Lanka, were screened for A. marginale using a major surface protein 5 (msp5) gene-based PCR assay. The findings showed that 32.7 and 57.5% of cattle and buffalo, respectively, were A. marginale-positive. The rate of positivity differed significantly among geographical regions. In conclusion, the high rates of A. marginale infection in cattle and buffalo highlight the importance of effective control measures in Sri Lanka.

Prevalence and molecular phylogenetic analysis of Babesia vogeli from dogs in Karnataka

Babesia vogeli infection is an important tick-borne haemoprotozoan disease of canids. The study was carried out to understand the prevalence and molecular phylogeny of B. vogeli in pet dogs. A total of 128 blood samples were collected during study period. The microscopy based prevalence of 7.81% was recorded for large form of Babesia spp. We developed and standardized heat shock protein (hsp) 70 genebased PCR assay with high analytical sensitivity and specificity tested with B. gibsoni, H. canis and D. repens. The PCR assay on all the blood samples revealed higher prevalence rate of 25% of B. vogeli infection. The correlation analysis for age, breed, sex and presence of ticks on animal body did not reveal significant association with B. vogeliinfection, and among the clinical factors, only pale mucus membrane was significantly associated with presence of infection. The sequence analysis revealed 99.8 - 99.9% identity among themselves and 99.8 - 100% similarity with the published sequences of B. vogeli. Phylogenetically, all the Indian isolates clustered together with Japan and Taiwan isolates, indicating genetically similar species circulation in Asian content. In conclusion, in addition to clinical and microscopic examination, the PCR assay is recommended for confirmatory diagnosis of B. vogeli infection as genetically similar species are circulating in dogs.

PCR-based detection of Plasmodium falciparum in saliva using mitochondrial cox3 and varATS primers

BackgroundSampling of saliva for diagnosing Plasmodium falciparum infections is a safe, non-invasive alternative to sampling of blood. However, the use of saliva presents a challenge because lower concentrations of parasite DNA are present in saliva compared to peripheral blood. Therefore, a sensitive method is needed for detection of parasite DNA in saliva. This study utilized two recently reported “ultra-sensitive” PCR assays based on detection of the P. falciparum mitochondrial cox3 gene and the multi-copy nuclear varATS gene. The ultra-sensitive assays have an advantage over standard 18S rRNA gene-based PCR assay as they target genes with higher copy numbers per parasite genome. Stored saliva DNA samples from 60 Cameroonian individuals with infections previously confirmed by 18S rRNA gene PCR in peripheral blood were tested with assays targeting the cox3 and varATS genes.ResultsOverall, the standard 18S rRNA gene-based PCR assay detected P. falciparum DNA in 62% of the stored saliva DNA samples, whereas 77 and 68% of the samples were positive with assays that target the cox3 and varATS genes, respectively. Interestingly, the ultra-sensitive assays detected more P. falciparum infections in stored saliva samples than were originally detected by thick-film microscopy (41/60 = 68%). When stratified by number of parasites in the blood, the cox3 assay successfully detected more than 90% of infections using saliva when individuals had > 1000 parasites/μl of peripheral blood, but sensitivity was reduced at submicroscopic parasitemia levels. Bands on electrophoresis gels were distinct for the cox3 assay, whereas faint or non-specific bands were sometimes observed for varATS and 18S rRNA that made interpretation of results difficult. Assays could be completed in 3.5 and 3 h for the cox3 and varATS assays, respectively, whereas the 18S rRNA gene assays required at least 7 h.ConclusionsThis study demonstrates that a PCR assay targeting the cox3 gene detected P. falciparum DNA in more saliva samples than primers for the 18S rRNA gene. Non-invasive collection of saliva in combination with the proposed cox3 primer-based PCR assay could potentially enhance routine testing of P. falciparum during disease surveillance, monitoring, and evaluation of interventions for malaria elimination.

Antibiotic-resistant Staphylococcus epidermidis isolated from patients and healthy students comparing with antibiotic-resistant bacteria isolated from pasteurized milk.

Antibiotic-resistant Staphylococci are a global issue affecting humans, animals, and numerous natural environments. Antibiotic-resistant Staphylococcus epidermidis is an opportunistic pathogen frequently isolated from patients and healthy individuals. This study aimed to examine the antibiotic resistance of S. epidermidis isolated from patients, healthy students and compare the results with antibiotic-resistant bacteria isolated from pasteurized milk. Clinical strain isolation was performed in several hospitals in the Riyadh. Skin swabs from 100 healthy undergraduate candidate students were obtained at King Saud University. The pasteurized milk samples were obtained from local market (company, X). After isolation, identification and susceptibility tests were performed using an automated system. A multiplex tuf gene-based PCR assay was used to confirm identification. Biofilm production and biofilm-related gene expression were studied. S. epidermidis represented 17% of clinical bacterial isolates, and 1.7% of isolates obtained from healthy students were multiantibiotic-resistant. All patient strains were teicoplanin- and vancomycin-susceptible, while all student strains were gentamicin-, levofloxacin-, moxifloxacin-, and trimethoprim/sulfamethoxazole-susceptible. All the bacteria isolated from pasteurized milk were benzylpenicillin and oxacillin-resistant strains. Of the S. epidermidis strains, 91% could produce biofilms, and mecA, icaADBR, ica-ADB, ica-AD, ica-A only, and ica-C only were expressed in 83, 17.1, 25.7, 37.1, 20, and 0% of the strains, respectively. This work demonstrates that S. epidermidis can be accurately identified using a multiplex tuf-based assay, and that multiantibiotic-resistant S. epidermidis strains are widespread amongst patients and healthy students.

Molecular Tests That Target the RTX Locus Do Not Distinguish between Kingella kingae and the Recently Described Kingella negevensis Species.

Kingella kingae is an important invasive pathogen in early childhood. The organism elaborates an RTX toxin presumably restricted to this species. Consequently, real-time quantitative PCR (qPCR) assays targeting the RTX locus have been developed in recent years and are gaining increasing use for the molecular diagnosis of K. kingae infections. However, the present study shows that Kingella negevensis, a Kingella species newly identified in young children, harbors an identical Kingella RTX locus, raising the question of whether K. negevensis can be misidentified as K. kingae by clinical microbiology laboratories. In silico comparison of Kingella sp. RTX and groEL genes and in vitro studies provided evidence that targeting the rtxA and rtxB genes could not differentiate between strains of K. kingae and K. negevensis, whereas targeting the groEL gene could. This prompted the design of a highly specific and sensitive qPCR assay targeting K. negevensis groEL (kngroEL). Ninety-nine culture-negative osteoarticular specimens from 99 children younger than 4 years of age were tested with a conventional 16S rRNA gene-based broad-range PCR assay and Kingella-specific rtxB, K. kingae-specific groEL (kkgroEL), and kngroEL qPCR assays. Forty-two specimens were rtxB positive, including 41 that were also kkgroEL positive and 1 (the remaining one) that was kngroEL positive. Thus, this study discloses an invasive infection caused by K. negevensis in humans and demonstrates that targeting the RTX locus cannot be used for the formal diagnosis of K. kingae infections. These findings stress the need for further studies on the epidemiology of asymptomatic carriage and invasive infections caused by K. negevensis in humans.