Mosquito-borne California serogroup orthobunyaviruses Inkoo (INKV) and Chatanga (CHATV) are known to be endemic in Finland with a high seroprevalence. We developed a novel multiplexed reverse transcription quantitative polymerase chain reaction method for discriminating between the INKV and CHATV. This assay was used along with traditional serological tests to study a set of summertime patients during the years 2021, 2023, and 2024 to assess the epidemiology and prevalence of acute INKV and CHATV infections in Finland. Altogether, 1470 samples were screened, and there were 16 patients who had an acute infection based on serological findings and/or nucleic acid test. The orthobunyavirus-IgG seroprevalences were 18% (2021), 20% (2023), and 30% (2024), being lower than that in studies from 20 years ago. Neutralization tests were carried out, and all but one acute case had more than four-fold higher titre to INVK vs. CHATV, indicating specificity to INKV infection. The results suggest that epidemiology has changed from previous studies, and INKV should be considered a causative agent of summertime infections in Finland. The symptom diversity in mild disease outcomes should be studied to guide orthobunyavirus recognition by clinicians. The use of molecular assay discriminating INKV and CHATV aids in understanding disease associations.

The question of whether PCR is reliable sounds strange at first. However, looking at the scientific literature from the 1950s and 60s, one will find many publications on the physicochemistry of DNA that have been forgotten meanwhile. Quite a few of these studies have shown that DNA is thermolabile, which consequently raises the question of whether this thermolability is relevant in the context of PCR, namely in the denaturation phase. However, it can be shown that this is not the case: losses due to thermal hydrolysis are irrelevant for the performance of contemporary PCR protocols and their specificity as well as for the significance of their results. There is now a huge amount of scientifically verified and published data on technical and molecular aspects of PCR, a small selection of which we quote here. In addition, we present some primary data that also clearly demonstrate the reliability of PCR.

The aim of this study was to describe how the detection of protozoan and helminth parasites has been affected by the introduction of polymerase chain reaction (PCR) and changes in test algorithms. We extracted data about faecal samples tested for parasites (n = 114839) at five Norwegian clinical microbiology laboratories. Samples were classified into prePCR or postPCR depending on whether they were submitted before or after the introduction of PCR, and into diagnostic episodes (n = 99320). The number of diagnostic episodes increased 3.7-fold from prePCR to postPCR. Giardia positive episodes doubled, the positivity rate decreased from 2.0% to 1.3%. Cryptosporidium was hardly detected prePCR and increased to a positivity rate of 1.2%. Entamoeba histolytica was rarely found. Episodes examined for helminths decreased 51%, the number of positive episodes decreased 34%. Samples from immigrants were more likely to be positive for Giardia, E. histolytica, or helminths and less likely to be Cryptosporidium positive. During the COVID-19 pandemic, the number of Giardia and helminth-positive episodes decreased. Cryptosporidium-positive episodes remained unchanged. The implementation of multiplex PCR for protozoa led to a doubling of Giardia cases and a better test for Cryptosporidium. Fewer microscopy examinations raise concerns that helminth infections may be overlooked.

Dientamoeba fragilis (D. fragilis) is an intestinal protozoan parasite with uncertain pathogenic potential. In the United States, data on D. fragilis in the era of molecular detection are limited. The aim of this retrospective chart review was to evaluate the epidemiology and clinical characteristics of D. fragilis cases identified using polymerase chain reaction assays between 2016 and 2024 at our academic medical centre located in Utah. We identified 28 unique cases with varying gastrointestinal symptomatology including diarrhoea, abdominal pain, nausea, vomiting, and bloating. Approximately half (52%) of patients with follow-up data demonstrated improvement in symptoms following initial treatment for D. fragilis. The overall prevalence of D. fragilis was low among those tested (0.6% positivity). Additional research, including case-control studies, is needed to better describe the etiologic role of D. fragilis.

Coinfection of a host by more than 1 parasite is more common than single infection in wild environments and can have differing impacts, although coinfections have relatively rarely been quantified. Host immune responses to coinfection can contribute to infection costs but are often harder to predict than those associated with single infection, due to the influence of within-host parasite–parasite interactions on infection virulence. To first quantify coinfection in a common bird species, and then to test for immune-related impacts of coinfection, we investigated the prevalence and immune response to avian haemosporidian (genera: Plasmodium, Haemoproteus and Leucocytozoon) coinfection in wild blackbirds. Coinfection status was diagnosed using a 1-step multiplex polymerase chain reaction, immune response was quantified through white blood cell counts and heterophil: lymphocyte ratios, and parasitaemia was quantified for each infected sample. We detected high rates of haemosporidian infection and coinfection, although neither impacted immune activity, despite a significantly higher parasitaemia in individuals experiencing double vs single infection. This suggests that immune-related costs of haemosporidian single and coinfection are low in this system. This could be due to long-term host–parasite coevolution, which has decreased infection virulence, or a consequence of reduced costs associated with chronic infections compared to acute infections. Alternatively, our results may obscure immune-related costs associated with specific combinations of coinfecting haemosporidian genera, species or lineages. Future research should investigate interactions that occur between haemosporidian parasites within hosts, as well as the ways in which these interactions and resulting impacts may vary depending on parasite identity.

Helminthiasis is a common infection in both humans and other animal populations and negatively affects the health of the host, causing a range of morbidity and even mortality, especially in young people and those with weakened immune systems. A variety of diagnostic procedures with a number of modifications are available to identify the sources of infection and to assess the epidemiological situation, the effectiveness of parasite control programs, anthelmintic treatment, and the prevention of drug resistance. These can be simple and cheap methods – for example, stool smear examination – or modern highly sensitive methods – for example, PCR analysis. This review attempts to summarize the advantages and limitations of each of these frequently used methods.

As astroviral infection rapidly increased in the summer of 2022 in Korea, this study aimed to determine the cause and genotype of astroviruses during this period. From January to December 2022, we tested 43,312 stool samples from patients with acute gastroenteritis utilizing multiplex PCR to detect HAstV. For the HAstV-positive samples, we determined the genotypes of the HAstVs by PCR and sequencing. The monthly positive rate from 2015 to 2022 showed a notable and abrupt increase of HAstV infection between June and August 2022, peaking at 9.8% in July 2022. The annual positivity rate of HAstV remained at 2–3% between 2015 and 2019, and then decreased to 0.5% in 2020, followed by an increase to 1.5% in 2021 and 3.6% in 2022.The genotyped astroviruses in 2022 were all identified as HAstV-1 type, and the nucleotide identity% among them was >99%. The GenBank accession number for the strain genetically closest to the strains identified in our study was ON571597.1, which was HAstV-1 isolated from Pingtan in 2019. Our results provide recent epidemiological data on HAstVs in Korea. The decline and surge in astrovirus positivity in recent years may be related to the COVID-19 pandemic.

One of the most recognizable cases of preimplantation genetic diagnosis (PGD) is X-linked diseases. Diagnosis of fetal sex is essential for couples who are known to be at risk of some X-linked disorders. The objective of this study was to discriminate between female (XX) and male (XY) embryos by detecting sex chromosomes-specific sequences in spent culture medium and comparing these results to PGD/CGH array results. It may open new window for the development of a non-invasive PGD method. 120 Embryo’s spent media from Day 3 and Day 5 embryos were collected. Modified phenol-chloroform solution was used for DNA extraction from spent media. Sex determination was performed using SRY, TSPY and AMELOGENIN evaluation through quantitative polymerase chain reaction (q-PCR) method. IBM SPSS and MedCalc were used for statistical analyses to compare sex determination of embryos by spent medium with PGD/CGH array results. Culture time was demonstrated to increase the DNA amount among day 5 embryos culture medium samples. Non-invasive PGD by means of spent culture medium gave a sensitivity, specificity, positive predictive value and negative predictive value of 100% for sex determination. Results of sex determination using spent medium by q-PCR were consistent with the results of PGD/CGH array. Improvements in cell-free DNA extraction and PCR amplification procedures provide us an effective method to perform a PGD test without biopsy in the future, especially about X-linked diseases.

Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

This study aimed to prospectively evaluate the risk factors of infection by Aelurostrongylus abstrusus in Brazilian cats with cough and/or radiographic changes, using as diagnostic tools the Baermann method (BM), polymerase chain reaction (PCR) of feces, bronchoalveolar lavage fluid (BALF), and cytology. Forty-three cats that were presented with cough or lung radiographic abnormalities compatible with bronchoalveolar disease were included in the study. After clinical evaluation, feces samples were collected to investigate lungworm parasitism through BM and PCR. BALF was performed to provide samples for cytology, bacteriology, and fungal culture. Stool PCR was considered the gold standard for diagnosis tests, and the other methods were evaluated by their agreement. PCR presented 74% (32/43) of positivity for A. abstrusus, while in the BM, 41% (18/43) were positive. BM showed sensitivity of 56.25% and specificity of 100% when compared with PCR. No larva was found in the cytological evaluation of 21 BALF samples. Lungworm is an important cause of bronchopulmonary disease in domestic cats in Brazil and should be included as a differential diagnosis when a cat is presented with cough or radiographic abnormalities. BM is a sensitive, non-invasive, and cheap technique to diagnose the disease, but it is not as sensitive as PCR.

Molecular techniques are an alternative for the diagnosis of strongyloidiasis, produced by Strongyloides stercoralis. However, it is necessary to determine the best amplification target for the populations of this parasite present in a geographical area and standardize a polymerase chain reaction (PCR) protocol for its detection. The objectives of this work were the comparison of different PCR targets for molecular detection of S. stercoralis and the standardization of a PCR protocol for the selected target with the best diagnostic results. DNA extraction was performed from parasite larvae by saline precipitation. Three amplification targets of the genes encoding ribosomal RNA 18S (18S rDNA) and 5.8S (5.8S rDNA) and cytochrome oxidase 1 (COX1) of S. stercoralis were compared, and the PCR reaction conditions for the best target were standardized (concentration of reagents and template DNA, hybridization temperature, and number of cycles). The analytical sensitivity and specificity of the technique were determined. DNA extraction by saline precipitation made it possible to obtain DNA of high purity and integrity. The ideal target was the 5.8S rDNA, since the 18S rDNA yielded non-reproducible results and COX1 never amplified under any condition tested. The optimal conditions for the 5.8S rDNA-PCR were: 1.5 mM MgCl2, 100 μM dNTPs, 0.4 μM primers, and 0.75 U DNA polymerase, using 35 cycles and a hybridization temperature of 60 °C. The analytical sensitivity of the PCR was 1 attogram of DNA, and the specificity was 100%. Consequently, the 5.8S rDNA was shown to be highly sensitive and specific for the detection of S. stercoralis DNA.

Four methods were compared for the diagnosis of human taeniasis caused by Taenia solium. Fecal samples from persons living in a T. solium endemic region of Madagascar were examined for taeniid eggs by the Kato–Katz method. Subsequently, samples positive (n = 16) and negative (n = 200) for T. solium eggs were examined by (i) amplification of the fragment of small subunit of the mitochondrial ribosomal RNA (rrnS) gene using conventional polymerase chain reaction (PCR) and (ii) a nested PCR of a fragment of the T. solium Tso31 gene. Additionally, 12 egg-positive and all egg-negative samples were tested for coproantigen detection. A further 9 egg-positive fecal samples were examined using both PCRs. Of the 12 egg-positive samples tested by PCRs and coproantigen methods, 9 (75%) were positive by rrnS PCR, 3 (25%) using Tso31-nested PCR and 9 (75%) by coproantigen testing. None of the 200 egg-negative fecal samples was positive in either rrnS or Tso31-nested PCR. Twenty of the 25 egg-positive samples (80%) were positive in rrnS PCR, and DNA sequencing of PCR amplicons was obtained from 18 samples, all confirmed to be T. solium. Twelve of the 25 egg-positive samples (48%) were positive in the Tso31-nested PCR, all of which were also positive by rrnS PCR. It is suggested that species-specific diagnosis of T. solium taeniasis may be achieved by either coprological examination to detect eggs or coproantigen testing, followed by rrnS PCR and DNA sequencing to confirm the tapeworm species in egg-positive or coproantigen-positive samples.

The aim of this paper is to describe the prevalence of Mycoplasma genitalium and Trichomonas vaginalis in patients who visited general practitioners in the Netherlands. Additionally, we describe the prevalence of M. genitalium resistance to azithromycin and moxifloxacin. We used data from 7,411 consecutive female patients who were screened for Chlamydia trachomatis, Neisseria gonorrhoeae, M. genitalium, and T. vaginalis and data from 5,732 consecutive male patients screened for C. trachomatis, N. gonorrhoeae, and M. genitalium. The prevalence of M. genitalium and T. vaginalis in female patients was 6.7% (95% CI: 6.2 to 7.4) and 1.9% (95%CI: 1.6 to 2.2%), respectively. M. genitalium prevalence in male patients was 3.7% (3.3 to 4.3). M. genitalium co-occurred with C. trachomatis in 1.4% (0.3 to 0.6%) of female and in 0.7% (0.5 to 0.9) of male patients. Macrolide resistance gene mutations and fluoroquinolone resistance gene mutations were detected in 73.8% and 9.9%, respectively. We concluded that M.genitalium is relatively infrequently found in a large general practitioner population in the Netherlands. It can co-occur with C. trachomatis, and is often resistant to azithromycin. Therefore, when treating sexually transmitted infections, these prevalence and resistance data should be taken into account.

Polycystic ovary syndrome is an endocrine disorder commonly found among females of reproductive age. Different factors have been correlated with this syndrome, although the aetiology of the disease is still unrecognized with both environmental and hereditary factors leading to the progression. Hormonal effects of the AKT pathway have made it an interesting study unit for PCOS cases. The aim of this study was to investigate the expression patterns of genes involved in the AKT pathway, including IRS1, IRS2, AKT1 and AKT2. In total, 13 human oocytes were collected for this study at the meiosis II stage, in which seven of them were collected from individuals with polycystic ovaries and the rest formed the control group of individuals with no signs of polycystic ovaries. RNA was extracted from oocytes and then the RNA was converted into cDNA for the real-time PCR process. Expression levels of four genes in the AKT pathway, in addition to housekeeping gene (ACTB), were evaluated. Expression levels of each gene were quantified using real-time PCR and statistical analysis was performed. The results of this study showed that there was no significant correlation between the expression of genes in oocyte samples obtained from patients with polycystic ovaries and the control group. This study is the first to evaluate the expression levels of genes involved in the AKT pathway in human oocyte samples. Therefore, it provides crucial information to form the basis of further studies.

In this research communication we describe a straightforward triplex-PCR protocol able to differentiate the origin of milk from three closely related species (goat, sheep and cow) in Halloumi, a cheese with Protected Designation of Origin (PDO), and yogurts. Halloumi must contain at least 51% sheep or goat milk, therefore, the fraudulent adulteration of this cheese with excess of cow milk must be routinely tested. The assay employs one universal forward primer and three species-specific reverse primers giving rise to 287 bp (cow), 313 bp (goat), and 336 bp (sheep) amplicons, under the same amplification conditions. This protocol, when used to test a small number of Cyprus commercial products, correctly detected mislabeling in Halloumi (2 out of 6 samples were adulterated) and yogurt brands (1 out of 4 was adulterated). The suggested protocol is a reliable tool for identifying the origin of milk in Halloumi cheeses and yogurts and can be used in any laboratory equipped with a thermocycler and an agarose gel electrophoresis apparatus.

Embryo quality determines the success of in vitro fertilization and embryo transfer (IVF-ET) treatment. Biomarkers for the evaluation of embryo quality have some limitations. Apoptosis in cumulus cells (CCs) is important for ovarian function. PTEN (phosphatase and tensin homolog) is a well known tumour suppressor gene that functions as a mediator of apoptosis and is crucial for mammalian reproduction. In the present study, we analyzed the expression level of PTEN in human CCs and aimed to investigate its association with embryo developmental competence in IVF treatment cycles. The PTEN mRNA level in CCs was measured using real-time fluorescence quantitative PCR. The association of the differential expression of PTEN with embryo quality was analyzed. Our data showed that PTEN mRNA levels were significantly decreased in CCs surrounding mature oocytes compared with immature oocytes. Similar changes were found in the analysis of fertilization and blastocyst formation. The speculation that the measurement of PTEN mRNA levels in human CCs would provide a useful tool for selecting oocytes with greater chances to implant into the uterus needs to be further verified through single-embryo transfer in the future. The proapoptotic mechanism of PTEN in human reproduction needs to be further studied.