Puparia are commonly found in tsetse fly larviposition sites during studies on larval ecology. This chitinous shell is representative of past or ongoing exploitation of these sites by tsetse flies. The morphological characteristics of the puparium are not sufficiently distinctive to allow identification of the species. This study explores the applicability of biomolecular techniques on empty puparia for tsetse fly species identification. Five techniques were compared for DNA extraction from tsetse fly puparia, 1/Chelex® 100 Resin, 2/CTAB, 3/Livak's protocol, 4/DEB + proteinase K and 5/QIAamp® DNA Mini kit, using two homogenisation methods (manual and automated). Using a combination of two primer pairs, Chelex, CTAB, and DEB + K proved the most efficient on fresh puparia with 90, 85, and 70% samples identified, respectively. Shifting from fresh to one- to nine-month-old puparia, the Chelex method gave the best result allowing species identification on puparia up to seven months old. The subsequent testing of the Chelex extraction protocol identified 152 (60%) of 252 field-collected puparia samples at species level. The results show that reliable genetic identification of tsetse flies species can be performed from empty puparia, what can prove of great interest for future ecological studies on larviposition sites. The Chelex technique was the most efficient for DNA extraction, though the age-limit of the samples stood at seven months, beyond which DNA degradation probably compromises the genetic analysis.

Fasciolosis, caused by Fasciola hepatica and F. gigantica, is an impediment to the livestock industry’s expansion and has a massively negative socio-economic impact due to its widespread prevalence in livestock. It is a waterborne zoonosis affecting human populations in the countries where rural economies are associated with livestock rearing. Conventional diagnosis of Fasciola infection is done by detecting parasite eggs in the faeces of infected animals or by immunological methods. Accurate and quick immunodiagnosis of Fasciola infection in animals and humans is based on the detection of antibodies and specific antigens expressed in the prepatent stage of the parasite. Both molecular and serodiagnostic tests developed thus far have enhanced the reliability of Fasciola diagnosis in both man and animals but are not widely available in resource-poor nations. A pen-side diagnostic test based on a lateral flow assay or a DNA test like loop-mediated isothermal amplification (LAMP) would be simple, fast, and cost-effective, enabling clinicians to treat animals in a targeted manner and avoid the development of drug resistance to the limited flukicides. This review focuses on the recent advances made in the diagnosis of this parasite infection in animals and humans.

An adult Indian buffalo (Bubalus bubalis) presented with corneal opacity, irritation, and excessive lacrimation from the left eye in the Referral Veterinary Polyclinic-Teaching Veterinary Clinical Complex (RVC-TVCC), Indian Veterinary Research Institute, Izatnagar. Clinical examination revealed a whitish thread-like worm in the left eye’s anterior chamber. The worm was surgically removed from the eye with supportive nerve blocks. Light microscopy was used for parasite morphological identification, which provided insight into the worm as female Setaria sp. Genomic DNA was isolated, and polymerase chain reaction amplification of 12S rRNA was conducted for molecular confirmation of the parasite. The amplicon was sequenced and analysed by bioinformatics software. Sequence data showed an amplicon size of 243 bp. Phylogenetic analysis with reference data from the NCBI Genbank database revealed the worm was S. digitata, with a similarity of 99.17%. The common predilection site of S. digitata is in the peritoneal cavity of natural hosts like cattle and buffalo and is mostly non-pathogenic. The aberrant migration of the parasite larva to the brain and eye commonly occurs in goats, sheep, and horses, causing clinical conditions like cerebrospinal nematodiasis (lumbar paralysis) and ocular setariasis, respectively. Nevertheless, until now, there have been no reports of ocular setariasis in buffalo. This report is the first unusual occurrence of ocular setariasis in buffalo and its molecular confirmation and phylogenetic analysis using 12S rRNA.

The understanding of the genetic architecture of psychiatric disorders has made significant advances in the last decade and some scientific findings can now be translated into clinical practice. The rise of genetic testing and the awareness of patients and their families motivate psychiatrists to examine this approach. The COST Action EnGagE (CA17130) is promoting these developments in Europe. Whereas the findings of common variants are the domain of research, screening for rare variants at the genome-wide level is already applicable in clinical practice. It is now possible to return meaningful results to the individual to help him/her understanding the disease and the comorbidities, to guide treatment and to perform genetic counseling. In this presentation, we will give meaningful examples for psychiatric practice. For instance, around one-third of the patients diagnosed with autism spectrum disorder can benefit from a molecular diagnosis (fragile X syndrome, SHANK3 deletion…). Microdeletion or microduplication may explain a fraction of schizophrenic cases (e.g. del22q11). Identification of rare variants causing the disease may decrease the stigma and feeling of guilt often reported by patients and families. This could also help to detect and manage other comorbidities. It is expected that treatment guidelines and clinical trials would be developed in the near future for patients carrying a rare variant, opening the way to personalized psychiatry. Finally, this effort has a huge impact on the family, by enhancing genetic counseling in psychiatry. The rise of psychiatric genetics might align our field more closely with the other medical specialties.

The parasitic species of the Echinococcus granulosus sensu lato (sl) complex are the causative agents of cystic echinococcosis in humans. The lifecycle of E. granulosus sl is essentially domestic, and is based on the consumption by dogs of hydatid cysts in viscera of livestock species. The aim of this study was to survey E. granulosus sensu lato in livestock in France. A 1-year national survey of E. granulosus sl in livestock at the slaughterhouse was organized in 2012 in France, with systematic molecular confirmation. The prevalence of E. granulosus ss nationally was 0.002% in sheep, mainly focused in the Alpine area, and 0.001% in cattle, with the distribution of cases throughout the country. Echinococcus canadensis G6/7 was observed only in Corsica in pigs, with a prevalence of nearly 1% in the island. A national prevalence of 0.0002% was estimated for E. ortleppi in cattle, due to seven cases distributed in two foci. The results of this survey are of particular interest because of the zoonotic risk associated with the presence of these parasite species, for which systematic control at the slaughterhouse should enable their elimination.

Visceral leishmaniasis (VL) was firstly reported in Armenia in 1913. Following a considerable increase of the number of cases until the mid 1950s, the disease disappeared after 1969 and re-emerged in 1999. Scientific literature about VL in Armenia is available only in Russian or Armenian. This paper presents a historical overview about leishmaniasis in Armenia based on this literature as well as an epidemiological update since the re-emergence of the disease. In 1999–2016, 116 indigenous VL cases were recorded mainly in children in 8 of the 11 districts, however, VL is underreported because of lack of trained medical personal and diagnostic facilities. The aim of this work was to apply for the first time molecular diagnosis of VL in Armenia. Out of 25 VL suspected patients, 22 were positive by microscopy and polymerase chain reaction (PCR). Genotyping using internal transcribed spacer 1-PCR-restriction fragment length polymorphism and sequencing identified the causative agent of VL in Armenia as Leishmania infantum. The present work is an important step towards the inclusion of molecular techniques in the current diagnosis of VL in Armenia and the establishment of local molecular diagnostic facilities.

Persistent infection with high-risk human papillomavirus (HPV) is the main cause of cervical cancer and the prevalence of HPV types varies depending on the geographic region. Therefore, this study assessed the prevalence of HPV types in women with cervical lesions from Sergipe state, Northeastern Brazil. A cross-sectional study was conducted in women with cervical lesions from March to December 2014. These lesions were investigated by PCR and HPV types were identified by DNA sequencing. 432 patients were included, of which 337 patients tested positive for HPV. Eighteen different HPV types were detected, and high-risk HPV types were detected in 69.2%. HPV 16 (63.4%) was the most prevalent HPV type found, followed by HPV 66 (4.6%), HPV 18 (1.6%) and HPV 45 (1.4%). These results highlight the importance of the high prevalence of HPV 66, which is a possibly carcinogenic virus type not covered by the available vaccines. The prevalence of HPV 16 was high in the studied population, reaffirming the importance of young vaccination. However, the high prevalence of HPV 66 found in this study shows the importance of monitoring the diversity of HPV types in different populations and geographic regions to better understand the impacts of current HPV vaccines.

There is limited evidence of the prevalence of enteric protozoon infections in developed settings. We estimated the prevalence of enteric protozoa and evaluated the outcome of testing algorithms used in hospital settings in Sydney, Australia. This retrospective study assessed microbiological data from four public clinical laboratories. Pooled data from the four hospitals revealed the most common enteric protozoon detected was Blastocystis spp. in an average of 5·4% of cases, followed by Giardia intestinalis (1·1%) and Dientamoeba fragilis (0·8%). Protozoon detection rates between hospitals were significantly different and could be based on multiple factors. The modified iron haematoxylin staining method, consistently detected higher rates of Blastocystis spp., and G. intestinalis in comparison with microscopy of wet preparations, as well as higher rates of G. intestinalis and Cryptosporidium when compared with enzyme immunoassay. The study concludes that there are multiple factors that contribute to the variability in protozoa detection rates in metropolitan hospitals, including widespread variability in the testing protocols for enteric protozoa, individual and population characteristics. A gold standard approach for diagnosis of enteric protozoa is recommended. Molecular diagnostic methods such as polymerase chain reaction would provide consistency across laboratories and yield more reliable estimates of the actual prevalence of enteric protozoa.

The current mainstay for control of the four major helminth diseases in humans (lymphatic filariasis, onchocerciasis, soil-transmitted helminthiases and schistosomiasis) is with preventive chemotherapy by mass administration of key anthelminthics. Following the London Declaration on Neglected Tropical Diseases in 2012, a roadmap for the elimination and control of these helminthiases by 2020 has been devised. With expected declines in prevalence and intensity of these infections, there is urgent need for implementing more sensitive, high-throughput and cost-effective diagnostic tools. Currently available diagnostic approaches for surveying, monitoring and evaluating helminth control programmes are based on microscopical observation of eggs/larvae, and/or detection of antibodies or parasite antigens in stool, urine or blood; all relatively low-throughput and of limited sensitivity and specificity. Newly proposed approaches for helminthiases diagnosis include the nucleic acid-based methods of (multiplex) real-time polymerase chain reaction assays, loop-mediated isothermal amplification and recombinase polymerase amplification. However, as well as sensitivity/specificity evaluation, their comparison to current ‘gold standard’ diagnostics and future application in individual-/community-based diagnosis, or in xenomonitoring requires consideration of relative costs, agreement of standard methods and strategic interpretation of resulting data before control/elimination programmes might best utilize molecular diagnostics to inform decision making. We review current nucleic-acid-based molecular diagnostic methods and highlight the needs and future research required to refine these tools for monitoring and evaluation of control and elimination programmes for four major human helminthiases.

Flat grain beetles of the genus Cryptolestes (Coleoptera: Laemophloeidae) are one of the economically most important stored-product pests which feed on many kinds of agricultural products, especially grains. Nine of more than 40 described Cryptolestes species are recognized as stored-product pests and two of the pest species have a cosmopolitan distribution. Given the rapid growth in global trade of food products, ecological barriers to the spread of pests are easily overcome. Therefore, development of reliable systems for routine quarantine inspection and early infestation detection is vital. In the present study, we established a new rapid and accurate cytochrome c oxidase subunit I-based system for molecular identification of five common stored-product Cryptolestes species, namely, Cryptolestes capensis, Cryptolestes ferrugineus, Cryptolestes pusilloides, Cryptolestes pusillus and Cryptolestes turcicus. Five species-specific primer pairs for traditional uniplex polymerase chain reaction assay are described and their specificity and sensitivity for the identification process is evaluated using larval samples of 12 different populations from three continents (Asia, Europe and North America).

Variation in clinical accuracy of molecular diagnostic methods for cutaneous leishmaniasis (CL) is commonly observed depending on the sample source, the method of DNA recovery and the molecular test. Few attempts have been made to compare these variables. Two swab and aspirate samples from lesions of patients with suspected CL (n = 105) were evaluated alongside standard diagnosis by microscopic detection of amastigotes or culture of parasites from lesion material. Three DNA extraction methods were compared: Qiagen on swab and aspirate specimens, Isohelix on swabs and Boil/Spin of lesion aspirates. Recovery of Leishmania DNA was evaluated for each sample type by real-time polymerase chain reaction detection of parasitic 18S rDNA, and the diagnostic accuracy of the molecular method determined. Swab sampling combined with Qiagen DNA extraction was the most efficient recovery method for Leishmania DNA, and was the most sensitive (98%; 95% CI: 91–100%) and specific (84%; 95% CI: 64–95%) approach. Aspirated material was less sensitive at 80% (95% CI: 70–88%) and 61% (95% CI: 50–72%) when coupled to Qiagen or Boil-Spin DNA extraction, respectively. Swab sampling of lesions was painless, simple to perform and coupled with standardized DNA extraction enhances the feasibility of molecular diagnosis of CL.

Strongyloidiasis is frequently asymptomatic and diagnosis of latent infection is difficult due to limitations of current parasitological and serological methods. This study aimed to verify the use of conventional polymerase chain reaction (PCR) assay for molecular diagnosis of Strongyloides stercoralis infection. Fresh stool samples were obtained from 103 individuals: 33 S. stercoralis positive, 30 positive for other parasites and 40 negative for parasitological methods. These samples were examined by the Lutz, Rugai and agar plate culture methods and conventional PCR assay. Two sets of primers (S. stercoralis species-specific and genus-specific sets), located in the 18S ribosomal RNA gene, were used for PCR. Of the 33 samples positive for S. stercoralis by parasitological methods, 28 (84·8%) were also detected by PCR assay using species-specific primers and 26 (78·8%) using genus-specific primers. Among the stool samples negative by parasitological methods, seven (17·5%) were positive by PCR using species-specific primers and two (5·0%) using genus-specific primers. In conclusion, the conventional PCR assay described in this study using a species-specific primer pair provided a molecular method for S. stercoralis diagnosis in human stool samples.

The guava fruit fly, Bactrocera correcta (Bezzi) (Diptera: Tephritidae), is an invasive pest of fruit and vegetable crops that primarily inhabits Southeast Asia and which has the potential to become a major threat within both the Oriental and Australian oceanic regions as well as California and Florida. In light of the threat posed, it is important to develop a rapid, accurate and reliable method to identify B. correcta in quarantine work in order to provide an early warning to prevent its widespread invasion. In the present study, we describe a species-specific polymerase chain reaction assay for the diagnosis of B. correcta using mitochondrial DNA cytochrome oxidase I (mtDNA COI) barcoding genes. A B. correcta-specific primer pair was designed according to variations in the mtDNA COI barcode sequences among 14 fruit fly species. The specificity and sensitivity of the B. correcta-specific primer pair was tested based on the presence or absence of a band in the gel profile. A pair of species-specific B. correcta primers was successfully designed and named BCOR-F/BCOR-R. An ∼280 bp fragment was amplified from specimens belonging to 17 geographical populations and four life stages of B. correcta, while no such diagnostic bands were present in any of the 14 other related fruit fly species examined. Sensitivity test results demonstrated that successful amplification can be obtained with as little as 1 ng μl−1 of template DNA. The species-specific PCR analysis was able to successfully diagnose B. correcta, even in immature life stages, and from adult body parts. This method proved to be a robust single-step molecular technique for the diagnosis of B. correcta with respect to potential plant quarantine.

Tall stature is a common feature of both Marfan syndrome and XYY syndrome. Differential diagnosis between these entities has important prognostic implications. We report the case of a 21-year-old young man with a previously known diagnosis of XYY syndrome, in whom the identification of a fibrilin-1 mutation was determinant to establish an appropriate diagnosis, medical follow-up, and genetic counselling.

Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in young chickens. Two serotypes of this double-stranded RNA virus exist but only serotype 1 viruses cause disease in chickens. Detection and strain identification of IBDV is important because antigenic subtypes found within serotype 1 make it necessary to tailor vaccination programs to the antigenic type found in the bird's environment. Because conventional virus isolation and characterization are not practical for routine diagnosis of IBDV, antigen-capture enzyme-linked immunosorbent assay (ELISA) and molecular assays based on reverse transcription–polymerase chain reaction (RT-PCR) technology were developed. Compared with antigen-capture ELISA, RT-PCR assays have greater versatility and are more sensitive and specific. Strain identification has been accomplished using a variety of post-RT-PCR assays, including restriction enzyme digestion of the RT-PCR products. The resulting restriction fragment length polymorphisms (RFLP) are used to differentiate viruses into molecular groups that correlate with antigenic and pathogenic types. Recently, two types of real-time RT-PCR have been used to identify and differentiate strains of IBDV. Both methods use distance-dependent interaction between two dye molecules, known as fluorescence resonance energy transfer (FRET). The dye molecules are attached to one or more nucleotide probes that detect specific nucleotide sequences of the virus. Our laboratory has used a two-probe assay to identify single-nucleotide mutations among IBDV strains. A mutation probe is used in this assay to detect substitution mutations in a region of the viral genome that encodes a neutralizing epitope of the virus. These assays are accurate, reliable and inexpensive compared with conventional RT-PCR because they do not require RFLP or other labor-intensive post-RT-PCR assays to distinguish viral strains.

Marteilia refringens is a paramyxean parasite which infects the flat oyster Ostrea edulis and mussels (Mytilus galloprovincialis), where it has been attributed to a separate species, Marteilia maurini, by several authors. Doubts persist though as to the existence or not of two species of Marteilia in Europe. We have devised a molecular method for the diagnosis of M. refringens based on 358 bp nested-PCR of the rDNA intergene spacer (rDNA IGS) which is capable of detecting 0·5 fg of M. refringens DNA. Molecular characterization of this spacer indicates that the Marteilia parasites which infect oysters and mussels are two different strains of the same species.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae that mainly affects the skin and peripheral nerves. Over recent years, many important advances have been made in developing molecular diagnostics, in identifying highly effective drugs and designing multidrug regimens for treatment, and in unravelling the genomic structure and functions of the leprosy bacillus. Using the new information about specific sequences of M. leprae, several gene probes and gene amplification systems for confirming diagnosis and monitoring treatment have been developed. Among these, polymerase chain reaction (PCR)-based methods have been useful in confirming the diagnosis in paucibacillary leprosy (where few bacilli are present). RNA-targeting systems for monitoring the progress of treatment, in situ hybridisation techniques for analysing specimens with nonspecific histological features, and molecular methods for direct detection of rifampicin/dapsone resistance are other major technological advances with immense applied value. Several effective regimens for the treatment of leprosy have been developed, which include rifampicin, clofazimine and dapsone as core drugs. Although these regimens are generally satisfactory, limitations in terms of persisting activity and late reactions/relapses in paucibacillary leprosy, and persistence of dead and/or live organisms in multibacillary forms of the disease, have been observed.

Infection with Entamoeba histolytica, the protozoan parasite that causes amoebic colitis and liver abscess, results in 34 million to 50 million symptomatic cases of amoebiasis (all illnesses caused by E. histolytica, including amoebic dysentery) worldwide each year, causing 40 thousand to 100 thousand deaths annually. As a result of accruing biochemical, genetic and immunological data, E. histolytica was re-defined in 1993 to recognise the existence of two morphologically identical but genetically distinct human parasites: E. histolytica, the aetiological agent of invasive intestinal and extraintestinal amoebiasis, and Entamoeba dispar, a non-pathogenic intestinal parasite. Because microscopy is unable to distinguish between these two organisms, it should no longer be relied upon to diagnose amoebiasis. Sensitive and specific molecular techniques that are able to distinguish E. histolytica from E. dispar have been developed recently; they include (1) the detection of an E. histolytica antigen using an enzyme-linked immunosorbent assay (ELISA), (2) the use of the polymerase chain reaction (PCR) to amplify amoebic DNA, and (3) the culture of stool samples followed by isoenzyme analysis. Of these three test methods, only antigen detection using ELISA can be performed rapidly and easily, making it the diagnostic test method of choice for clinical use in the developing world, where the morbidity and mortality caused by E. histolytica are greatest. However, the PCR method is a powerful tool for the genetic typing of different amoebic strains. Together these two methods should result in both improved clinical diagnosis and treatment of amoebiasis, and a greater understanding of the epidemiology of E. histolytica. Such knowledge will not only assist public health efforts to control amoebiasis, but also facilitate the careful testing of the anti-amoebic vaccines that are currently being developed.

This study reports the development of a novel multiplex PCR assay based on SCAR (Sequence-Characterised Amplified Region) markers for the simultaneous diagnosis of the 7 Eimeria species that infect domestic fowl. Primer pairs specific for each species were designed in order to generate a ladder of amplification products ranging from 200 to 811 bp. Sensitivity tests for each species were carried out, showing a detection threshold of 1–5 pg, which corresponds approximately to 2–8 sporulated oocysts. Distinct isolates of the 7 Eimeria species from different geographical sources were tested and successfully detected by the assay. All the species were amplified homogeneously, whether or not one of them was present in a high quantity, indicating that there was no cross-interference. The assay was also tested with different sources of Taq DNA polymerase and thermocycler models, confirming the high reproducibility of the reaction. The economy of consumables and labour represented by a single-tube reaction greatly facilitates the molecular diagnosis of a large number of samples, making it appropriate for field epizootiological surveys. We propose the use of this multiplex PCR assay as a rapid and cost-effective diagnostic method for the detection and discrimination of the 7 Eimeria species that infect domestic fowl.

Due to their widespread distribution and virulence, protozoan species of the genus Perkinsus are especially worrisome parasites for shellfish farmers. In the present paper, we investigate the organization and the structural features of the nuclear ribosomal genes of Perkinsus atlanticus as well as the use of DNA sequence information from this region for phylogenetic analyses. This information has been useful, further, for the development of a diagnostic test based on the amplification by the polymerase chain reaction (PCR) technique. We have isolated a high-copy DNA sequence in this species, and, after its characterization, we have determined that it corresponds to the ribosomal RNA (rRNA) genes 28S–5S–18S and the intergenic spacers. By comparing the complete sequence of the 5S rRNA gene and a partial sequence of the 18S rRNA gene of P. atlanticus with the sequences of those genes in other Alveolates, we have found additional support for the hypothesis that Perkinsus is more closely related to species of Dinoflagellata than to species of Apicomplexa. The intergenic spacer sequence between the 5S and the 18S rRNA genes was used to design a pair of primers to be used as a PCR-based diagnostic test.The nucleotide sequence data reported in this paper are available in the EMBL database with the accession number AJ238400.The commercial use of the diagnostic test presented here is protected by a patent. This patent is pending upon the application number P9801511.