Blood Polymerase Chain Reaction Research Articles

Polymerase chain reaction blood tests for the diagnosis of invasive aspergillosis in immunocompromised people

This is an update of the original review published in the Cochrane Database of Systematic Reviews Issue 10, 2015.Invasive aspergillosis (IA) is the most common life-threatening opportunistic invasive mould infection in immunocompromised people. Early diagnosis of IA and prompt administration of appropriate antifungal treatment are critical to the survival of people with IA. Antifungal drugs can be given as prophylaxis or empirical therapy, instigated on the basis of a diagnostic strategy (the pre-emptive approach) or for treating established disease. Consequently, there is an urgent need for research into both new diagnostic tools and drug treatment strategies. Increasingly, newer methods such as polymerase chain reaction (PCR) to detect fungal nucleic acids are being investigated. To provide an overall summary of the diagnostic accuracy of PCR-based tests on blood specimens for the diagnosis of IA in immunocompromised people. We searched MEDLINE (1946 to June 2015) and Embase (1980 to June 2015). We also searched LILACS, DARE, Health Technology Assessment, Web of Science and Scopus to June 2015. We checked the reference lists of all the studies identified by the above methods and contacted relevant authors and researchers in the field. For this review update we updated electronic searches of the Cochrane Central Register of Controlled Trials (CENTRAL; 2018, Issue 3) in the Cochrane Library; MEDLINE via Ovid (June 2015 to March week 2 2018); and Embase via Ovid (June 2015 to 2018 week 12). We included studies that: i) compared the results of blood PCR tests with the reference standard published by the European Organisation for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG); ii) reported data on false-positive, true-positive, false-negative and true-negative results of the diagnostic tests under investigation separately; and iii) evaluated the test(s) prospectively in cohorts of people from a relevant clinical population, defined as a group of individuals at high risk for invasive aspergillosis. Case-control and retrospective studies were excluded from the analysis. Authors independently assessed quality and extracted data. For PCR assays, we evaluated the requirement for either one or two consecutive samples to be positive for diagnostic accuracy. We investigated heterogeneity by subgroup analyses. We plotted estimates of sensitivity and specificity from each study in receiver operating characteristics (ROC) space and constructed forest plots for visual examination of variation in test accuracy. We performed meta-analyses using the bivariate model to produce summary estimates of sensitivity and specificity. We included 29 primary studies (18 from the original review and 11 from this update), corresponding to 34 data sets, published between 2000 and 2018 in the meta-analyses, with a mean prevalence of proven or probable IA of 16.3 (median prevalence 11.1% , range 2.5% to 57.1%). Most patients had received chemotherapy for haematological malignancy or had undergone hematopoietic stem cell transplantation. Several PCR techniques were used among the included studies. The sensitivity and specificity of PCR for the diagnosis of IA varied according to the interpretative criteria used to define a test as positive. The summary estimates of sensitivity and specificity were 79.2% (95% confidence interval (CI) 71.0 to 85.5) and 79.6% (95% CI 69.9 to 86.6) for a single positive test result, and 59.6% (95% CI 40.7 to 76.0) and 95.1% (95% CI 87.0 to 98.2) for two consecutive positive test results. PCR shows moderate diagnostic accuracy when used as screening tests for IA in high-risk patient groups. Importantly the sensitivity of the test confers a high negative predictive value (NPV) such that a negative test allows the diagnosis to be excluded. Consecutive positives show good specificity in diagnosis of IA and could be used to trigger radiological and other investigations or for pre-emptive therapy in the absence of specific radiological signs when the clinical suspicion of infection is high. When a single PCR positive test is used as the diagnostic criterion for IA in a population of 100 people with a disease prevalence of 16.3% (overall mean prevalence), three people with IA would be missed (sensitivity 79.2%, 20.8% false negatives), and 17 people would be unnecessarily treated or referred for further tests (specificity of 79.6%, 21.4% false positives). If we use the two positive test requirement in a population with the same disease prevalence, it would mean that nine IA people would be missed (sensitivity 59.6%, 40.4% false negatives) and four people would be unnecessarily treated or referred for further tests (specificity of 95.1%, 4.9% false positives). Like galactomannan, PCR has good NPV for excluding disease, but the low prevalence of disease limits the ability to rule in a diagnosis. As these biomarkers detect different markers of disease, combining them is likely to prove more useful.

Molecular diagnosis of acute and chronic infection of Trypanosoma evansi in experimental male and female mice.

Trypanosoma evansi is enzootic in camels in Egypt, and water buffaloes act as a reservoir for camel infection. Molecular techniques have contributed towards understanding the epidemiology of T. evansi. Trypanosoma evansi was detected in acute and chronic stages of the disease in male and female mice by polymerase chain reaction (PCR) using two primers. Two experiments were conducted. In experiment I, two groups consisting of 26 female and 26 male mice received 104 trypanosome by I/P inoculation for each mouse. In experiment II, 42 female and 42 male mice were inoculated I/P with 102 trypanosome/mouse. In addition, five mice were kept as uninfected control for each group. Mice were monitored daily for parasitaemia level during the pre-patent period using the micro-haematocrit centrifugation technique (MHCT) and conventional PCR. The primer pairs, (Trypanosoma brucei) TBR1/2 and TeRoTat1.2 (T. evansi Rode Trypanozoon antigen type [RoTat] 1.2), detected the infection after 24 hours earlier than MHCT in both experiments. The course of infection that was detected by MHCT revealed three waves of parasitaemia in female mice and two waves in male mice in the chronic stage of infection. In addition, PCR was able to detect T. evansi in different organs in the chronic stage (i.e. disappearance of parasite from blood). Application of the two primer sets on blood samples from camels showed that all samples were positive by TBR1/2 primers and only 32 of 44 were positive by TeRoTat1.2 primers. Acutely and chronically Trypanosoma-infected mice were detected by PCR in blood and organs. TBR1/2 primers were more sensitive than TeRoTat1.2 primers in detecting Trypanosoma-infected mice, and more reliable in detecting field-infected camels and excluding carrier animals.

Rapid diagnosis of neonatal sepsis by PCR for detection of 16S rRNA gene, while blood culture and PCR results were similar in E.coli-predominant EOS cases.

PurposeTo determine the bacteriological pattern and antibiotic susceptibility of bacterial isolates causing neonatal sepsis in Qena University Hospitals and compare polymerase chain reaction (PCR) and blood culture results in a trial for rapid diagnosis.Patients and methodsBlood samples from 75 clinically suspected cases of neonatal sepsis were subjected to identification of bacteria and determination of their antibiotic sensitivity through blood culture, and rapid detection of 16S rRNA and the uidA gene (to confirm the presence of E. coli) by PCR from extracted bacterial DNA.ResultsMost patients were preterm (64%) and low birth weight (LBW) (68%). In total, 42.7% presented with early onset sepsis (EOS). LBW was significantly associated with EOS (P-value=0.03). Although the blood culture and PCR results were similar in EOS, the PCR results were significantly higher than those of blood culture in detecting bacteria (85.3% vs 68%, respectively, P-value=0.001). Blood culture showed 100% specificity. The most common pathogen was E. coli (86.2%) in EOS and Staphylococcus spp. (45.5%) in late-onset sepsis (LOS) (P-value=0.001 and 0.02, respectively). The most effective antibiotics against Gram-negative bacteria were ofloxacin, ciprofloxacin, imipenem, and amikacin, while vancomycin, oxacillin, and imipenem were the most effective antibiotics against Gram-positive bacteria.ConclusionEOS was mainly caused by E. coli, while LOS was mainly caused by Staphylococcus spp. The 16S rRNA PCR showed higher sensitivity with rapid and accurate diagnosis. Blood culture is the most suitable method for antimicrobial sensitivity testing.

Visceral leishmaniasis

Visceral leishmaniasis is aworldwide distributed infectious zoonotic disease caused by protozoan pathogens of the genus Leishmania which are transmitted by sandflies. The main hosts are dogs. The prevalence in Germany is low. Predominantly affected are migrants and travelers returning from Mediterranean countries. The main clinical symptoms are fever, hepatosplenomegaly and pancytopenia. The diagnosis is made by polymerase chain reaction of peripheral blood or direct detection of Leishmania in bone marrow aspirates. Lesions of the liver and spleen can easily be misinterpreted as numerous benign or malignant differential diagnoses. Treatment is always systemic with antiparasitic drugs. Immunosuppressed patients with HIV co-infection or after solid organ transplantation are prone to infection as well as atypical and severe courses.

Meningitis and epididymitis caused by Toscana virus infection imported to Switzerland diagnosed by metagenomic sequencing: a case report

BackgroundWe report a rare case of Toscana virus infection imported into Switzerland in a 23-year old man who travelled to Imperia (Italy) 10 days before onset of symptoms. Symptoms included both meningitis and as well epididymitis. This is only the fourth case of Toscana virus reported in Switzerland.Case presentationThe patient presented with lymphocytic meningitis and scrotal pain due to epididymitis. Meningitis was initially treated with ceftriaxone. Herpes simplex, tick-borne encephalitis, enterovirus, measles, mumps, rubella and Treponema pallidum were excluded with specific polymerase chain reaction (PCR) or serology. In support of routine diagnostic PCR and serology assays, unbiased viral metagenomic sequencing was performed of cerebrospinal fluid and serum. Toscana virus infection was identified in cerebrospinal fluid and the full coding sequence could be obtained. Specific PCR in cerebrospinal fluid and blood and serology with Immunoglobulin (Ig) M and IgG against Toscana virus confirmed our diagnosis. Neurological symptoms recovered spontaneously after 5 days.ConclusionsThis case of Toscana virus infection highlights the benefits of unbiased metagenomic sequencing to support routine diagnostics in rare or unexpected viral infections. With increasing travel histories of patients, physicians should be aware of imported Toscana virus as the agent for viral meningitis and meningoencephalitis.

P84-T Neuromyositis associated with cytomegalovirus infection in an immunocompetent patient with antineutrophil cytoplasmic antibodies-associated vasculitis

The role of CMV in ANCA-positive vasculitis is controversial. A 40-year-old man, with an unremarkable medical history, presented with progressive muscle weakness of all four limbs, accompanied by diffuse myalgia, arthralgia, and a 10 kg weight loss 2 months before admission to the Neurology Department of our hospital. Clinical examination revealed a flaccid tetraparesis with absent reflexes on the lower limbs and marked muscle atrophy. Electrophysiological investigations were consistent with a demyelinating motor polyradiculoneuropathy. Laboratory data showed AST = 1137 IU/L (normal < 37 IU/L), ALT = 582 IU/L (normal < 41 IU/L), C-reactive protein (CRP) = 19 mg/L (normal < 8 mg/L), CPK = 13779 IU/L (normal < 397). and LDH = 911 IU/L (normal < 192). CMV ELISA (serum) was strongly positive and CMV DNA by polymerase chain reaction (PCR) in blood and CSF also was positive. Immunologic screening revealed positive anti-myeloperoxidase antibodies anti-MPO = 56,9 IU/mL (normal < 9 IU/mL; ELISA). The inflammatory process involving the vessel walls and muscle fibers was histologically found in biopsy specimen of the muscle, with positive staining for CMV. The patient was treated initially with corticosteroids and intravenous immunoglobulins, but these therapies had very poor effect. Immunosuppressive therapy was instituted, resulting in a progressive partial recovery of all four limb weakness without relapses. We hypothesize that the CMV infection triggered an ANCA-positive vasculitis and its particular severe neurological involvement. An awareness of the association between CMV infection and vasculitis may have important therapeutic consequences.

Direct PCR-RFLP based detection and differentiation of Anaplasma species in naturally infected goats of eastern Haryana, India

The present investigation was designed to develop a novel, rapid and cost effective direct PCR-RFLP assay, as a specific diagnostic tool for detection and differentiation of two Anaplasma species (A. ovis and A. marginale). Blood samples were collected randomly from 83 goats. A direct blood polymerase chain reaction (DT-PCR) for amplifying a fragment of the major surface protein 5 (msp5) gene of A. ovis/A. marginale from whole blood was developed and standardized. Blood smear examination revealed 24 samples (28.91%) positive for Anaplasma inclusion bodies. While, 39 (47%) samples were positive by DT-PCR. The results revealed that DT- PCR was 100% sensitive and 74.57% specific compared to microscopy based detection (k =0.62). Additionally DT-PCR showed 94.44% sensitivity and 100% specificity compared to conventional PCR results with suspected blood samples (k=0.94). All DT-PCR positive samples were confirmed to be A. ovis by restriction fragments length polymorphism (RFLP) analysis. The phylogenetic tree and sequence analysis revealed msp5 gene of Anaplasma species Indian isolate had maximum distance from A. phagocytophilum followed by A. centrale and A. marginale and 100% sequence identity with A. ovis isolates of Chinese origin which further confirmed the sequence identified in native goats to be of A. ovis. The simplified DT-PCR assay as a viable alternative to conventional PCR could be helpful for fast and accurate diagnosis of Anaplasma species and suitable for screening a large number of samples. Furthermore, results revealed that DT-PCR-RFLP of the msp5 gene might be a useful method for simultaneous detection and differentiation of A. ovis and A. marginale in goats.

The yield of monitoring adenovirus in pediatric hematopoietic stem cell transplant patients

Human adenovirus (HAdV) is recognized as a serious pathogen after allogeneic hematopoietic stem cell transplantation (HSCT), causing morbidity and mortality. Currently, there is no universal agreement regarding routine HAdV surveillance after HSCT. We assessed the impact of HAdV weekly monitoring by polymerase chain reaction (PCR) on HAdV viremia rates and the risk factors that influence survival. Three-hundred and fifty-six pediatric allogeneic HSCT were done between 2007 and 2015. Until July 2011, HAdV testing was performed based on clinical suspicion (cohort 1, n = 175) and from August 2011, weekly blood-HAdV monitoring was done (cohort 2, n = 181) until day +100. Twenty-three patients (4 [2.3%] from cohort 1 and 19 [10.5%] from cohort 2, p = .001) were found with HAdV viremia and seven of them died. Both cohorts had a similar incidence of HAdV-associated mortality (3/175; 1.7% in cohort 1 and 4/181; 2.2% in cohort 2). Respiratory failure was the cause of death in all patients. Clinical symptoms appeared prior to or within 5 days of HAdV detection in cohort 2. In summary, weekly monitoring was associated with higher detection of HAdV. The study could not assess survival benefit due to small numbers of HAdV-positive cases. In many instances, symptoms occurred with the development of positive HAdV blood PCR results and hence, symptomatology could have triggered the test. Future studies are needed to provide data that help establishing a uniform approach for regular monitoring of HAdV post-transplant.

Clinical Correlation of Cytomegalovirus Infection With CMV-specific CD8+ T-cell Immune Competence Score and Lymphocyte Subsets in Solid Organ Transplant Recipients.

Control of cytomegalovirus (CMV) infection after solid organ transplantation (SOT) requires a functional immune system. We assessed the association between quantitation and function of CMV-specific CD8+ T cells and CMV infection in SOT recipients. During a 10-year period, selected kidney, heart, lung, pancreas, liver, and composite tissue recipients were tested for CMV-specific CD8+ T cells immune competence (CMV-CD8+), as measured by enumeration, interferon-gamma production, and CD107a/b degranulation. Quantitative and functional data were used to assemble T-cell immune competence (TIC) score. CMV infection was diagnosed by polymerase chain reaction in blood and other samples or histopathology. Of 130 patients tested, 59 had CMV infection or disease. The median onset to CMV infection was 10.5 months (interquartile range [IQR], 5.5-18.7). Gastrointestinal disease (28.8%), pneumonia (20.3%), and CMV syndrome (17%) were most common presentation. An impaired nonspecific or CMV-CD8+ TIC score was associated with tissue-invasive disease (hazard risk, 2.84, 95% confidence interval, 1.03-11.81; P = 0.04). Patients with impaired CMV-CD8+ TIC score had longer viremia duration (42.4 days vs 18.8 d; P < 0.001). Patients with impaired nonspecific or CMV-CD8+ TIC score had higher risk of relapse (68.8% vs 27.9%; hazard risk, 2.56; 95% confidence interval, 1.09-5.89; P = 0.03). Patients with CMV infection or disease had lower median absolute lymphocyte count (380 [IQR, 240-540] vs 940 [IQR, 551-1210] cells/mm; P < 0.0001) and CD4+ T cell count (29 cells/mm [IQR, 1.3-116.0] vs 325.5 cells/mm [IQR, 151.5-589.8]; P < 0.0001). Nonspecific and CMV-specific CD8+ T-cell function correlated with the course of CMV after SOT, and measuring these has the potential to assist in its clinical management.

Leishmania infantum-specific IFN-\u03b3 production in stimulated blood from cats living in areas where canine leishmaniosis is endemic

BackgroundFeline leishmaniosis caused by Leishmania infantum is considered a rare disease in endemic areas, whereas subclinical infections are common. Immune response plays a key role in driving the course of L. infantum infection in other host species; however, the feline cell-mediated immune response to L. infantum infection has not yet been investigated. The aim of this study was to determine the cell-mediated immune response specific to L. infantum by means of interferon (IFN)-γ release in whole blood assay from cats living in endemic areas (66 in Sicily and 113 in Catalonia) and to compare with antibody levels to L. infantum [enzyme-linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT)], blood parasite load and retroviral infections.ResultsMost cats (n = 140) were L. infantum antibody negative and only 22% (n = 39) were positive. Only 9 and 2% of tested cats had a feline immunodeficency virus (FIV) infection or a feline leukemia virus (FeLV) infection, respectively. Thirty-two cats out of 179 (18%) produced IFN-γ after stimulation with L. infantum soluble antigen (LSA) while the majority of cats (93%) produced IFN-γ after stimulation with concanavalin A (ConA). Six LSA-IFN-γ-producer cats were seropositive (three to ELISA and five to IFAT) but they were polymerase chain reaction (PCR) negative, while only one cat was antibody- and PCR-positive. Significant positive correlations were found between IFN-γ concentrations after stimulation with LSA and ConA, and between serology and PCR testing. No association was found between FIV status and LSA or ConA-IFN-γ production. Combining PCR, serology and specific IFN-γ concentration results, we found that 36% of cats studied were exposed to L. infantum.ConclusionsAs expected, cats from endemic areas produce IFN-γ after ex vivo blood stimulation with LSA and therefore are able to activate a cell-mediated adaptive immune response against the parasite that is variably associated with antibody or blood PCR positivity. The association of this assay to serological and molecular tests provides a better estimate of cat exposure to L. infantum.

CD4+ T Cell Immune Response to VP1 and VP3 in BK Virus Infected Recipients of Renal Transplantation.

To investigate the characteristics of BK virus (BKV) specific cellular immune response in the recipients who have early infection with BKV after renal transplantation. The recipients of renal allografts (n = 30) were divided into groups of BK virus nephropathy (BKVN), viruria, and viremia. The BKV load was observed with real-time fluorescence quantitative polymerase chain reaction in urine and blood every three months. The values of serum creatinine (SCr) were detected. The peripheral blood mononuclear cells (PBMCs) were cultivated with overlapping peptide pool containing BKV structural proteins VP1, VP2, and VP3, and regulatory proteins large tumor antigen (LT-Ag) and small tumor antigen (st-Ag), to stimulate in vitro specific cellular immunoresponse. Flow cytometry was used to measure the proliferation of CD3+/CD4+/CD8+ T and interferon [INF]-γ/interleukin [IL]-2/tumor necrosis factor [TNF]-α T cell subsets. The BKV infection increased SCr values in recipients of renal transplantation. CD4+ T cells were dominant (>90%) in the in vitro cellular immunoresponse to VP1, VP2, VP3, LT-Ag, and st-Ag. At the presence of viremia and BKVN, IL-2/IFN-γ+/TNF-α+ CD4+ T cells showed significantly decreased in vitro cellular immunoresponse to VP1, VP2, and VP3 (p < 0.05), but insignificantly changed to LT-Ag and st-Ag (p > 0.05). For the cases of viruria and viremia, IL-2/IFN-γ+/TNF-α+ CD4+ T cells showed significantly higher in vitro cellular immunoresponse to VP1, VP2, and VP3 than to LT-Ag and st-Ag (p < 0.05). The immunogenicity of VP1 and VP3 was significantly higher than that of VP2 (p < 0.05). The BKV infection increases SCr values, and CD4+ T cells are dominant in the in vitro BKV specific cellular immunoresponse in the recipients of renal transplantation. Viremia significantly decreased the immunoresponse to VP1, VP2, and VP3. There is the significantly stronger immunoresponse to VP1 and VP3 when compared with that to VP2, LT-Ag, and st-Ag, suggesting that VP1 and VP3 may be the major targets for the BKV specific immune response.

Impact of Post-Transplant High-Dose Cyclophosphamide and Donor Sources on CMV Reactivation.

Introduction Cytomegalovirus (CMV) reactivation remains a common cause of morbidity and mortality following allogeneic hematopoietic cell transplant (HCT) despite attempts at prophylaxis. Following transplantation, surveillance of the peripheral blood CMV viral load with early aggressive treatment is commonly employed and has been an effective strategy at preventing overt infection. While a matched related donor (MRD) is often the preferred transplant type, the use of alternative donors including matched unrelated donors (MUD), mismatched unrelated donors (MMUD) and haploidentical (HI) transplants, has greatly increased over the past several years. The data regarding CMV infection among these alternative donor types is scarce. Objectives To better characterize the extent and risk factors for CMV reactivation among patients who received an allogeneic HCT for hematologic malignancies, particularly among HI transplant recipients and those who received post-transplant high-dose cyclophosphamide (PTCy) for graft-versus-host disease (GVHD) prophylaxis. Methods A retrospective analysis was performed on 88 adults with hematologic malignancies who received an allogeneic HCT at our institution from 2012-2017. Only cases with CMV seropositive donors and/or recipients were included. We compared donor types/sources, conditioning regimens, GVHD prophylaxis regimens, and assessed for CMV reactivation utilizing the peripheral blood polymerase chain reaction (PCR) values that are monitored in post-transplant clinic visits. Results Nineteen (22%) patients received MRD transplants, while the remainder received alternative donor sources, including 17 (19%) HI transplants. Overall, the rate of CMV reactivation was 27% with a significant difference noted between donor sources (p= Conclusion HI HCT transplant recipients are at high risk for CMV reactivation. The use of myeloablative conditioning regimens or PTCy did not affect the rate of CMV reactivation.

Molecular Epidemiology and Prevalence of Theileria lestoquardi and Theileria ovis Infection in Goats Infested With Tick Vectors From Multan, Pakistan.

Caprine theileriosis is a major production problem in regions of the world that rely on goats as a major source of milk, meat, and other means of economic income. Theileria ovis and Theileria lestoquardi are responsible for caprine theileriosis in Pakistan. The present study examined the prevalence of Theileria spp. infection using polymerase chain reaction (PCR) and light microscopy of blood collected from goats from Multan, Pakistan. In addition, the prevalence and identity of the genus of ticks feeding on these goats was determined on specimens collected at the time of blood sampling. The current project hypothesized that Theileria spp. prevalence would be higher in goats infested with ticks than goats without tick infestation. Four hundred and sixty-three blood samples from goats were analyzed, and the prevalence of infection was 16.0% (95% confidence interval [CI] = ±5.96) 74 of 463 by PCR and 5.4% (95% CI = ±7.79) 25 of 463 by microscopic examination. Thirty-six (48.6%, 95% CI = ±4.96) and 30 (40.5%, 95% CI = ±7.10) of 74 samples were positive by PCR for T. ovis and T. lestoquardi, respectively. Eight samples (10.0%, 95% CI = ±9.61) had mixed infections of these Theileria species. Infection was observed significantly (P < 0.05) more often in male 22.8%, 95% CI = ±3.85 (23/101) than in female 14%, 95% CI = ±2.36 (51/362) goats. No statistical (P > 0.05) difference in prevalence was present among the three age groups of ≤1 yr (12.6%, 95% CI = ±6.70), 1-4 yr (16.2%, 95% CI = ±8.24), and ≥4 yr (17.7%, 95% CI = ±11.30) goats examined. Two hundred and one (43.4%, 95% CI = ±7.45) goats were infested with ticks, Rhipicephalus spp. and Haemaphysalis spp., which are capable of transmitting Theileria spp. Tick infestation was found to be a significant (P < 0.05) risk factor for infection. Prevalence of Theileria infection in goats was significantly higher (P < 0.05) in the summer compared with the other three seasons. Data from this study support our hypothesis and demonstrate that both T. ovis and T. lestoquardi are highly prevalent in goats from Multan, Pakistan.

Mutations in FGFR3 gene associated with maxillary retrognathism.

Understanding the role of fibroblast growth factor receptor (FGFR) in the regulation of bone development and disease will ultimately lead to better prevention and treatment of related bone deformities and disorders. To evaluate the role of gene FGFR3 in individuals with retrognathic maxilla by polymerase chain reaction (PCR) technique at molecular level and evaluate the significance of the same. Hospital based fundamental research involving individuals having maxillary retrognathism. A total of 62 individuals (30M and32F) who were willing to take part in the study were selected from cephalometric measurements of N I A and the length PNS to ANS. The institution based basic genetic research study involved collection of fresh blood samples, DNA extraction, PCR analysis, and amplification using the specifically designed forward and reverse primers for targeting the commonly occurring mutations in FGFR3 gene. Further the products were sequenced to evaluate the presence of any novel mutations. The targeted single-nucleotide polymorphisms, at position 1138 in exon 10 of the FGFR3 gene were not identified in the analyzed blood samples. The detailed sequencing of full gene revealed the presence of 2 novel mutations, Exon 3: A213G and Exon 3: A223A/G in one individual. The present study indicated 2 novel mutations in gene FGFR3 in individual with maxillary retrognathism. The genetic-environmental interactions might have played a significant role in the expression of retrognathic maxilla.

The Association Analysis of Vascular Endothelial Growth Factor -2549 Insertion/ Deletion Variant and Endometriosis Risk.

Endometriosis is a debilitating disorder, defined as the presence of endometrial gland and stroma outside of the uterus. It may affect angiogenesis and vascular endothelial growth factor (VEGF) is one of the angiogenic factors that plays an important role in both physiological and pathological angiogenesis. The present study aimed to evaluate the association of VEGF -2549 insertion/deletion (I/D) polymorphism with endometriosis. This case-control study enrolled 244 (100 cases and 144 controls) women who were admitted for laparoscopy or laparotomy for gynecological procedures. Genomic DNA was separated from peripheral blood leukocytes and polymerase chain reaction (PCR) amplification was performed for genotyping of the VEGF gene Insertion/Deletion (I/D) polymorphism. The frequency of the II, ID, and DD genotype was 14%, 52% and 34% in patients versus 18.8%, 47.8% and 34% in controls. The results did not provide any evidence supporting the endometriosis risk related to the VEGF polymorphism in a group of Iranian women population.

Development and validation of direct PCR-RFLP assay for rapid detection and differentiation of Anaplasma species in naturally infected goats

This study was carried out with the objective to develop a novel, rapid and cost effective direct PCR-RFLP assay for detection and differentiation of two Anaplasma species (A. ovis and A. marginale). Blood samples were collected randomly from 112 goats. DNA was extracted from blood samples and a direct blood polymerase chain reaction (DT-PCR) for amplifying a fragment of the major surface protein 5 (msp5) gene of A. ovis/A. marginale from whole blood was developed and standardized. Additionally, the 16srRNA and msp4 genes were analysed by nested PCR, semi nested PCR and PCR-RFLP methods. Blood smear examination revealed 33 samples (29.46%) to be positive for Anaplasma inclusion bodies. On the contrary, 54 (48.21%) samples were positive by DT-PCR. The results revealed that DT-PCR was 100% sensitive and 73.41% specific when compared with microscopy based detection (k =0.63). All DT-PCR positive samples were confirmed as A. ovis by RFLP analysis. DT-PCR showed 94.44% sensitivity and 100% specificity compared to conventional PCR results with suspected blood samples (k=0.94). The phylogenetic tree and comparative sequence analysis revealed msp5 gene of Anaplasma species of Indian isolate had maximum distance from A. phagocytophilum followed by A. centrale and A. marginale and 100% sequence identity with A. ovis isolates of Chinese origin. The in-house standardized DT-PCR-RFLP method based on msp5 gene is an easy and reliable diagnostic tool to identify and discriminate between these closely related pathogens in ruminants. This type of study is the first of its kind as it depicts the molecular detection, differentiation and phylogenetic characterization of Anaplasma ovis among goat flocks in India.

The clinical features and mortality risk factors of cytomegalovirus infection in patients with systemic lupus erythematosus.

The clinical features and outcomes of cytomegalovirus (CMV) diseases in patients with systemic lupus erythematosus (SLE) are unknown. We analyzed such data from a medical center in Taiwan. We retrospectively reviewed the medical records of patients with SLE who were diagnosed with CMV diseases between 2006 and 2016 in Taipei Veterans General Hospital Taiwan. Clinical and laboratory parameters and treatment outcomes were analyzed. The study enrolled 56 eligible patients with CMV diseases and separated them into survival (n=24) and mortality (n=32) groups. All cases showed a significantly high incidence of pneumonitis (71.43%). The patients in the mortality group had a higher SLE disease activity index (SLEDAI)-2000 (p=0.009), more cases of recent methylprednisolone pulse therapy (p=0.013) and pancytopenia (p=0.001), stronger evidence of CMV infection demonstrated by polymerase chain reaction (PCR) in blood (p<0.001) and bronchoalveolar lavage (p=0.021), and more concurrent infections (bacteremia p=0.026; fungemia p<0.001). Recent pulse therapy, pancytopenia, and concurrent infections constituted risk factors for mortality in patients with SLE and CMV infection. Among mortality patients, PCR rather than serological tests (IgM antibodies) helped to arrive at an earlier diagnosis.

High percentage of microbial colonization of osteosynthesis material in clinically unremarkable patients.

Stabilization of fractures with internal fixation devices is a common procedure and implant‐associated infections are a dreaded complication. The exact pathomechanism is not completely understood; however, microbial colonization of osteosynthesis material is considered a trigger for infection. This study aimed to determine the colonization rate of osteosynthesis implants in patients with no clinical or laboratory signs of infection, using two methods, conventional culture and polymerase chain reaction (PCR) of sonication fluid. Fifty‐seven patients aged between 18 and 79 years without signs of infection who underwent routine removal of osteosynthesis devices between March 2015 and May 2017 were included in this study. Osteosynthesis material was investigated by sonication followed by cultivation of the sonication fluid in blood culture bottles and PCR analysis, simultaneously. Additionally, electron scanning microscopy was performed in nine representative implants to evaluate biofilm production. Thirty‐two (56.1%) implants showed a positive result either by culture or PCR with coagulase‐negative staphylococci being the most commonly identified microorganism (68.1%). Furthermore, the detection rate of the culture (50.9%) was significantly higher compared to PCR (21.1%). The scanning electron microscopy imaging demonstrated biofilm‐like structures in four of six culture and/or PCR‐positive samples. This study is the first, to the best of our knowledge, to demonstrate bacterial colonization of osteosynthesis implants in healthy patients with no clinical or laboratory signs of infection. Colonization rate was unexpectedly high and conventional culture was superior to PCR in microbial detection. The common understanding that colonization is a trigger for infection underlines the need for strategies to prevent colonization of implant material like antibiotic‐loaded coating or intraoperative gel application.

PCR sensitivity of peripheral blood of dogs co-infected with Leishmania spp. and Ehrlichia spp. in endemic area of Brazil.

Peripheral blood of 400 dogs infected with Leishmania and Ehrlichia were analyzed using polymerase chain reaction (PCR), and clinical signs were characterized. PCR and parasitological tests were conducted. PCR was positive for Leishmania in 84.75%, and parasitological tests showed that 63.25% and 31.75% were positive for Leishmania and Ehrlichia, respectively. All animals showed more than three clinical signs. PCR results were negative for Leishmania in 15.25% of the samples. Conventional PCR of peripheral blood can be used for diagnosing canine visceral leishmaniasis in combination with other techniques, especially in uncertain cases that need species identification.

Toxoplasma gondii seroprevalence among pregnant women attending antenatal clinic in Northern Tanzania

BackgroundAcute Toxoplasma gondii infection during pregnancy represents a risk for congenital disease, especially among women without previous exposure to infection. There is, however, a paucity of information about the epidemiology of T. gondii infection in pregnant women in Tanzania. This study aimed to determine the seroprevalence of T. gondii infection and associated demographic, clinical, and behavioral risk factors in pregnant women attending ante-natal clinic (ANC) at Kilimanjaro Christian Medical Center (KCMC), a referral medical center in Northern Tanzania.MethodsA hospital-based cross-sectional study was carried out from 1 February to 30 April 2017. Data on maternal demographic characteristics, obstetric history, knowledge, and practices related to T. gondii infection were collected from 254 pregnant women attending antenatal care at KCMC. A sample of 4 mL of blood was collected from each participant and sera prepared from each sample. Serum samples were tested for the presence of specific T. gondii IgG and IgM antibodies by indirect Enzyme-Linked Immunosorbent Assay (ELISA). DNA was extracted from whole blood for polymerase chain reaction (PCR) testing, targeting the DNA sequence coding for the Internal Transcribed Spacer 1 (ITS1).ResultsThe overall T. gondii seroprevalence, including both IgM- and IgG-positive individuals, was 44.5%. Of the 254 tested women, 102 and 23 were seropositive for T. gondii-specific IgG and IgM antibodies respectively and 113 individuals had antibodies of either or both classes. All IgM-positive samples were also tested by PCR, and all were negative. The majority (90%) of the women surveyed had never heard about toxoplasmosis. Consumption of raw vegetables [aOR = 0. 344; 95% CI 0.151–0.784; p = 0.011] and having regular contact with soil [aOR = 0.482; 95% CI 0.268–0.8681; p = 0.015] were both associated with T. gondii antibody status. Inverse relationships with probability of T. gondii exposure were observed, such that these practices were associated with reduced probability of antibody detection.ConclusionBased on serology results, we report widespread exposure to T. gondii infection among pregnant women attending ANC in KCMC. The complex interaction of risk factors for T. gondii infection needs to be studied in larger longitudinal studies.