Serum PCR Research Articles

Detection of Borrelia burgdorferi sensu lato DNA by PCR in serum of patients with clinical symptoms of Lyme borreliosis

Lyme borreliosis is a disease caused by spirochaetes belonging to the genospecies complex Borrelia burgdorferi sensu lato (s.l.) transmitted by Ixodes ticks. At present, serology remains the main diagnostic tool for laboratory diagnosis of Lyme borreliosis. Recently, the PCR technique has been applied for diagnosis of B. burgdorferi s.l., but, until now, a reliable, easy-to-perform and sensitive method has not been described. Here we present a new PCR-based method for the detection of both B. burgdorferi s.l. and Borrelia genospecies DNAs in serum samples collected from patients showing Lyme disease symptoms. Of 265 serum samples of patients included in this study, 7.5% were positive, 1.9% was borderline and 90.6% were negative for antibodies against B. burgdorferi by enzyme-linked immunosorbent assay and Western blotting. The B. burgdorferi s.l. 16S rRNA gene was detected by PCR in all serum-positive and in two borderline samples. None of the serum-negative samples nor serum samples collected from healthy subjects gave positive PCR reactions. Of PCR-positive serum samples, 50% gave a positive reaction for Borrelia afzelii, 18% for Borrelia garinii and 23% for two Borrelia species. Two samples (9%) were not identified to species level. The new protocol could be considered to be reliable as neither false-positive nor false-negative reactions were recorded, and to be sensitive as it detects DNA from one bacterial cell.

A soluble Fc receptor in channel catfish, Ictalurus punctatus, binds IgM

An Fc‐receptor (FcR) homolog, IpFcRI, representing the first such receptor from an ectothermic vertebrate, has been identified in the channel catfish. It consists of three immunoglobulin (Ig) domains (D1–3) but lacks transmembrane (TM) and cytoplasmic regions. Due to this lack of a TM, IpFcRI is suggested to be a soluble FcR. The soluble IpFcRI is readily detected in catfish serum and quantitative PCR analyses demonstrate that kidney leukocytes express high levels of IpFcRI message. Recombinant (r) IpFcRI immobilized onto latex beads for Ig binding studies was found to bind both native catfish IgM and IgM‐immune complexes. In addition, rIpFcRI was also found to recognize reduced catfish IgM heavy (H) chain and Ig from other species, including rainbow trout, mouse, rabbit, goat, and chicken. To identify the IpFcRI domain(s) involved in IgM binding, various combinations of rIpFcRI domains were expressed. Recombinant IpFcRI proteins containing two domains, D1–2 or D2–3, reacted with reduced catfish IgM H chain, while the individual D1, D2 and D3 domains did not. Furthermore, the IpFcRI‐binding site appears to be localized on Cμ3 and Cμ4 domains. Taken together, the IpFcRI‐IgM binding studies suggest an immunoregulatory role for IpFcRI. Supported by NIH grant R01 AI19530.

Dengue encephalopathy in children in Northern India: Clinical features and comparison with non dengue

Acute febrile encephalopathy (AFE) is a common cause of childhood hospital admissions in Lucknow. In recent years, many patients have been hospitalized with AFE and hemorrhagic manifestations, some of whom were proven to have dengue viral infection. To (i) define the role of dengue encephalopathy (DE) as a cause of AFE in children in Lucknow, (ii) document features of dengue hemorrhagic fever (DHF) in them and (iii)compare clinical features of definite dengue and non dengue AFE. Prospective study at a teaching hospital in northern India. Children between 1-12 years of age hospitalized with fever and altered consciousness of 2 weeks or less duration were enrolled after excluding bacterial and tuberculous meningitis and frank hepatic encephalopathy. Clinical and laboratory details were charted. Haemagglutination inhibition (HI) test for dengue and Japanese encephalitis viruses in paired sera and IgM antibody capture ELISA for dengue were done. Real time PCR was done in those samples testing positive for dengue IgM. Those with either positive HI test or positive dengue PCR in CSF or serum were considered definite dengue infection and features of DHF were charted in them. Those negative for IgM antibodies after 5 days of illness or whenever done, HI test, were considered definite non dengue. Clinical and laboratory features were compared between definite dengue and non dengue groups. A total of 265 patients of AFE were enrolled over a 2 year period. HI test was positive in 15/49 (30.6%) and IgM in 52/238 (21.8%) patients thus tested. A total of 62 patients were positive for dengue antibodies by either test. Real time PCR assay for dengue virus genome was positive in 28/42 (69%) tested -- 21/29 (72.4%) in CSF and 9/15 (60%) in serum. A total of 39 patients met the criteria for definite dengue infection of which only 2 fulfilled the WHO criteria for DHF. Comparing DE and non DE, rash, bleeding, swelling over body, and hepatomegaly were significantly more common and meningeal signs less frequent in DE. Mean platelet counts and serum albumen were lower and liver enzymes and INR were significantly higher in DE. Dengue viral infection is a cause of AFE in children in this region. Majority of DE here appears to be due to viral invasion of brain as suggested by high PCR positivity in CSF and lack of WHO criteria for DHF. Differentiating features of DE include swelling and hepatic dysfunction.

787 NEGATIVE TISSUE PCR DOES NOT CONFER REDUCED RISK OF RELAPSE AFTER TREATMENT OF HCV IN PATIENTS POST-LIVER TRANSPLANT

Background: Treatment of recurrent HCV after liver transplant (LT) with interferon (PEG) and ribavirin (RBV) is complicated by poor tolerance, risk of rejection, low SVR (15−35%), and high relapse (18−50%). Ability to predict relapse may prompt extended therapy. Tissue PCR may be useful in identifying residual hepatic HCVRNA when unable to detect in serum. We postulated that negative tissue PCR during PEG/RBV would predict low risk of relapse. Methods: Patients with recurrent HCV following LT were given PEG/RBV for a minimum 48 weeks. In 14 patients that achieved serum PCR negativity, treatment was extended until serum TMA negative, tissue PCR negative, and absence of moderate necroinflammation on biopsy. Tissue PCR (NGI, Los Angeles, CA) was performed on liver biopsy specimens frozen in liquid nitrogen at bedside. Results: 11/14 patients eventually achieved SVR (79%) while 3/14 patients had relapse or breakthrough on treatment. One patient required 2 treatment courses for SVR (initially relapsed), and another patient received 2 treatment courses, with one relapse and one breakthrough, for a total of 16 treatment courses for 14 patients. SVR was noted in 11/16 courses; relapse or breakthrough were noted in 5/16 courses. The baseline viral load, HAI, stage, duration of PEG, and duration of PCR or TMA negativity on treatment were no different between the groups. The Delta HAI trended toward more improved in those with SVR (p = 0.07). 20 biopsies for tissue PCR from 14 patients were obtained. 18/20 biopsy PCRs were negative while 2 were positive. All patients with negative serum PCR/TMA at time of biopsy also had negative tissue PCR, including the 3 patients that relapsed. The only 2 positive tissue PCRs had viremia (breakthrough) at time of biopsy. Positive tissue PCR identified breakthrough, but not relapse. Conclusions: Treatment of HCV recurrence following LT appears to require protracted treatment courses with prolonged negative molecular testing and a decrease in necroinflammation before sustained response is likely. Tissue PCR does not appear to add additional information regarding risk of relapse in post-transplant patients undergoing treatment for HCV.

Detection of Aspergillus fumigatus‐specific DNA, (1–3)‐β‐d‐glucan and galactomannan in serum and bronchoalveolar lavage specimens of experimentally infected rats

The aim of this study was to detect Aspergillus fumigatus-specific DNA by nested PCR (nPCR) in serum and bronchoalveolar lavage (BAL) specimens of experimentally infected rats and compare the results with (1-3)-beta-D-glucan (BDG) and galactomannan (GM) detection. Sixty Wistar rats, immunosuppressed with an intraperitoneal injection of cyclophosphamide (70 mg kg(-1)) were infected with 1 x 10(6)A. fumigatus conidia. The rats were killed on days 1, 3, 5, 7 and 9 postinfection in groups of six each and their BAL, blood and lungs were cultured. The A. fumigatus-specific DNA, BDG and GM in serum and BAL were detected by nPCR, Fungitell kit and Aspergillus Platelia kit respectively. Base line values were obtained by using sera from six healthy rats. Except the lungs, blood and BAL specimens of all the infected rats were negative for A. fumigatus culture. The BDG, GM and nPCR positivity in serum specimens was 80%, 77% and 63% respectively. The sensitivity of GM and nPCR tests in BAL specimens was 77% and 70% respectively. The data suggest that BDG and GM appear early in the course of infection, and have similar kinetics (r = 0.483, P = 0.007). Hence, their combined detection could be useful in the early diagnosis of invasive aspergillosis.

Detection of HCV by PCR in serum and PBMC of patients with hepatitis C after treatment

Although hepatitis C is mainly hepatotropic, some studies suggest that hepatitis C virus (HCV) infects peripheral blood mononuclear cells (PBMC), using them as a reservoir, which might contribute to the development of resistance to treatment. Fifty-four hepatitis-C patients, who had been submitted to treatment, were selected. Blood samples were collected on the same day for the detection of HCV RNA in serum and PBMC by PCR, using the Amplicor HCV 2.0 assay (Roche Diagnostics). HCV genotyping was performed using the INNO-LiPA HCV kit (Versant, Bayer Diagnostics). HCV RNA was detected in both serum and PBMC in 35 (64%) patients and no RNA in 16 (29.6%). Disagreement between the serum and PBMC results was observed for three patients (5.6%), with HCV RNA being detected in PBMC but not in serum. Four months later, new serum and PBMC samples were collected from one of these patients and HCV RNA was detected in both samples, showing that PBMC can reveal signs of a lack of response to treatment. We conclude that the absence of HCV in the serum of patients with chronic hepatitis C by the end of treatment does not mean that there is no circulating virus. HCV in mononuclear cells may be an indicator of the persisting infection.

Management of invasive pulmonary aspergillosis in non-neutropenic critically ill patients.

During recent years, a rising incidence of invasive pulmonary aspergillosis (IPA) in non-neutropenic critically ill patients has been reported. Critically ill patients are prone to develop disturbances in immunoregulation during their stay in the ICU, which render them more vulnerable for fungal infections. Risk factors such as chronic obstructive pulmonary disease (COPD), prolonged use of steroids, advanced liver disease, chronic renal replacement therapy, near-drowning and diabetes mellitus have been described. Diagnosis of IPA may be difficult and obtaining histo- or cytopathological demonstration of the fungus in order to meet the gold standard for IPA is not always feasible in these patients. Laboratory markers used as a non-invasive diagnostic tool, such as the galactomannan antigen test (GM), 1,3-β-glucan, and Aspergillus PCR, show varying results. Antifungal therapy might be considered in patients with persistent pulmonary infection who exhibit risk factors together with positive cultures or sequentially positive GM and Aspergillus PCR in serum, in whom voriconazole is the drug of choice. The benefit of combination antifungal therapy lacks sufficient evidence so far, but this treatment might be considered in patients with breakthrough infections or refractory disease.

Bovine viral diarrhoea virus infection of alpacas (Vicugna pacos) in the uk

Three alpacas (Vicugna pacos) aged two to 22 months with a history of illthrift and diarrhoea were examined postmortem, and tissues were collected for histology, including immunohistochemical labelling for pestivirus...

The histopathologic and clinical analysis of viral chronic hepatitis patients with negative serological viral markers

To analyze the histopathological and clinical features of viral chronic hepatitis patients with negative serological viral markers. 62 hepatitis patients with negative serological markers were assayed with serological viral hepatitis markers, liver function test and liver biopsies were enrolled in the study. Serum HBV DNA of HBV cases was analyzed by PCR. Liver specimens were examined by immunohistochemistry for HBsAg and HBcAg. The fit rate of histopathological diagnosis with clinical diagnosis is 53.2%, the fit rate is 69.1% in moderate chronic hepatitis group. The immunohistochemistry showed that HBsAg and/or HBeAg positive rate was 45.2%, 53.6% had moderate chronic hepatitis and 25% had mild hepatitis. 13 (46.4%) had G1 hepatitis, 10 (35.7%) had G2 hepatitis, 3 (10.8%) had G3 hepatitis and 2 (7.1%) had G4 hepatitis, and serum HBV DNA positive rate was 35.7%. There were no differences in HBV DNA levels between different hepatitis group and fibrosis stage group (P > 0.05). There were no differences in all indexes between HBV DNA negative group and HBV DNA positive group (P > 0.05). There were no differences in all indexes between HBV patients and other patients (P > 0.05). Occult HBV infection may account for a high proportion of the cases with chronic hepatitis of unknown etiology. Most patients are chronic mild hepatitis, but they still have HBV replication and can progress to liver cirrhosis. Serum PCR test, liver biopsy and immunohistochemistry are helpful for the diagnosis.

Detection of neutralizing antibodies in postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs

The notion that postweaning multisystemic wasting syndrome (PMWS)-affected pigs develop an impaired humoral response against porcine circovirus type 2 (PCV2) has been reported in several studies. However, little information is available regarding the presence of neutralizing antibodies (NA) in PCV2-infected pigs and their role in the pathogenesis of the disease. The aim of the present work was to further characterize the humoral response, and in particular the production of NA, in pigs with different PCV2-infection status. Seventy-two conventional pigs from different farms were classified into three groups based on PCV2 infection and clinico-pathological status, namely: PCV2-negative, non-PMWS PCV2-positive and PMWS-affected animals. In addition, 9-week old pigs from an experimental infection (6 controls and 14 PCV2-inoculated pigs) were also studied. NA and total PCV2 antibodies (TA) as well as viral load in serum were determined and correlated with the clinico-pathological status of pigs. Results indicated that PMWS-affected pigs had lower NA titres, if any, than healthy animals. NA titres were also inversely correlated with PCV2 load in serum. NA and TA titres were positively correlated; however, correlation differed among infection status, being lower in PCV2-positive pigs. Also, the diagnostic performance of each test was evaluated, indicating that the combination of viral neutralization and quantitative PCR in serum was useful to discard PMWS (specificity 92%). In experimentally infected animals, the evolution of NA paralleled the course TA, although a slight delay in NA production was seen in some animals. The increase of NA coincided with the drop in viral load. Results from this work further support that PMWS-affected pigs show an impaired humoral immune response and, particularly, an inefficient NA response against PCV2.

Rapid quantification of hepatitis B virus DNA by direct real-time PCR from serum without DNA extraction

The purpose of this study was to quantify hepatitis B virus DNA by direct real-time PCR from serum without the need for DNA extraction. Crossing point (Cp) values were determined automatically using the second derivative maximum mode. Since serum samples from patients are inevitably haemolysed, lipaemic or icteric, the interference of endogenous substances from the serum in real-time PCR was evaluated. The result showed that, although serum protein quenched the intensity of fluorescence, the Cp value adopted to calculate the quantity of DNA copies remained unchanged. Importantly, real-time PCR from serum with or without DNA extraction reached a high level of concordance. This direct serum PCR method without the DNA extraction and gel electrophoresis allows for substantial labour and cost savings. In addition, it is also suitable for rapid DNA quantification during clinical diagnosis.

Épidémie estivale de méningites à entérovirus en 2005 : expérience d'un service de pédiatrie générale

Objective To describe the clinical and biological characteristics of children presenting with enteroviral (EV) meningitis in a French paediatric unit during summer 2005. Methods Retrospective study of children with EV meningitis from May to September 2005, diagnosed by PCR and/or viral culture in cerebrospinal fluid (CSF), serum or throat. Results We reported 99 cases of EV meningitis (96 confirmed and 3 probable). The sex ratio was 2/1, and the median age was 5 years. Peak incidence was reached during the second week of July. The predominant symptom was meningism. ENT (16%), digestive (10%), cutaneous (15%) or respiratory (4%) symptoms were rare. Blood leucocyte count found a predominance of neutrophils (73%), and lymphopenia in half of the children. The mean value of CRP was 25,5 mg/l. The median leukocyte count in CSF was 65 cells/mm 3, with a prevalence of neutrophils in 60% of cases. Pleiocytosis was absent in 20 children. CSF protein level was increased in 20% of cases. The rate of hospitalization was 57,5%. Intravenous antibiotic treatment, initiated among 18 patients, was stopped in 66,6% of the cases on reception of PCR result. The latter result was obtained in 2,3 days on average. Conclusion The epidemic of 2005 EV meningitis was as widespread as that of summer 2000. Characteristics of these meningitis are strong proportion of CSF without pleiocytose and high prevalence of neutrophils in blood and CSF.

Diagnostic significance of Neospora caninum DNA detected by PCR in cattle serum

A nested PCR that successfully detected Neospora caninum DNA in serum of cattle was used for investigation of selected abortion cases and in a study of healthy pregnant cows at an abattoir. N. caninum DNA was not detected in serum from antibody positive dams that aborted due to N. caninum, but was present in serum of some antibody negative dams that aborted due to other causes. N. caninum DNA was also found in the serum of about half of the animals that aborted of undetermined cause, but was not detected in cow sera from two beef cattle herds in Western Australia with no recent history of abortion. In the abattoir study of 79 dams and their foetuses N. caninum DNA was found in serum of 3 dams and in material from 11 foetuses. The majority of the cows and all foetuses were antibody negative. Our findings suggest that there is no obvious relationship between the presence or absence of N. caninum DNA in serum and the presence of antibodies to N. caninum in dams, the presence of N. caninum DNA in foetuses or abortion due to N. caninum. This is the first report of the detection of N. caninum DNA in serum of cattle rather than the white blood cell fraction. It indicates the presence of free tachyzoites and/or parasite DNA in circulation. The results suggest that persistent infection in the absence of antibodies is a possible outcome of N. caninum infection. Infection of foetuses in the absence of antibodies supports the possibility of persistent infection due to immunotolerance to an early in utero infection. It is therefore important to test for N. caninum DNA as well as antibodies for the detection of exposed and/or infected animals. However, the presence or absence of N. caninum antibodies or DNA did not support nor exclude N. caninum as the cause of abortion. Additional criteria are required for a positive diagnosis of abortion caused by N. caninum.

Marked stem cell factor expression in the airways of lung transplant recipients

BackgroundAirways repair is critical to lung function following transplantation. We hypothesised that the stem cell factor (SCF) could play a role in this setting.MethodsWe studied 9 lung transplant recipients (LTx recipients) during their first year postgraft, and evaluated SCF mRNA expression in bronchial biopsy specimens using on-line fluorescent PCR and SCF protein levels in bronchoalveolar lavage (BAL) and serum using ELISA. The expression of SCF receptor Kit was assessed using immunostaining of paraffin-embedded bronchial sections.ResultsSCF mRNA was highly expressed during the early postgraft period [Month (M)1-M3] (300% increase vs controls: 356 vs 1.2 pg SCF/μg GAPDH cDNA, p < 0.001) and decreased thereafter (M4-M12: 187 pg/μg), although remaining at all times 10–100 times higher than in controls. While SCF protein levels in BAL were similar in LTx recipients and in controls, the SCF serum levels were at all times higher in LTx recipients than in controls (p < 0.05), with no relationship between these levels and the acute complications of the graft. Finally, Kit was strongly expressed by the mast cells as well as by the bronchial epithelium of LTx recipients.ConclusionSCF and Kit are expressed in bronchial biopsies from lung transplant recipients irrespective of the clinical status of the graft. A role for these factors in tissue repair following lung transplantation is hypothesised.

418 Occult hepatitis C infection in patients with histological hepatitis and negative serum PCR for HCV

418 Occult hepatitis C infection in patients with histological hepatitis and negative serum PCR for HCV

Syndrome d’hypersensibilité médicamenteuse associé à une primo-infection HHV6

Drug hypersensitivity syndrome is a severe life-threatening drug reaction. An association between this syndrome and HHV6 reactivation has been hypothesized.A 45 year-old women was treated with sulfasalazine for polyarthralgia. One month after beginning the treatment, she developed a drug hypersensitivity syndrome with severe acute hepatic failure. HHV6 serology and serum PCR revealed a primary HHV6 infection.We report the second case of drug hypersensitivity syndrome associated with a primary HHV6 infection. An immunological disorder may explain such an association by increasing viral replication. Detection and titration of anti-HHV6 antibodies in each case of drug hypersensitivity syndrome should help to confirm this association and possibly modify treatment strategy.

らい菌の遺伝子診断の現状

Laboratory tests necessary for the diagnosis of leprosy have not been well introduced in general hospitals and clinical laboratories. Therefore, several tests have been performed in Leprosy Research Center, National Institute of Infectious Diseases since July, 1997, as a part of administrative examinations (tests done by request of Ministry of Health, Labour and Welfare). These examinations include histopathology, serum antibody titers (anti-PGL-I antibody), PCR test and bioactivity of anti-bacterial agents.

P16.15: First trimester prenatal diagnosis of fetal RhD status and gender by real‐time PCR in maternal serum

P16.15: First trimester prenatal diagnosis of fetal RhD status and gender by real‐time PCR in maternal serum

Invasive meningococcal disease and latex agglutination test — Is it still beneficial for diagnosis?

We showed current clinical usefulness of the latex agglutination (LA) test for confirmation of meningococcal etiology on 32 cerebrospinal fluid, 77 serum and 93 urine samples collected during the first week of hospitalization from 19 patients with laboratory confirmed invasive meningococcal disease. The positivity of the LA test in cerebrospinal fluid was 47%, in serum 42% and in urine 24%, while the PCR of cerebrospinal fluid and serum was positive in 95 and 47% cases, respectively. The latest positivity of the LA test was detected on day 2 in cerebrospinal fluid, on day 3 in serum and on day 4 in urine. In the group of patients who had received antibiotic therapy we found nonsignificant reduction of LA positivity and also statistically significant reduction of culture positivity in CSF (p = 0.04); the PCR positivity changed minimally. In blood samples, nonsignificant reduction of culture positivity and no difference in LA and PCR positivity was found. We did not find any statistically significant relationship between test results and clinical forms. The LA test can be therefore considered to be an auxiliary diagnostic method, rapid and easily practicable but less sensitive than PCR. It can be recommended especially for local laboratories where PCR is not available and the patient already received antibiotics before admission to the hospital.

Identification of a DAF-7 ortholog from the hookworm Ancylostoma caninum

Infective hookworm L3 encounter a host specific signal during invasion that re-activates suspended developmental pathways. Response to this cue is critical for the successful infection and completion of the life cycle in the host. In the free-living nematode Caenorhabditis elegans, recovery from the developmentally arrested dauer stage in response to environmental cues is analogous to the resumption of development in invading hookworm L3. Transforming growth factor β (TGF-β) and insulin-like signalling pathways mediate dauer formation and recovery. An insulin-like signalling pathway mediates L3 activation in hookworms. To determine the role of TGF-β signalling in hookworm infection, an ortholog of the C. elegans TGF-β signalling molecule daf-7 was cloned and characterised. Sequence from a hookworm expressed sequence tag was used to design specific primers for PCR amplification of Ac-daf-7 from Ancylostoma caninum infective L3 cDNA. Amplicons from the 5′ and 3′ ends were cloned, sequenced, and combined to create a full-length composite Ac- daf-7 cDNA sequence. The 1634 nucleotide cDNA encoded a 355 amino acid open reading frame with significant homology to Ce-DAF-7 and other TGF-β signalling molecules. The deduced amino acid sequence contained seven conserved cysteines characteristic of TGF-β family members, as well as two additional conserved cysteines found in members of the TGF-β/activin subfamily. Ac-DAF-7 contains a characteristic C-terminal ligand domain that is predicted to be released from a propeptide by proteolytic cleavage at a tetrabasic cleavage site. Ac-daf-7 mRNA was strongly detected by reverse transcriptase PCR in L3 and serum stimulated L3 cDNA, and weakly in cDNA from L1 and adult life cycle stages. Antiserum against Escherichia coli expressed recombinant Ac-DAF-7 detected the mature protein in L3 and adult soluble extracts, but not in excretory/secretory products from serum stimulated L3 or adults. Increased expression in arrested L3 stages suggests that Ac-daf-7 is important for developmental arrest.