Usage of polymerase chain reaction in complex diagnosis of hepatitis B

Sera from 926 patients were analyzed by PCR using universal primers to surface gene of hepatitis B virus (HBV). HBV DNA was detected in 195 specimens. There were no serological markers of HBV in 8.2% of these sera, but later they were detected in patients' sera. In patients without HBV DNA in the serum, specific HBV antigens and antibodies were detected in 62%. Only in 14% of them the clinical picture of the disease corresponded to acute viral hepatitis, although the blood for PCR analysis was collected during the late stages of the infection. The rest cases were referred to mixed hepatitis C + D. Comparison of the results of PCR test and detection of serological virus replication markers HBeAg and HBeAb showed the presence of HBV DNA in 28% cases without HBeAg.     

Whipple disease: a 15-year retrospective study on 36 patients with positive polymerase chain reaction for Tropheryma whipplei

Our institution has performed microbiological diagnosis of Tropheryma whipplei since 2001, initially with a PCR targeting 16S rRNA before the development of a quantitative PCR in 2012. Here we report the clinical characteristics of a cohort of patients suffering from Whipple disease (WD) and evaluate the impact of these molecular techniques. Patients with a positive PCR for T. whipplei between 2001 and 2016 were retrospectively collected from microbiological databases. Two infectious diseases specialists reviewed their medical records and classified them as definite WD, probable WD or carriage of T. whipplei without disease. A total of 1153 samples were tested for T. whipplei; 76 samples taken from 36 patients were positive. Fifteen were considered as presenting a definite WD, seven as a probable WD and 14 as carriers. Median age was 56.4 years (extremes, 6.6–76.1). Median time from symptoms to diagnosis was 3 years (2.5 months to 13.3 years). About 60% were immunosuppressed. The most frequent clinical presentations were joint pain (16/22), weight loss (15/22) and/or digestive tract disorder (15/22); 41% had neurological manifestations, 32% pulmonary involvement and 32% lymphadenopathies. Bacterial load in faeces or saliva were 88 425 copies/mL (IQR 6175-292 725) in definite and probable WD and 311 copies/mL (IQR 253–2090) in carriers, respectively. We observed a 90% PPV above 32 200 copies/mL in faeces. WD is a chronic multisystemic disease with frequent pulmonary involvement. Underlying immunodeficiency is commonly observed leading to more complex clinical presentation. Positive T. whipplei PCR in both stool and saliva has a high positive predictive value. Moreover, patients with WD present higher bacterial load in faeces with a threshold of >32 200 copies/mL predicting ongoing infection.     

Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 10⁶–10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.     

Analysis of group B streptococcal types associated with disease in human infants and adults.

It is important to resolve existing differences of opinion regarding group B streptococcal type distribution in human disease because of the relevance of type prevalence to future programs of prevention. This report compares data obtained from typing 392 group B streptococci isolated from systemic infections in both infants and adults in the United States from 1972 through 1975. The data showed a substantial predominance of type III among strains isolated from cases of infant meningitis and from "late-onset" septicemia but did not confirm a prior report that type Ia causes most cases of "early-onset" infant septicemia. Type II was the predominant serotype among 11 cerebrospinal fluid isolates from adults. The fact that over one-fourth of the isolates were types other than Ia or III means that future epidemiological studies, including definition of immunological factors, must include all five group B types.     

Commercial latex agglutination test for rapid diagnosis of group B streptococcal infection in infants.

Although latex agglutination assays for detection of a variety of bacterial antigens in body fluids from patients with systemic infection have been shown to be useful as rapid diagnostic techniques, lack of commercial availability has restricted their application. The Streptex latex test kit for the detection of group B streptococcal (GBS) antigen in admission body fluid specimens was evaluated for sensitivity and specificity in 54 infants with meningitis and in 10 infants with normal cerebrospinal fluid (CSF) parameters. GBS antigen was detected in 22 of 28 (78.6%) CSF specimens by latex agglutination and in 23 of 28 (82.1%) by countercurrent immunoelectrophoresis. Antigen was present in 21 of 28 (latex agglutination) and 19 of 26 (countercurrent immunoelectrophoresis) CSF specimens after the initiation of antimicrobial therapy. Heat-labile factors accounted for nonspecific agglutination reactions with latex suspensions other than group B in 3 of 28 CSF samples from patients with GBS meningitis. These nonspecific reactions were readily eliminated by heating specimens for 10 min at 100 degrees C. Fifteen patients with GBS meningitis had admission serum and urine samples collected in addition to CSF. Antigen was detected by latex agglutination and countercurrent immunoelelectrophoresis in 14 of 15 (93.3%) and 13 of 15 (86.7%) concentrated urine specimens, respectively, and in 12 of 15 (80%) CSF specimens and 4 of 15 (27%) sera by each method. These findings indicate that the Streptex latex test is a rapid, sensitive, and readily available method for detection of GBS antigen in admission body fluid specimens from infants with meningitis.     

Method of polymerase chain reaction in toxoplasmosis diagnosis

Since introduction of polymerase chain reaction for the detection of DNA Toxoplasma gondii 398 biological samples from 301 patients were examined from 2000 to 2003. Positive finding of toxoplasmosis DNA we noted in 23 cases. Polymerase chain reaction enables exact and fast diagnosis of the actual parasitemia Toxoplasma gondii, which is important especially in the pregnant patients, new-born with suspicion on congenital toxoplasmosis and in the patients with immunosupression.     

Human parvovirus B19 infections: Routine diagnosis by a new nested polymerase chain reaction assay

A nested primer PCR assay was developed to detect human parvovirus B19 in various clinical specimens in a routine diagnostic laboratory. Under optimized conditions the highly specific assay had a sensitivity of less than 10 genome units. For practical reasons, however, this sensitivity was adjusted to 10–100 virus genomes in diagnostic applications. Using clinical specimens from 200 patients with suspected B19 infection, nested PCR was shown to have important diagnostic advantages over the detection of B19 specific antibodies. The data suggest that on the basis of serological data as obtained with currently available test systems a considerable proportion of B19 infections would be misdiagnosed. Examples for the usefulness of the PCR assay in routine diagnosis are given. © 1993 Wiley-Liss, Inc.     

Real-time polymerase chain reaction and laser capture microdissection for the diagnosis of BK virus infection in renal allografts

We used real-time polymerase chain reaction (PCR) technology to detect BK virus (BKV) in H and E-stained kidney biopsy sections, using laser capture microdissection. Renal allograft biopsy specimens from 4 patients with the histopathologic diagnosis of BKV-associated nephropathy (BKVAN; group 1) and 3 patients suspected to have BKVAN but without diagnostic histologic features (group 2) were retrieved. Diagnostic inclusion-bearing cells were microdissected by laser capture microscopy from group 1. Renal tubular epithelial cells were microdissected randomly in group 2. DNA was extracted and real-time amplification performed using primers targeting the large "T" and small "t" regions of the BKV and JC virus genomes. Tubular epithelial cells from a case without evidence of BKV infection were used as negative controls in a similar reaction. BKV presence was demonstrated only in epithelial cells containing typical viral inclusions. Group 2 and negative control samples were confirmed as negative for BKVAN. Real-time PCR technology can be used to detect BKV in H and E-stained, paraffin-embedded tissue sections. This technique detected BKV in tubular epithelial cells of renal allografts. To our knowledge, this is the first report of detecting BKV in laser capture microdissected renal biopsy specimens using real-time PCR.     

Prenatal diagnosis of haemophilia B by the use of polymerase chain reaction and direct sequencing

Summary A second prenatal diagnosis of severe haemophilia B was carried out in a family with no prior history of the disease. The first prenatal diagnosis was based on linkage analysis and showed the male fetus not to be affected because he had inherited the same X-chromosome as his healthy brother. Carrier status in the female at risk could not be assessed by restriction fragment length polymorphisms (RFLPs). She was found to have inherited the same marker constellation as her affected brother. However, due to the fact that a pedigree with no prior history of haemophilia B has been examined diagnosis was impossible. In addition factor IX coagulant and antigen values gave no definitive clue to a haemophilia B carriership. The problems with RFLP analysis in this pedigree were circumvented by polymerase chain reaction (PCR) based direct sequencing of the factor IX gene. A previously unknown mutation could be detected in patient haemophilia B (Kleve) and the carrier status in the female at risk could be confirmed. The second prenatal diagnosis showed that the male fetus had inherited the mutation and will therefore be afflicted with haemophilia B.Abbreviations bp basepairs - FIX:Ag factor IX antigen - FIX:C factor IX activity - kb kilobasepairs - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism     

Comparison of polymerase chain reaction with culture and enzyme immunoassay for diagnosis of pertussis.

A polymerase chain reaction (PCR) assay amplifying a segment of a repeated gene element of Bordetella pertussis was compared with culture and enzyme immunoassay (EIA) for the diagnosis of pertussis. The PCR assay was specific for B. pertussis in tests with a panel of other bacteria and with an extensive collection of specimen material from healthy persons and children with respiratory infections other than pertussis. The PCR assay was used in the analysis of 117 nasopharyngeal swabs collected from children at an elementary school at which a pertussis outbreak occurred. Fifty-six (48%) of the 117 swabs were positive, including those for all six culture-positive cases. The PCR method was then applied to analyze another pertussis outbreak. Of 40 nasopharyngeal aspirates taken from 37 clinically susceptible pertussis patients and from three asymptomatic contacts, the PCR identified 18 (45%), including all 3 culture-positive and 5 (35%) of the 14 seropositive patients. The most consistent and reliable diagnosis by positive PCR result was observed with those patients experiencing symptoms within 1 to 6 weeks of sample collection. We conclude that PCR is a rapid, sensitive, and specific means of diagnosing pertussis, especially during the first weeks of disease. The assay can be performed with both nasopharyngeal swabs and aspirates.     

Detection and diagnosis of parapoxvirus by the polymerase chain reaction

The genus Parapoxvirus includes four members, bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV), orf virus (ORFV) and parapoxvirus of red deer in New Zealand (PVNZ). A set of primers for polymerase chain reaction (PCR) was designed to detect viral DNA from cells infected with each of the four parapoxviruses. The set of primers resulted in the amplification of appropriately sized products from cells infected with BPSV, PCPV, ORFV and PVNZ, respectively. The PCR method was applied for the detection of seven field isolates of parapoxvirus from cattle, sheep and free-ranging wild Japanese serows. The expected size of DNA was amplified from cells infected with each of the seven isolates. No specific PCR products were detected from vaccinia virus-, fowlpox virus- and mock-infected cells. Moreover, by a semi-nested PCR with an inner primer and Southern blot analysis, viral DNA was detected from lesions of clinically affected cattle, sheep and Japanese serows. These results suggested that the PCR method used in this study was specific for the detection of parapoxviruses and thus useful for diagnosis of parapoxvirus infections, especially in discrimination from diseases with similar clinical symptoms.     

Emergence of invasive serotype VIII group B streptococcal infections in Denmark

Serotype VIII group B streptococcus has only rarely been described outside Japan. The Streptococcus Unit, Statens Serum Institut, performed national surveillance of invasive group B streptococcal (GBS) diseases in Denmark in 1999 to 2002 and identified seven clinical GBS isolates of serotype VIII in blood from seven patients admitted to different hospitals.     

Chlamydia trachomatis infection in a high-risk population: comparison of polymerase chain reaction and cell culture for diagnosis and follow-up.

A study to compare the polymerase chain reaction (PCR) test with the cell culture method in diagnosing urogenital Chlamydia trachomatis infections was performed. From 497 patients (212 women, 285 men) attending an outpatient clinic for sexually transmitted diseases, a total of 814 samples (female patients, cervix and urethra; male patients, urethra) were collected. This total included follow-up samples from 35 women and 35 men positive for C. trachomatis by cell culture and/or PCR test, which were collected 2 weeks after treatment with doxycycline (two 100-mg doses per day for 7 days). The PCR test was performed directly on clinical samples without performing phenol-chloroform extraction and ethanol precipitation of DNA. The prevalence of C. trachomatis as measured by positive cell culture was 64 of 497 (12.9%) for all patients, 31 of 212 (14.6%) for women, and 33 of 285 (11.6%) for men. The prevalences as measured by positive PCR test were 71 of 497 (14.3%), 36 of 212 (17.0%), and 35 of 285 (12.3%), respectively. The sensitivities of the cell culture and the PCR test compared with that of true-positive samples were 77.5 to 78.4% and 99.0 to 100.0%, respectively. Discrepancies between cell culture and the PCR test were found for 23 of 497 patients (4.9%), 19 of 212 females (9.0%), and 4 of 285 males (1.4%). Nineteen pretreatment samples from 19 patients (4 female endocervical, 13 female urethral, and 2 male urethral samples) were cell culture negative and PCR test positive, while 1 pretreatment female endocervical sample was cell culture positive and PCR test negative. The posttreatment samples from all patients were cell culture negative, but the PCR test remained positive for 3 of 70 patients (1 female endocervical and 2 male urethral samples). One of these samples became spontaneously negative in three more weeks. The medical history of the individual patient and the negative PCR tests after treatment for nearly all patients support our hypothesis that the positive PCR test results were clinically relevant for the cell culture-negative but PCR test-positive but PCR test-positive patients of the population studied.     

Comparison of in situ hybridisation and polymerase chain reaction in the diagnosis of B cell lymphoma.

AIM: To compare the sensitivity of the detection of immunoglobulin light chain messenger RNA (mRNA) restriction by in situ hybridisation (ISH) and clonal immunoglobulin heavy chain gene rearrangements by polymerase chain reaction (PCR) in the diagnosis of B cell lymphoma. METHODS: Analyses were applied to formalin fixed, paraffin wax embedded, routine diagnostic specimens from cases with a provisional diagnosis of reactive lymph node (n = 23), B cell lymphoma (n = 21), and T cell lymphoma (n = 4). Nonisotopic ISH for kappa and lambda immunoglobulin light chain mRNA was performed using both fluorescein and digoxigenin labelled oligodeoxynucleotide probe cocktails. PCR was carried out on DNA extracted from sections using primers to framework 3 (Fr3) of the V segments and to conserved sequences from the J regions of the immunoglobulin heavy chain genes. RESULTS: All reactive lymph nodes showed a polyclonal pattern of light chain mRNA by ISH, although one showed an excess of kappa positive cells. Nineteen of 21 (90%) cases of B cell lymphoma showed light chain restriction, and a further case showed a vast excess of kappa positive cells. By PCR, 20 of 23 reactive nodes (87%) showed a polyclonal pattern. In 13 of 21 B cell lymphomas (62%) a clonal band was detected. CONCLUSION: In the diagnosis of B cell lymphoma in routinely processed diagnostic material ISH for light chain mRNA was more sensitive (90%) than PCR for heavy chain gene rearrangement using Fr3 and J region primers (62%).     

Specificity of the histopathological triad for the diagnosis of toxoplasmic lymphadenitis: polymerase chain reaction study

Toxoplasmosis is a common cause of lymphadenopathy, but toxoplasmic cysts are not usually found in histological sections used for establishing diagnosis, except on extremely rare occasions. The histopathological triad of florid reactive follicular hyperplasia, clusters of epithelioid histiocytes, and focal sinusoidal distention by monocytoid B cells has been considered to be diagnostic of toxoplasmic lymphadenitis, but the validity of the histopathological triad is based indirectly on serological correlation only. The demonstration of Toxoplasma gondii DNA in lymph nodes displaying the histopathological triad will indicate the validity of the histopathological triad as the criterion for the histopathological diagnosis of toxoplasmic lymphadenitis. We used frozen tissues of 12 lymph nodes with the histopathological triad and tissues of 27 lymph nodes from patients with various other conditions (including 13 cases of follicular lymphoid hyperplasia, FLH; three cases of dermatopathic lymphadenopathy, DPL; two cases of plasmacytosis; two cases of Castleman's disease; two cases of metastatic adenocarcinoma; and five cases of lymphoma) to detect T. gondii DNA by polymerase chain reaction. Ten out of 12 lymph nodes with the triad and six out of 27 lymph nodes without the triad were positive for T. gondii DNA. Thus, the sensitivity of the triad was 62.5% (10/16) and the specificity was 91.3% (21/23). The predictive value of positive tests was 83.3% (10/12) and the predictive value of negative tests was 77.7% (21/27). The six cases positive for T. gondii DNA without the triad were four cases of FLH, one case of DPL, and one case of plasmacytosis. None of the neoplastic diseases was positive. The false positive and negative cases could be due to sampling problems or past T. gondii infection. The results confirm that the histopathological triad is highly specific for the diagnosis of toxoplasmic lymphadenitis and can be used confidently.     

The polymerase chain reaction in the human immunodeficiency virus diagnosis

The PCR technique detects HIV1 and HIV2 DNA and RNA sequences in mononuclear cells with high sensitivity. It allows therefore to analyse the mother to child HIV1 transmission. Moreover, in the near future, the quantification of the PCR products could allow the follow-up of the patients treated for HIV infection.