Monoclonal antibodies for the identification of herpesvirus simiae (B virus)

Summary To differentiate between B virus and HSV isolates from monkeys and man monoclonal antibodies (mabs) were produced to herpesvirus simiae (B virus) and herpes simplex type 1 and 2 (HSV-1 and HSV-2). Mabs were tested by indirect immunofluorescence (IFAT) for reactivity against herpesviruses from Asiatic monkeys (B virus), African monkeys (SA 8 virus), and man (HSV-1, HSV-2, varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus). Mabs could be divided into groups A-E displaying specific reactivity for B virus (A); reactivity with both B virus and SA 8 but not HSV (B); reactivity with B virus, SA 8 virus and HSV strains (C); specific reactivity with HSV-1 (D); and specific reactivity with HSV-2 (E). Two of the B virus specific mabs were able to differentiate between cynomolgus and rhesus strains of B virus. None of the mabs reacted with human varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. A panel of mabs for the unequivocal identification of B virus isolates from monkey or man is proposed.     

Polymerase chain reaction assay of parvovirus B19 DNA in clinical specimens.

The polymerase chain reaction (PCR) was used to detect parvovirus B19 DNA in a panel of sera from individuals recently infected with B19, as demonstrated by the presence of anti-B19 immunoglobulin M. Of 95 serum samples, 60 (63%) were found positive by PCR, whereas only 1 was also found positive by dot hybridization. In a control panel of 100 serum samples from individuals with other infections, only 1 serum sample was found positive by PCR, and this was also found positive by dot hybridization. This was probably just a fortuitous discovery of viremia. Placental tissues from women (n = 89) who had proven B19 infections in pregnancy but who gave birth to healthy infants at term were also tested. A total of 74 (83%) were found positive for B19 DNA by PCR. The high rate of detection by PCR probably represents "decay" of viral DNA after the peak of viremia and is not a clinically significant phenomenon.     

Polymerase chain reaction for detection ofChlamydia pneumoniae in gargled-water specimens of children

The objective of the present study was to establish the occurrence ofChlamydia pneumoniae by direct detection in gargled-water specimens obtained from 193 children suffering from acute or chronic respiratory infections. Specimens were analyzed by an indirect immunofluorescence test (IIF), a genus-specific antigen enzyme immunosorbent assay (EIA) and the polymerase chain reaction (PCR). The pathogen was detected in three children by PCR only. As underlying disease, chronic obstructive bronchitis resistant to therapy was reported. In two of the children, the presence of pneumonia could be verified by X-ray. With a detection threshold of target DNA obtained from two inclusion forming units (IFU), the PCR proved clearly more sensitive than EIA becoming positive at levels of 100 IFU and above. No interpretable results could be obtained for the IIF.     

Rapid identification of herpesvirus simiae (B virus) DNA from clinical isolates in nonhuman primate colonies

A rapid, simple technique, based on restriction endonuclease analysis of radioactively labeled infected cell DNA, is described for identification of Herpesvirus simiae (B virus) infection in clinical isolates from nonhuman primates. Isolates can be screened within 2-3 days from the time of collection of a specimen from a suspect lesion to final viral identification. Isolates were obtained from eight animals with suspected B virus infections. The results indicated the presence of B virus in each of the eight animals, each isolate unique from the others, but with the dominant prototypic pattern of the laboratory strain of B virus (E2490) and not HSV-1 (KOS), HSV-2 (186), or SA8 (3264).     

Polymerase chain reaction for detection of Mycobacterium leprae in nasal swab specimens.

The polymerase chain reaction based on the selective amplification of a 531-bp fragment of the gene encoding the proline-rich antigen of Mycobacterium leprae was applied to nasal swab specimens from leprosy patients, occupational contacts, and endemic and nonendemic controls. To prevent false-positive amplification, we used dUTP and uracil-DNA-glycosylase in all polymerase chain reactions. False-negative reactions were detected by using a 531-bp modified template as an internal control. Amplification products were found in 55% of untreated patients, in 19% of the occupational contacts, in 12% of endemic controls, and in none of the nonendemic controls. This study strongly suggests that not only leprosy patients but also healthy persons may carry M. leprae. We concluded that polymerase chain reaction is a reliable method to detect M. leprae in nasal specimens. The method holds promise for studying the spread and transmission of M. leprae within a population.     

Polymerase chain reaction for detection of herpes simplex virus encephalitis.

A patient with herpes simplex virus encephalitis is described. The polymerase chain reaction (PCR) was used to confirm the diagnosis in cerebrospinal fluid. PCR allows the rapid diagnosis of many infectious organisms, such as HSV, in which prompt diagnosis is essential.     

Polymerase chain reaction for detection of Mycoplasma genitalium in clinical samples.

We have used the polymerase chain reaction to detect Mycoplasma genitalium in artificially seeded human throat swab samples as well as in clinical material. On the basis of the published nucleotide sequence of the M. genitalium 140-kDa adhesin gene, primers were chosen to produce an amplified fragment of 281 bp. Five different previously isolated strains, including the type strain of M. genitalium, could all be detected by the polymerase chain reaction, and DNAs from other mycoplasmal and bacterial species yielded negative results. The detection limits were estimated to be approximately 50 organisms by inspection of ethidium bromide-stained agarose gels and 4 organisms when a biotinylated oligonucleotide probe was used in filter hybridization. The amplified DNA fragments were subjected to restriction enzyme analysis. DNAs from the five different isolates all possessed EcoRI, SspI, AluI, Sau3AI, and DdeI restriction sites, as predicted from the published sequence. A total of 150 urogenital swabs collected from 100 patients for culturing of Chlamydia trachomatis were tested for the presence of M. genitalium DNA. Ten samples from eight patients were found positive. The amplified DNA fragments from all of our clinical samples possessed the AluI, Sau3AI, and DdeI restriction sites, but three samples from two patients did not contain the SspI site and none of the samples contained the EcoRI site.     

Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus

A polymerase chain reaction (PCR) amplification method was developed and evaluated to detect porcine parvovirus (PPV). A pair of 20-base primers and an oligonucleotide probe were derived from the DNA sequences common to two isolates of PPV, NADL-8 and NADL-2. The primers flanked 118-bp nucleotides within the region coding for the major structural protein VP2. After DNA amplification of PPV replicative form (RF), a 158-bp fragment was detected in agarose gels. This amplified fragment was shown to be specific for PPV DNA after Southern transfer and hybridization to a 20-base internal probe. The amplified fragment also contained a single EcoRI cleavage site. Various conditions, such as number of cycles and annealing temperature, were examined to optimize the conditions for detecting viral DNAs from infected cell cultures and swine fetal tissues. Four different isolates of PPV, NADL-8, NADL-2, KBSH and Kresse, and two other viruses, canine parvovirus (CPV) and pseudorabies virus (PRV), were included to determine specificity of amplification. Slot blot hybridization with a radiolabeled probe was used to evaluate the sensitivity of PCR amplification. The optimized protocol was specific for PPV detecting equally all four strains of PPV, but failing to amplify CPV or PRV sequences. The PCR method could detect at least 100 fg of viral replicative form (RF) DNA or the equivalent of 1 PFU of infectious virus. The applications of this method include routine detection of PPV in clinical samples and as a contaminant in mammalian cell lines.     

Polymerase chain reaction for detection of Leptospira spp. in clinical samples.

A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative.     

Nested polymerase chain reaction for detection of human immunodeficiency virus type 1 DNA in clinical specimens.

A highly sensitive two-step polymerase chain reaction (PCR) method was evaluated for detection of human immunodeficiency virus type 1 (HIV-1) DNA in clinical specimens. The product resulting from the first amplification reaction is used as the template for the second PCR with an internal (nested) primer pair. Even when starting from a single copy of HIV-1 DNA, the double PCR product was readily detected by direct visualization in ethidium bromide-stained agarose gels. Amplification of minute amounts of HIV-1 DNA was successful in a considerable excess of HIV-1 negative DNA than reported previously. All of 85 HIV-1-infected individuals were PCR-positive with at least two of the three sets of primers used, 252 of 255 amplifications allowing unambiguous visualization of a unique DNA fragment of the expected size. The two-step amplification protocol is simple and rapid and fulfills the requirements of sensitivity and specificity for use in a clinical laboratory.     

Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples.

AIMS: To evaluate the use of a cppB gene derived polymerase chain reaction (PCR) assay for direct detection of Neisseria gonorrhoeae in clinical samples. METHODS: A PCR assay was performed on 33 N gonorrhoeae strains and 12 other Neisseria species and other normal genital flora to evaluate the specificity of the chosen cppB primers. The assay was subsequently evaluated with 52 clinical swab samples collected from China. RESULTS: An amplified product of 390 base pairs (bp) was observed with all the N gonorrhoeae strains, each of these products on digestion with the restriction enzyme MspI produced two bands of 250 bp and 140 bp respectively. This set of primers did not produce any amplified product of the expected length with the other non-gonococcal strains tested. For the 52 clinical swabs, 34 were culture positive and PCR successfully detected all these positives. In addition the PCR was positive for two swabs which were culture negative but positive for N gonorrhoeae antigens when tested with the ELISA method (Gonozyme). CONCLUSIONS: This PCR assay is a promising diagnostic tool for detection of gonococci directly from clinical swab samples. Further evaluation is necessary.     

Polymerase chain reaction for detection of Toxoplasma gondii

A DNA-based assay has been developed for the detection of Toxoplasma gondii. The assay makes use of the polymerase chain reaction (PCR) to amplify part of the P30 gene on the parasite's DNA. Following gel electrophoresis, the amplified DNA can be detected either directly on the gel or by Southern hybridisation with radioactive or non-radioactive DNA probes. The assay has been used to detect the DNA from different isolates of T. gondii in a background of human or mouse DNA. Together with other information such as clinical data, CT scans and serology, the PCR assay should improve the diagnosis of toxoplasmosis in immunosuppressed and immunocompromised patients as well as in fetal tissues.     

Quantitative real-time PCR for detection of monkey B virus (Cercopithecine herpesvirus 1) in clinical samples

A TaqMan based real-time PCR assay was developed for rapid detection and quantitation of herpes B virus (Cercopithecine herpesvirus 1) in clinical samples. The assay utilizes B virus-specific primers and a probe to the non-conserved region of the gG gene to discriminate B virus from closely related alphaherpesviruses. Fifty copies of B virus DNA could be detected with 100% sensitivity with a wide range of quantitation spanning 6 logs. The assay was highly reproducible with intra- and inter-assay coefficients of variation of 0.6 and 2.4%, respectively. Clinical utility of the developed real-time PCR was evaluated by testing genomic DNA prepared from B virus clinical isolates (n=23) and human and monkey clinical specimens (n=62). This novel method was also compared with conventional cell culture with respect to sensitivity and specificity. TaqMan PCR assay was shown to be equally specific and more sensitive than culture method (culture vs. PCR sensitivity 50%) and was able to identify all B virus clinical isolates tested. Fast, reliable assessment of B virus DNA in infected cells and tissues makes real-time PCR assay a valuable tool for diagnosis and management of B virus infections.     

A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens

Objective: Because presently used methods for diagnosis of Legionella pneumonia lack sufficient sensitivity and sometimes specificity and rapidity, the detection of Legionella spp. by amplification of nucleic acids might be valuable. However, performing polymerase chain reaction (PCR) on clinical samples such as sputum is difficult because of the presence of extraneous DNA and inhibitors of the reaction. An attempt to circumvent these problems was made.
Method: A nested PCR method was devised using primers from the mip gene of Legionella pneumophila. This PCR was tested on pure cultures of legionellae and clinical isolates of other bacteria. Clinical samples (bronchoalveolar lavage fluid, bronchial aspirate and sputum) from patients who suffered from legionellosis and samples from patients who suffered from other causes of pneumonia were also tested.
Results: The PCR was specific for L. pneumophila and no non- Legionella bacteria reacted. Ten to 50 colony forming units of Legionella in the sample could be detected. Twenty-two of 25 clinical samples were positive among patients suffering from pneumonia proven to be due to L. pneumophila serogroups 1, 3, 4, 5 and 6. Two of the three negative samples were from patients who had been treated with adequate therapy for at least 2 days and were culture negative. However, nine other culture-negative samples were PCR positive, of which seven came from patients who had been treated for 3–7 days. All pneumonia patients in the control group proved negative in PCR. A commercial kit for DNA preparation from clinical samples, based on absorption of nucleic acids to silica gel, was superior to the traditional phenol/chloroform extraction and increased the rapidity, simplicity and sensitivity of the procedure.
Conclusions: A nested, simplified and rapid PCR method using mip primers proved to be more sensitive than culture and as sensitive and specific as other PCR procedures previously reported.     

Polymerase chain reaction for detection ofMycobacterium tuberculosis in sputum

The polymerase chain reaction (PCR) was evaluated in a trial which, with respect to the positive-to-negative ratio, approximated the situation of a diagnostic laboratory in a tuberculosis-endemic area. Three hundred sputum samples were included in the study, of which one-third were known to contain mycobacteria as judged by direct microscopy. The repetitive insertion sequence IS6110/IS986 ofMycobacterium tuberculosis was used as a target. The samples were spiked with DNA from a modified IS6110/IS986 sequence, which gives rise to PCR products easily distinguished from PCR products amplified from chromosomalMycobacterium tuberculosis DNA. This allowed identification of samples that contained substances inhibitory to theTaq polymerase. The detection limit of the assay was 0.05 pg to 0.5 pg of purifiedMycobacterium tuberculosis DNA, corresponding to 10 to 100 organisms. The sensitivity and specificity of the PCR was compared with that of conventional microscopy and culture. It was concluded that this method is fast and sensitive, but that culture currently is crucial for assessing viability and thus infectivity.