Optimized detection of respiratory viruses in nasopharyngeal secretions.

Nasopharyngeal secretions (NPS) from 121 (110 pediatric) patients with acute respiratory infections were examined for respiratory virus detection by: i) conventional virus isolation in cell cultures (CC) using HEp-2, LLC-MK2, and MDCK cells; ii) rapid virus isolation using shell vial cultures (SVC) of a mixture (MIX) of mink lung epithelial cells (Mv1Lu) and human lung carcinoma (A549) cells in comparison to LLC-MK2 and MDCK cells; iii) direct fluorescent antibody (DFA) assay on NPS cells. A pool of monoclonal antibodies (MAbs) to influenzavirus A and B, parainfluenzavirus types 1 to 3, adenoviruses and respiratory syncytial virus (RSV), as well as single MAbs to the same viruses, were used for virus identification in all three procedures. Results on 101 NPS examined in parallel showed a sensitivity of 89.5%, 73.7%, and 81.6% for CC, SVC, and DFA, respectively, with the relevant negative predictive values of 94.0%, 86.3%, and 90.0%. Specificity and positive predictive values were 100%. However, the combination of DFA and SVC gave best results in terms of sensitivity (94.7%) and negative predictive value (95.5%). Use of the new MIX cell culture system in the SVC procedure enhanced virus detection, while use of the MAb pool allowed prompt identification of negative samples and saving of reagents and time for all three procedures. The combination of DFA and SVC allows diagnosis of the large majority of viral respiratory infections within 48h, while conventional virus isolation on CC may be limited to laboratories involved in research and epidemiological studies.

Standardization of the human cytomegalovirus antigenemia assay by means of in vitro-generated pp65-positive peripheral blood polymorphonuclear leukocytes.

We generated in vitro human cytomegalovirus (HCMV) pp65-positive polymorphonuclear leukocytes (PMN) resembling those detected in vivo, following cocultivation of PMN from healthy donors and wild-type HCMV-infected endothelial cells or fibroblasts. After purification, PMN are suitable for preparation of cytospots which can be used for the antigenemia assay. Cytospin preparations containing a predetermined number of in vitro-generated pp65-positive PMN were used to test some of the major parameters involved in performing the antigenemia assay. The results showed or confirmed that (i) formalin fixation followed by permeabilization is the best fixation procedure developed to date, (ii) the test performance levels provided by different pools of pp65-specific monoclonal antibodies may be significantly different, and (iii) long-term storage (for an unlimited time) is best achieved by keeping fixed slides at -80 degreesC, whereas short-term storage (for up to 1 month) is best achieved by keeping unfixed slides at room temperature. This finding signifies that slides can be shipped all over the world at room temperature. In conclusion, the newly developed procedure for in vitro generation of pp65-positive PMN will provide the basis for standardization of the HCMV antigenemia assay and development of quality control programs.

Diagnostic significance and clinical impact of quantitative assays for diagnosis of human cytomegalovirus infection/disease in immunocompromised patients.

In recent years several assays have been developed for quantitation of human cytomegalovirus (HCMV) in blood of immunocompromised (transplanted and AIDS) patients. It is currently agreed that the only reliable indication of the degree of dissemination of HCMV infection/disease is the measurement of HCMV in blood. Diagnosis of HCMV end-organ disease (organ localizations) often does not benefit from quantitation of virus in blood, but requires detection and quantification of virus in samples taken locally. The most important and clinically useful diagnostic assays for HCMV quantitation in blood are: i) viremia, quantifying infectious HCMV carried by peripheral blood leukocytes (PBL); ii) pp65-antigenemia, quantifying the number of PBL positive for HCMV pp65 in the nucleus; iii) circulating cytomegalic endothelial cell (CEC) viremia (CEC-viremia) measuring the number of circulating CEC carrying infectious HCMV (during the antigenemia assay); iv) leuko- and plasma-DNAemia, quantifying the number of HCMV genome equivalents present in PBL or plasma, respectively, by quantitative polymerase chain reaction (Q-PCR). Other less widely used assays are: i) determination of immediate early and late gene transcripts (mRNA) to detect active viral infection; ii) in situ hybridization to detect viral nucleic acid (DNA or RNA) in tissue sections or cell smears; iii) in situ PCR to detect a low DNA copy number in single cells. Monitoring of HCMV infection/disease in transplant recipients and AIDS patients has established threshold values for different assays above which HCMV-related clinical symptoms are likely to appear. These values are approximately 10 for viremia, 100 for antigenemia and 1,000 GE for leukoDNAemia, and are valid for both solid organ and bone marrow transplant recipients as well as AIDS patients, whereas presence of even a single circulating CEC is sufficient to suggest the presence of a disseminated HCMV infection with potential organ involvement. Monitoring of antiviral treatment of HCMV infection/disease with either ganciclovir or foscarnet has aimed at keeping virologic parameters below the threshold values reported above. On the other hand, rising levels of the same virologic parameters during antiviral treatment have mostly revealed emergence of resistant HCMV strains to either ganciclovir (mutations in the UL97 or DNA polymerase gene) or foscarnet (mutations in the UL54 gene) or both drugs (double resistance with both types of mutations). Rapid assays for chemosensitivity testing of virus directly in clinical specimens have been developed to allow timely (4-6 days) detection of resistance to a drug and provide clinicians with the rationale for shifting to an alternative treatment.

Rising levels of human cytomegalovirus (HCMV) antigenemia during initial antiviral treatment of solid-organ transplant recipients with primary HCMV infection.

In 7 of 18 solid-organ transplant recipients with primary human cytomegalovirus (HCMV) infection, HCMV antigenemia levels were unexpectedly found to rise significantly (P = 0.018) during a mean time of 7.3 +/- 3.2 days after initiation of specific antiviral treatment, whereas corresponding levels of viremia dropped significantly (P = 0.043). Thus, shifting to an alternative antiviral drug based solely on increasing antigenemia levels is not justified in this group of patients.

Prenatal diagnosis of rubella virus infection by direct detection and semiquantitation of viral RNA in clinical samples by reverse transcription-PCR.

A reverse transcription-nested PCR (RT-nPCR) method for prenatal diagnosis of rubella virus (RV) infection was developed. In the first step of RT-nPCR a synthetic RNA molecule (pRRV) differing from the RV target sequence by having a 21-nucleotide insertion was used as the internal control of amplification for the detection of PCR inhibitors. In addition, comparison of pRRV and RV-specific PCR signals allowed for the semiquantitation of RV input target sequences (range, 10 to > and = 1,000 RV genomes). In parallel, a complete RT-nPCR assay was performed with the same samples in the absence of the internal control to confirm the results of the first step and to detect RV RNA-positive samples containing < 10 RV genomes. Subsequently, the RT-nPCR method was used to examine retrospectively clinical samples (direct RT-nPCR) from eight congenitally infected and eight uninfected fetuses for RV RNA. RT-nPCR was also used to detect RV RNA in cell cultures (culture-RT-nPCR) 96 h after inoculation with the same specimens. With amniotic fluid (AF) samples, direct RT-nPCR identified eight of eight cases of RV transmission (sensitivity, 100%), whereas culture-RT-nPCR and virus isolation detected only six of eight cases (sensitivity, 75%). However, when the culture-RT-nPCR results were positive, culture-RT-nPCR confirmed the direct RT-nPCR results 3 days to 3 weeks earlier than virus isolation. The specificity of direct RT-nPCR was 100%, with eight of eight uninfected fetuses being negative. Semiquantitation showed only small amounts (< and = 100 copies) of viral RNA in clinical samples. In conclusion, direct RT-nPCR with AF samples (i) shows 100% sensitivity and specificity for prenatal diagnosis of RV infection and (ii) is a rapid technique, giving results in 24 to 48 h after sampling.

Diagnosis of human cytomegalovirus infections in the immunocompromised host.

BACKGROUND: In the last decade several major advances have been made in the rapid diagnosis of human cytomegalovirus (HCMV) infections and disease in immunocompromised patients both at the immunological and molecular level. OBJECTIVES: The objective was to review in some detail the new diagnostic tools allowing determination and quantitation of HCMV infection in blood of transplanted and AIDS patients. STUDY DESIGN: The determination and quantitation as well as the clinical significance of antigenemia, viremia, HCMV-infected circulating endothelial cells (CEC) and DNAemia will be discussed in view of the therapeutic management of HCMV disease. Levels of viremia represent the number of p72-positive cultured fibroblasts inoculated with 2 x 10(5)PBL, while levels of antigenemia represent number of pp65-positive PBL/2 x 10(5) PBL examined. The number of CEC is determined simultaneously and in parallel with antigenemia. DNAemia, both qualitative and quantitative, can be determined by polymerase chain reaction (PCR) per 1 x 10(5)PBL. The clinical utility of determining either immediate-early or late mRNA is still debated. RESULTS: In solid organ transplant recipients mean levels of viremia of 100 and of antigenemia of 400 correlate with onset of clinical symptoms. The time between first HCMV positivity and the onset of symptoms (>/= 10 days), together with the observation that most patients with reactivated infection clear virus without treatment, allowed the establishment of an antigenemia cut-off of 100 for the initiation of treatment. On the other hand, seronegative recipients of solid organs from seropositive donors must be treated preemptively, i.e. at first appearance of HCMV positivity in blood. Due to the risk of early appearance of HCMV pneumonia, the same preemptive approach must be used in bone-marrow transplant recipients. In acquired immunodeficiency syndrome (AIDS) patients with HCMV infection/disease, general criteria for initiation of treatment are more difficult to establish and treatment must be maintained. CEC are detected only in untreated disseminated HCMV infections with organ involvement. Qualitative DNA determination is useful only in special cases, such as in aqueous or vitreous humor of AIDS patients with HCMV retinitis. Quantitative DNA levels obtained by PCR are much more helpful for diagnosing HCMV disease and establishing initiation of treatment. CONCLUSIONS: New diagnostic procedures currently ensure fine monitoring of HCMV infections/diseases and evaluation of the effect of specific antiviral treatment in the immunocompromised host.

Microneutralization as a reference method for selection of the cut-off of an enzyme-linked immunosorbent assay for detection of IgG antibody to human cytomegalovirus.

In view of developing an enzyme-linked immunosorbent assay (ELISA) for the determination of IgG antibody to human cytomegalovirus, a rapid microneutralization (Nt) assay was used to test five positive standard sera containing increasing amounts of specific antibody and a negative standard serum. The standard serum containing the minimal amount of detectable Nt antibody was selected as a cut-off standard for the ELISA test. Following preliminary testing on previously characterized sera which gave expected results, the ELISA assay was tested in the field on 992 sera from blood donors. In parallel, sera were tested by Nt and complement fixation (CF). ELISA detected 82 negative and 910 positive sera. Nt gave concordant result except for two ELISA-negative sera, which showed Nt antibody titers of 1:10. The absorbance value of these two sera was just below that of the cut-off. Thus, for ELISA, the sensitivity was 99.8% (910/912) and specificity 100% (80/80). CF gave results concordant with ELISA and Nt, except for 23 sera (2 ELISA- and Nt-negative, and 21 ELISA- and Nt-positive) showing anticomplementary activity. Quantitation of specific ELISA antibody was achieved by interpolation from a calibation curve. Nt appears to be the reference test to establish the ELISA cut-off.

Group- and type-specific serologic response in infants and children with primary rotavirus infections and gastroenteritis caused by a strain of known serotype.

Antibody response to group A rotavirus (RV), investigated in paired sera from 72 infants and young children with acute gastroenteritis caused by an RV infection, was diagnosed on the basis of a fourfold or greater rise in group A common RV IgG antibody titer. Virus-specific IgM was detected in sera from 64 patients showing seroconversion; these were considered primary infection. RV was detected in stools of 56 (77.8%) patients with serologic evidence of infection and 54 were considered primary infection isolates: 39, serotype 1; 11, serotype 4; and 2, serotype 2. Two could not be typed. Neutralizing antibody studies showed that in primary infections serotype 1 induced an antibody response to serotype 4 at least fourfold lower than the homotypic response; serotype 2 elicited antibody titers to serotypes 1 and 4 at least fourfold lower than homotypic titer; and serotype 4 infections produced a response to serotype 1 as high as the homotypic response. Of 12 patients with primary infection, virus was not typed in 2 or detected in 10; however, the infecting serotype was identified on the basis of distinct patterns of homotypic and heterotypic antibody response.

Rapid detection of human rotavirus strains in stools by single-sandwich enzyme-linked immunosorbent assay systems using monoclonal antibodies.

Using murine monoclonal antibodies (MAbs) raised against the common antigen of group A rotavirus (RV), two single-sandwich ELISA systems were developed for detection of RV in stools: one using polyclonal antibody (PAb) as capture and a MAb as detector antibody (referred to as PAb-MAb assay); and the other based on the use of two different MAbs as capture and detector antibodies (referred to as MAb-MAb assay). In each single-sandwich ELISA system, samples and peroxidase-labeled MAb were incubated sequentially (two-step method) or simultaneously (one-step method). Using the two-step procedure on purified RV, 50 pg of protein was detected in the PAb-MAb as well as in the MAb-MAb assay, whereas the one-step method detected 0.4 ng and a conventional double-sandwich ELISA detected 3.2 ng of viral protein. Titration of RV samples from stools and cell cultures showed that single-sandwich ELISA titers were, on the average, 10-100-fold higher than those obtained by electron microscopy (EM), but 10-100-fold lower than those obtained by solid-phase immune EM (SPIEM). However, when 200 stool samples previously examined by EM or SPIEM were tested by the single-sandwich ELISA systems, specificity and sensitivity of these assays were 100%, and comparable to SPIEM. No false positive results were obtained when 54 samples of meconium and 91 stools from newborns in the first five days of life were tested. The two-step procedure appeared to be somewhat preferable over the one-step method, which, although faster, gave a marked prozone with a few samples in the MAb-MAb assay. The use of MAbs in rapid single-sandwich ELISA systems for RV detection in stools appears highly convenient, due to reliable results and short test performance times.

Characterization of rotavirus subgroup-specific monoclonal antibodies and use in single-sandwich ELISA systems for rapid subgrouping of human strains.

Two subgroup-specific monoclonal antibodies (MAb) raised in mice against group A human rotavirus were shown to react by immunoblotting with the trimeric form of VP6 of the homologous subgroup and successfully applied to development of new single-sandwich ELISA systems for rapid subgrouping of human strains. All of the 344 strains tested could be subgrouped, but for two of them prior propagation in cell cultures was required.

Comparative sensitivities of solid-phase immune electron microscopy and enzyme-linked immunosorbent assay for serotyping of human rotavirus strains with neutralizing monoclonal antibodies.

Suspensions of 24 rotavirus strains, 6 for each known human rotavirus serotype, were serially diluted and titrated by (i) enzyme-linked immunosorbent assay (ELISA) for rotavirus detection, using monoclonal antibodies (MAbs) specific for group-specific sites of the VP6 inner capsid protein; (ii) ELISA for subgrouping, using MAbs reactive with subgroup-specific determinants of rotavirus VP6; (iii) ELISA for serotyping, using MAbs directed to serotype-specific sites of the VP7 outer capsid glycoprotein; and (iv) solid-phase immune electron microscopy (SPIEM) for serotyping, using VP7-specific MAbs. In addition, in each preparation the proportion of double-shelled rotavirus particles were determined by direct electron microscopy. Results showed that SPIEM was 2- to 16-fold more sensitive than ELISA for serotyping of rotavirus. The titers in VP7-specific tests correlated well with the proportion of double-shelled virus particles in each of the samples. Titers obtained by ELISA for serotyping of suspensions containing 20% or fewer complete particles were up to 4,096-fold lower than those obtained by ELISA for detection. ELISA serotyping titers of samples containing 20 to 80% double-shelled rotavirus particles were up to 128-fold lower than ELISA detection titers, whereas preparations with nearly 100% complete particles had ELISA titers that were less different from each other. ELISA subgrouping titers were four- to eightfold lower than corresponding rotavirus detection titers. It was concluded that, although SPIEM appears to be more sensitive than ELISA, the amount of complete virus particles in the specimens is of critical importance for successful serotyping of human rotavirus strains. Samples rich in single-shelled particles but containing low amounts of VP7 outer capsid glycoprotein might even be strongly reactive in assays for rotavirus detection and subgrouping but virtually unreactive in tests for serotyping.

Synergistic neutralization of rubella virus by monoclonal antibodies to viral haemagglutinin.

Using murine monoclonal antibodies (MAbs) to rubella virus haemagglutinin, five epitopes were identified in competitive ELISA binding assays: A, B, D and E by haemagglutination-inhibiting (HI) MAbs with no neutralizing (Nt) activity, and C by a MAb with neither activity. However, when HI and Nt activities were determined in the presence of anti-mouse immunoglobulins, epitopes A, B and D were defined by both HI and Nt MAbs, whereas epitopes C and E were identified by HI MAbs without Nt activity. A synergistic Nt activity, in the absence of anti-mouse immunoglobulins, was displayed by mixtures of antibodies of different epitope groups. Analysis of mixtures of MAb pairs each belonging to a different epitope class, showed that synergistic Nt activity was elicited primarily by the group A epitope, secondarily by groups B and D and only minimally by groups C and E.

Comparative evaluation of a commercial enzyme-linked immunoassay and solid-phase immune electron microscopy for rotavirus detection in stool specimens.

Using solid-phase immune electron microscopy (SPIEM) as a reference test, we examined 151 stool specimens from infants and young children with acute gastroenteritis for rotavirus detection by a one-step commercial enzyme-linked immunosorbent assay (ELISA) with labeled monoclonal antibody. Of the 83 samples determined to be positive for rotavirus by SPIEM, 82 were detected as positive by the monoclonal antibody ELISA (sensitivity, 98.7%), while 67 of the 68 specimens determined to be negative by SPIEM were correctly detected as negative by the ELISA (specificity, 98.5%). The diagnostic accuracy of the ELISA kit was 98.6%. Thus, the one-step monoclonal antibody ELISA, which can be completed in less than 90 min, appears to be highly suitable for the rapid and reliable detection of rotavirus in stools.

Subgrouping of human rotavirus strains by complement fixation, indirect double-antibody sandwich enzyme-linked immunosorbent assay and solid-phase immune electron microscopy.

Complement fixation (CF), indirect double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) and solid-phase immune electron microscopy (SPIEM) were compared for their ability to subgroup 73 human rotavirus (HRV) strains from infants and young children with gastroenteritis admitted to one or the other of two different hospitals of Northern Italy. By both indirect DAS ELISA and SPIEM all 73 HRV strains were classified into one or the other of two subgroups. By CF only 67 strains could be subgrouped, as six HRV-positive stools showed anticomplementary activity which could not be eliminated. Indirect DAS ELISA required subgroup-specific, unabsorbed antisera from two different animal species. For SPIEM two antisera from a single animal species were needed, but they had to be absorbed with single-shelled bovine rotavirus for HRV subgrouping to be reliable. Indirect DAS ELISA appeared to be the technique most suitable for extensive application in epidemiological studies of HRV infections by different subgroups. However, SPIEM allowed rapid subgrouping of HRV in stool specimens showing anticomplementary activity in the CF test or non-specific reactions in the ELISA test. In one area of Northern Italy the prevalence of subgroup I HRV infections was 7.8 per cent, while in another it reached 68.1 per cent in the same period.

Diagnosis of acute non-bacterial gastroenteritis by rotavirus detection and serology.

In 127 infants and young children suffering from acute non-bacterial gastroenteritis, diagnosis of rotavirus infection was done by virus detection and serology. Human rotavirus (HRV) detection was performed by direct electron microscopy (EM), conventional immune electron microscopy (IEM) and/or solid phase immune electron microscopy ( SPIEM ); rotavirus antigens were detected by indirect double-antibody sandwich (DAS) ELISA and HRV isolation was attempted in MA-104 or LLC-MK2 cell cultures. HRV serology was done on paired sera from all the patients by the indirect immunoperoxidase antibody (IPA) technique for HRV IgG determination, and by an indirect ELISA method using a purified HRV Wa strain as a solid phase. HRV particles were detected by EM and/or IEM in 53 cases (41.7%) and by SPIEM in 5 additional cases; HRV antigens were demonstrated by indirect DAS ELISA in the same 53 cases, whereas 40 cases (31.4%) were positive for HRV isolation in cell cultures. Sixty-four patients (50.3%) seroconverted by IPA and ELISA, including all the cases (58) positive for rotavirus detection in stools and 6 additional cases. Thus, SPIEM appears to be the most sensitive technique for detecting a few virus particles in stool specimens, but HRV serology is the most sensitive method for diagnosing HRV infections retrospectively, when paired sera are drawn at an appropriate time. However, EM possess the great advantage of detecting in fecal specimens viral agents other than rotaviruses, such as adenoviruses, enteric coronaviruses, small round viruses, astroviruses and others.