Serial Isolates Research Articles

Molecular subtypes and antifungal susceptibilities of serial Cryptococcus neoformans isolates in human immunodeficiency virus-associated Cryptococcosis. Cryptococcal Disease Active Surveillance Group.

Serial isolates of Cryptococcus neoformans from 33 human immunodeficiency virus-infected patients with cryptococcosis were analyzed to determine whether persistence might result from reinfection with a new cryptococcal strain or acquisition of antifungal resistance. Isolates were subtyped by multilocus enzyme electrophoresis (MEE), electrophoretic karyotyping (EK), random-amplified polymorphic DNA (RAPD), and the CNRE-1 DNA probe, MICs of amphotericin B, fluconazole, and 5-fluorocytosine were determined. No changes in MEE or RAPD subtypes were detected in serial isolates from any patient. Isolates from 8 patients (24%) showed alterations in EK only (mobility change in two or more bands) but not with any other subtyping method. MICs did not change significantly in isolates from 30 patients. In 1 case, the fluconazole MIC increased stepwise over 18 months, suggesting development of resistance. These overall invariant subtyping and MIC results confirm previous studies suggesting that persistent cryptococcal infection is due to relapse rather than reinfection or antifungal drug resistance.

Relationship of virologic, immunologic, and clinical parameters in infants with vertically acquired human immunodeficiency virus type 1 infection.

We have investigated the relationship among the HIV-1 biologic phenotype, replicative capacity of virus isolates, HIV-RNA copy number in plasma, p24 antigenemia, CD4+ T lymphocyte counts in peripheral blood, and the clinical status in a cohort of 13 HIV-infected children younger than 12 mo of age, born of HIV-1 seropositive mothers. Six out of 13 HIV-1 isolates from these patients were classified as rapid/high and seven as slow/low. We have found a significantly positive correlation between the replication rate of HIV isolates and their capacity to induce syncytia in vitro. Most of the serial HIV-1 isolates obtained from infants with AIDS had the rapid/high phenotype and induced syncytia, whereas only two out of 23 HIV-1 isolates obtained from infants without AIDS showed these properties. In sequential analysis of HIV-1 isolates from infants with AIDS, the presence of viral isolates with rapid/high and SI phenotype was associated with higher levels of HIV-1 RNA in plasma, CD4+ T cell depletion, and clinical progression. By contrast, infants whose viruses exhibited nonsyncytium-inducing phenotype throughout the follow-up showed lower levels of HIV RNA, stable CD4+ T cell counts, and mild symptomatic HIV infection. Our findings indicate that infants who carried viruses with more cytopathic biologic phenotype and who had higher viral RNA coy numbers in blood were more likely to have lower CD4+ T cell counts and more likely to have AIDS.

Molecular karyotyping of multiple yeast species isolated from nine patients with AIDS during prolonged fluconazole therapy.

Variations in molecular karyotype and fluconazole susceptibility of serial yeast isolates from the oral cavities of nine patients with AIDS receiving fluconazole for single or multiple episodes of oropharyngeal candidiasis were monitored. Multiple yeast species were isolated from the initial oral specimens in six patients. Molecular karyotyping identified at least eight different DNA subtypes of C. albicans, at least eight of T. glabrata and only one DNA subtype each of C. krusei, C. tropicalis and C. parapsilosis. Among isolates of T. glabrata, fluconazole MICs in each patient were consistently within one or two dilutions, regardless of strain variations. Similarly, among five patients monitored during one course of therapy, the MICs of fluconazole of C. albicans isolates of either the same or different DNA subtypes remained within two dilutions. However, increases in MICs of fluconazole of C. albicans were observed in four patients who received two or more courses of fluconazole, three of whom had the same DNA subtype and one of whom changed from one DNA subtype to another.

Comparison of M and T Type Antigen Testing to Field Inversion Gel Electrophoresis in the Differentiation of Strains of Group A Streptococcus

Recent clusters of patients with acute rheumatic fever and invasive group A Streptococcus (GAS) have stimulated renewed interest in the epidemiology of streptococcal infections. We compared conventional serotyping for M and T antigens and serum opacity factor with field inversion gel electrophoresis (FIGE) for distinguishing among GAS. Fifteen pairs of throat isolates obtained from children positive for GAS before and after therapy were evaluated by conventional serotyping and by FIGE after SmaI digestion. Ten of the 15 pairs were identical by serotyping. FIGE correctly identified the 10 concordant and 5 discordant pairs. Individual clones were identified within each M type tested, including analysis performed on additional isolates of M1 and M3 obtained from the Centers for Disease Control and Prevention. This preliminary experience suggests that FIGE can successfully determine whether serial isolates from a given patient represent persistence of one strain or acquisition of a new strain of GAS and that this method might provide an alternative typing system for GAS.

Long PCR-ribotyping of nontypeable Haemophilus influenzae.

PCR-ribotyping, a new typing method based on long PCR, has been developed for nontypeable Haemophilus influenzae (NTHi). Ribosomal operons of NTHi were amplified by long PCR and were found to be highly polymorphic for internal HaeIII sites. The technique was applied to 49 isolates previously subjected to conventional ribotyping, and the two methods showed a high level of concordance for serial isolates from individual subjects. PCR-ribotyping provides a powerful new typing tool for strain characterization in epidemiological investigations of NTHi.

Stability of karyotype in serial isolates of Candida albicans from neutropenic patients.

Serial isolates of Candida albicans were obtained from 20 patients with leukemia over periods of up to 8 months. The fingerprinting of these isolates by interrepeat PCR and random amplified polymorphic DNA PCR has been described previously (A. van Belkum, W. Melchers, B. E. de Pauw, S. Scherer, W. Quint, and J. F. Meis, J. Infect. Dis. 169:1062-1070, 1994). Contour-clamped homogeneous electric field gel electrophoresis was used to examine the chromosomes of these isolates. When changes in the karyotype were seen in a series of isolates, additional interrepeat PCR and Southern blotting with a repeat DNA sequence from the 27A family were performed. These two genotyping tools were used to determine if karyotypic changes seen in a series of isolates were due to chromosome rearrangements in a single strain or due to colonization with more than one strain. It was determined that changes in karyotype in a series of strains indicated infection by a new strain.

Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins.

The protein and antigen profiles of 11 isolates of Mycoplasma bovis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of whole organisms. The isolates examined included the type strain PG45 and 10 other filter-cloned strains or purified isolates both from animals without clinical signs and from clinical cases of bovine mastitis, arthritis, or pneumonia. While the overall protein patterns visualized by silver staining were very similar, marked differences in the antigen banding profiles were detected by rabbit antiserum prepared against whole organisms from one of the strains analyzed. This antigenic heterogeneity was shown to be independent of the geographical origin, the type of clinical disease, and the site of isolation and was also observed among serial isolates from a single animal. Antigen profiles were further monitored throughout sequentially subcloned populations of the PG45 strain. This clonal analysis revealed a high-frequency variation in the expression levels of several prominent antigens. All of these variable antigens were defined by detergent-phase fractionation with Triton X-114 as amphiphilic integral membrane proteins. A subset of different-sized membrane proteins was identified by a monoclonal antibody raised against a PG45 subclone expressing a 63- and a 46-kDa variant antigen within that set. The selective susceptibility of these proteins to trypsin treatment of intact organisms and their ability to bind the monoclonal antibody in colony immunoblots demonstrated that they were exposed on the cell surface. In addition, their preferential recognition by serum antibodies from individual cattle with naturally induced M. bovis mastitis or arthritis confirmed that they were major immunogens of this organism. These studies establish that the apparent antigenic heterogeneity among M. bovis isolates reported here does not represent stable phenotypic strain differences generated from accumulated mutational events but reflects distinct expression patterns of diverse, highly variable membrane surface proteins.

Transmissibility of Pseudomonas cepacia infection in clinic patients and lung-transplant recipients with cystic fibrosis.

In patients with cystic fibrosis, infection with Pseudomonas cepacia is associated with poor outcomes. However, the extent of person-to-person transmission and the source of P. cepacia infection after lung transplantation are not well defined. Using DNA-based typing systems, we sought to determine the genetic relatedness of P. cepacia infection at one cystic fibrosis center. We analyzed 65 P. cepacia isolates gathered over a period of eight years at a single cystic fibrosis center from 17 clinic patients and from 5 patients who underwent double-lung transplantation. The isolates were analyzed by ribotyping and chromosomal fingerprinting based on pulsed-field gel electrophoresis. Analyses of serial isolates revealed that each clinic patient and transplant recipient harbored a different P. cepacia clone that was persistent. In the transplant recipients, the preoperative and postoperative isolates were identical. In the two patients with disseminated infection after lung transplantation, isolates from multiple sites were identical and indicated clonal expansion of the previous respiratory P. cepacia strain. Pulsed-field gel electrophoresis proved both more discriminative and more practical than ribotyping as a means of defining the genetic relatedness of the P. cepacia isolates. Our serial analyses in patients with cystic fibrosis at one center found distinct strains of P. cepacia persistently infecting each patient and no evidence of person-to-person transmission of this organism. P. cepacia infection after lung transplantation was due to the persistence of the strain present before transplantation.

Susceptibilities of serial Cryptococcus neoformans isolates from patients with recurrent cryptococcal meningitis to amphotericin B and fluconazole

Amphotericin B and fluconazole susceptibilities of 13 Cryptococcus neoformans isolates from five patients with recurrent cryptococcal meningitis were determined. For each patient, serial isolates showed no increase in antibiotic resistance relative to the initial isolate. For these patients, recurrent disease was not due to drug resistance but may reflect changes in immune function and/or poor compliance.

Restriction endonuclease analysis of clinical Pseudomonas aeruginosa strains: useful epidemiologic data from a simple and rapid method

Newer genetic techniques have replaced phenotypic methods of subtyping Pseudomonas aeruginosa strains. Widespread application of newer methodologies, however, may be limited by technologic complexity and the cost of equipment. We conducted restriction endonuclease analysis (REA) of sheared genomic DNAs from 48 clinical P. aeruginosa strains using the enzyme SalI and electrophoresis in horizontal, low-concentration (0.3 to 0.6%) agarose gels. Each REA profile consisted of a smear of lower-molecular-mass bands as well as a countable number of well-resolved bands in the 8.3- to 48.5-kbp range which could easily be compared when isolates were run side-by-side on the same gel. In general, the REA patterns of strains recovered from different patients differed by at least seven bands, and those of serial isolates from individual patients were identical or differed by, at most, two bands over this 8.3- to 48.5-kbp range. REA of strains already subtyped by field inversion gel electrophoresis revealed that the two techniques generally paralleled each other. Overall, some unrelated strains had similar REA profiles, but the relative simplicity and low cost of the approach coupled with the ability to demonstrate differences between most unrelated strains should make this type of REA an attractive first step in the investigation of institutional P. aeruginosa problems.

Exogenous reinfection with multidrug-resistant Mycobacterium tuberculosis in patients with advanced HIV infection.

In the United States there have been recent outbreaks of multidrug-resistant tuberculosis. These outbreaks have primarily involved persons infected with the human immunodeficiency virus (HIV). We collected clinical information on 17 patients seen at a New York City hospital who had repeatedly positive cultures for Mycobacterium tuberculosis. Analysis of restriction-fragment--length polymorphisms (RFLPs) was performed on serial isolates of M. tuberculosis obtained from these patients. Six patients had isolates that remained drug-susceptible, and the RFLP patterns of these isolates did not change over time. Eleven patients had isolates that became resistant to antimicrobial agents. The RFLP patterns of the isolates from six of these patients remained essentially unchanged (two strains showed one additional band) despite the development of drug resistance. In five other patients, however, the RFLP patterns of the isolates changed dramatically at the time that drug resistance was detected. The change in the RFLP pattern of the isolate from one patient appeared to be the result of contamination during processing in the laboratory. In the remaining four patients, all of whom had advanced HIV disease, the clinical and microbiologic evidence was consistent with the presence of active tuberculosis caused by a new strain of M. tuberculosis. Resistance to antituberculous drugs can develop not only in the strain that caused the initial disease, but also as a result of reinfection with a new strain of M. tuberculosis that is drug-resistant. Exogenous reinfection with multidrug-resistant M. tuberculosis can occur either during therapy for the original infection or after therapy has been completed.

Increased viral burden and cytopathicity correlate temporally with CD4+ T-lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals.

The rate of clinical progression is variable among individuals infected with human immunodeficiency virus type 1 (HIV-1). Changes in viral burden which correlate with disease status have been demonstrated in cross-sectional studies; however, a detailed longitudinal study of the temporal relationship between viral burden, CD4+ T-cell numbers, and clinical status throughout the course of infection has not been reported. Multiple longitudinal blood samples were obtained from four HIV-1-infected individuals with clinically divergent profiles. Levels of HIV-1 were measured in sequential samples of peripheral blood mononuclear cells, using both end-point dilution cultures and quantitative polymerase chain reaction methods. Serial HIV-1 isolates from each case were also evaluated to determine their biological properties in vitro. For the three patients with clinical progression, a dramatic increase in the level of HIV-1 was observed concurrent with or prior to a marked drop in CD4+ T lymphocytes. This increase in viral burden was temporally associated with the emergence of a more cytopathic viral phenotype. In contrast, consistently low levels of HIV-1 were observed in the one patient who was clinically and immunologically stable for more than a decade. Moreover, viral isolates from this patient were less cytopathic in vitro compared with HIV-1 isolates from those patients with disease progression. The temporal association between increased viral burden and CD4+ T-cell decline suggests a direct role for HIV-1 in the cytopathology of CD4+ T cells in vivo. Our results indicate that the pathogenic mechanisms responsible for CD4+ T-cell depletion may be related to both quantitative and qualitative changes in HIV-1.

Comparison of genome fingerprinting with conventional typing methods used on Pseudomonas aeruginosa isolates from cystic fibrosis patients

Two hundred Pseudomonas aeruginosa serial isolates from 61 cystic fibrosis patients were examined using restriction fragment length polymorphism in connection with pulsed field gel electrophoresis. A comparison was made of results obtained by genome fingerprinting and by conventional typing methods. It was possible to subdivide the majority of the genome types with the conventional typing methods, indicating the likelihood of bacterial phenotypes occurring in the lung of the cystic fibrosis patient. Two clusters of strains were observed among the 200 P. aeruginosa isolates from the 61 patients. Strains belonging to one cluster were present in 26 (42.6%) of the 61 patients. Strains belonging to the other cluster were present in 11 (18.0%) of the 61 patients. The occurrence of these clusters indicates that cross-infection has taken place among CF patients attending the Danish Cystic Fibrosis Centre. Conventional typing methods are based on the presence of specific bacterial surface structures. Therefore, conventional typing methods may sometimes lead to wrong classification of isolates from cystic fibrosis patients, especially if applied alone. A combination of two to six methods decreased the reproducibility of the typing results. The best combination was genome fingerprinting and phage typing, which yielded a reproducibility of 82.5%. Each time a method is added, the reproducibility decreases by an average of 14.4%.

Whole-cell protein electrophoresis for typing Mycobacterium tuberculosis.

A method of discriminating between strains of Mycobacterium tuberculosis by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins combined with a sensitive silver stain is described. Thirty-five isolates of M. tuberculosis and five isolates from other species of Mycobacterium were examined, including serial isolates from the same patients and isolates from a small cluster of hospital cases. Different species of Mycobacterium were clearly distinguished, and within the species M. tuberculosis, different fingerprints were obtained, allowing discrimination of some strains from different patients. The reproducibility and discrimination of the technique are discussed.

Development of resistance to zidovudine in HIV strains isolated from CD4+ lymphocytes and plasma during therapy

An assay based on production of HIV antigen in cultures of CD4+ lymphocytes infected ‘in vitro’ with cell-free virus was established. Using this assay it was possible to isolate, propagate and reliably determine the zidovudine susceptibility of HIV isolates from all patients despite differences in cellular tropism and syncytium inducing capacity. Using this assay, differences in zidovudine susceptibility of 52 serial isolates obtained from 16 patients before and after initiation of therapy were examined. HIV with a 10- to 100-fold reduced susceptibility to zidovudine were isolated from 13 patients as early as 4 months after initiation of therapy. Number of months of zidovudine treatment was strongly associated with development of viral resistance, and high CD4 cell counts tended to be associated with lower rates of development of resistance. That patients can harbor mixtures of virus strains with different susceptibility to zidovudine was confirmed by the differences in susceptibility between isolates obtained simultaneously from CD4+ lymphocyte and plasma, and by the differences in susceptibility between virus strains isolated from clones of CD4+ lymphocytes.

Ordered appearance of zidovudine resistance mutations during treatment of 18 human immunodeficiency virus-positive subjects.

The order of appearance in the reverse transcriptase gene of four mutations implicated in the development of resistance to zidovudine was investigated by selective polymerase chain reaction. Serial human immunodeficiency virus isolates were studied from 18 initially asymptomatic individuals who had been treated with zidovudine for 2 years. Most subjects had similar patterns. The first mutation occurred transiently at codon 70; its disappearance was paralleled by the appearance of a mutation at codon 215. Subsequently, in some individuals, the mutation at codon 70 reappeared. During the 2 years of treatment, no mutations developed at codon 219 and only one at codon 67, suggesting that most individuals developed only partly resistant virus. This was confirmed by plaque-reduction assay. Six subjects progressed to AIDS within the 2-year study period, confirming that the development of highly resistant isolates is not required for progression in treated individuals. No clear temporal relationship was found between the development of partial resistance and progression.

Human Immunodeficiency Virus isolates from asymptomatic homosexual men and from AIDS patients have distinct biologic and genetic properties

Human immunodeficiency virus type 1 (HIV-1) isolates from asymptomatic homosexual men and AIDS patients were compared for their in vitro biologic and genetic properties. Most of the HIV-1 isolates from asymptomatic men, but not from AIDS patients, failed to infect CD4 + H9 cells and phytohemagglutinin-stimulated peripheral blood lymphocytes. In a longitudinal study, serial HIV-1 isolates obtained from men who seroconverted to HIV-1 and later developed AIDS were able to infect H9 cells. In contrast, longitudinal isolates from men who remained asymptomatic did not infect H9 cells. HIV-1 isolates from AIDS patients in general exhibited increased production of intracellular viral DNA, RNA, and protein as compared to isolates from asymptomatic men. Cells infected with HIV-1 isolates from asymptomatic men produced very little gp120, p24, and p55 proteins as compared to those from AIDS patients. The overall restriction patterns of HindIII, Sac-1, Pst-1, EcoR1, and BamH1 were very similar between HIV-1 isolates from asymptomatic men and those from AIDS patients. However, the restriction endonuclease pattern of BgIII was quite distinct for isolates from asymptomatic men as compared to AIDS patients. Preliminary studies mapped a unique BgIII site in the gag region of most of the isolates from asymptomatic men, approximately 2.0 kb from the 5′ end. Thus, HIV-1 isolates from asymptomatic subjects and from AIDS patients have distinct biologic and genetic properties which may be related to the various clinical outcomes of HIV-1 infection.

Emergence of Teicoplanin Resistance During Therapy of Staphylococcus aureus Endocarditis

Serial isolates of Staphylococcus aureus showing two- to eightfold increases in teicoplanin minimum inhibitory concentrations (MICs) and twofold or less increases in MICs of other glycopeptides were recovered from the blood of a patient with endocarditis in whom drug therapy was unsuccessful. Comparable resistance emerged during teicoplanin treatment of rabbits with endocarditis caused by the original susceptible parent strain. For the parent strain, spontaneous resistance to teicoplanin at concentrations of 2-10 times the MIC was detected in vitro at frequencies of 10(-7) to 10(-9). Similar results were found for isolates of S. aureus from other geographic locations. Resistance was constitutive and not plasmid mediated, and its acquisition was not associated with changes in cytoplasmic membrane proteins. Teicoplanin was less effective than vancomycin at inhibiting peptidoglycan synthesis in resistant strains, suggesting that there is differential interference with the access of teicoplanin to or interaction with its target(s). Alternatively, teicoplanin and vancomycin may differ in some detail(s) of their mechanism of action against S. aureus.

Emergence of acyclovir-resistant varicella zoster virus in an AIDS patient on prolonged acyclovir therapy

We demonstrate for the first time the appearance of acyclovir resistance in serial varicella zoster isolates from a patient treated with acyclovir. We recovered varicella zoster virus three times over a period of 5 months from the skin lesions of this patient with AIDS who was treated with three courses of intravenous acyclovir and prolonged low-dose oral acyclovir. The isolate recovered from a typical zoster lesion before acyclovir, and one obtained from a hyperkeratotic lesion 2 months later, after intravenous and oral acyclovir, were sensitive to acyclovir and produced normal amounts of thymidine kinase. In contrast, virus recovered from lesions 5 months after the onset, when the patient had received repeated courses of acyclovir, was acyclovir-resistant and thymidine-kinase-deficient. Resistance to acyclovir was associated with persistence of lesions which failed to improve with intravenous acyclovir, but was not associated with new lesion formation.

Occurrence of a common lipopolysaccharide antigen in standard and clinical strains of Pseudomonas aeruginosa

The lipopolysaccharide (LPS) of Pseudomonas aeruginosa PAO1 contains two species of O polysaccharide termed A and B bands. The high-molecular-weight B-band LPS determines the O specificity of the bacterium, while the antigenically distinct A-band LPS consists of only shorter-chain polysaccharides. Seven hybridomas secreting A-band-specific monoclonal antibodies were produced and used to study the LPS of standard and clinical strains. Although A-band antibodies did not agglutinate any of the serotype strains presently in the International Antigenic Typing Scheme, Western immunoblots revealed that 11 of the 17 serotype strains possessed A-band LPS. In a group of 250 clinical isolates from patients with cystic fibrosis, 170 (68%) had A-band LPS on the basis of agglutination tests, but in silver-stained gels all were shown to be deficient in O-antigen-containing B band. Investigation of serial isolates from a single patient revealed a pattern of antigenic variation. During the course of the infection, serotypeable isolates became nontypeable, and the O antigen was replaced with A band as the major LPS antigen. These results suggest that A-band LPS may be the major LPS antigen in nontypeable clinical isolates and a common antigen among other P. aeruginosa strains.