Epidemiologic trends in Clostridioides difficile isolate ribotypes in United States from 2011 to 2016.

BACKGROUND: Geographic and temporal trends in the distribution of PCR ribotypes for Clostridioides difficile associated diarrheal isolates obtained in the United States (US) are changing. As part of a US national surveillance program of C. difficile susceptibility to fidaxomicin, we quantified the distribution of PCR ribotypes of stool isolates collected from 2011 to 2016. METHODS: C. difficile isolates or C. difficile toxin + stools from patients with C. difficile infection (CDI) were submitted for testing to Tufts Medical Center from 6 geographically distinct medical centers. Following isolation and confirmation as C. difficile, approximately 35% of the isolates were randomly sampled, stratified by center, for PCR ribotyping by capillary gel electrophoresis. Toxin gene profiling was performed on all isolates. RESULTS: 939 isolates from a total of 2814 (33.4%) isolated over the 6 years were analyzed. Seventy unique ribotypes were observed, including 19 ribotypes observed 10 or more times. Sixteen ribotypes were not previously observed in our data base. Ribotype 027 declined by more than 60% over the 6 years of the survey from 35.3% to 13.1% (p < 0.001). Ribotype 106 was the most common in 2016, followed by 027 and 014-020. There were strong correlations between 027 and binary toxin with the 18 base pair deletion of tcdC and ribotype 078-126 had 100% concordance with the previously described tcdC 39 base pair deletion. CONCLUSIONS: The frequency of ribotypes in the US has changed with a marked decline in 027. Each of the geographical areas had variations which differed from each other, but collectively, these results suggest that the changing epidemiology of C. difficile in the US is consistent with what is being seen in Europe. Continued surveillance and monitoring of changes in ribotype distributions of C. difficile are warranted.

U.S.-Based National Surveillance for Fidaxomicin Susceptibility of Clostridioides difficile-Associated Diarrheal Isolates from 2013 to 2016.

In 2011, we initiated a sentinel surveillance network to assess changes in Clostridioides (formerly Clostridium) difficile antimicrobial susceptibility to fidaxomicin from 6 geographically dispersed medical centers in the United States. This report summarizes data from 2013 to 2016. C. difficile isolates or toxin-positive stools from patients were referred to a central laboratory. Antimicrobial susceptibility was determined by agar dilution. CLSI, EUCAST, or FDA breakpoints were used, where applicable. Toxin gene profiles were characterized by multiplex PCR on each isolate. A random sample of approximately 40% of isolates, stratified by institution and year, was typed by restriction endonuclease analysis (REA). Among 1,889 isolates from 2013 to 2016, the fidaxomicin MIC90 was 0.5 µg/ml; all isolates were inhibited at ≤1 µg/ml. There were decreases in metronidazole and vancomycin MICs over time. Clindamycin resistance remained unchanged (27.3%). An increase in imipenem resistance was observed. By 2015 to 2016, moxifloxacin resistance decreased in all centers. The proportion of BI isolates decreased from 25.5% in 2011 to 2012 to 12.8% in 2015 to 2016 (P < 0.001). The BI REA group correlated with moxifloxacin resistance (BI 84% resistant versus non-BI 12.5% resistant). Fidaxomicin MICs have not changed among C. difficile isolates of U.S. origin over 5 years post licensure. There has been an overall decrease in MICs for vancomycin, metronidazole, moxifloxacin, and rifampin and an increase in isolates resistant to imipenem. Moxifloxacin resistance remained high among the BI REA group, but the proportion of BI isolates has decreased. Continued geographic variations in REA groups and antimicrobial resistance persist.

U.S.-Based National Sentinel Surveillance Study for the Epidemiology of Clostridium difficile-Associated Diarrheal Isolates and Their Susceptibility to Fidaxomicin.

In 2011 a surveillance study for the susceptibility to fidaxomicin and epidemiology of Clostridium difficile isolates in the United States was undertaken in seven geographically dispersed medical centers. This report encompasses baseline surveillance in 2011 and 2012 on 925 isolates. A convenience sample of C. difficile isolates or toxin positive stools from patients were referred to a central laboratory. Antimicrobial susceptibility was determined by agar dilution (CLSI M11-A8). Clinical and Laboratory Standards Institute (CLSI), Food and Drug Administration, or European Union of Clinical Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied where applicable. Toxin gene profiles were characterized by multiplex PCR on each isolate. A random sample of 322 strains, stratified by institution, underwent restriction endonuclease analysis (REA). The fidaxomicin MIC90 was 0.5 µg/ml for all isolates regardless of REA type or toxin gene profile, and all isolates were inhibited at ≤1.0 µg/ml. By REA typing, BI strains represented 25.5% of the isolates. The toxin gene profile of tcdA, tcdB, and cdtA/B positive with a tcdC 18-bp deletion correlated with BI REA group. Moxifloxacin and clindamycin resistance was increased among either BI or binary toxin-positive isolates. Metronidazole and vancomycin showed reduced susceptibility (EUCAST criteria) in these isolates. Geographic variations in susceptibility, REA group and binary toxin gene presence were observed. Fidaxomicin activity against C. difficile isolated in a national surveillance study did not change more than 1 year after licensure. This analysis provides baseline results for future comparisons.

Acceptability of low-volume, induced sputum specimens for diagnosis of Pneumocystis carinii pneumonia.

Two milliliters of induced sputum is the minimum volume recommended for microscopic examination for Pneumocystis carinii by direct fluorescent antibody (DFA) stain. Many specimens received in our laboratory do not meet this criterion. Rejection of these inadequate specimens increases cost and discomfort to the patient because additional specimens must be obtained. To determine whether volumes less than 2 mL were acceptable for microscopic analysis, we examined 177 consecutive induced sputum specimens submitted for P. carinii DFA stain. Eighty-four (47.4%) specimens were less (0.5-1.9 mL) than the 2-mL volume recommended by the manufacturer. Overall, 33 specimens were positive. The positivity rates for specimens 2 mL or more and less than 2 mL were 18% and 19%, respectively. Induced sputum volumes between 0.5 and 2 mL may be acceptable for DFA examination.

Critical assessment of gene amplification approaches on the diagnosis of tuberculosis.

The resurgence of tuberculosis prompted the development of a number of new options for the rapid laboratory diagnosis of Mycobacterium tuberculosis (MTB). One of the most promising and exciting methodologies has been the introduction of assays employing amplification technology to detect MTB directly in clinical specimens. Although amplification assays hold significant promise to improve the laboratory diagnosis of tuberculosis, the decision to perform or not perform these assays is complicated. The performance of in-house polymerase chain reaction (PCR) assays and two commercially-prepared assays. GenProbe's AMTD test and Roche's AMPLICOR PCR assay are reviewed. Regardless of the amplification format, all assays have decreased sensitivity with specimens that are acidfast bacilli (AFB) stain-negative. Data from these studies and others indicate possible potential pitfalls of amplification assays, those being sampling errors, the presence of substances in clinical specimens that inhibit the amplification assay, and clinical utility. In light of these findings, the possible roles for these assays in the clinical microbiology laboratory are reviewed. In addition, factors such as cost, assay performance, etc. are discussed in order to facilitate the decision-making process concerning whether an amplification assay would be appropriate in a particular laboratory setting.

Substances interfering with direct detection of Mycobacterium tuberculosis in clinical specimens by PCR: effects of bovine serum albumin.

Interfering substances have been reported to inhibit PCR assays for the direct detection of Mycobacterium tuberculosis in clinical specimens. Using an internal control, we determined that 52% of respiratory specimens interfered with our PCR assay. On the basis of these findings, we tried to circumvent the problem by simply diluting prepared sediments. With sediment from a routinely processed sputum known to be inhibitory to PCR, one aliquot was prepared in a routine manner for PCR. Remaining sediment was diluted in phosphate-buffered saline, Middlebrook 7H10 broth, or BACTEC 12B broth; an internal control was added to all reaction mixtures and controls. Internal control was detected only in the sample diluted with BACTEC 12B medium. Components of the BACTEC 12B medium including PANTA reagent (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin), reconstituting fluid, 0.2% glycerol, 0.05% Tween 80, and 0.05% bovine serum albumin (BSA) were tested in a similar manner. Only 0.05% BSA resulted in amplification of the internal control DNA. Varying concentrations of BSA were added to 11 aliquots of a respiratory sediment known to be inhibitory to the PCR. Internal control was detected in all reaction mixtures containing 0.00038 to 0.1% BSA. To determine the ability of BSA to override inhibition, respiratory specimens were run in triplicate: undiluted, diluted 1:2 with BACTEC 12B medium, or diluted with 0.026% BSA. For 21 of 22 inhibitory specimens, BSA was able to override the presence of interfering substances. These data suggest that the presence of BSA in a PCR assay is critical for the direct detection of M. tuberculosis in respiratory specimens.

Evaluation of the polymerase chain reaction for the detection of human papillomavirus from urine.

The ability to detect the presence of human papillomavirus (HPV)-DNA sequences in urine was evaluated using polymerase chain reaction (PCR). DNA was purified and extracted from urine samples, and subjected to 40 cycles of amplification using the consensus primer pair MY11 and MY09. Coamplification using the beta-globin primers, GH20 and PC04, was performed as an internal reaction control. Following assay optimization, urine samples from 22 women undergoing examination for cervical dysplasia were tested for the presence of HPV-DNA. PCR assay results were correlated with cytologic and histologic findings as well as ViraType assay results. Overall, HPV was detected by PCR in 16 (76%) of the interpretable samples. HPV sequences were detected in 13 (87%) of the 15 specimens from women showing evidence of condylomata, dysplasia, or invasive carcinoma. HPV was detected in 3 (50%) of the women whose cytologic or histologic results were either negative or showed benign atypia. Although the sample size in this study is small, our results show that HPV can be detected by PCR in a majority of individuals showing evidence of HPV infection. The method described provides a means for the clinical laboratory to detect a broad range of HPV types from using a sample obtained by noninvasive techniques. The ability to easily obtain urine would allow for increased numbers of individuals to be tested, and thus, aid in our understanding of HPV.

Ability of PCR assay to identify Mycobacterium tuberculosis in BACTEC 12B vials.

Introduction of PCR to directly detect Mycobacterium tuberculosis in clinical specimens has shown promise; however, interfering substances in clinical material have contributed to lowered assay sensitivities. We evaluated the ability of a PCR assay to detect M. tuberculosis in BACTEC 12B broth cultures. Clinical specimens were processed and inoculated into BACTEC 12B vials. Evaluation was approached in two phases, starting with an initial evaluation in which an aliquot of 12B broth was removed when the growth index (GI) was > or = 10 and stored at 4 degrees C until assayed by PCR. Of the 290 specimens initially assayed, 129 were culture negative for mycobacteria as well as PCR negative for M. tuberculosis. Except for one, cultures (n = 102) which grew mycobacteria other than M. tuberculosis were all PCR negative. The remaining 59 broths were all culture and PCR positive for M. tuberculosis; 39% (n = 23) of these cultures when assayed by PCR had GIs of < or = 50. Following initial evaluation, 200 12B BACTEC vials with GIs of > or = 10 were assayed in a similar manner except that specimens were amplified twice weekly to determine PCR's impact on the length of time to identification of M. tuberculosis as compared with standard laboratory practices. Utilization of PCR resulted in a mean time to detection of M. tuberculosis of 14 days, compared with 29 days by using commercially available nucleic acid probes to identify M. tuberculosis complex from growth of BACTEC 12B subcultures on solid media. In light of an overall sensitivity and specificity of 100 and 99.7%, respectively, coupled with the ability to identify M. tuberculosis days or weeks before other methods can be applied, we conclude that PCR might prove to be a rapid alternative for identification of M. tuberculosis in culture and allow for earlier setup of susceptibility testing.

Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction.

The emergence of epidemic multiple-drug-resistant (MDR) strains of Mycobacterium tuberculosis in conjunction with an increase in the number of reported cases of tuberculosis (TB) represents a major public health problem. In light of a recent outbreak of MDR M. tuberculosis at our center, we began the development of a polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary TB using two sets of primers, one based on the IS6110 repeated sequence of M. tuberculosis and the other based on the protein antigen b (PAB). Reaction conditions were first optimized as to the appropriate extraction protocol and the concentrations of primer pairs, nucleotides, and MgCl2. Following a preliminary evaluation of the assay with clinical specimens, extraction and amplification procedures were further modified. PAB and IS6110 primers detected between 2 and 23 and 0.023 and 0.23 CFU of M. tuberculosis, respectively, in pooled, M. tuberculosis-negative sputa by our optimized PCR assay. After routine processing for mycobacteria, 734 specimens were subsequently amplified. DNA for amplification was obtained by boiling and beating the sediments with Tween 20. For each reaction, DNA (10 microliters) was added to an amplification mixture containing 12 pmol of IS6110 primers, 20 pmol of PAB primers, 2 mM MgCl2, 200 microM nucleotides, and 2.5 U of Taq polymerase and the mixture was then amplified for 40 cycles. The sensitivity and specificity of our PCR assay were 87.2 and 97.7%, respectively. We were unable to interpret the results for seven specimens (1%). In our experience, PCR proved to be a useful rapid diagnostic test for TB in a clinical setting and a valuable epidemiological tool for determining exposure groups in the hospital setting. Our findings also underscore the need for the systematic optimization of PCR assay conditions.

Immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue sections. A comparison with genotypic analysis.

Several monoclonal antibodies (MoAbs) are now available for immunophenotyping non-Hodgkin's lymphomas (NHLs) in paraffin-embedded tissue sections. To determine the reliability of these reagents in predicting the genotype, 44 cases of NHL were studied with the alkaline phosphatase-anti-alkaline phosphatase technique with the use of the following MoAbs: leukocyte common antigen (CD45), Mac 387, L26, 4KB5, MB1, MB2, LN2, UCHL1, MT1, and MT2. The lineage of the neoplastic cells was determined in all cases by gene rearrangement studies for immunoglobulin heavy chain and for the T-cell receptor beta-chain. Genotypic results showed B-cell lineage in 33 cases (75%), T-cell lineage in 6 cases (14%), and mixed or undetermined lineage in 5 cases (11%). A concordance of lineage assignment by paraffin section immunophenotyping with gene rearrangement studies was observed in 37 of 39 (95%) lymphomas with an unequivocally defined genotype. MoAb L26 was the most sensitive in detecting B-cell genotype; MoAbs MT1 and UCHL1 were the most sensitive and specific, respectively, in detecting T-cell genotype. The authors conclude that lineage assignment of NHLs in paraffin sections is reflective of the corresponding genotype when an appropriate panel of MoAbs is used.

Evaluation of two commercially available nucleic acid hybridization assays for the detection and typing of human papillomavirus in clinical specimens.

Southern blot (Oncor, Gaithersburg, MD) and dot blot (Life Technologies, Gaithersburg, MD) nucleic acid hybridization assays were compared for their ability to detect and type human papillomavirus (HPV) DNA in 50 cervical swab specimens and 11 biopsy specimens. Overall agreement between the two methods was 78.7%. With the use of Southern blot analysis, HPV 6, 11, 16, or 18 was detected in 22 specimens, however, 4 were untypable because of abnormal or smeared band patterns. Dot blot analysis detected HPV 6/11, 16/18, or 31/33/35 in those same 22 specimens and in 9 additional specimens. Eight of the 13 specimens in which HPV was not detected or untypable by Southern blot contained type 31/33/35 by dot blot. Based on convenience of specimen collection and transport, ease of performance and the ability to detect HPV types 31, 33, and 35, the authors are currently using the dot blot assay for the detection and typing of HPV in clinical specimens.

The effects of a promoter of cell differentiation and selected hormones on human cytomegalovirus infection using an in vitro cell system.

The influence of factors that can regulate cellular developmental or metabolic processes in host tissue on cytomegalovirus (CMV) replication in vitro was determined. Hydrocortisone treatment of cells before viral infection resulted in a 12- to 13-fold increase in the expression of immediate early proteins at 4 h after virus inoculation. The addition of a phorbol diester 1.5 h after CMV infection resulted in an 8- to 13-fold increase in production of viral progeny. In contrast, beta-human chorionic gonadotropin treatment generally resulted in a 25%-72% suppression of both CMV-specific proteins and progeny. Effects on CMV infection with either progesterone or estradiol were minor and generally suppressive. The stimulating or suppressive effects of these factors on CMV replication in vitro may be important to CMV reactivation in humans. Further study of regulatory factors may lead to the development of therapeutic approaches to the prevention of CMV reactivation in patients at risk for severe disease.

Successful use of shell vial centrifugation and 16 to 18-hour immunofluorescent staining for the detection of influenza A and B in clinical specimens.

The rapid diagnosis of influenza A and B infections is beneficial for the proper management of patients with acute respiratory illness. The authors evaluated a shell vial centrifugation method to detect these viruses 16-18 hours postinoculation and compared it with conventional tube cell culture. Rhesus monkey kidney cells were used in both methods. Conventional culture of 334 respiratory specimens recovered 64 influenza isolates; the average time to positivity was 4.1 days. Low-speed shell vial centrifugation with polyclonal immunofluorescent staining 16-18 hours postinoculation was performed on 96 fresh specimens and on an additional 38 frozen specimens. These 134 specimens contained 49 of the 64 total influenza-positive specimens. The shell vial method yielded a sensitivity of 90.9% and 87.5% for fresh and frozen specimens, respectively, as compared with conventional tube cell culture. The authors conclude that the shell vial method is an important adjunct to conventional culture for the rapid detection of influenza A and B in clinical specimens.

Detection of specific IgG and IgA antibodies to Chlamydia trachomatis in women with salpingitis confirmed by laparoscopy.

The sera from 12 consecutive symptomatic women with laparoscopy-confirmed salpingitis were screened for the presence of specific IgG and IgA antibodies to Chlamydia trachomatis by a single antigen (L-2) immunoperoxidase assay. All women were found to have IgG and IgA antibodies to C trachomatis. Six women had positive endocervical cultures for C trachomatis, and one of these had positive cultures from the conjunctiva and fallopian tubes. Serum chlamydial IgA antibodies may serve as markers for active infection with C trachomatis regardless of whether organisms can be identified by culture or direct fluorescent antibody techniques from endocervical or fallopian tube samples.

Acquisition of cytomegalovirus infection: an update.

Human cytomegalovirus (CMV) is a ubiquitous deoxyribonucleic acid virus that commonly infects a majority of individuals at some time during their life. Although most of these CMV infections are asymptomatic, certain patient groups are at risk to develop serious illness. Understanding the epidemiology of this virus is a key element in the development of strategies for preventing CMV disease. However, a number of features of this virus complicate such understanding. Following infection, CMV can remain latent, with subsequent reactivation; the factors controlling latency and reactivation and those factors which determine whether a CMV infection will be symptomatic are unknown. CMV disease can be acquired by natural routes, including horizontal and vertical transmission. Due to the ubiquity of CMV, the delineation of CMV transmission by these natural routes is complicated by the myriad of possible sources. Moreover, concerns over the risk of CMV transmission to the seronegative pregnant female have been raised in relation to preventing CMV transmission. By using molecular biologic techniques, much knowledge has been gained regarding the transmission of CMV disease by natural routes; however, a number of questions remain unanswered. The transmission of CMV infection by natural routes is therefore reviewed and the issues are highlighted. Primary infection, reactivation, and reinfection are the types of active CMV infections that can occur in an immunocompromised patient. In addition to natural routes of infection, introduction of presumably latently infected organs and requirements for multiple blood transfusions increase potential exposure to CMV in the immunocompromised patient. Understanding the epidemiology of CMV infections in the immunocompromised patient is difficult and in some instances controversial due to the complexity and interdependency of a number of factors which lead to CMV infection. In an immunocompromised individual, a major risk factor in developing overt CMV-related disease is associated with the serological status of an organ donor, the recipient, and the blood product given to these patients. In addition, a large body of inferential data supports the transmission of CMV by blood products or organs from seropositive donors; however, the mechanisms by which transmission occurs remain unclear. The possible sources and mechanisms of transmission of CMV infections in the immunocompromised host are reviewed. Lastly, strategies for the ultimate prevention of CMV disease are discussed in light of the epidemiology of CMV infections. To date, these strategies have included use of CMV-seronegative blood products or organs, antiviral agents, and vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of cytomegalovirus-specific IGM in renal transplant recipients.

We have compared two IgM-specific cytomegalovirus (CMV) antibody assays, an immunofluorescence assay (IFA-M) and an enzyme-linked antigen immunoassay (ELA-M), with an assay for CMV total antibody (ELISA) and viral culture for the detection of active CMV infection in renal transplant recipients. Of 75 patients (49 ELISA negative pretransplant, 26 ELISA positive), CMV-specific IgM was detected in 35 (27 ELISA negative pretransplant, 8 ELISA positive) using the IFA-M assay and in 25 (16 ELISA negative pretransplant, 9 ELISA positive) using the ELA-M test. Of the 25 patients identified as positive by ELA-M, 21 had positive viral cultures post-transplant, two seronegative patients had evidence of infection indicated by post-transplant seroconversion, and two patients were seropositive pretransplant but remained viral culture negative throughout the follow-up period. ELA-M and CMV total antibody ELISA detected primary infection in renal transplant recipients equally well, but ELA-M was found to be superior to ELISA and IFA-M for detecting reinfection and reactivation infections.

Prevalence of IgA and IgG antichlamydial antibodies in women in the third trimester of pregnancy.

The prevalence of serum antichlamydial IgA and IgG antibodies was investigated by screening 77 randomly selected patients who were in the third trimester of pregnancy. An indirect immunoperoxidase assay that quantitates IgA and IgG was used for screening. Twenty-five women had both IgA and IgG antibodies; an additional ten women had only IgG antibodies. These findings suggest that greater than 45 percent of pregnant women tested had been exposed to Chlamydia trachomatis, and more than 32 percent had evidence of active infection.

Detection of cytomegalovirus in clinical specimens using shell vial centrifugation and conventional cell culture.

Shell vial centrifugation and conventional cell culture methods for detecting cytomegalovirus (CMV) were compared in clinical specimens. Our data confirm that the shell vial centrifugation method is more sensitive and rapid than conventional cell culture; however, due to the shell vial method's problems with toxicity to the fibroblast monolayer, both methods must be performed if all specimens positive for CMV are to be detected.

A comparison of culture, direct fluorescent antibody test, and a quantitative indirect immunoperoxidase assay for detection of Chlamydia trachomatis in pregnant women.

An indirect immunoperoxidase assay that quantitates antichlamydial immunoglobulin G (IgG) and IgA antibodies in serum was compared with endocervical culture and direct fluorescent antibody (MicroTrak) for detection of Chlamydia trachomatis in asymptomatic pregnant women. Of the 64 women tested by the three methods, 22 (34%) had antichlamydial IgG and IgA in their serum. The culture was positive in nine patients (14%) and the MicroTrak was positive in eight (12.5%). All positive cultures were immunoperoxidase-positive. One positive MicroTrak was immunoperoxidase- and culture-negative. Thirteen patients had IgG and IgA antichlamydial antibodies with no organisms detected in the endocervix by culture or by direct fluorescent antibody screen.