Optimized Protocol for Testing Multipurpose Contact Lens Solution Efficacy Against Acanthamoeba.

OBJECTIVES: To evaluate the interlaboratory and intralaboratory reproducibility of a proposed protocol for multipurpose contact lens solution (MPS) disinfection efficacy against Acanthamoeba. METHODS: Acanthamoeba castellanii and Acanthamoeba polyphaga and four MPS with different biocidal agents were used to evaluate the protocol in two different laboratories. In addition to the negative control, a positive control and neutralization control were used. One experiment was performed in triplicate, and all other experiments were performed in duplicate in each laboratory. Acanthamoeba trophozoites were grown axenically, and cysts were generated using the starvation method. Trophozoites and cysts at a concentration of 2.0 × 10 to 2.0 × 10 organisms per milliliter were exposed to the test MPS for 0, 4 or 6 (manufacturer's recommended soak time [MRST]), 8, and 24 hr. Survivors were determined by a limiting dilution method that used a most probable number evaluation. RESULTS: The positive and negative controls displayed consistent results and trends both within each laboratory and between each laboratory for trophozoites and cysts of both A. castellanii and A. polyphaga. The neutralization control consistently demonstrated the ability of the neutralizing agents to neutralize the MPS and the positive control and demonstrated no inhibition of Acanthamoeba by the negative control. Testing in triplicate and duplicate demonstrated the reproducibility of the protocol both within each laboratory and between the laboratories. Our results demonstrated that the MPS at the MRST and at 8 hr (likely overnight soak time) are generally more effective against trophozoites than they are against cysts. Only the MPS with hydrogen peroxide as the biocidal agent was able to provide a greater than three-log kill of cysts at the MRST and longer. Among the MPS we tested, trophozoites of A. castellanii and A. polyphaga showed similar responses. Some variability was observed when testing cysts of both species. In both laboratories, one nonhydrogen peroxide containing MPS had some effect (>1 log kill) on A. polyphaga cysts. This solution had no effect (<1 log kill) on A. castellanii cysts, A. castellanii trophozoites, and A. polyphaga trophozoites. CONCLUSIONS: The protocol that we have revised and evaluated is a well-controlled and reproducible procedure that can effectively evaluate the efficacy of MPS against Acanthamoeba trophozoites. Some variability was observed when testing the cyst stage.

The utility of bronchoalveolar lavage beta-D-glucan testing for the diagnosis of invasive fungal infections.

OBJECTIVES: To investigate the utility of beta-D-glucan (BDG) testing in bronchoalveolar lavage (BAL) fluid for the diagnosis of invasive fungal infection (IFI), as compared to BAL galactomannan (GM). METHODS: We retrospectively reviewed medical records of 132 consecutive patients at the National Institutes of Health (NIH) in whom BAL BDG testing was performed for diagnosis of pneumonia. Using the European Organization for Research and Treatment of Cancer/Mycoses Study Group guidelines, we determined which patients had proven or probable IFI, and assessed the diagnostic performance of BAL BDG testing, relative to BAL GM. We also determined the reproducibility of the BDG assay in BAL via repeat testing of patient samples. RESULTS: Ten patients had Pneumocystis pneumonia, and 34 patients had proven/probable IFI, including 14 with invasive aspergillosis (IA). BAL BDG was 100% sensitive for Pneumocystis. Although BAL BDG had similar sensitivity to BAL GM for the diagnosis of IA and IFI, it exhibited inferior specificity. Repeat testing demonstrated poor reproducibility of the BDG assay in BAL but not in serum. CONCLUSIONS: BDG testing exhibits poor specificity and reproducibility in BAL. Identification of the BAL-specific factors that may interfere with the performance of the assay could improve the clinical usefulness of BAL BDG testing.

Peripheral blood stem cell transplant-related Plasmodium falciparum infection in a patient with sickle cell disease.

BACKGROUND: Although transmission of Plasmodium falciparum (Pf) infection during red blood cell (RBC) transfusion from an infected donor has been well documented, malaria parasites are not known to infect hematopoietic stem cells. We report a case of Pf infection in a patient 11 days after peripheral blood stem cell transplant for sickle cell disease. STUDY DESIGN AND METHODS: Malaria parasites were detected in thick blood smears by Giemsa staining. Pf HRP2 antigen was measured by enzyme-linked immunosorbent assay on whole blood and plasma. Pf DNA was detected in whole blood and stem cell retention samples by real-time polymerase chain reaction using Pf species-specific primers and probes. Genotyping of eight Pf microsatellites was performed on genomic DNA extracted from whole blood. RESULTS: Pf was not detected by molecular, serologic, or parasitologic means in samples from the recipient until Day 11 posttransplant, coincident with the onset of symptoms. In contrast, Pf antigen was retrospectively detected in stored plasma collected 3 months before transplant from the asymptomatic donor. Pf DNA was detected in whole blood from both the donor and the recipient after transplant, and genotyping confirmed shared markers between donor and recipient Pf strains. Lookback analysis of RBC donors was negative for Pf infection. CONCLUSIONS: These findings are consistent with transmission by the stem cell product and have profound implications with respect to the screening of potential stem cell donors and recipients from malaria-endemic regions.

Use of adherence monitors as part of a team approach to control clonal spread of multidrug-resistant Acinetobacter baumannii in a research hospital.

BACKGROUND: Multidrug-resistant Acinetobacter baumannii (MDRAB) is difficult to treat and eradicate. Several reports describe isolation and environmental cleaning strategies that controlled hospital MDRAB outbreaks. Such interventions were insufficient to interrupt MDRAB transmission in 2 intensive care unit-based outbreaks in our hospital. We describe strategies that were associated with termination of MDRAB outbreaks at the National Institutes of Health Clinical Center. METHODS: In response to MDRAB outbreaks in 2007 and 2009, we implemented multiple interventions, including stakeholder meetings, enhanced isolation precautions, active microbial surveillance, cohorting, and extensive environmental cleaning. We conducted a case-control study to analyze risk factors for acquiring MDRAB. In each outbreak, infection control adherence monitors were placed in MDRAB cohort areas to observe and correct staff infection control behavior. RESULTS: Between May 2007 and December 2009, 63 patients acquired nosocomial MDRAB; 57 (90%) acquired 1 or more of 4 outbreak strains. Of 347 environmental cultures, only 2 grew outbreak strains of MDRAB from areas other than MDRAB patient rooms. Adherence monitors recorded 1,330 isolation room entries in 2007, of which 8% required interventions. In 2009, around-the-clock monitors recorded 4,892 staff observations, including 127 (2.6%) instances of nonadherence with precautions, requiring 68 interventions (1.4%). Physicians were responsible for more violations than other staff (58% of hand hygiene violations and 37% of violations relating to gown and glove use). Each outbreak terminated in temporal association with initiation of adherence monitoring. CONCLUSIONS: Although labor intensive, adherence monitoring may be useful as part of a multifaceted strategy to limit nosocomial transmission of MDRAB.

Disseminated Strongyloides stercoralis infection in HTLV-1-associated adult T-cell leukemia/lymphoma.

A 55-year-old woman with human T-cell lymphotropic virus type-1 (HTLV-1)-associated adult T-cell leukemia (ATL) and a history of previously treated Strongyloides stercoralis infection received anti-CD52 monoclonal antibody therapy with alemtuzumab on a clinical trial. After an initial response, she developed ocular involvement by ATL. Alemtuzumab was stopped and high-dose corticosteroid therapy was started to palliate her ocular symptoms. Ten days later, the patient developed diarrhea, vomiting, fever, cough, skin rash, and a deteriorating mental status. She was diagnosed with disseminated S. stercoralis. Corticosteroids were discontinued and the patient received anthelmintic therapy with ivermectin and albendazole with complete clinical recovery.

A species-specific approach to the use of non-antimony treatments for cutaneous leishmaniasis.

We used a species-specific approach to treat 10 patients with cutaneous leishmaniasis diagnosed using polymerase chain reaction. Non-antimony treatments (oral miltefosine, ketoconazole, and liposomal amphotericin B) were chosen as an alternative to pentavalent antimony drugs based on likely or proven drug efficacy against the infecting species. Leishmania Viannia panamensis was diagnosed in three patients and treated successfully with oral ketoconazole. Miltefosine treatment cured two patients with L. infantum chagasi. A wide variety of Leishmania responded to liposomal amphotericin B administered for 5-7 days. Three patients with L. V. braziliensis, one patient with L. tropica, and two patients with L. infantum chagasi were treated successfully. One person with L. V. braziliensis healed slowly because of a resistant bacterial superinfection, and a second patient with L. infantum chagasi relapsed and was retreated with miltefosine. These drugs were reasonably well-tolerated. In this limited case series, alternative non-antimony-based regimens were convenient, safe, and effective.

Legionella feeleii serotype 2 pneumonia in a man with chronic lymphocytic leukemia: a challenging diagnosis.

Legionella feeleii has rarely been reported as causing pneumonia in patients with hematologic malignancies. We present a case of Legionella feeleii serotype 2 pneumonia with empyema in a man with chronic lymphocytic leukemia and describe the methods of identifying this organism using both standard methods and newer diagnostic techniques.

An enhanced method for the identification of Leishmania spp. using real-time polymerase chain reaction and sequence analysis of the 7SL RNA gene region.

The accurate identification of Leishmania spp. is important for the treatment of infected patients. Molecular methods offer an alternative to time-consuming traditional laboratory techniques for species determination. We redesigned a 7SL RNA gene-based polymerase chain reaction and sequence assay for increased species identification. DNA extracted from 17 reference strains and 10 cultured clinical isolates was examined. Sequence comparison was used successfully to identify organisms to the complex level with intercomplex similarity ranging from 77.5% to 98.4%. Many species within each complex were discriminated accurately by this method including Leishmania major, Leishmania tropica, Leishmania aethiopica, Leishmania guyanensis, and the previously indistinguishable Leishmania braziliensis and Leishmania panamensis. The Leishmania donovani complex members remain indistinguishable by this method, as are the representatives of Leishmania amazonensis/Leishmania garnhami and Leishmania mexicana/Leishmania pifanoi.

Loss of erythromycin resistance genes from strains of Streptococcus pyogenes that have developed resistance to levofloxacin.

In the past 2 to 3 decades, erythromycin resistance in Streptococcus pyogenes has been decreasing, whereas fluoroquinolone resistance (or reduction in its susceptibility) has been reported often. Although a shift of M-type prevalence and decreased pressure from macrolides have been suggested for the decrease in erythromycin resistance, we hypothesized that this might also be a result of increased antimicrobial pressure from fluoroquinolone use. Levofloxacin resistance for 4 erythromycin-resistant parent strains was induced in vitro. Their mutants became highly resistant to the fluoroquinolones but lost their erythromycin resistance trait. Erythromycin resistance was fully restored by transconjugation with respective parent strains with either mefA- or ermTR-mediated mechanisms.

An intrinsic pattern of reduced susceptibility to fluoroquinolones in pediatric isolates of Streptococcus pyogenes.

A total of 116 clinical isolates collected in 2003 from a tertiary pediatric hospital and a primary pediatric department in Chicago, IL, were screened for reduced susceptibility to selected fluoroquinolones by disc diffusion. Correlation between reduced susceptibility and point mutations in the quinolone resistance-determining region of parC and gyrA genes was evaluated, and point mutations were compared with other reports of isolates derived from adult or mixed patient populations. Nine percent of isolates had reduced susceptibility to 1 or more of these fluoroquinolones by Etest: ciprofloxacin, levofloxacin, and moxifloxacin. A single point mutation (Ser-79) in parC seemed responsible for the reduced susceptibility. Resistant Streptococcus pyogenes isolates were compared using M/emm type, repetitive sequence-based PCR (rep-PCR), and pulsed-field gel electrophoresis (PFGE). Rep-PCR provided no more separation of strains than M/emm typing, and PFGE results with SgrAI were more discriminatory than with SmaI. The majority of these isolates were M/emm type 6. PFGE analysis using SgrAI demonstrated 2 different resistant strains among the M/emm type 6 isolates. The findings suggest that a population of S. pyogenes with an intrinsic reduced susceptibility to fluoroquinolones exists in pediatric clinical isolates. Monitoring of amino acid changes in both parC and gyrA will assist in the prediction of emergence of high-level fluoroquinolone resistance.

Identification and antimicrobial susceptibility of lactic acid bacteria from retail fermented foods.

One important safety criterion of using lactic acid bacteria (LAB) in food applications is to ensure that they do not carry transferable antimicrobial resistance (AR) determinants. In this study, 63 LAB belonging to six genera, Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Leuconostoc, and Pediococcus, were recovered from 28 retail fermented food products in Maryland, identified to species with 16S-23S rRNA spacer PCRs, and characterized for antimicrobial susceptibility against eight antimicrobials. Besides intrinsic resistance to ciprofloxacin or vancomycin in some lactobacilli, tetracycline resistance was observed in two Streptococcus thermophilus isolates from one cheese and one sour cream sample and was associated with the presence of a nonconjugative tet(S) gene. The results indicated a low level of AR among naturally occurring and starter LAB cultures in fermented dairy and meat products in the United States; therefore, the probability for foodborne LAB to serve as reservoirs of AR is low. Further studies involving a larger sample size are needed to assess the potential risk of AR gene transfer from LAB in fermented food products.

Detection of vaccinia virus DNA, but not infectious virus, in the blood of smallpox vaccine recipients.

The authors of a recent study [Savona MR, Dela Cruz WP, Jones MS, Thornton JA, Xia D, Hadfield TL, et al. Detection of vaccinia DNA in the blood following smallpox vaccination. JAMA 2006; 295:1898-1900] suggested that the duration of deferral for blood donations by smallpox vaccinees should be extended, based on detection of vaccinia virus DNA in five blood samples by polymerase chain reaction (PCR) and the potential for viremia. We found that 4 of 202 blood specimens (from 3 of 27 smallpox vaccinees) were positive for vaccinia virus DNA by PCR; none were positive for virus by culture. Throat swabs were negative by PCR and culture. Thus, while some blood specimens contained vaccinia virus DNA, infectious virus was not detected. Current guidelines for deferral of blood donation in vaccinees seem appropriate.

In vitro induction and selection of fluoroquinolone-resistant mutants of Streptococcus pyogenes strains with multiple emm types.

OBJECTIVES: To perform a systematic analysis of point mutations in the quinolone resistance determining regions (QRDRs) of the DNA gyrase and topoisomerase genes of emm type 6 and other emm types of Streptococcus pyogenes strains after in vitro exposure to stepwise increasing concentrations of levofloxacin. METHODS: Twelve parent strains of S. pyogenes, each with a different emm type, were chosen for stepwise exposure to increasing levels of levofloxacin followed by selection of resistant mutants. The QRDRs of gyrA, gyrB, parC and parE correlating to mutants with increased MICs were analysed for point mutations. RESULTS: Multiple mutants with significantly increased MICs were generated from each strain. The amino acid substitutions identified were consistent regardless of emm type and were similar to the mechanisms of resistance reported in clinical isolates of S. pyogenes. The number of induction/selection cycles required for the emergence of key point mutations in gyrA and parC was variable among strains. For each parent-mutant set, when MIC increased, serine-81 of gyrA and serine-79 of parC were the primary targets for amino acid substitutions. No point mutations were found in the QRDRs of gyrB and parE in any of the resistant mutants sequenced. CONCLUSIONS: Despite its intrinsic polymorphism in the QRDR of parC, emm type 6 is not more likely to develop high-level resistance to fluoroquinolones when compared with other emm types. All emm types seem equally inducible to high-level fluoroquinolone resistance.

Comparison of methods for detection of vaccinia virus in patient specimens.

We analyzed a shell vial culture assay (SVA), real-time PCR, and a direct fluorescent antibody assay (DFA) for rapid detection of vaccinia virus from vaccination sites of Dryvax vaccine recipients. Of 47 samples assayed, 100% were positive by PCR, 89% were positive by SVA, and 40% were positive by DFA. DFA was limited by the need for adequate numbers of cells, with 32% of samples inadequate for interpretation. DFA performed better with specimens from patients who had not previously received the vaccine. PCR was positive for longer times postvaccination than was SVA. Infectious virus could be recovered after 45 min of acetone fixation of shell vial coverslips. Commercially available polyclonal antibodies cross-reacted with other orthopoxviruses and herpes simplex 1, but commercially available monoclonal antibodies were specific for vaccinia virus. In summary, PCR was the most sensitive test for detecting vaccinia virus in clinical specimens, while the DFA was the most rapid but the least sensitive test.

Evaluation of 7SL RNA gene sequences for the identification of Leishmania spp.

We evaluated the use of 7SL RNA gene sequences for the identification of Leishmania spp. A fragment (approximately 137 basepairs) of the 7SL RNA gene from 13 reference strains and 18 clinical isolates of 11 different Leishmania species was amplified and sequenced using conserved primers. Reference strains from each Leishmania spp. complex showed unique sequences. The nucleotide sequences were compared pairwise and a range of 81.0-99.3% intercomplex similarity was observed. Clinical isolates of the same species had sequences identical to the corresponding reference strains; thus, the intraspecies similarity was 100%. A phylogenetic tree derived from the 7SL RNA gene partial sequences was constructed and is in agreement with accepted phylogenetic schemes.

Large-scale screening of nasal swabs for Bacillus anthracis: descriptive summary and discussion of the National Institutes of Health's experience.

In October 2001, a letter containing a large number of anthrax spores was sent through the Brentwood post office in Washington, D.C., to a United States Senate office on Capitol Hill, resulting in contamination in both places. Several thousand people who worked at these sites were screened for spore exposure by collecting nasal swab samples. We describe here a screening protocol which we, as a level A laboratory, used on very short notice to process a large number of specimens (3,936 swabs) in order to report preliminary results as quickly as possible. Six isolates from our screening met preliminary criteria for Bacillus anthracis identification and were referred for definitive testing. Although none of the isolates was later confirmed to be B. anthracis, we studied these isolates further to define their biochemical characteristics and 16S rRNA sequences. Four of the six isolates were identified as Bacillus megaterium, one was identified as Bacillus cereus, and one was an unidentifiable Bacillus sp. Our results suggest that large-scale nasal-swab screening for potential exposure to anthrax spores, particularly if not done immediately postexposure, may not be very effective for detecting B. anthracis but may detect a number of Bacillus spp. that are phenotypically very similar to B. anthracis.

Controversies in Clostridium difficile testing.

Recent reports of two nosocomial outbreaks of Clostridium difficile-associated disease caused by toxin A-deficient strains emphasize that these strains can cause disease. Laboratories using an assay that detects only toxin A as their primary diagnostic test risk misdiagnosis of cases or outbreaks in the institutions they serve. Repeat testing can account for a significant portion of a laboratory's C. difficile testing workload. Published data are available to support laboratory rules for rejection of repeat stool specimens within 7 days of an initial specimen. There are also substantial published data to support laboratory rejection of formed stools sent to the laboratory for C. difficile testing.