Practical Guidance for Clinical Microbiology Laboratories: Mycobacteria.

Mycobacteria are the causative organisms for diseases such as tuberculosis (TB), leprosy, Buruli ulcer, and pulmonary nontuberculous mycobacterial disease, to name the most important ones. In 2015, globally, almost 10 million people developed TB, and almost half a million patients suffered from its multidrug-resistant form. In 2016, a total of 9,287 new TB cases were reported in the United States. In 2015, there were 174,608 new case of leprosy worldwide. India, Brazil, and Indonesia reported the most leprosy cases. In 2015, the World Health Organization reported 2,037 new cases of Buruli ulcer, with most cases being reported in Africa. Pulmonary nontuberculous mycobacterial disease is an emerging public health challenge. The U.S. National Institutes of Health reported an increase from 20 to 47 cases/100,000 persons (or 8.2% per year) of pulmonary nontuberculous mycobacterial disease among adults aged 65 years or older throughout the United States, with 181,037 national annual cases estimated in 2014. This review describes contemporary methods for the laboratory diagnosis of mycobacterial diseases. Furthermore, the review considers the ever-changing health care delivery system and stresses the laboratory's need to adjust and embrace molecular technologies to provide shorter turnaround times and a higher quality of care for the patients who we serve.

Surveillance of carbapenem-resistant Klebsiella pneumoniae: tracking molecular epidemiology and outcomes through a regional network.

Carbapenem resistance in Gram-negative bacteria is on the rise in the United States. A regional network was established to study microbiological and genetic determinants of clinical outcomes in hospitalized patients with carbapenem-resistant (CR) Klebsiella pneumoniae in a prospective, multicenter, observational study. To this end, predefined clinical characteristics and outcomes were recorded and K. pneumoniae isolates were analyzed for strain typing and resistance mechanism determination. In a 14-month period, 251 patients were included. While most of the patients were admitted from long-term care settings, 28% of them were admitted from home. Hospitalizations were prolonged and complicated. Nonsusceptibility to colistin and tigecycline occurred in isolates from 7 and 45% of the patients, respectively. Most of the CR K. pneumoniae isolates belonged to repetitive extragenic palindromic PCR (rep-PCR) types A and B (both sequence type 258) and carried either blaKPC-2 (48%) or blaKPC-3 (51%). One isolate tested positive for blaNDM-1, a sentinel discovery in this region. Important differences between strain types were noted; rep-PCR type B strains were associated with blaKPC-3 (odds ratio [OR], 294; 95% confidence interval [CI], 58 to 2,552; P < 0.001), gentamicin nonsusceptibility (OR, 24; 95% CI, 8.39 to 79.38; P < 0.001), amikacin susceptibility (OR, 11.0; 95% CI, 3.21 to 42.42; P < 0.001), tigecycline nonsusceptibility (OR, 5.34; 95% CI, 1.30 to 36.41; P = 0.018), a shorter length of stay (OR, 0.98; 95% CI, 0.95 to 1.00; P = 0.043), and admission from a skilled-nursing facility (OR, 3.09; 95% CI, 1.26 to 8.08; P = 0.013). Our analysis shows that (i) CR K. pneumoniae is seen primarily in the elderly long-term care population and that (ii) regional monitoring of CR K. pneumoniae reveals insights into molecular characteristics. This work highlights the crucial role of ongoing surveillance of carbapenem resistance determinants.

Evaluation of the Verigene Gram-positive blood culture nucleic acid test for rapid detection of bacteria and resistance determinants.

Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results.

Longitudinal analysis of the temporal evolution of Acinetobacter baumannii strains in Ohio, USA, by using rapid automated typing methods.

Genotyping methods are essential to understand the transmission dynamics of Acinetobacter baumannii. We examined the representative genotypes of A. baumannii at different time periods in select locations in Ohio, using two rapid automated typing methods: PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), a form of multi-locus sequence typing (MLST), and repetitive-sequence-based-PCR (rep-PCR). Our analysis included 122 isolates from 4 referral hospital systems, in 2 urban areas of Ohio. These isolates were associated with outbreaks at 3 different time periods (1996, 2000 and 2005-2007). Type assignments of PCR/ESI-MS and rep-PCR were compared to each other and to worldwide (WW) clone types. The discriminatory power of each method was determined using the Simpson's index of diversity (DI). We observed that PCR/ESI-MS sequence type (ST) 14, corresponding to WW clone 3, predominated in 1996, whereas ST 12 and 14 co-existed in the intermediate period (2000) and ST 10 and 12, belonging to WW clone 2, predominated more recently in 2007. The shift from WW clone 3 to WW clone 2 was accompanied by an increase in carbapenem resistance. The DI was approximately 0.74 for PCR/ESI-MS, 0.88 for rep-PCR and 0.90 for the combination of both typing methods. We conclude that combining rapid automated typing methods such as PCR/ESI-MS and rep-PCR serves to optimally characterize the regional molecular epidemiology of A. baumannii. Our data also sheds light on the changing sequence types in an 11 year period in Northeast Ohio.

Multisite reproducibility of the broth microdilution method for susceptibility testing of Nocardia species.

Antimicrobial susceptibility testing (AST) of clinical isolates of Nocardia is recommended to detect resistance to commonly used antimicrobial agents; such testing is complicated by difficulties in inoculum preparation and test interpretation. In this study, six laboratories performed repetitive broth microdilution testing on single strains of Nocardia brasiliensis, Nocardia cyriacigeorgica, Nocardia farcinica, Nocardia nova, and Nocardia wallacei. For each isolate, a total of 30 microdilution panels from three different lots were tested at most sites. The goal of the study was to determine the inter- and intralaboratory reproducibility of susceptibility testing of this group of isolates. Acceptable agreement (>90% agreement at ±1 dilution of the MIC mode) was found for amikacin, ciprofloxacin, clarithromycin, and moxifloxacin. After eliminating MIC values from single laboratories whose results showed the greatest deviation from those of the remaining laboratories, acceptable agreement was also found for amoxicillin-clavulanic acid, linezolid, minocycline, and tobramycin. Results showed unsatisfactory reproducibility of broth microdilution testing of ceftriaxone with N. cyriacigeorgica and N. wallacei, tigecycline with N. brasiliensis and N. cyriacigeorgica, and sulfonamides with N. farcinica and N. wallacei. N. nova ATCC BAA-2227 is proposed as a quality control organism for AST of Nocardia sp., and the use of a disk diffusion test for sulfisoxazole is proposed as a check of the adequacy of the inoculum and to confirm sulfonamide MIC results.

Increased rate of spontaneous bacterial peritonitis among cirrhotic patients receiving pharmacologic acid suppression.

BACKGROUND & AIMS: Patients with cirrhosis frequently receive proton pump inhibitor (PPI) or H2-receptor antagonist therapies. We investigated whether acid-suppressive therapy is associated with spontaneous bacterial peritonitis (SBP) in cirrhotic patients with ascites. METHODS: We compared data from 65 hospitalized cirrhotic patients with paracentesis-proven SBP, collected from 2006 to 2009, with those of 65 contemporaneous, hospitalized cirrhotic patients without SBP (controls). We evaluated PPI use and analyzed the effects of covariates. RESULTS: Patients with SBP had a significantly higher incidence of recent (past 7 days) PPI use (71%) than controls (42%). Of patients with SBP, 68% had no documented indication for PPI therapy. Based on multivariable logistic regression analysis, subjects who had not taken PPIs in the past 90 days were almost 70% less likely to develop SBP than those who had taken PPIs in the previous 7 days. Subjects who took PPIs within 8 to 90 days before hospitalization were 79% less likely to develop SBP than those who took PPIs within 7 days before hospitalization. There was no significant difference between patients who received no PPI therapy in the previous 90 days versus those who had taken PPIs in the previous 8 to 90 days (P = .58). Hyponatremia was associated significantly with SBP. There were no significant differences in length of hospital stay or 30-day survival for the SBP and control groups. CONCLUSIONS: Pharmacologic acid suppression is associated with SBP in patients with advanced cirrhosis. Prospective studies are needed to determine the mechanism of this association and to determine whether reduced use of PPIs and H2-receptor antagonists reduce the incidence of SBP.

New fecal occult blood tests may improve adherence and mortality rates.

Several new fecal occult blood tests have advantages over older ones when used for colorectal cancer screening. Fecal immunochemical tests can detect antibodies to human globin in the stool and can be used without the dietary restrictions needed with traditional guaiac tests. Although colonoscopy is often considered the gold standard, we hope that these new tests will allow more people to be screened and more cases of colorectal cancer to be detected early.

Progression to bacteremia in critical care patients colonized with methicillin-resistant Staphylococcus aureus expressing Panton-Valentine leukocidin.

The role of Panton-Valentine leukocidin (PVL) in methicillin-resistant Staphylococcus aureus (MRSA) infections is unclear. PVL has been long associated with soft tissue infections and necrotizing pneumonia, but inconsistently with other site infections or mortality. The retrospective cohort study explores the association between PVL and bacteremia in colonized medical intensive care unit (ICU) patients with surveillance isolates and blood cultures. A total of 840 patients were screened by nasal swab, with 266 patients found to be colonized and 46 with bacteremia. Colonization by PVL(+) MRSA increased the odds of bacteremia (odds ratio, 2.40; confidence interval, 1.23-4.57), and invasive infection developed earlier in these patients (relative risk, 0.44; confidence interval 0.25-0.85) compared to those colonized with PVL(0) MRSA. PVL was not associated with infections at other sites, length of ICU stay, or mortality. PVL decreases the time to bacteremia in colonized patients but does not otherwise contribute to disease course or clinical outcome.

The role of Stenotrophomonas maltophilia in refractory chronic rhinosinusitis.

BACKGROUND: Stenotrophomonas maltophilia is a multidrug-resistant Gram-negative bacillus that has been implicated in serious nosocomial infections. This microbe has also been isolated from sinus cultures in refractory chronic rhinosinusitis (CRS). The goal of this study was to elucidate the implications of S. maltophilia-positive cultures in the setting of CRS. The objectives of the study were (1) to define clinical and microbiological characteristics and (2) to assess management strategy and overall outcomes of S. maltophilia-positive sinus cultures in CRS patients. METHODS: A retrospective review was performed of 101 patients over a 5-year period. RESULTS: The mean age was 56.9 years with a female/male ratio of 1.1:1. Previous sinus surgery had been performed in 90.1% of patients. Greater than 97% of patients had been given antibiotics in the previous 6 months. The most common presenting symptom was discolored nasal drainage (76.2%) with endoscopic evidence of pus or crusting in 83.2% of cases. Monotherapy with trimethoprim sulfamethoxazole (TMX) or fluoroquinolones was used in 41 and 26%, respectively. This intervention resulted in overall symptom and endoscopic improvement in 70% of cases. CONCLUSION: The presence of S. maltophilia isolates on culture are associated with clinically relevant symptoms and endoscopic findings in patients with refractory CRS. Despite its multidrug-resistant nature, monotherapy with TMX or fluoroquinolones appears to be effective in providing patients with bacterial infection with symptom relief in a majority of cases.

Nontuberculous mycobacterial blood stream and cardiac infections in patients without HIV infection.

Nontuberculous mycobacteria rarely cause bacteremia in HIV-negative patients. We describe 16 cases, including the first Mycobacterium neoaurum endocarditis. Nine cases were line related. Most patients were immunocompromised secondary to hematologic malignancy or other comorbid conditions. Amikacin had the most reliable in vitro activity. Combination therapy was frequently used. Mortality was 25%.

Pyrosequencing for rapid detection of Mycobacterium tuberculosis resistance to rifampin, isoniazid, and fluoroquinolones.

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.

Solitary Nocardia farcinica brain abscess in an immunocompetent adult mimicking metastatic brain tumor: rapid diagnosis by pyrosequencing and successful treatment.

BACKGROUND: Nocardia brain abscess carries a higher morbidity and mortality rate than other bacterial cerebral abscesses, with reported mortality rates of 55% and 20% in immunocompromised and immunocompetent patients, respectively. To prevent a delay in diagnosis and treatment, an aggressive therapeutic approach is required. In the present study, a rapid and accurate molecular diagnostic approach using pyrosequencing (PS), a semiautomated molecular genotyping method of nucleotide sequencing-by-synthesis, was performed. CASE DESCRIPTION: A 53-year-old man developed word-finding difficulties, followed by confusion and disorientation. On examination, the patient had a mixed aphasia; the receptive component was greater than the expressive component. The remainder of his neurologic examination findings was normal. Gadolinium-enhanced magnetic resonance imaging of the brain revealed a 2.0-cm multilobular, partially cystic, peripheral-enhancing mass in the posterior left temporal-parietal region with significant vasogenic edema and localized mass effect. A detailed laboratory investigation revealed that this patient was immunocompetent. An awake left posterior temporal-parietal craniotomy with cortical motor and speech mapping, using frameless stereotactic image guidance and intraoperative real-time ultrasound, was performed. Frozen section was consistent with cerebral abscess and methenamine silver staining revealed many beaded, thin-branching gram-positive bacilli. Colonies suspicious for Nocardia sp were seen within 2 days, and PCR followed by pyrosequencing (PS) identified Nocardia farcinica. CONCLUSIONS: We report a nocardial cerebral abscess mimicking a metastatic brain tumor, and we demonstrate that PS technology can be used for the accurate and rapid identification of N farcinica isolated from a brain abscess -- facilitating a rapid diagnosis and successful, durable treatment.

Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae isolates possessing blaKPC in the United States.

The presence of plasmid-mediated quinolone resistance genes [i.e., qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA] was evaluated among 42 bla(KPC)-containing Klebsiella pneumoniae isolates collected in the eastern United States. One isolate carried the bla(KPC-3) and qnrB19 genes on the same conjugative plasmid, whereas another carried the bla(KPC-3) and qnrA1 genes on separate plasmids.

Treatment of chronic rhinosinusitis exacerbations due to methicillin-resistant Staphylococcus aureus with mupirocin irrigations.

INTRODUCTION: Chronic rhinosinusitis (CRS) exacerbations due to methicillin-resistant Staphylococcus aureus (MRSA) are routinely encountered. Treatment often involves intravenous antibiotics that provide only transient benefits. Mupirocin has well-recognized antistaphylococcal activity, and its nasal formulation is approved by the Food and Drug Administration for the eradication of nasal colonization with MRSA. OBJECTIVE: The aim of this study was to describe the use of mupirocin nasal irrigations for the treatment of CRS exacerbations due to MRSA. MATERIALS AND METHODS: Charts of patients who received mupirocin nasal irrigations for MRSA exacerbations of CRS between January 2000 and October 2003 were reviewed. RESULTS: Forty-two MRSA-positive cultures were obtained from 24 patients (mean age, 61 years; range, 38-82 years; 15 women and 6 men). Twenty-eight episodes were treated with mupirocin nasal irrigations and doxycycline; 4 were treated with mupirocin nasal irrigations and trimethoprim-sulfamethoxazole, and 7 episodes were treated with mupirocin nasal irrigations alone. Patients were reevaluated at approximately 4 to 6 weeks. Repeat cultures were not obtained in 12 patients (because of clinical and endoscopic resolution). Adequate follow-up was unavailable for 3 patients, and of the 27 repeat cultures, only 1 grew MRSA. Twelve patients had at least one recurrence, with a mean number of episodes of 1.75 (range, 1-8 episodes). The mean follow-up was 11.8 months (range, 3-27 months). CONCLUSIONS: Mupirocin nasal irrigations may avoid the need for intravenous antibiotics, which often provide temporary benefits and entail greater cost and morbidity. Thus, mupirocin nasal irrigations may provide a relatively simple means for the management of MRSA exacerbations of CRS.

Daptomycin-resistant Enterococcus faecium in a patient with acute myeloid leukemia.

Daptomycin is a lipopeptide antimicrobial used for the treatment of aerobic gram-positive skin and soft tissue infections. We describe a patient with acute myeloid leukemia whose febrile neutropenia was treated with daptomycin and who later developed daptomycin-resistant Enterococcus faecium infection. Defervescence and negative blood cultures ensued after treatment with linezolid. Guidelines for testing daptomycin susceptibillities of enterococci include breakpoints only for vancomycin-susceptible Enterococcus faecalis, making interpretation of minimum inhibitory concentrations for common clinical infections difficult. No enterococcal cross-resistance has been reported among daptomycin, linezolid, or quinupristin-dalfopristin, and these agents may be viable alternatives.

Broad-range (pan) Salmonella and Salmonella serotype typhi-specific real-time PCR assays: potential tools for the clinical microbiologist.

We describe broad-range salmonellae (ie, Salmonella) and Salmonella serotype Typhi-specific LightCycler (Roche Diagnostics, Indianapolis, IN) real-time polymerase chain reaction assays. We validated these with a battery of 280 bacteria, 108 of which were salmonellae representing 20 serotypes. In addition, 298 isolates from 170 clinical specimens that were suspected to possibly represent Salmonella were tested with the pan- Salmonella assay. Finally, the pan-Salmonella assay also was used to test DNA extracts from 101 archived, frozen stool specimens, 55 of which were culture-positive for salmonellae. Both assays were 100% sensitive and specific when cultured isolates of the battery were tested. The pan- Salmonella assay also characterized correctly all salmonellae on the primary isolation agar and was 96% sensitive (53/55) and 96% specific (49/51) when nucleic acid extracts from direct stool specimens were tested. These assays represent potential tools the clinical microbiologist could use to screen suspect isolates or stool specimens for Salmonella.

Antimicrobial resistance in Cairo, Egypt 1999-2000: a survey of five hospitals.

Antimicrobial resistance among bacterial pathogens is a global problem, but in Egypt data are sparse. We reviewed the antimicrobial susceptibility patterns of bloodstream isolates of Gram-positive cocci and Gram-negative bacilli in five hospitals in Cairo, Egypt, from 1999 to 2000. In addition, susceptibilities of non-bloodstream isolates of Streptococcus pneumoniae and Enterococcus spp. were analysed. High rates of resistance were found in most of the bacteria studied. In the hospitals, a variety of methods were used for identification and susceptibility testing, but in the laboratories quality controlled strains were utilized routinely, to ensure accurate performance of the assays. Only 29% of Staphylococcus aureus isolates and 23% of coagulase-negative staphylococcal isolates were oxacillin susceptible. Both groups of staphylococci were also highly resistant to erythromycin, co-trimoxazole, clindamycin and doxycycline; all isolates were susceptible to vancomycin. Susceptibility of S. pneumoniae isolates to penicillin, ceftriaxone and fluoroquinolones was 63%, 84% and 82%, respectively. Vancomycin susceptibility of the enterococci was 96%; susceptibility to high-level gentamicin and streptomycin was 54% and 48%, respectively. Resistance to most relevant antimicrobials was commonplace among the Gram-negative bacilli; however, most remained susceptible to imipenem. The percentage of bloodstream isolates of Escherichia coli susceptible to common antimicrobial agents was as follows: ampicillin (6%), ampicillin-sulbactam (38%), co-trimoxazole (38%) and aminoglycosides (52%). The susceptibility of isolates of E. coli, Klebsiella and Enterobacter spp. to ceftazidime was 62%, 40% and 46%, respectively. This suggests a potentially high rate of extended-spectrum beta-lactamase (ESBL) and/or Amp-C enzyme production. These results call for a nationwide surveillance programme to monitor microbial trends and antimicrobial resistance patterns in Egypt.