Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test.

Emergence of colistin-resistance in extremely drug-resistant Acinetobacter baumannii containing a novel pmrCAB operon during colistin therapy of wound infections.

BACKGROUND: Colistin resistance is of concern since it is increasingly needed to treat infections caused by bacteria resistant to all other antibiotics and has been associated with poorer outcomes. Longitudinal data from in vivo series are sparse. METHODS: Under a quality-improvement directive to intensify infection-control measures, extremely drug-resistant (XDR) bacteria undergo phenotypic and molecular analysis. RESULTS: Twenty-eight XDR Acinetobacter baumannii isolates were longitudinally recovered during colistin therapy. Fourteen were susceptible to colistin, and 14 were resistant to colistin. Acquisition of colistin resistance did not alter resistance to other antibiotics. Isolates had low minimum inhibitory concentrations of an investigational aminoglycoside, belonged to multi-locus sequence type 94, were indistinguishable by pulsed-field gel electrophoresis and optical mapping, and harbored a novel pmrC1A1B allele. Colistin resistance was associated with point mutations in the pmrA1 and/or pmrB genes. Additional pmrC homologs, designated eptA-1 and eptA-2, were at distant locations from the operon. Compared with colistin-susceptible isolates, colistin-resistant isolates displayed significantly enhanced expression of pmrC1A1B, eptA-1, and eptA-2; lower growth rates; and lowered fitness. Phylogenetic analysis suggested that colistin resistance emerged from a single progenitor colistin-susceptible isolate. CONCLUSIONS: We provide insights into the in vivo evolution of colistin resistance in a series of XDR A. baumannii isolates recovered during therapy of infections and emphasize the importance of antibiotic stewardship and surveillance.

Detection of New Delhi metallo-ß-lactamase (encoded by blaNDM-1) in Acinetobacter schindleri during routine surveillance.

A carbapenem-resistant Alcaligenes faecalis strain was isolated from a surveillance swab of a service member injured in Afghanistan. The isolate was positive for bla(NDM) by real-time PCR. Species identification was reevaluated on three identification systems but was inconclusive. Genome sequencing indicated that the closest relative was Acinetobacter schindleri and that bla(NDM-1) was carried on a plasmid that shared >99% identity with one identified in an Acinetobacter lwoffii isolate. The isolate also carried a novel chromosomally encoded class D oxacillinase.

Update on ocular tuberculosis.

PURPOSE OF REVIEW: Despite recent downtrends, tuberculosis remains a worldwide public health concern. This review provides an update on recent demographic data, clinical and experimental data, and diagnostic modalities. RECENT FINDINGS: Quantitative PCR showing mycobacterial load in intraocular fluids may have an emerging role in the diagnosis of ocular tuberculosis when used in combination with ophthalmic features of tuberculosis. Recent investigations in porcine models of ocular tuberculosis have provided valuable insight into the microbiologic, histologic, and clinical features of Mycobacterium tuberculosis infection of the choroid. Differentiating features between sarcoidosis and tuberculosis include tuberculin skin test status, the presence of ocular surface disease, and the anatomic relationship between vasculitis and choroiditis. SUMMARY: The diagnosis of presumed ocular tuberculosis remains a clinical challenge with currently available diagnostic modalities. Although newer interferon-γ release assays can distinguish exposure to M. tuberculosis from the Bacille-Calmette-Guérin vaccine strain, they currently lack the specificity to distinguish between latent tuberculosis infection and active tuberculosis. Continued improvement in the currently available molecular diagnostic techniques including quantitative PCR may be valuable in our ability to establish an earlier etiologic diagnosis and institute appropriate antimycobacterial therapy.

Ocular tuberculosis: a clinicopathologic and molecular study.

OBJECTIVE: To analyze the clinical profiles, histopathologic features, and Mycobacterium tuberculosis polymerase chain reaction testing in patients with ocular tuberculosis. DESIGN: Retrospective case series. PARTICIPANTS: Forty-two patients. METHODS: This retrospective study was approved by the Armed Forces Institute of Pathology (AFIP) Institutional Review Board. The AFIP data banks were screened for cases with diagnosis of ocular tuberculosis using key words such as mycobacterium; tuberculosis; and acid-fast bacilli. Files and slides stained with hematoxylin-eosin and acid-fast staining were reviewed by the Division of Ocular Pathology and by the Infectious Diseases and Parasitic Diseases Pathology Branches. When available; blocks and unstained slides were sent to the Doheny Eye Institute; Los Angeles; California; for quantitative polymerase chain reaction (qPCR) analysis to detect Mycobacterium tuberculosis-specific DNA. MAIN OUTCOME MEASURES: Tuberculin skin test (TST) results, as well as the chest radiograph results, were recorded. When acid-fast bacilli were identified in tissue, their locations-ocular or extraocular sites-were recorded. Emphasis was placed on lymph node involvement and any systemic diseases. RESULTS: In the histopathologic specimens, microscopy revealed a paucity of organisms, and often there were only 1 or 2 organisms associated with or near a giant cell or near an area of necrosis. The qPCR analysis was performed on 6 biopsy specimens. These specimens showed necrotizing granulomatous inflammation from 6 different patients; 3 had positive qPCR results. In 2 of the 3 cases with positive qPCR results, acid-fast bacilli were not found in the tissue sections. In 17 patients, TST results were available; 10 had positive results (60%) and 7 had negative results (40%). Fourteen chest radiograph results were submitted, and 8 (57%) of 14 patients had normal chest films. CONCLUSIONS: This study suggests that in dealing with those populations at increased risk of tuberculosis (e.g., immigrants from endemic areas and human immunodeficiency virus-infected patients) or patients receiving biologic therapy, the ophthalmologist should endeavor to entertain this diagnosis and to rely on the support of infectious disease specialists and pulmonologists to help solidify the diagnosis, because the current methods for the diagnosis have limited sensitivity.

In vitro activity of the aminoglycoside antibiotic arbekacin against Acinetobacter baumannii-calcoaceticus isolated from war-wounded patients at Walter Reed Army Medical Center.

We determined the in vitro MIC of arbekacin against 200 Acinetobacter isolates recovered from wounded soldiers. The median MIC was 2 microg/ml (range, 0.5 to > 64 microg/ml). A total of 97.5% of the isolates had arbekacin MICs of < 8 microg/ml and 86.5% had MICs of < or = 4 microg/ml. There was no association between the arbekacin MIC and susceptibility to 16 other antibiotics or the specimen source (P = 0.7239). Synergy testing suggested an enhanced effect of arbekacin-carbapenem combinations.

Persistent Detection and Infectious Potential of SARS-CoV-2 Virus in Clinical Specimens from COVID-19 Patients.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) that emerged in December 2019 as the causative agent of Coronavirus 2019 (COVID-19) and was declared a pandemic by the World Health Organization in March 2020 has several distinctive features, including extensive multiorgan involvement with a robust systemic inflammatory response, significant associated morbidity and mortality, and prolonged persistence of viral RNA in the clinical specimens of infected individuals as detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR) amplification. This review begins with an overview of SARS-CoV-2 morphology and replication and summarizes what is known to date about the detection of the virus in nasal, oropharyngeal, and fecal specimens of patients who have recovered from COVID-19, with a focus on the factors thought to contribute to prolonged detection. This review also provides a discussion on the infective potential of this material from asymptomatic, pre-symptomatic, and convalescing individuals, to include a discussion of the relative persistence and infectious potential of virus in clinical specimens recovered from pediatric COVID-19 patients.

Infectious complications of damage control orthopedics in war trauma.

BACKGROUND: War-trauma, especially due to blast injury, can be associated with long bone fracture. Immediate external fixation of fractures, followed by internal fixation when the patient is medically stabilized (damage control orthopedics [DCO]), is the U.S. Army policy for war-related fractures. Data on infectious outcomes when DCO is used for war-trauma fractures are scant. METHODS: A retrospective review of U.S. war-trauma patients from 2003 to 2007 with femoral or tibial fractures treated by DCO was conducted. Fisher's Exact and Mann-Whitney tests were used for comparisons. RESULTS: Fifty-eight soldiers were identified. Fifty-five were males with a median age of 26 years (19-54 years) and a median time to internal fixation by intramedually nailing of 9 days (4-414 days). Eighty-eight percent of fractures were open, and 57% were femoral fractures. The median duration of follow-up was 447 days (20-1,340 days). Fracture site infection occurred in 40% (23 of 58), with suspected osteomyelitis in 17% (10 of 58). Of infected nails, fracture union occurred in 70% and nail retention in 57%. Median time to infection after nail placement was 15 days (0-717 days) with 75% of infections occurring by day 113. Multiple bacterial pathogens including Acinetobacter baumannii and Staphylococcus spp. were causative organisms. Blast injuries occurred in 91% of infected versus 47% of uninfected (p = 0.005). There was no difference between infections occurring in femoral (61%) versus tibial (39%) (p = 0.620) location. CONCLUSIONS: Infection was associated with 40% of DCO-associated intramedullary nails. Blast injury was a predictor of infection. Despite infection, fracture union and nail retention rates were high, suggesting a good outcome.

Isolation of Rapidly Growing Nontuberculous Mycobacteria in Wounds Following Combat-Related Injury.

OBJECTIVES: Rapidly growing nontuberculous mycobacteria (RGNTM) have yet to be described in combat-related injuries. This study investigates the epidemiology, clinical findings, treatment, and outcomes of RGNTM infections among combat casualties wounded in Afghanistan from 2010 to 2012. METHODS: Patients with RGNTM were identified from the Department of Defense Trauma Registry through the Trauma Infectious Disease Outcomes Study. Trauma history, surgical management, and clinical data were collected. Six isolates from patients requiring antimycobacterial therapy were sequenced. RESULTS: Seventeen cases were identified. Six cases, predominantly associated with Mycobacterium abscessus, required aggressive debridement and a median of 180 days of multidrug antimycobacterial therapy that included clofazimine. M. abscessus isolates expressed the erythromycin resistance methylase (erm(41)) gene for inducible macrolide resistance, yet there were no clinical treatment failures when macrolides were utilized in combination therapy. No clonal similarity between M. abscessus isolates was found. Eleven cases had positive wound cultures, but did not require antimycobacterial therapy. The median duration of time of injury to first detection of a RGNTM was 57 days. CONCLUSIONS: This represents the first report of RGNTM infections in war-wounded patients. RGNTM should be recognized as potential pathogens in grossly infected combat wounds. Surgical debridement and multidrug antimycobacterial therapy, when clinically indicated, was associated with satisfactory clinical outcomes.

Rapid identification of Leishmania complexes by a real-time PCR assay.

A real-time PCR assay for the detection of four Leishmania complexes (L. Viannia, L. mexicana, L. donovani/infantum, and L. major) was developed and evaluated. The assay was developed to detect the glucosephosphate isomerase gene and capitalizes on DNA sequence variability within that gene for Leishmania complex identification. Primer/probe sets were created and tested against a panel of 21 known negative controls and on DNA extracted from cultured promastigotes or from tissue biopsies from patients with cutaneous leishmaniasis. The assay was highly specific, as no amplification products were detected in the negative control samples while simultaneously retaining a high degree of complex-specific diagnostic accuracy for cultured organisms and patient clinical samples. Real-time PCR offers rapid (within hours) identification of Leishmania to the complex level and provides a useful molecular tool to assist both epidemiologists and clinicians.

Apparent exacerbation of Hansen's disease despite appropriate antimicrobial therapy.

In 1991, the World Health Organization and its member states resolved to reduce the global prevalence of Hansen's disease (leprosy) by at least 90%. Despite its waning prevalence, however, the disease remains endemic in parts of Africa, Asia, and Latin America. Moreover, imported cases are occasionally encountered innonendemic areas. This fact, coupled with the current tempo of overseas deployments, makes it imperative that military physicians familiarize themselves with both the presentation and potential complications of Hansen's disease. Here, we present the case of a soldier referred to Walter Reed Army Medical Center for apparent worsening of his disease despite appropriate antibiotic therapy. On evaluation, the patient was diagnosed with reversal reaction, an immune-mediated phenomenon resembling disease exacerbation and requiring prompt intervention to avert lasting sequelae. Following the case presentation, we discuss the clinical manifestations, pathophysiology, and management of reversal reactions as well as the related entity erythema nodosum leprosum.

Aerosolized biologic toxins as agents of warfare and terrorism.

Incomplete participation in the 1972 Biological Weapons Convention treaty and noncompliance by several signatory nations makes the malicious use of aerosolized biologic toxins a continuing threat. Unfortunately,prompt diagnosis of toxin exposure may be confounded by a paucity of pathognomonic features and limited diagnostics, but the scenario of patients presenting en masse with a similar pulmonary syndrome should alert the clinician to the possibility of aerosolized toxin exposure. As knowledge of these agents increases, so too should the capabilities for detection,protection, diagnosis, and therapy. This improved understanding, coupled with ongoing public education and awareness and with detailed proactive planning will provide the framework for a prepared community.

Curious marks on an Iraqi man with respiratory complaints.

A 26-year-old suspected Iraqi insurgent is brought to a Troop Medical Clinic in Baghdad, Iraq, with curious marks on his body. On questioning, he describes productive cough, subjective fever, drenching night sweats, and unintentional weight loss. This case shows a condition with which Western physicians are generally unfamiliar but one which quite possibly could be encountered by the deploying provider.

Use of qPCR and genomic sequencing to diagnose Plasmodium ovale wallikeri malaria in a returned soldier in the setting of a negative rapid diagnostic assay.

Plasmodium ovale is one of several clinically relevant malaria species known to cause disease in humans. However, in contrast to Plasmodium falciparum and Plasmodium vivax, which are responsible for most cases of human malaria, P. ovale has a wide distribution but low prevalence in tropical regions. Here, we report the case of a soldier returning from Liberia with P. ovale wallikeri malaria. This case highlights the limitations of both microscopy and the malaria rapid diagnostic test for diagnosing infection with P. ovale and for distinguishing P. ovale wallikeri from P. ovale curtisi. To our knowledge, this is the first case report in which quantitative real-time polymerase chain reaction amplification using the Cytochrome B gene, coupled with genomic sequencing of the potra locus, was used for definitive diagnosis of P. ovale wallikeri malaria.

Lipsosomal amphotericin B for treatment of cutaneous leishmaniasis.

Treatment options for cutaneous leishmaniasis in the United States are problematic because the available products are either investigational, toxic, and/or of questionable effectiveness. A retrospective review of patients receiving liposomal amphotericin B through the Walter Reed Army Medical Center for the treatment of cutaneous leishmaniasis during 2007-2009 was conducted. Twenty patients who acquired disease in five countries and with five different strains of Leishmania were treated, of whom 19 received a full course of treatment. Sixteen (84%) of 19 experienced a cure with the initial treatment regimen. Three patients did not fully heal after an initial treatment course, but were cured with additional dosing. Acute infusion-related reactions occurred in 25% and mild renal toxicity occurred in 45% of patients. Although the optimum dosing regimen is undefined and the cost and toxicity may limit widespread use, liposomal amphotericin B is a viable treatment alternative for cutaneous leishmaniasis.

Emergence of multidrug resistance in bacteria and impact on antibiotic expenditure at a major army medical center caring for soldiers wounded in Iraq and Afghanistan.

Since the invasions of Iraq and Afghanistan, the epidemiologic traits of clinical isolates at Walter Reed Army Medical Center have shifted toward drug-resistant strains of microorganisms, particularly among the gram-negative bacteria. Moreover, antibiotic prescribing patterns during this period have changed remarkably and mirror the emergence of these organisms at our institution.

Infectious diseases during wartime.

PURPOSE OF REVIEW: The infectious disease challenges of war include pathogens endemic to the geographic area of operations as well as wound infections with common environmental microorganisms. This review summarizes papers, unpublished data and personal communications from 2004-2005 pertaining to infectious diseases during war with a focus on the current operations in Iraq and Afghanistan. RECENT FINDINGS: To date, there have been several hundred cases of cutaneous leishmaniasis and five cases of visceral leishmaniasis among US military personnel serving in southwest Asia. There have been reports of malaria in soldiers serving in Afghanistan and an outbreak of acute eosinophilic pneumonia among soldiers serving in or near Iraq. Diarrheal illness is a well-known threat to military operations and remains problematic for combatants throughout the theater of operations. Infectious complications caused by multiply drug-resistant Acinetobacter baumannii have been particularly challenging for healthcare providers managing the wounded evacuated from Iraq. We are now facing outbreaks of nosocomial infection with this pathogen in military treatment facilities in Europe and the USA. SUMMARY: Historically, infectious diseases have had significant impact on the conduct of military operations, and the conflict in southwest Asia is no exception. Physicians caring for returning military personnel should be aware of the diseases prevalent in this campaign, particularly cutaneous leishmaniasis and infections with multiply drug-resistant A. baumannii.