David H. Persing

Rapid Molecular Detection of Tuberculosis and Rifampin Resistance

BACKGROUND Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect. METHODS We assessed the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), with fully integrated sample processing in 1730 patients with suspected drug-sensitive or multidrug-resistant pulmonary tuberculosis. Eligible patients in Peru, Azerbaijan, South Africa, and India provided three sputum specimens each. Two specimens were processed with N-acetyl-L-cysteine and sodium hydroxide before microscopy, solid and liquid culture, and the MTB/RIF test, and one specimen was used for direct testing with microscopy and the MTB/RIF test. RESULTS Among culture-positive patients, a single, direct MTB/RIF test identified 551 of 561 patients with smear-positive tuberculosis (98.2%) and 124 of 171 with smear-negative tuberculosis (72.5%). The test was specific in 604 of 609 patients without tuberculosis (99.2%). Among patients with smear-negative, culture-positive tuberculosis, the addition of a second MTB/RIF test increased sensitivity by 12.6 percentage points and a third by 5.1 percentage points, to a total of 90.2%. As compared with phenotypic drug-susceptibility testing, MTB/RIF testing correctly identified 200 of 205 patients (97.6%) with rifampin-resistant bacteria and 504 of 514 (98.1%) with rifampin-sensitive bacteria. Sequencing resolved all but two cases in favor of the MTB/RIF assay. CONCLUSIONS The MTB/RIF test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than 2 hours with minimal hands-on time. (Funded by the Foundation for Innovative New Diagnostics.)

Clinical Characteristics and Treatment Outcome of Early Lyme Disease in Patients with Microbiologically Confirmed Erythema Migrans

Context Lyme disease is the most common vector-borne disease in the United States. The traditional clinical presentation, an expanding erythematous rash with partial central clearing, sometimes accompanied by systemic symptoms, was described in patients who usually had clinically manifest Lyme disease for several days. Contribution This study describes 118 patients who acquired Lyme disease while under surveillance in a vaccine trial. Fifty-nine percent of rashes were homogeneous lesions, 32% had dense central erythema, and only 9% had classic central clearing. Signs and symptoms usually resolved within 3 weeks of antibiotic treatment. Implications Early Lyme disease may present with homogeneous or dense central erythematous lesions rather than classic erythema migrans. With antibiotic treatment, the prognosis is excellent. Early Lyme disease may present with homogeneous or dense central erythematous lesions rather than classic erythema migrans. With antibiotic treatment, the prognosis is excellent. The Editors Lyme disease in the United States is caused by the tick-transmitted spirochete Borrelia burgdorferi sensu stricto (1). This infection is the most common vector-borne disease in the country (2). The illness usually presents with localized infection of the skin, erythema migrans, which is often followed days to a few weeks later by dissemination of the spirochete to multiple sites, particularly to other skin sites, the nervous system, the heart, or the joints (3). Borrelia burgdorferi has been cultured readily from skin biopsy samples of erythema migrans early in the illness (4, 5), but later culture from other sites has been difficult. As a substitute for culture, B. burgdorferi DNA may be detected by polymerase chain reaction (PCR) in most patients with erythema migrans (6, 7) and in the joint fluid of patients with Lyme arthritis (8, 9). However, PCR has had low sensitivity in samples from other sites, including cerebrospinal fluid and blood. Serodiagnosis is not sensitive during the first several weeks of infection, but patients often seroconvert during convalescence (10). The initial clinical series of patients with early Lyme disease (11) was described before identification of the causative agent. Researchers used clinical criteria for study entry, particularly the classic appearance of erythema migrans, a slowly expanding erythema with partial central clearing. In that series of 314 patients, most had systemic symptoms and nearly half also had multiple annular erythemas, suggesting dissemination of the spirochete to multiple sites. In a subsequent study, 79 patients from Westchester County, New York, with early Lyme disease had erythema migrans from which B. burgdorferi was cultured (12). Most of the patients in this series, who were often seen earlier than those in the original study, had systemic symptoms, but only 18% had multiple erythemas. In Europe, where erythema migrans more often results from infection with B. afzelii or B. garinii rather than B. burgdorferi sensu stricto, inflammation of erythema migrans is less intense and migration is slower; in addition, patients generally have fewer systemic symptoms (13). In the southeastern United States, patients have been reported to have erythema migranslike skin lesions, but laboratory evidence of B. burgdorferi infection has been lacking. This suggests that another agent, perhaps even from the Borrelia genus, may cause the infection in this geographic area (14-16). Moreover, the same tick that transmits the Lyme disease agent may transmit other infectious agents, including the agent of human granulocytic ehrlichiosis and Babesia microti, and co-infection may influence the clinical presentation of Lyme disease (17, 18). Thus, microbiological confirmation is beneficial in describing the clinical features of Lyme disease in endemic areas in the United States. The recent phase III study of SmithKline Beechams Lyme disease vaccine provided an exceptional opportunity to assess the clinical picture of early Lyme disease in a large cohort of patients who acquired the infection in major endemic locations throughout the United States (19). As part of the protocol, patients who had symptoms of Lyme disease were extensively evaluated for that infection. We describe the clinical presentation, serologic results, and treatment outcome of early Lyme disease in 118 patients with microbiologically confirmed cases of erythema migrans. Methods Patients A total of 10 936 participants 15 to 70 years of age were enrolled in a double-blind, placebo-controlled study of the efficacy and safety of an outer surface protein A Lyme disease vaccine (LYMErix, recombinant outer surface protein A, SmithKline Beecham [now GlaxoSmithKline], Collegeville, Pennsylvania) at 31 sites in 10 endemic states. The Human Investigations Review Committees at all participating centers approved the study protocol. The complete protocol, as well as all members of the Lyme Disease Vaccine Study Group, have been reported elsewhere (19). Briefly, study participants received a packet with information about Lyme disease. Participants were requested to report promptly to their clinician-investigator any symptoms that suggested infection, including onset of a new rash or flu-like illness (without predominant respiratory or gastrointestinal symptoms), arthralgias or arthritis, facial palsy, radiculopathy, or syncope. All participants who were thought to have possible Lyme disease underwent a focused history, physical examination, and laboratory testing. All clinical data were entered on an electronic data form for central analysis. Antibiotic treatment was prescribed according to recommended guidelines (20), but the actual antibiotic and the duration of treatment were at the discretion of the investigator or the patients personal physician. Of the 10 936 participants, 146 met study criteria for definite Lyme disease and 118 had an acute illness with culture-proven or PCR-confirmed erythema migrans. Two clinicians independently reviewed photographs of erythema migrans from case-patients. Laboratory Methods Serologic testing was done at the New England Medical Center exclusively by Western blot (MarDx, San Diego, California), since the standard enzyme-linked immunosorbent assay would be expected to yield positive results in patients vaccinated with outer surface protein A (21). A baseline sample was obtained at study entry, and acute and convalescent samples were obtained when the patient had symptoms suggestive of Lyme disease. The diagnosis of Lyme disease was serologically supported by IgM or IgG seroconversion, or both, between baseline and the acute phase of the illness or between the acute and convalescent phases of the illness. Samples from the same participant were always tested together in the same assay. Western blot results were interpreted according to the criteria of the Centers for Disease Control and Prevention and of the Association of State and Territorial Public Health Laboratory Directors (22). On Western blot, outer surface protein A antibodies bind to a 31-kilodalton band; this is not included in the Centers for Disease Control and Prevention criteria. Following local anesthesia, skin biopsy specimens from erythema migrans were obtained by using 2-mm punch biopsy. Half of each sample was placed directly into a 15-mL tube of BarbourStoennerKelly medium (BSK-H medium, Sigma-Aldrich, St. Louis, Missouri) with ciprofloxacin (0.4 g/mL) and rifampin (40 g/mL); the other half was placed in a 2-mL polypropylene plastic tube for PCR testing. Specimens were shipped the same day by Federal Express to New England Medical Center. On arrival at the laboratory, half of the medium was replaced with fresh medium. Skin samples for culture were placed in an incubator at 33 C and were examined weekly for 1 month by darkfield microscopy for motile spirochetes. Polymerase chain reaction assays of skin biopsy samples were performed as described elsewhere (8). Role of the Funding Source SmithKline Beecham provided data and gave the authors permission to review them, compile them, and independently present results. Dr. Parenti was employed as a research physician with SmithKline Beecham during the early phases of the study. Results During the 20-month study period, which covered two summers of Lyme disease transmission, 1917 of the 10 936 study participants were evaluated for possible Lyme disease (19). Of the 1917 patients, 146 (7.6%) met study criteria for definite Lyme disease, and 118 (6.2%) had microbiological confirmation of this infection by culture or PCR testing of erythema migrans. The mean age of these 118 patients was 51 years (range, 17 to 71 years); 53% were men, and 47% were women. Forty-seven percent were from New England, 51% were from mid-Atlantic states, and 2% were from Wisconsin, reflecting the locations of the study sites. June and July were the peak months of disease onset, which correlated with the expected peak questing period of nymphal Ixodes scapularis ticks. However, cases occurred from March through October, suggesting that adult ticks may also transmit the disease. Vaccine and placebo recipients did not differ in the size of erythema migrans, persistence of symptoms after treatment, and morphologic characteristics of the lesions. In addition, no clinical differences were noted in different geographic areas. Therefore, we present data from vaccine and placebo recipients and from different geographic areas together. Characteristics of Erythema Migrans One hundred eighteen patients had erythema migrans in which B. burgdorferi was detected by culture (88%); by PCR testing (72%); or, in most instances, by both methods (60%). Rashes, which were evaluated a median of 3 days after onset (range, 1 to 30 days), were a median of 10 cm in diameter (range, 5 to 37 cm) at the time of diagnosis. In this adult population, nearly half of the lesions were located on the groin, buttocks, or lo

Persistent Parasitemia after Acute Babesiosis

BACKGROUND Babesiosis, a zoonosis caused by the protozoan Babesia microti, is usually not treated when the symptoms are mild, because the parasitemia appears to be transient. However, the microscopical methods used to diagnose this infection are insensitive, and few infected people have been followed longitudinally. We compared the duration of parasitemia in people who had received specific antibabesial therapy with that in silently infected people who had not been treated. METHODS Forty-six babesia-infected subjects were identified from 1991 through 1996 in a prospective, community-based study designed to detect episodes of illness and of seroconversion among the residents of southeastern Connecticut and Block Island, Rhode Island. Subjects with acute babesial illness were monitored every 3 months for up to 27 months by means of thin blood smears, Bab. microti polymerase-chain-reaction assays, serologic tests, and questionnaires. RESULTS Babesial DNA persisted in the blood for a mean of 82 days in 24 infected subjects without specific symptoms who received no specific therapy. Babesial DNA persisted for 16 days in 22 acutely ill subjects who received clindamycin and quinine therapy (P=0.03), of whom 9 had side effects from the treatment. Among the subjects who did not receive specific therapy, symptoms of babesiosis persisted for a mean of 114 days in five subjects with babesial DNA present for 3 or more months and for only 15 days in seven others in whom the DNA was detectable for less than 3 months (P<0.05); one subject had recrudescent disease after two years. CONCLUSIONS When left untreated, silent babesial infection may persist for months or even years. Although treatment with clindamycin and quinine reduces the duration of parasitemia, infection may still persist and recrudesce and side effects are common. Improved treatments are needed.

A Fatal Case of Babesiosis in Missouri: Identification of Another Piroplasm That Infects Humans

Human cases of the tick-borne disease babesiosis are caused by the bovine parasite Babesia divergens in Europe [1, 2], by the rodent parasite B. microti in the northeastern and upper midwestern United States [2, 3], and by WA1-type piroplasms in Washington and California [4-6]. We describe the first reported zoonotic case of babesiosis acquired in Missouri and provide evidence to show that this fatal case was caused by an intraerythrocytic piroplasm (MO1) that is probably distinct from but shares morphologic, antigenic, and molecular characteristics with B. divergens. Case Report A 73-year-old man was hospitalized on 1 July 1992 because of fever, a rigor, and thrombocytopenia. He had developed a dry cough, mild headache, sore throat, and joint pain 4 days before admission and had had a temperature of 38.9 C and a platelet count of 70 109/L (baseline count, 100 109/L to 150 109/L) 2 days before admission. He began receiving erythromycin therapy on an outpatient basis but did not improve. His medical history included systemic lupus erythematosus, which had been diagnosed in 1979 and for which he was taking prednisone (10 mg/d). He had had a splenectomy in 1979 because of hemolytic anemia and thrombocytopenia and had had an intracerebral hemorrhage in 1989 because of thrombocytopenia. Except for proteinuria due to membranous glomerulonephritis, his systemic lupus erythematosus had been quiescent since that time. His medical history was also notable for a myocardial infarction in 1986 and recurrent supraventricular tachycardia, for which he was taking digoxin. The patient lived with his wife on 1 acre of land (all mowed or gardened) in a rural area of southeastern Missouri (Cape Girardeau County). He primarily stayed indoors, but he mowed the lawn with a riding mower and did some gardening. He had not traveled outside Missouri in the previous 3 to 4 years, had not traveled outside the midwestern United States in the previous 10 years, and had never been in a country other than the United States. He had no pets or known tick exposures. He had intermittently been employed to feed dairy cattle, which neighbors kept about 1 mile from his home, until 8 years before his hospitalization on 1 July 1992. On admission to the hospital, the patient was febrile Table 1, had a small effusion in one knee joint, and had slight pain on shoulder rotation. He was thrombocytopenic Table 1, his total bilirubin and lactase dehydrogenase levels were elevated, a 24-hour urine specimen contained 11.4 g of protein, and his creatinine clearance was 0.95 mL/s (57 mL/min). Complement levels were normal, no anti-DNA antibody was detectable, and his antinuclear antibody titer was 80 (speckled pattern), suggesting that his systemic lupus erythematosus was quiescent. The patient tested negative for antibody to the human immunodeficiency virus, and blood and urine cultures obtained on 1 July and periodically thereafter also tested negative. He was treated with aztreonam, cefazolin, and an increased dosage of prednisone (80 mg/d). Table 1. Clinical Data for a Patient Who Acquired Babesiosis in Missouri On 2 July, babesiosis was diagnosed after intraerythrocytic ring forms were noted on the patients blood smear Table 1, Figure 1. The antibiotic regimen was changed to oral quinine sulfate, 650 mg three times daily, and intravenous clindamycin, 600 mg three times daily. The patient became afebrile on 3 July, but his parasitemia level continued to increase. His lactate dehydrogenase, total bilirubin, and creatinine levels also increased. Hemodialysis was instituted on 6 July and was provided periodically thereafter. By 11 July, the prednisone dosage had been reduced to 10 mg/day. By 13 July, the 12th day of therapy for babesiosis, the patients parasitemia level had markedly decreased. Figure 1. Giemsa-stained blood smear obtained on 2 July from a patient who acquired babesiosis in Missouri. On 15 July, the patient had a cardiopulmonary arrest that was attributed to hypoxemia; he was intubated and resuscitated. Diffuse pulmonary infiltrates were noted and were thought to be at least partly due to volume overload. After his arrest, the patient had a generalized seizure and never fully regained his baseline mental status. Intravenous methylprednisolone therapy (10 mg three times daily) was started. The quinine dosage was decreased to 650 mg twice daily because of high quinine levels (as high as 7.1 g/mL). On 16 July, the patient again became febrile, but no parasites were noted on his blood smear; ceftazidime therapy was started because of Enterobacter cloacae pneumonia. On 17 July, the patient developed ventricular tachycardia and was cardioverted. On 20 July, the 20th day of hospitalization, supportive therapy was discontinued, and the patient died. No autopsy was done. Methods Serologic Assays At the Centers for Disease Control and Prevention, indirect immunofluorescent antibody testing was used to assay serum specimens, in serial fourfold dilutions, for reactivity to B. microti and WA1 antigens [4, 7]. At the Laboratoire de Biologie Cellulaire, serum specimens were tested for antibody to B. divergens and B. canis (a canine piroplasm). Indirect immunofluorescent antibody testing and immunoprecipitation assays were done as previously described [8, 9]; however, the immunoprecipitation assays were done on long-term cultures of B. divergens (human isolate Rouen 1987) [9] but on short-term cultures of B. canis (isolate Gignac 1994) [10]. Babesia divergens had been obtained from a naturally infected human and maintained in jirds (Mongolian gerbils [Meriones unguiculatus]) by syringe passage twice weekly [11] or in long-term in vitro culture [9]. Babesia canis had been obtained from a naturally infected dog. At the U.S. Department of Agriculture, serum specimens were tested for antibody to bovine isolates of B. divergens (German isolate) and B. bovis (Mexican isolate); a Babesia species from desert bighorn sheep (Ovis canadensis nelsoni; California isolate) that is morphologically similar to B. divergens and serologically cross-reacts with it to some degree [12]; and B. odocoilei (Texas isolate), a parasite of white-tailed deer (Odocoileus virginianus) [13, 14]. The techniques for obtaining in vitro-derived antigens from these isolates have been described previously [12, 15-18]. Indirect immunofluorescent antibody testing was done as previously described [19], except that fluorescein-conjugated recombinant protein G was used to detect specific IgG [20]. When human serum specimens were tested, fluorescein-conjugated goat antihuman IgG (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland) was used. Animal Inoculations Whole blood from the patient was inoculated into hamsters (Mesocricetus auratus), jirds (some of which were immunosuppressed with dexamethasone), and calves and bighorn sheep that had had splenectomy and were immunosuppressed (Table 2). Hamsters and jirds are suitable animal hosts for both B. microti and WA1 [4]; jirds and calves are suitable hosts for B. divergens [2, 11]; and bighorn sheep (or the bighorn sheep culture system) are suitable hosts for various Babesia species that infect wild ruminants [12, 23]. Sheep BHR-32 Table 2 was challenged intravenously on day 63 after inoculation with a stabilate of the bighorn Babesia species (2 108 merozoites) that had been cryopreserved in polyvinylpyrollidone-40 (Sigma, St. Louis, Missouri). On day 27, calf C-03 was challenged intravenously with a cryopreserved stabilate of B. bovis (2 108 merozoites), and sheep BHR-34 was challenged intravenously with a cryopreserved stabilate of the bighorn Babesia species (2 108 merozoites). Table 2. Inoculations of Animals with Blood from a Patient Who Acquired Babesiosis in Missouri* In Vitro Culturing At the Laboratoire de Biologie Cellulaire, 0.5-mL aliquots of whole blood obtained from the patient before treatment on 2 July and cryopreserved in 10% dimethyl sulfoxide were used for each of two in vitro culture systems: B. divergens [9] and B. canis [10]. Human erythrocytes were used for the former; canine erythrocytes were used for the latter. At the U.S. Department of Agriculture, in vitro culturing of blood from bighorn sheep (before and after inoculation with the patients blood; Table 2) was attempted as previously described [16, 17], except that bighorn sheep erythrocytes and medium supplemented with bighorn sheep serum were used. The sheep blood was cultured fresh, with the exception of the specimen taken before inoculation from the sheep inoculated in May (Table 2); this specimen had been cryopreserved in polyvinylpyrollidone-40. The cultures were monitored for 30 days. Molecular Studies At the Mayo Clinic, MO1 DNA was isolated [24] from whole blood that had been obtained from the patient on 2 July and cryopreserved in 10% dimethyl sulfoxide. Broad-range amplification with the polymerase chain reaction, to recover piroplasm-specific nuclear small-subunit ribosomal DNA, and DNA sequence analysis of a 144 base-pair region of the amplification product were done as previously described [5, 6]. Phylogenetic analysis was done by maximum parsimony analysis in PAUP (phylogenetic analysis using parsimony) version 3.1.1 [25]; the analysis included 119 alignable nucleotides and 28 phylogenetically informative positions. The sequences for the other pathogens included in the analysis were previously known [5, 6, 26]; the GenBank accession number for the nuclear small-subunit ribosomal RNA gene for B. odocoilei is U16369 [26]. Results Morphologic Analysis Most of the intraerythrocytic parasites noted on the patients blood smear Figure 1 were in a subcentral position; those in a subperipheral position did not protrude from the erythrocytes. Most erythrocytes were multiply infected. The parasites were polymorphic. Punctiform (< 1 m in diameter), annular (1 m to 2.5 m in diameter), piriform (1 m to 2.5 m in length), and tetrad (Maltese cross) forms were noted. T

Propionibacterium acnes Types I and II Represent Phylogenetically Distinct Groups

ABSTRACT Although two phenotypes of the opportunistic pathogen Propionibacterium acnes (types I and II) have been described, epidemiological investigations of their roles in different infections have not been widely reported. Using immunofluorescence microscopy with monoclonal antibodies (MAbs) QUBPa1 and QUBPa2, specific for types I and II, respectively, we investigated the prevalences of the two types among 132 P. acnes isolates. Analysis of isolates from failed prosthetic hip implants (n = 40) revealed approximately equal numbers of type I and II organisms. Isolates from failed prosthetic hip-associated bone (n = 6) and tissue (n = 38) samples, as well as isolates from acne (n = 22), dental infections (n = 8), and skin removed during surgical incision (n = 18) were predominately of type I. A total of 11 (8%) isolates showed atypical MAb labeling and could not be conclusively identified. Phylogenetic analysis of P. acnes by nucleotide sequencing revealed the 16S rRNA gene to be highly conserved between types I and II. In contrast, sequence analysis of recA and a putative hemolysin gene (tly) revealed significantly greater type-specific polymorphisms that corresponded to phylogenetically distinct cluster groups. All 11 isolates with atypical MAb labeling were identified as type I by sequencing. Within the recA and tly phylogenetic trees, nine of these isolates formed a cluster distinct from other type I organisms, suggesting a further phylogenetic subdivision within type I. Our study therefore demonstrates that the phenotypic differences between P. acnes types I and II reflect deeper differences in their phylogeny. Furthermore, nucleotide sequencing provides an accurate method for identifying the type status of P. acnes isolates.

Bordetella holmesii-Like Organisms Associated with Septicemia, Endocarditis, and Respiratory Failure

We recovered an unusual bacterial strain from blood or sputum of three patients with septicemia, endocarditis, and/or respiratory failure. The three isolates were thin, curved, gram-negative, light brown, pigment-producing bacilli with variable catalase activity. They were asaccharolytic, oxidase-negative, nonmotile, and fastidious. Identification was not possible on the basis of these characteristics alone or in combination with cellular fatty acid profiles. Nucleic acid amplification and sequence analysis of the 16S rRNA gene revealed that all three isolates were identical and most closely related to the emerging pathogen Bordetella holmesii, diverging from the published sequence at three nucleotide positions (99.8% similarity). Isolation of a B. holmesii-like pathogen from sputum suggests that, in addition to producing septicemia, the organism may inhabit the respiratory tract like other Bordetella species.

Characterization of Nasal and Blood Culture Isolates of Methicillin-Resistant Staphylococcus aureus from Patients in United States Hospitals

ABSTRACT A total of 299 nares and 194 blood isolates of methicillin-resistant Staphylococcus aureus (MRSA), each recovered from a unique patient, were collected from 23 U.S. hospitals from May 2009 to March 2010. All isolates underwent spa and staphylococcal cassette chromosome mec element (SCCmec) typing and antimicrobial susceptibility testing; a subset of 84 isolates was typed by pulsed-field gel electrophoresis (PFGE) using SmaI. Seventy-six spa types were observed among the isolates. Overall, for nasal isolates, spa type t002-SCCmec type II (USA100) was the most common strain type (37% of isolates), while among blood isolates, spa type t008-SCCmec type IV (USA300) was the most common (39%). However, the proportion of all USA100 and USA300 isolates varied by United States census region. Nasal isolates were more resistant to tobramycin and clindamycin than blood isolates (55.9% and 48.8% of isolates versus 36.6% and 39.7%, respectively; for both, P < 0.05). The USA300 isolates were largely resistant to fluoroquinolones. High-level mupirocin resistance was low among all spa types (<5%). SCCmec types III and VIII, which are rare in the United States, were observed along with several unusual PFGE types, including CMRSA9, EMRSA15, and the PFGE profile associated with sequence type 239 (ST239) isolates. Typing data from this convenience sample suggest that in U.S. hospitalized patients, USA100 isolates of multiple spa types, while still common in the nares, have been replaced by USA300 isolates as the predominant MRSA strain type in positive blood cultures.

Laboratory Diagnosis of Clostridium difficile Infection: Can Molecular Amplification Methods Move Us Out of Uncertainty?

The laboratory diagnosis of Clostridium difficile infection (CDI) continues to be challenging. Recent guidelines from professional societies in the United States note that enzyme immunoassays for toxins A and B do not have adequate sensitivity to be used alone for detecting CDI, yet the optimal method for diagnosing this infection remains unclear. Nucleic acid amplification tests (NAATs) that target chromosomal toxin genes (usually the toxin B gene, tcdB) show high sensitivity and specificity, provide rapid results, and are amenable to both batch and on-demand testing, but these tests were not universally recommended for routine use in the recent guidelines. Rather, two-step algorithms that use glutamate dehydrogenase (GDH) assays to screen for C. difficile in stool specimens, followed by either direct cytotoxin testing or culture to identify toxin-producing C. difficile isolates, were recommended in one guideline and either GDH algorithms or NAATs were recommended in another guideline. Unfortunately, neither culture nor direct cytotoxin testing is widely available. In addition, this two-step approach requires 48 to 92 hours to complete, which may delay the initiation of therapy and critical infection control measures. Recent studies also show the sensitivity of several GDH assays to be <90%. This review considers the role of NAATs for diagnosing CDI and explores their potential advantages over two-step algorithms, including shorter time to results, while providing comparable, if not superior, accuracy.

Direct Identification of Bacteria from Positive Blood Cultures by Amplification and Sequencing of the 16S rRNA Gene: Evaluation of BACTEC 9240 Instrument True- Positive and False-Positive Results

ABSTRACT In a previous study which evaluated the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), we noted a 1.3% “instrument false-positive” rate. That is, the BACTEC system signaled that a bottle (BACTEC Plus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culture was positive but a Gram stain was negative and there was no growth of bacteria or yeasts on subculture to chocolate agar. Furthermore, from the same sample of blood, cultures for fungi using the Isolator blood culture system (Wampole Laboratories, Cranbury, N.J.) were negative for growth. For the present study, we evaluated 76 instrument false-positive samples for the presence of 16S ribosomal DNA using the MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). These samples also were negative for fungi by the Isolator method. This kit has a PCR module and sequencing module for the amplification and sequencing of the 16S RNA gene and provides a database for sequence alignment and identification of bacteria. To optimize the assay, we evaluated the effect of adding 0.5% bovine serum albumin to the sample from blood culture bottles and found that it decreased the effects of inhibitors on the PCR. Two control groups of blood culture specimens were also evaluated. One group (n = 45) were “instrument true positives”; the instrument signaled positive, and subsequent Gram stains were positive and subcultures on chocolate agar grew bacteria. The other group (n = 20) were “instrument true negatives”; the instrument signaled negative, the Gram stain was negative, and subcultures on chocolate agar and from the Isolator tube on fungal media showed no growth. None of the 76 instrument false-positive samples had evidence for 16S rRNA gene sequences. All of the instrument true-positive samples and all of the instrument true-negative specimens were positive and negative, respectively, using the MicroSeq 500 kit. Total peripheral white blood cell counts were statistically significantly higher for patients who had instrument false-positive results than for patients who had instrument true-positive or true-negative results (P = 0.001). We conclude that instrument false positives signaled by the BACTEC 9240 system are not due to bacteria in the blood culture samples.

Evaluation of a Polymerase Chain Reaction-Based Universal Heteroduplex Generator Assay for Direct Detection of Rifampin Susceptibility of Mycobacterium tuberculosis from Sputum Specimens

In a double-blind study, 655 sputum specimens were obtained from individuals suspected of having tuberculosis and were analyzed for the presence of Mycobacterium tuberculosis and rifampin susceptibility with use of a polymerase chain reaction (PCR)-based universal heteroduplex generator assay (PCR/UHG-Rif). Of the specimens containing viable M. tuberculosis, 100% of the smear-positive (n = 41) and 50% of the smear-negative (n = 6) specimens tested positive for the organism by PCR/UHG-Rif. Nineteen of 537 culture-negative specimens tested positive for M. tuberculosis by PCR/UHG-Rif and were from patients with confirmed tuberculosis who were receiving antituberculosis therapy at the time of specimen collection. Thirty-five specimens contained nontuberculous mycobacteria and were negative by PCR/UHG-Rif. Genotypic evidence of rifampin resistance in five of six culture-confirmed, rifampin-resistant isolates was obtained by PCR/UHG-Rif, yielding a sensitivity and specificity for the assay of 83% and 98.2%, respectively. These results demonstrate the feasibility of using a PCR-based assay directly on sputum specimens for simultaneous detection of M. tuberculosis and rifampin susceptibility, and they suggest that patients with smear-positive, untreated tuberculosis and those presenting with suspected drug-resistant tuberculosis are the most appropriate groups for testing by PCR/UHG-Rif.