Ribosomal DNA Polymerase Chain Reaction Research Articles

Microbiology Profile of COVID-19-Associated Rhino-Orbital Mucormycosis Pathogens in South India.

This study describes the microbiological and histopathological features of patients with COVID-19-associated rhino-orbital mucormycosis (ROM) seen at the L V Prasad Eye Institute between May and August 2021. Diagnosed clinically and radiologically, 24 patients with ROM were included in the study. Deep nasal swabs or endoscopically collected nasal swabs or orbital tissues were submitted for microbiological evaluation and invitro susceptibility testing by microbroth dilution for natamycin, amphotericin B, caspofungin, posaconazole, ketoconazole, and voriconazole. Cultures were processed by 28S ribosomal DNA polymerase chain reaction and molecular sequencing. A portion of orbital tissues was also sent for histopathological evaluation. The age of the patients ranged from 27 to 75 (mean 48.58 ± 14.09) years and the majority (79%) were male. Nineteen patients were known to be diabetic prior to developing ROM and 18 patients had recovered from active COVID-19 infection. Thirteen patients had a history of hospitalization during COVID-19 infection and eight received steroids. Of the 24 samples, microbiological evaluation identified Rhizopus arrhizus in 12, Rhizopus microsporus in 9, Lichtheimia ramosa in 2, and Rhizopus delemar in 1. Twelve isolates were tested for antifungal susceptibility and all were susceptible to natamycin and amphotericin B. The susceptibility to posaconazole was high, with minimum inhibitory concentration (MIC) < 2 µg/mL for 10/12 (84%) isolates, whereas the MIC of other drugs varied. Histopathological examination of tissues showed acute fulminant disease, granuloma formation, and vascular invasion by the fungal pathogens in these specimens. Rhizopus arrhizus was predominantly associated with ROM and most isolates were susceptible to amphotericin B and posaconazole. Further studies are needed to corroborate the findings and explain possible underlying links.

Comparison of Fungal Fluorescent Staining and ITS rDNA PCR-based Sequencing with Conventional Methods for Diagnosis of Onychomycosis

Accurate identification of fungal causes for onychomycosis is essential for proper treatment. Presently available laboratory methods show unreliable sensitivity; so there is a requirement for innovative detection techniques. The aim for this work was to assess the efficiencies of fluorescent staining and internal transcribed spacer (ITS) ribosomal DNA (rDNA) polymerase chain reaction (PCR)-based sequencing in comparison to conventional techniques for diagnosis of onychomycosis. Nail specimens obtained from 100 patients with clinically- diagnosed onychomycosis were analyzed. Nail scrapings or clippings were subjected to direct microscopic examination by KOH mount, culture by using Sabouraud’s dextrose agar and histopathological examination with periodic-acid Schiff (PAS). Collected specimens were subsequently examined by fluorescent staining and PCR-based sequencing (30 specimens only) to compare the feasibility, sensitivity and diagnostic accuracy for these two methods. The most frequently isolated fungi were yeasts (39/76: 51.3%), dermatophytes (24/76; 31.6%) and non-dermatophyte molds (NDMs) (13/76; 17.1%). Mixed mycotic infections were recovered from 6% of the collected nail specimens. The positive detection rates were significantly different between KOH examinations (52%), nail plate histology (55%), fungal culture (70%) and fluorescent staining (80%). Considering fungal culture as the gold standard, the most sensitive technique was PCR (100%) followed by fluorescent staining (89%), PAS staining (69%) while the least sensitive technique was KOH mount (53%). Fluorescence staining can be used as a rapid and high-yield technique for identification of fungi in the specimens. PCR-based sequencing was highly sensitive and faster compared to culture. Whenever possible, it enables species identification with higher adequacy.

An Infected Bicuspid Aortic Valve, an Anomalous Coronary Artery, and a Dog-Bitten Postman

Risk factors for infective endocarditis (IE) include congenital heart defects, poor dentition, immunosuppression, or recent instrumentation. The occupational hazard of a dog bite, combined with bicuspid aortic valve (BAV) led to IE. 16S ribosomal DNA was able to pinpoint the causative organism. A healthy 33-year-old postman presented in profound heart failure and sepsis due to aortic regurgitation and an aortic root abscess. He underwent emergency aortic valve replacement and was found to have a BAV and anomalous right coronary artery. Blood cultures remained negative. 16S ribosomal DNA polymerase chain reaction (PCR) revealed the causative organism was Capnocytophaga canimorsus. On review, he recalled receiving a dog bite followed by a febrile illness a few days later. Congenital BAVs may become infected by seemingly innocuous injuries. 16S rDNA PCR is a more sensitive and specific diagnostic test than culture. This case demonstrates its utility in providing appropriate antimicrobial management for IE.

Capnocytophaga canimorsus Meningitis – Diagnosis with 16S rDNA PCR when Conventional Methods Failed to Identify the Causative Agent

Meningitis whether bacterial or viral, poses many challenges to clinicians as the causative agent is often not found. According to guidelines, it is standard to start empiric treatment before a Cerebrospinal Fluid (CSF) sample is obtained. Meningitis, if not diagnosed and treated early, can lead to high morbidity and mortality rates with serious neurological sequelae. While the most common cases of bacterial meningitis are related to Streptococcus pneumoniae and Neisseria meningitidis, this clinical case report found a rare case of meningitis caused by a zoonotic pathogen, Capnocytophaga canimorsus; a commensal found as part of the normal flora of dogs and cats. This rare organism was identified with the help of broad range 16S ribosomal DNA Polymerase Chain Reaction (rDNA PCR), an emerging technique that is now increasingly useful in rapid diagnosis especially if the offending agent is not timely identified and conventional methods have failed, making diagnosis and management difficult for physicians.

Decoy Receptor 3 as a Biomarker for Diagnosis of Bacterial Sepsis

Background: Sepsis is a leading cause of morbidity and mortality that has a global burden. Early recognition of sepsis and differentiating it from similar conditions is crucial. Objective: In the present study we aimed to measure the serum level of decoy receptor 3 (DcR3) in sepsis patients to study its role as a promising biomarker for bacterial sepsis. Methodology: The present study included 30 patients, divided into a sepsis group (n=15) and a systemic inflammatory response syndrome (SIRS) group (n=15), and 15 healthy controls. Sepsis patients were identified by positive blood culture or positive 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) results. SIRS patients were identified by negative blood culture or negative 16S rDNA PCR results. Serum DcR3 level was measured by quantitative enzyme-linked immunosorbent assay (ELISA). Receiver-operating characteristic (ROC) curve analysis was performed for DcR3 and C-reactive protein (CRP) alone and in combination. Results: The serum DcR3 level was significantly higher in sepsis than SIRS patients and healthy controls (5.21 ± 2.28 ng/mL, 1.96 ± 0.90 ng/mL, and 0.95 ± 0.79 ng/mL, respectively). The ROC area under the curve (AUC) of DcR3 for sepsis versus SIRS was 0.920 at a cut-off &gt;2.4 ng/mL, with 93.33% sensitivity and 86.67% specificity. The AUC of combined positive DcR3 and positive CRP for sepsis versus SIRS was 0.967 with 93.33% sensitivity and 100% specificity. Conclusion: DcR3, alone or in combination with CRP, is a promising biomarker for distinguishing sepsis from SIRS patients and may efficiently guide physicians to identifying sepsis patients, for whom the further usage of new diagnostics can be cost-effective.

Development of a Diagnostic Marker for Phlebotomus papatasi to Initiate a Potential Vector Surveillance Program in North America.

Phlebotomus papatasi, an Old World sand fly species, is primarily responsible for the transmission of leishmaniasis, a highly infectious and potentially lethal disease. International travel, especially military rotations, between domestic locations and P. papatasi-prevalent regions in the Middle East poses an imminent threat to the public health of US citizens. Because of its small size and cryptic morphology, identification of P. papatasi is challenging and labor-intensive. Here, we developed a ribosomal DNA-polymerase chain reaction (PCR)-based diagnostic assay that is capable of detecting P. papatasi genomic DNA from mixed samples containing multiple sand flies native to the Americas. Serial dilution of P. papatasi samples demonstrated that this diagnostic assay could detect one P. papatasi from up to 255 non-target sand flies. Due to its simplicity, sensitivity and specificity, this rapid identification tool is suited for a long-term surveillance program to screen for the presence of P. papatasi in the continental United States and to reveal geographical regions potentially vulnerable to sand fly-borne diseases.

Molecular Characterization and Identification of Stubby Root Nematode Species From Multiple States in the United States.

Stubby root nematodes (SRN) are important plant parasites infecting many crops and widely distributed in many regions of the United States. SRN transmit Tobacco rattle virus, which causes potato corky ringspot disease, thereby having a significant economic impact on the potato industry. In 2015 to 2017, 184 soil samples and 16 nematode suspensions from North Dakota, Minnesota, Idaho, Oregon, Washington, South Carolina, North Carolina, and Florida were assayed for the presence of SRN. SRN were found in 106 soil samples with population densities of 10 to 320 SRN per 200 g of soil and in eight of the nematode suspensions. Sequencing of ribosomal DNA (rDNA) or species-specific polymerase chain reaction assays revealed the presence of four SRN species, including Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus. Accordingly, their rDNA sequences were characterized by analyzing D2-D3 of 28S rDNA, 18S rDNA, and internal transcribed spacer (ITS) rDNA obtained in this study and retrieved from GenBank. Both intra- and interspecies variations were higher in ITS rDNA than 18S rDNA and D2-D3 of 28S rDNA. Based on phylogenetic analysis, the four SRN species formed a monophyletic group, with P. allius more closely related to P. porosus than P. minor and T. obtusus. Indel variation of ITS2 rDNA was present in P. allius populations from the same geographic regions. This study documented the occurrence of SRN species across multiple states. The intra- and interspecies genetic diversity of rDNA in this study will provide more information for understanding the evolutionary relationships of SRN and will be valuable for future studies of SRN species identification and management.

Eimeria taggartiN. Sp., a Novel Coccidian (Apicomplexa: Eimeriorina) in the Prostate of anAntechinus flavipes

A novel coccidian species was discovered in the prostate of an Antechinus flavipes (yellow-footed antechinus) in South Australia during the period of postmating male antechinus immunosuppression and mortality. This novel coccidian is unusual because it develops extraintestinally and sporulates endogenously within the prostate gland of its mammalian host. Histological examination of prostatic tissue revealed dense aggregations of spherical and thin-walled tetrasporocystic, dizoic, sporulated coccidian oocysts within tubular lumina, with unsporulated oocysts and gamogonic stages within the cytoplasm of glandular epithelial cells. This coccidian was observed occurring concurrently with dasyurid gammaherpesvirus 1 infection of the antechinus' prostate. Eimeria-specific 18S small-subunit ribosomal (r)DNA polymerase chain reaction amplification was used to obtain a partial 18S rDNA nucleotide sequence from the antechinus coccidian. Bayesian phylogenetic analysis based on 18S rDNA gene sequences revealed that the novel coccidian clusters with reptile-host coccidians, forming an ancestral basal lineage of the eimeriid clade. The species has been named Eimeria taggarti n. sp. on the basis of both sporulated oocyst morphology and molecular characterization. It is suspected that E. taggarti is sexually transmitted via excretion of sporulated oocysts or free sporocysts with prostatic secretions in semen.

Cutaneous Leishmaniasis in Khyber Pakhtunkhwa Province of Pakistan: Clinical Diversity and Species-Level Diagnosis.

This study primarily aimed to identify the causative species of cutaneous leishmaniasis (CL) in the Khyber Pakhtunkhwa Province of Pakistan and to distinguish any species-specific variation in clinical manifestation of CL. Diagnostic performance of different techniques for identifying CL was assessed. Isolates of Leishmania spp. were detected by in vitro culture, polymerase chain reaction (PCR) on DNA extracted from dried filter papers and microscopic examination of direct lesion smears from patients visiting three major primary care hospitals in Peshawar. A total of 125 CL patients were evaluated. Many acquired the disease from Peshawar and the neighboring tribal area of Khyber Agency. Military personnel acquired CL while deployed in north and south Waziristan. Leishmania tropica was identified as the predominant infecting organism in this study (89.2%) followed by Leishmania major (6.8%) and, unexpectedly, Leishmania infantum (4.1%). These were the first reported cases of CL caused by L. infantum in Pakistan. PCR diagnosis targeting kinetoplast DNA was the most sensitive diagnostic method, identifying 86.5% of all samples found positive by any other method. Other methods were as follows: ribosomal DNA PCR (78.4%), internal transcribed spacer 2 region PCR (70.3%), culture (67.1%), and microscopy (60.5%). Clinical examination reported 14 atypical forms of CL. Atypical lesions were not significantly associated with the infecting Leishmania species, nor with “dry” or “wet” appearance of lesions. Findings from this study provide a platform for species typing of CL patients in Pakistan, utilizing a combination of in vitro culture and molecular diagnostics. Moreover, the clinical diversity described herein can benefit clinicians in devising differential diagnosis of the disease.

Are atrophic long-bone nonunions associated with low-grade infections?

Impaired fracture healing, especially when associated with bacterial infection, is a severe complication following long-bone fractures and requires special treatment. Because standard diagnostic techniques might provide falsely negative results, we evaluated the sonication method for detection of bacteria on implants of patients with fracture nonunions. A total of 49 patients with a nonunion (group NU) and, for comparison, 45 patients who had undergone routine removal of osteosynthetic material (group OM), were included in the study. Five different diagnostic methods (culture of tissue samples, culture of intraoperative swabs, histopathology of tissue samples, culture of sonication fluid, and 16S ribosomal DNA polymerase chain reaction of sonication fluid) were compared and related to clinical data. Among the diagnostic tests, culture of sonication fluid demonstrated by far the highest detection rate of bacteria (57%) in group NU, and rather unexpectedly 40% in group OM. Culture of sonication samples also revealed a broad spectrum of bacteria, in particular Propionibacterium spp. In conclusion, our results indicate that more bacteria can be detected on implants of patients with atrophic nonunions of long-bone fractures by means of the sonication procedure, which provides a valuable additional diagnostic tool to decide on a surgical procedure (eg, two-step procedure) and to further specify antimicrobial therapy.

Sterility Testing of Stem Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction.

To evaluate broad-range 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) as a rapid screening tool to detect bacterial contamination of stem-cell products. We performed the evaluation using whole blood spiked with serially diluted bacterial-type strains. Detection sensitivity was defined as the bacterial concentration for which all replicates were positive at each concentration (100% detection). We tested the sterility of 29 bags of autologous peripheral blood stem cell (PBSC) products harvested at our facility using the 16S rDNA PCR method. The detection sensitivity of 16S rDNA PCR in spiked whole blood was 10¹ to 10² colony-forming units (CFU) per mL, depending on the bacterial strain. We detected no amplified 16S rDNA among the PBSCs we used in this study. The BacT/ALERT automated bacterial culture system that we used also showed no positive signals in any of the PBSCs tested. Our data indicate that bacterial 16S rDNA PCR is a useful alternative for rapid sterility testing, not only for blood products used in transfusion medicine but also for stem-cell products used in regenerative medicine.

Sterility Testing of Stem-Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction

Sterility Testing of Stem-Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction

Epidemiology of meningitis in an HIV-infected Ugandan cohort.

There is limited understanding of the epidemiology of meningitis among human immunodeficiency virus (HIV)-infected populations in sub-Saharan Africa. We conducted a prospective cohort study of HIV-infected adults with suspected meningitis in Uganda, to comprehensively evaluate the etiologies of meningitis. Intensive cerebrospiral fluid (CSF) testing was performed to evaluate for bacterial, viral, fungal, and mycobacterial etiologies, including neurosyphilis,16s ribosomal DNA (rDNA) polymerase chain reaction (PCR) for bacteria, Plex-ID broad viral assay, quantitative-PCR for HSV-1/2, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Toxoplasma gondii; reverse transcription-PCR (RT-PCR) for Enteroviruses and arboviruses, and Xpert MTB/RIF assay. Cryptococcal meningitis accounted for 60% (188 of 314) of all causes of meningitis. Of 117 samples sent for viral PCR, 36% were EBV positive. Among cryptococcal antigen negative patients, the yield of Xpert MTB/RIF assay was 22% (8 of 36). After exclusion of cryptococcosis and bacterial meningitis, 61% (43 of 71) with an abnormal CSF profile had no definitive diagnosis. Exploration of new TB diagnostics and diagnostic algorithms for evaluation of meningitis in resource-limited settings remains needed, and implementation of cryptococcal diagnostics is critical.

Prosthetic infection: improvement of diagnostic procedures using 16S ribosomal deoxyribonucleic acid polymerase chain reaction

Prosthetic infection is the worst complication in joint arthroplasty. The diagnostic procedure is time consuming and in many cases unrewarding. The aim of this investigation was to raise the sensitivity of the diagnostic procedure. Altogether, 229 implants were removed from 229 patients. Complete data from 157 patients could be analysed. On explantation of the respective arthroplasty, tissue was removed, puncture fluid aspirated and biofilm scratched from the implant surface with a surgical knife. Specimens were investigated with conventional culture methods and with 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) and sequencing. In 123 cases, no pathogen could be identified by routine culture methods. In three of these culture-negative cases, bacteria could be identified with 16S rDNA sequencing of the removed biofilm. In 34 cases, bacteria could be identified with culture methods. In two of these cases, sequencing detected additional pathogens. The process of 16S ribosomal deoxyribonucleic acid polymerase chain reaction (rDNA PCR) and sequencing of biofilm removed from the explanted prosthesis is an important addition to conventional culture methods in prosthetic joint infection. Polymerase chain reaction detects additional pathogens and improves diagnostic sensitivity. The examination of tissue, puncture fluid and biofilm should be performed in cases of prosthesis loosening and explantation.

Incidence of Pulmonary Aspergillosis and Correlation of Conventional Diagnostic Methods with Nested PCR and Real-Time PCR Assay Using BAL Fluid in Intensive Care Unit Patients

Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients. Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting β-tubulin gene were used for the detection of A. fumigatus and A. flavus. Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5. Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group.

Livestock trypanosomosis in Uganda: parasite heterogeneity and anaemia status of naturally infected cattle, goats and pigs

The prevalence and pathogenic effects of trypanosomosis were determined in cattle, goats and pigs reared in Kasese, Jinja and Rakai districts, Uganda; presence of trypanosomes was detected by buffy coat technique (BCT). The overall prevalence of trypanosomosis in cattle was 7.6% (144/1,891), 0.7% in goats (4/573) and 2.3% in pigs (9/386). Internal transcribed spacer 1 (ITS1) of ribosomal DNA polymerase chain reaction was utilised to identify trypanosomes to species level and revealed infections in 108 of the 144 trypanosome-positive cattle while all infected goats and pigs gave amplicons. Trypanosoma vivax was the most prevalent trypanosome species in cattle in single and mixed infections compared to infections involving Trypanosoma congolense and Trypanosoma brucei; in pigs, eight were mixed infections with one single T. vivax infection. No predominant trypanosome species was detected in goats. Anaemia, the main trypanosomosis pathological feature, was investigated by determining packed cell volume (PCV). Mean PCV values by t test in infected individuals were significantly lower than non-infected individuals (P<0.05) for all animal species. However, the proportion of anaemic animals was not significantly different in infected and non-infected individuals. In addition, the percent of infected animals by Fisher's exact test depended on district of origin and species but not sex. These findings show that trypanosomosis is a major cause of anaemia in livestock in endemic areas. Cattle were the major animal species affected by trypanosomosis; similar genotypes of trypanosomes were detected in the three animal species. BCT was more effective than ITS1 rDNA detecting trypanosomes in naturally infected cattle.

Use of 18S ribosomal DNA polymerase chain reaction–denaturing gradient gel electrophoresis to study composition of fungal community in 2 patients with intestinal transplants

Fungi form a diverse microbial community in the human intestine. Little is known about the succession of species after intestinal transplantation. We investigated the alterations of the gut fungal population in 2 patients with intestinal allografts. The ileal effluent and feces were fingerprinted using denaturing gradient gel electrophoresis, with confirmation by DNA sequencing. Analysis of 18S ribosomal DNA indicated that the phylogenetic diversity of the fungal communities was higher soon after transplantation; less diversity was observed at the later time points in patient 1. The shifts in the denaturing gradient gel electrophoresis banding patterns over time were similar in the effluent and feces in this patient. Similar changes in the fungi in the effluent and feces also were observed in patient 2. Sequence analysis of denaturing gradient gel electrophoresis bands showed that Saccharomyces cerevisiae and Kluyveromyces waltii dominated the fungal microbiota in both patients. Some species, including Candida spp, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus clavatus, and Trichophyton verrucosum, were present early. We report for the first time the temporal alterations in fungal communities in patients with an intestinal allograft. This information may provide novel insight into the roles of the fungal microbiota in the pathophysiology of the transplanted intestine.

Heterogeneity of Anopheles Mosquitoes in Nyabushozi County, Kiruhura district, Uganda

The diversity of Anopheles mosquitoes was studied for seven months in eight villages in Nyabushozi County, Kiruhura District, Uganda. The aim of the study was to identify sibling species of Anopheles gambiae S.L, and the Anopheles funestus group using ribosomal DNA polymerase chain reaction assays. A total of 2566 female A. gambiae S.L, and 270 A. funestus group mosquitoes were collected in 24 households using pyrethrum spray catches, and from goat pens and cattle housing (kraals), using CDC light traps. More A. gambiae S.L, and A. funestus were caught in goat’s houses than in cattle kraals, probably due to the effect of weekly spraying of cattle. The densities of female A. gambiae S. L and A. funestus in all the eight villages studied were significantly influenced by rainfall. A. gambiae S.S. was the only sibling species within the A. gambiae complex identified by polymerase chain reaction assay. A. funestus was also identified by polymerase chain reaction and was found to be in sympathy with two other sibling species within the group, A. nopheles leesoni and A. nopheles parensis. Overall,A. gambiae S.S. was the most abundant species and probably contributed to most of the malaria transmission in Nyabushozi County, Kiruhura district. Key words: Anopheles mosquitoes, heterogeneity, polymerase chain reaction, kiruhura district, Uganda.

Diagnostic Strategy for Blood Culture–Negative Endocarditis

result of empirical antibiotic treatment before blood culture sampling. 2. Zoonotic agents, especially Coxiella and Bartonella species, are important causes of BCNE and may be prevalent. In the first case, streptococci, being the most common and antibiotic-susceptible agents of infective endocarditis overall, are cleared from the bloodstream, and valve tissue, from patients undergoing surgery, may be used for diagnosis through histopathological analysis and molecular biology tools. Considering all etiologies, the molecular biology tools used provided a diagnosis for 14% of blood samples and two-thirds of the valves studied, through broad-range polymerase chain reaction (PCR) (16S ribosomal DNA and 18S ribosomal DNA PCR assays). Concerning the zoonotic agents Bartonella and Coxiella species, results of serological analysis were positive, with titers compatible with endocarditis in nearly half the patients (47.7%) in the series. Because treatment for Bartonella endocarditis should include an aminoglycoside, since this affects the outcome, and because treatment for Coxiella endocarditis is long and specific (doxycycline plus cloroquine for many months, with close serological follow-up to determine cure), it is mandatory to rule in or rule out these diagnoses as early as possible in the workup. This study also brings new messages to the field: sophisticated methods, such as primer extension enrichment reaction (PEER) identified very few additional cases, and this was also the case for autoimmunohistochemistry of valvular tissue. Also, cell culture did not provide any additional diagnoses beyond those determined by serological analysis and PCR. Therefore, these tests should be used in exceptional circumstances. Histopathological analysis remains very important, and several diagnoses that were not infective were made on the basis of such analysis, including angiosarcoma and myxoma, as well as Libman-Sachs endocarditis, marantic endocarditis, and Behcet-associated endocarditis.

Actinomyces in Chronic Granulomatous Disease: An Emerging and Unanticipated Pathogen

Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system that causes defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections, mostly by catalase-producing organisms. We report for the first time, to our knowledge, chronic infections with Actinomyces species in 10 patients with CGD. Actinomycosis is a chronic granulomatous condition that commonly manifests as cervicofacial, pulmonary, or abdominal disease, caused by slowly progressive infection with oral and gastrointestinal commensal Actinomyces species. Treatment of actinomycosis is usually simple in immunocompetent individuals, requiring long-term, high-dose intravenous penicillin, but is more complicated in those with CGD because of delayed diagnosis and an increased risk of chronic invasive or debilitating disease. Actinomyces was identified by culture, staining, 16S ribosomal DNA polymerase chain reaction, and/or a complement fixation test in 10 patients with CGD. All 10 patients presented with a history of fever and elevated inflammatory signs without evident focus. Diagnosis was delayed and clinical course severe and protracted despite high-dose intravenous antibiotic therapy and/or surgery. These results suggest an unrecognized and unanticipated susceptibility to weakly pathogenic Actinomyces species in patients with CGD because these are catalase-negative organisms previously thought to be nonpathogenic in CGD. Actinomycosis should be vigorously sought and promptly treated in patients with CGD presenting with uncommon and prolonged clinical signs of infection. Actinomycosis is a catalase-negative infection important to consider in CGD.