Development of monoclonal antibodies against Rhodococcus equi virulence-associated protein N and their application to pathological diagnosis.

IMPORTANCE: Rhodococcus equi can cause infection in ruminants, and its pathogenicity is suggested to be associated with VapN. Despite its wide distribution, no immunological diagnostic method has been developed for VapN-producing R. equi. Against this background, we attempted to develop monoclonal antibodies targeting VapN and assess their application in immunostaining. In the study, mice were immunized with recombinant VapN, and cell fusion and cloning by limiting dilution permitted the generation of three antibody-producing hybridomas. The utility of the antibodies produced from the hybridomas in immunostaining was demonstrated using an infected mouse model, and the antibodies were further applied to previously reported cases of R. equi infection in goats and cattle. Although the 4H4 antibody induced the strongest reactions, the reactivity of two other antibodies was improved by antigen retrieval. Our monoclonal antibodies will be utilized to support the definitive diagnosis of suspected R. equi infection, including cases that were previously missed.

Successful treatment of Rhodococcus lung abscess and empyema thoracis in a heart transplant recipient.

Rhodococcosis is an uncommon cause of pulmonary infection in thoracic organ transplant recipients. We describe a heart transplant recipient diagnosed with Rhodococcus equi left upper lung abscess with empyema thoracis complicated by bacteremia. The patient was successfully treated with appropriate antibiotics, adequate surgical resection, and optimization of immunosuppressants.

Evaluation of serum concentration of acute-phase proteins (haptoglobin and serum amyloid A) in the affected Arabian foals with rhodococcosis.

BACKGROUND: Early detection of Rhodococcus equi pneumonia in foals is essential for horse health and for veterinarians. OBJECTIVES: This study aimed to demonstrate the usefulness of assessing the serum concentration of acute-phase proteins (APPs) in the early diagnosis of pneumonia. METHODS: The study evaluated APPs in 19 Arabian foals with R. equi pneumonia and compared them with 18 normal Arabian foals in equestrian clubs in Tabriz, Iran. Affected foals were identified through history, clinical findings and bacterial culture of tracheal washing. Biochemical methods and polymerase chain reaction tests were performed by examining the 16S rRNA and vapA genes to confirm the diagnosis of bacterial isolates. Blood samples were taken from all sick and healthy horses, and their serum was isolated. APPs in the serum were measured in all the samples. RESULTS: Rhodococcosis increased the serum concentration of haptoglobin (Hp) and serum amyloid A (SAA) (p < 0.001). The relationship between SAA and Hp was meaningful in the infected group (r = 0.933) but not in the healthy group. In cases where there are clinical findings of R. equi pneumonia, the concentration of SAA and Hp can help the effectiveness of treatment. - CONCLUSIONS: Serum concentration analysis of APPs can be helpful in early diagnosis and successfully treating foals with R. equi pneumonia.

Tuberculosis with cavities? Rapid diagnosis of Rhodococcus equi pulmonary infection with cavities by acid-fast staining: A case report.

Rhodococcus equi is a conditionally pathogenic bacterium widely distributed in soil, water, and marine environments, which can cause respiratory infections, pleurisy, blood and even bone marrow infections in immunocompromised people, and particularly in patients with acquired immunodeficiency syndrome (AIDS). This case report describes a patient with initially suspicion of tuberculosis (TB) as an outpatient in a TB clinic. However, laboratory findings identified R. equi in his sputum sample based on a positive acid-fast stain, which was highly suggestive of a pulmonary infection caused by R. equi. The patient was subsequently admitted to the respiratory unit for treatment. Once the source of infection was identified, the patient was treated with a combination of antibiotics for 2 weeks and was discharged with a significant improvement in symptoms.

Arcanobacterium haemolyticum: A case series.

Arcanobacterium haemolyticum formerly known as Corynebacteria haemolyticum is a Gram positive bacilli. It is a fastidious, facultative anerobic, catalase negative, beta haemolytic and non motile bacterium. Gram positive bacilli are usually considered to be non-pathogenic as the majority are part of normal flora of human skin and mucous membranes. Hence, diagnosis of such infection and its treatment may be delayed by a failure of recognition. However, this bacterium has been implicated in wound, superficial and deep-seated soft tissue infections, endocarditis, osteomyelitis, meningitis, pneumonia, and also septicemia. The early diagnostic evaluation of this organism is emphasized. We report a case series which illustrates the significance of Arcanobacterium haemolyticum in skin and soft tissue infections.

Fecal concentration of Rhodococcus equi determined by quantitative polymerase chain reaction of rectal swab samples to differentiate foals with pneumonia from healthy foals.

BACKGROUND: Diagnostic accuracy of real-time, quantitative PCR (qPCR) assays to quantify virulent Rhodococcus equi using rectal swab samples has not been systematically evaluated. OBJECTIVE: To evaluate the accuracy of qPCR of rectal swab samples to differentiate foals with pneumonia from healthy foals of similar age from the same environment. ANIMALS: One hundred privately owned foals born in 2021 from 2 farms in New York. METHODS: An incident case-control study design was used. Rectal swabs were collected from all foals diagnosed with R. equi pneumonia at 2 horse-breeding farms (n = 47). Eligible pneumonia cases (n = 39) were matched by age to up to 2 healthy (n = 53) control foals; rectal swabs were collected from control foals on the day of diagnosis of the index case. DNA was extracted from fecal swabs and the concentration of virulent R. equi (ie, copy numbers of the virulence-associated protein A gene [vapA] per 100 ng fecal DNA) was estimated by qPCR. RESULTS: The area under the ROC curve for qPCR of fecal swabs was 83.7% (95% CI, 74.9-92.6). At a threshold of 14 883 copies of vapA per 100 ng fecal DNA, specificity of the assay was 83.0% (95% CI, 71.7-92.4) and sensitivity was 79.5% (95% CI, 66.7-92.3). CONCLUSIONS AND CLINICAL IMPORTANCE: Although fecal concentrations of virulent R. equi are significantly higher in pneumonic foals than healthy foals of similar age in the same environment, qPCR of rectal swabs as reported here lacks adequate diagnostic accuracy for clinical use.

An Uncommon Case of Trueperella pyogenes Infection in an Adult Backyard Rooster and a Retrospective Study; 2000-20.

Trueperella pyogenes is an opportunistic Gram-positive bacterium that induces purulent lesions and abscesses in cattle, small ruminants, and swine. In birds, T. pyogenes infections have been linked to lameness and osteomyelitis in turkeys (Phasianidae) and hepatic fibriscess in turkeys and pigeons (Columbidae). An 18-mo-old backyard rooster with a history of progressive emaciation was submitted to the California Animal Health and Food Safety (CAHFS) laboratory system. At necropsy, unusual numerous miliary granulomas were identified, primarily in the spleen, but granulomas were also observed in air sacs and lungs. Microscopically, few to moderate numbers of granulomas with giant cells were observed in the spleen, lung, air sacs, and crop composed of necrosis and mixed inflammatory cell inflammation including multinucleated giant cells, fibrin deposition, and fibrosis. Trueperella pyogenes was isolated from the air sacs and trachea. Avibacterium paragallinarum PCR was positive from the tracheal swab. A retrospective analysis of CAHFS data on T. pyogenes between 2000 and 2020 identified 24 cases in avian species: chickens (Gallus gallus domesticus; 16/24), turkeys (5/24), Pekin duck (Anas platyrhynchos domesticus; 1/24), parrot (Psittaciformes; 1/24), and pheasant (Phasianidae; 1/24). Although T. pyogenes infection in birds is rare, the clinical signs and gross lesions might be indistinguishable from avian mycobacteriosis in some cases and should be considered in the differential diagnosis.

Reporte de caso­Un caso no común de infección por Trueperella pyogenes en un gallo adulto de traspatio y un estudio retrospectivo; entre los años 2000-20. Trueperella pyogenes es una bacteria grampositiva oportunista que induce lesiones purulentas y abscesos en bovinos, pequeños rumiantes y porcinos. En las aves, las infecciones por T. pyogenes se han relacionado con cojera y osteomielitis en pavos (Phasianidae) y fibrosis hepática en pavos y palomas (Columbidae). Un gallo de traspatio de 18 meses de edad con antecedentes de emaciación progresiva fue enviado al sistema de Laboratorios de Salud Animal y Seguridad Alimentaria de California (CAHFS). En la necropsia, se identificaron numerosos granulomas miliares inusuales, principalmente en el bazo, pero también se observaron granulomas en los sacos aéreos y los pulmones. Microscópicamente, se observaron pocos a moderados granulomas con células gigantes en el bazo, pulmón, sacos aéreos y buche compuesto por necrosis e inflamación celular inflamatoria mixta, incluidas células gigantes multinucleadas, depósito de fibrina y fibrosis. Trueperella pyogenes se aisló de los sacos aéreos y la tráquea. Un método de PCR para Avibacterium paragallinarum fue positivo realizado a partir de hisopos traqueales. Un análisis retrospectivo de los datos de CAHFS sobre T. pyogenes entre los años 2000 y 2020 identificó 24 casos en especies aviares: pollos (Gallus gallus domesticus; 16/24), pavos (5/24), pato Pekín (Anas platyrhynchos domesticus; 1/24), loro (Psittaciformes; 1/24) y faisán (Phasianidae; 1/24). Aunque la infección por T. pyogenes en aves es poco común, los signos clínicos y las lesiones macroscópicas pueden ser indistinguibles de micobacteriosis aviar en algunos casos y debe considerarse como diagnóstico diferencial.

Rhodococcus equi infection as inaugural manifestation of idiopathic CD4+ lymphopenia: A rare entity and a therapeutic challenge.

We report a case of disseminated infection by Rhodococcus equi as the inaugural manifestation of idiopathic T-CD4+ lymphopenia. We aim to demonstrate our diagnostic and therapeutic approach and focus on the major dilemmas arising from the lack of scientific evidence regarding best clinical practice of this infection in humans.

Bacteremia and sepsis by Arcanobacterium haemolyticum in a young immunocompetent patient / Bacteriemia y sepsis por Arcanobacterium haemolyticum en un paciente joven inmunocompetente

Abstract We report the case of a twenty-year-old immunocompetent male patient presenting to the emergency room with pharyngitis and fever. Blood cultures were drawn and Arcanobacterium haemolyticum (rough biotype) was recovered. The presence of the arcanolysin gene was investigated at the molecular level and the upstream region was amplified and sequenced in order to correlate it with the smooth or rough biotype. Although the isolate was susceptible to penicillin, vancomycin and gentamicin, empirical treatments first with amoxicillin/clavulanic acid (1g/12h) and then with ceftriaxone (1g/12h) failed and the infection evolved to sepsis. Finally, treatment with vancomycin (1 g/12 h) plus piperacillin/tazobactam (4.5g/8h) was effective. Lemierre's syndrome was ruled out. To the best of our knowledge, this is the first case of bacteremia by A. haemolyticum reported in Argentina.

Resumen Se describe el caso de un paciente varón inmunocompetente de veinte anos de edad que se presentó en la sala de emergencias con faringitis y fiebre. Se extrajeron muestras para realizar hemocultivos y se recuperó Arcanobacterium haemolyticum (biotipo rugoso). Se investigó la presencia del gen de la arcanolisina por un método molecular, y se amplificó y Faringitis; secuenció la región upstream de dicho gen para determinar su correlación con los biotipos lisos Bacteriemia; o rugosos. Aunque el aislamiento fue sensible a la penicilina, la vancomicina y la gentamicina, Sepsis; los tratamientos empíricos primero con amoxicilina/ácido clavulánico (1 g/12 h) y luego con Síndrome de Lemierre ceftriaxona (1 g/12 h) no fueron efectivos, y la infección evolucionó a sepsis. Finalmente, el tratamiento con vancomicina (1 g/12 h) más piperacilina/tazobactam (4,5g/8h) fue efectivo. Se descartó la presencia del síndrome de Lemierre. Según nuestro conocimiento, este es el primer caso de bacteriemia por A. haemolyticum reportado en Argentina.

A case report of the differential diagnosis of Cellulosimicrobium cellulans-infected endocarditis combined with intracranial infection by conventional blood culture and second-generation sequencing.

BACKGROUND: Cellulosimicrobium cellulans is a gram-positive filamentous bacterium found primarily in soil and sewage that rarely causes human infection, especially in previously healthy adults, but when it does, it often indicates a poor prognosis. CASE PRESENTATION: We report a case of endocarditis and intracranial infection caused by C. cellulans in a 52-year-old woman with normal immune function and no implants in vivo. The patient started with a febrile headache that progressed to impaired consciousness after 20 days, and she finally died after treatment with vancomycin combined with rifampicin. C. cellulans was isolated from her blood cultures for 3 consecutive days after her admission; however, there was only evidence of C. cellulans sequences for two samples in the second-generation sequencing data generated from her peripheral blood, which were ignored by the technicians. No C. cellulans bands were detected in her cerebrospinal fluid by second-generation sequencing. CONCLUSIONS: Second-generation sequencing seems to have limitations for certain specific strains of bacteria.

Rhodococcus equi, primer reporte de un caso de enfermedad diseminada en el Perú / Rhodococcus equi, first case report of disseminated disease in Peru

Resumen Presentamos el caso de un paciente con infección por virus de inmunodeficiencia humana (VIH) con recuento de LTCD4+ 49 céls/mm3, que consultó por un cuadro de siete meses de baja de peso, dolor abdominal, diarrea crónica y lesiones cutáneas gomosas. El mielocultivo y hemocultivos fueron positivos para Rhodococcus equi. Además, se observaron lesiones histológicas en piel e intestino compatibles con este agente como malacoplaquia, reacción granulomatosa y cuerpos de Michaelis-Gutmann. Se descartó compromiso pulmonar mediante tomografía de tórax. Recibió terapia antibacteriana combinada con claritromicina, imipenem y vancomicina. A pesar del tratamiento, el paciente evolucionó desfavorablemente y falleció.

Abstract We present the case of a patient with human immunodeficiency virus (HIV) with a LTCD4 + 49 cells/mm3, who was admitted due to a seven-month period of weight loss, abdominal pain, chronic diarrhea and rubbery skin lesions. Myeloculture and blood cultures were positive for Rhodococcus equi. In addition, histological lesions in the skin and intestine compatible with this agent were observed, such as malacoplachy, granulomatous reaction and Michaelis-Gutmann bodies. Pulmonary involvement was ruled out by chest tomography. The patient received antibacterial therapy combined with clarithromycin, imipenem, and vancomycin. Despite the treatment, the patient evolved unfavorably and died.

Paradoxical worsening of chest radiographs secondary to immune reconstitution syndrome (IRIS) in a patient with advanced HIV infection and Rhodococcus pneumonia.

We present a rare case of post-antiretroviral therapy (ART) paradoxically worsening of radiological findings in a patient with advanced HIV-infection on treatment for Rhodococcus pneumonia who was misdiagnosed with pulmonary tuberculosis. Despite clinical improvement, serial chest radiographs showed deteriorations a month after starting ART. This was attributed to Immune Reconstitution Inflammatory Syndrome (IRIS) which spontaneously resolved without any treatment.

Bacteremia and sepsis by Arcanobacterium haemolyticum in a young immunocompetent patient.

We report the case of a twenty-year-old immunocompetent male patient presenting to the emergency room with pharyngitis and fever. Blood cultures were drawn and Arcanobacterium haemolyticum (rough biotype) was recovered. The presence of the arcanolysin gene was investigated at the molecular level and the upstream region was amplified and sequenced in order to correlate it with the smooth or rough biotype. Although the isolate was susceptible to penicillin, vancomycin and gentamicin, empirical treatments first with amoxicillin/clavulanic acid (1g/12h) and then with ceftriaxone (1g/12h) failed and the infection evolved to sepsis. Finally, treatment with vancomycin (1g/12h) plus piperacillin/tazobactam (4.5g/8h) was effective. Lemierre's syndrome was ruled out. To the best of our knowledge, this is the first case of bacteremia by A. haemolyticum reported in Argentina.

[Rhodococcus equi, first case report of disseminated disease in Peru]. / Rhodococcus equi, primer reporte de un caso de enfermedad diseminada en el Perú.

We present the case of a patient with human immunodeficiency virus (HIV) with a LTCD4 + 49 cells/mm3, who was admitted due to a seven-month period of weight loss, abdominal pain, chronic diarrhea and rubbery skin lesions. Myeloculture and blood cultures were positive for Rhodococcus equi. In addition, histological lesions in the skin and intestine compatible with this agent were observed, such as malacoplachy, granulomatous reaction and Michaelis-Gutmann bodies. Pulmonary involvement was ruled out by chest tomography. The patient received antibacterial therapy combined with clarithromycin, imipenem, and vancomycin. Despite the treatment, the patient evolved unfavorably and died.

Soft tissue infection caused by Mycolicibacter kumamotonensis.

Mycolicibacter kumamotonensis (M. kumamotonensis), formerly Mycobacterium kumamotonense, is a nontuberculous mycobacteria species, which was first separated from Mycobacterium terrae complex in 2006. Reports about infections caused by M. kumamotonensis are extremely rare, with most of them being lung infection. Here, we report the case of a 68-year-old man with a hobby of gardening who developed swelling in his right middle finger. He underwent surgical debridement at a previous hospital and was diagnosed with nontuberculous mycobacteria infection based on positive findings of acid-fast staining of pus obtained from the surgical specimen. He was treated with rifampicin, ethambutol, and clarithromycin, but the swelling worsened. Therefore, he was referred to our hospital for further examination and treatment. We performed a second debridement and added isoniazid to the treatment regimen, but the swelling continued to worsen. We then administered levofloxacin, but his condition did not change. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and DNA sequencing analysis confirmed M. kumamotonensis as the causative bacterium. Since the finger swelling did not improve, the patient underwent a third debridement and amikacin was added to the treatment regimen. Finally, the infection was controlled. He completed amikacin therapy and will continue treatment with the other five antibiotics for a total of 24 months. To the best of our knowledge, this is the first report of a patient with M. kumamotonensis soft tissue infection. We consider this case might provide important insights into the diagnosis and treatment of soft tissue infections caused by M. kumamotonensis.

Half of 'Micrococcus spp.' cases identified by conventional methods are revealed as other life-threatening bacteria with different drug susceptibility patterns by 16S ribosomal RNA gene sequencing.

Bacterial infection during chemotherapy is a fatal complication, therefore precise identification of the pathogenic microorganism is required for treatment. We report that 2 of 4 pediatric patients with malignancy who were diagnosed with Micrococcus spp. infection by conventional methods were finally revealed to have Kytococcus schroeteri and Kocuria marina infection by 16S ribosomal RNA gene sequence analysis (16S rRNA analysis). Although K. schroeteri is morphologically similar to Micrococcus spp., its drug susceptibility profile is quite different from that of Micrococcus spp. K. schroeteri is resistant to penicillin and cephalosporin, which are effective for Micrococcus spp. In fact, penicillin-resistant lethal pneumonia caused by K. schroeteri has been reported in compromised hosts. Based on our results, Micrococcus spp. determined by conventional methods could contain other life-threatening bacteria with different drug susceptibility patterns from Micrococcus spp. To develop an effective empirical treatment for immunocompromised hosts, accumulation of pathogen data by 16S rRNA analysis is required.

Development of a novel Trueperella pyogenes-specific PCR assay.

Trueperella pyogenes is an opportunistic pathogen that causes a wide variety of purulent infections. We recently isolated a T. pyogenes strain unable to be identified by the previously reported T. pyogenes pyolysin gene (plo)-specific PCR from the lung of a sheep with astasia. Sequence comparison of plo among representative strains revealed several nucleotide substitutions in the primer-annealing regions. As such substitutions were considered to be a reason for the low PCR specificity, we designed novel primers in conserved regions of plo. Under optimized conditions, the novel primers precisely identified all T. pyogenes strains tested, and no products were generated from any other bacterial strains, suggesting the usefulness of the novel PCR assay for the diagnosis of T. pyogenes infections.