Extrapulmonary Legionella micdadei infection in a previously healthy child

A previously healthy 9-year-old girl presented with a 1-week history of left neck mass and low grade fever. Surgical drainage specimens from the mass were bacterial culture-negative. Legionella micdadei infection was diagnosed by a two step PCR-nucleic acid sequencing method and supported by organism-specific serology. A backyard hot tub was suspected as the source of infection. Infection with Legionella organisms usually presents as pneumonia and occurs primarily in immunocompromised patients. 1–4 There are >30 different species of Legionella, with Legionella pneumophila as the most common disease-causing agent and Legionella micdadei as the second. Legionella spp. have also been recovered from sternal wound infections in cardiac patients 5 as well as from cases of lung abscess. 6, 7 Extrathoracic infections with L. micdadei, however, occur rarely. Ampel et al. 8 reported a case of a cutaneous leg abscess in an immunocompromised patient that grew L. micdadei. Kilborn et al. 9 isolated L. micdadei in a renal transplant patient who initially had a purulent lesion of the finger that progressed to cellulitis of the arm. L. micdadei is a Gram-negative bacillus that can stain weakly acid-fast when in tissue or sputum specimens. It is a fastidious organism that will not grow on routinely used microbiologic media. Buffered charcoal yeast extract, rich in l-cysteine, is the recommended medium for isolation of Legionella organisms, and colonies on a primary charcoal yeast extract agar plate have a mottled, cut glass appearance under low magnification (e.g. dissecting microscope). Unless Legionella is suspected and the clinical laboratory alerted, isolation of these organisms is unlikely. We present a case of extrapulmonary L. micdadei infection in a healthy child and demonstrate how the diagnosis was made in the clinical microbiology laboratory by molecular techniques. Case report. The patient is a previously healthy 9-year-old girl with a 1-week history of fever and enlarging left neck mass. She had been treated with amoxicillin/clavulanic acid 3 days before admission, but the mass had continued to enlarge. The patient had no history of trauma, animal bite or scratch, travel outside of the country or contact with unpasteurized milk products. She had no known risk factors for HIV and no known contacts with individuals with tuberculosis or positive purified protein derivative skin tests. On admission the patient had a 3- by 4-cm high posterior cervical neck mass on the left side. Physical examination was otherwise unremarkable. Admission laboratory studies, including complete blood count, were normal. Bartonella serology was negative. A sonogram of the left neck revealed a 2.2- by 1.9-cm mass superficial to the sternocleidomastoid muscle, containing thick septations and central hypoechoic areas, as well as multiple reactive lymph nodes. The patient was treated empirically for cervical adenitis with intravenous clindamycin. During the next 3 days she continued to have intermittent low grade fever to 38.5°C, low energy and poor appetite. Because of poor clinical response to medical therapy, computed tomography of the neck was performed on Hospital Day 3, which demonstrated enhancing lymphadenopathy on the left side, with two distinct areas of low density compatible with abscess. The patient underwent incision and drainage with removal of 10 ml of yellow pus, which was sent for bacterial (aerobic and anaerobic), fungal and mycobacterial cultures, as well as for Bartonella PCR; all tests were negative. Postoperatively there was some improvement in left neck swelling despite continued fevers to 38.5°C. The patient was discharged on Postoperative Day 3 to complete a 10-day course of oral azithromycin. Two weeks after discharge the patient had improved clinically in terms of energy and appetite, but her fevers persisted. Her neck incision had partially reopened. On closer examination the incision margins appeared red and edematous with some serosanguineous drainage, and a mass ∼2 by 2 cm was palpable beneath the incision. A sample of drainage material was sent for bacterial culture, which was again sterile. The next day the bacterial broad spectrum PCR 10 and sequencing data indicated the presence of L. micdadei DNA in the original surgical specimen initially tested for Bartonella DNA. The patient was treated with oral clarithromycin 500 mg twice daily. At follow-up 6 weeks post-incision and drainage, the patient had returned to her baseline state of good health, with normal appetite and energy. The incision was well-healed, with only very subtle fullness at the site. Paired sera were sent for L. micdadei-specific serology (Specialty Laboratories, Santa Monica, CA) and were strongly positive; specific antibody titers rose from 1/128 for both IgG and IgM in the first set (taken during the hospital admission) to 1/256 for both in the second set (taken 3 weeks later; reference levels <1/64). Confirmatory PCR for L. micdadei was performed on the original archived surgical specimen material (purulent drainage material stored sterile in –70°C) with the use of the L. micdadei-specific mip (macrophage infectivity potentiator) gene signature sequences. 11 Primers MIP-f 5′-GAT GAG ATT GGT CGC TG-3′ and MIP-r 5′-CCT TCA TCT GGT CGT CG-3′ were specifically designed for the Step 2 molecular confirmation of the case diagnosis. A 260-bp mip amplicon was obtained and sequenced and was found to be 100% identical with the respective GenBank database sequences. Acridine orange stain of a reserved smear later revealed clusters of rod-shaped organisms. Discussion. The most common clinical Legionella infection, Legionnaires’ disease, presents with primarily pulmonary manifestations, whereas Pontiac fever presents as an influenza-like febrile illness without pneumonia. Extrathoracic infections by Legionella spp. are rare. Beyond outbreak settings legionellosis (whether pulmonary or extrathoracic) has not been documented in children; specifically Legionella lymphadenitis has not previously been reported. In our case of cervical adenitis unresponsive to empiric therapy and surgical management, the differential diagnosis included cat-scratch disease, nontuberculous mycobacterial adenitis, actinomycosis, nocardiosis, mononucleosis and wound infection (e.g. Mycoplasma). Appropriate culture, staining and Bartonella-specific PCR of surgical specimens were nondiagnostic, as was Bartonella serology. A molecular research technique using broad spectrum bacterial 16S ribosomal DNA primers has played an important role in culture-negative bacterial diagnosis in our microbiology laboratory. Using this technique we have been able to screen successfully for specific bacterial etiologies with properly collected specimens such as heart valves from clinically suspected endocarditis and sterile body fluids. 12 To reduce potential false positive results caused by contamination and indiscriminate DNA amplification by the broad spectrum bacterial 16S ribosomal DNA primers, we have established a molecular confirmatory PCR step tailored to the initial findings. In this case a L. micdadei-specific signature sequence of the mip gene was used in a split drainage sample for result confirmation. In support of the diagnosis paired sera also suggested L. micdadei infection. We believe that legionellosis in this healthy child was caused by the presence of L. micdadei in the patient’s home environment, and we specifically suspect a backyard hot tub as the source of infection. Because Legionella organisms can persist for prolonged periods in plumbing fixtures, 13 aquatic devices such as whirlpools and hot tubs can serve as reservoirs. Without proper disinfection (whether the result of disinfection system failure or suboptimal technique), whirlpool spas may become hazards to health, with the accumulation of organisms in unusually high concentrations; several documented outbreaks have been traced back to such sources. 2, 13–15 We were, however, unable to access the home environment to evaluate this hypothesis. Further we suspect that the oral cavity provided the portal of entry in this case, via either the oropharyngeal mucosa or tonsillar tissue, although no lesions were appreciated on presentation and dentition was normal. Alternatively organisms could have gained entry into the lymphatic system through undetected minor skin breaks in the scalp. 8, 9 A pulmonary portal of entry seems unlikely in our case in the absence of any signs or symptoms referable to the respiratory system. Although such an unusual infection raises the specter of underlying immunodeficiency, we consider this patient to be an immunologically normal host. Her growth and development have been normal, with no previous invasive infections, hospitalizations, or transfusions. Studies concerning the microbiology of lymphadenitis and head-neck abscesses have indicated that the spectrum of bacterial isolates includes both aerobic and anaerobic organisms, with Staphylococcus aureus and group A streptococci isolates constituting the majority of positive cultures, whereas the incidence of culture negativity can range from 15 to 25%. 16, 17 With increased use of molecular techniques, however, pathogens associated with culture-negative infections (e.g. Bartonella spp., Yersinia spp. and nontuberculous mycobacterial species) are being identified more frequently. Physicians should be aware of such emerging pathogens and, when suspicious, should contact clinical microbiologists so as to optimize the likelihood of recovering causative organisms. The popularity of hot tubs and Jacuzzis in residential homes calls for an increased clinical index of suspicion for Legionella infection where a suggestive exposure history is obtained. Our case demonstrates the growing role of molecular techniques in the diagnosis of infections caused by fastidious organisms, as well as in expanding our appreciation for the spectrum of diseases in which they may be involved.